Real-time PCR in the microbiology laboratory

  • I.M. Mackay
    Correspondence
    Corresponding author and reprint requests: I. M. Mackay, CVRU, SASVRC, Royal Children's Hospital, Herston Road, Herston, Queensland 4029, Australia
    Affiliations
    Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre

    Department of Paediatrics, Royal Children's Hospital, Brisbane, Queensland, Australia
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      ABSTRACT

      Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.

      Keywords

      BACKGROUND

      Diagnostic microbiology is in the midst of a new era. Rapid nucleic acid amplification and detection technologies are quickly displacing the traditional assays based on pathogen phenotype rather than genotype. The polymerase chain reaction (PCR) [
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      ] has increasingly been described as the latest gold standard for detecting some microbes, but such claims can only be taken seriously when each newly described assay is suitably compared to its characterised predecessors [
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      Effect of delayed processing of blood samples on performance of cytomegalovirus antigenemia assay.
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      Discrepant analysis: how can we test a test?.
      ,
      • Sternberg M
      Discrepant analysis is still at large.
      ,
      • Jeffery KJM
      • Read SJ
      • Peto TEA
      • Mayon‐White RT
      • Bangham CRM
      Diagnosis of viral infections of the central nervous system: clinical interpretation of PCR results.
      ,
      • Pfaffl MW
      • Horgan GW
      • Dempfle L
      Relative expression software tool (REST©) for group‐wise comparison and statistical analysis of relative expression results in real‐time PCR.
      ,
      • Bustin SA
      Absolute quantification of mRNA using real‐time reverse transcription polymerase chain reaction assays.
      ,
      • Nagano M
      • Kelly PA
      Tissue distribution and regulation of rat prolactin receptor gene expression.
      ]. PCR is the most commonly used nucleic acid amplification technique for the diagnosis of infectious disease, surpassing the probe and signal amplification methods. The PCR can amplify DNA or, when preceded by a reverse transcription (RT) incubation at 42-55 °C, RNA. RT-PCR is the most sensitive method for the detection and quantitation of mRNA, especially for low-abundance templates [
      • Pfaffl MW
      • Horgan GW
      • Dempfle L
      Relative expression software tool (REST©) for group‐wise comparison and statistical analysis of relative expression results in real‐time PCR.
      ,
      • Bustin SA
      Absolute quantification of mRNA using real‐time reverse transcription polymerase chain reaction assays.
      ,
      • Nagano M
      • Kelly PA
      Tissue distribution and regulation of rat prolactin receptor gene expression.
      ,
      • Gause WC
      • Adamovicz J
      The use of the PCR to quantitate gene expression.
      ]. The PCR process can be divided into three steps. First, double-stranded DNA (dsDNA) is separated at temperatures above 90 °C. Second, oligonucleotide primers generally anneal at 50–60 °C, and, finally, optimal primer extension occurs at 70–78 °C. The temperature at which the primer anneals is usually referred to as the TM. This is the temperature at which 50% of the oligonucleotidetarget duplexes have formed. In the case of real-time PCR, the oligonucleotide could represent a primer or a labelled probe. The TM differs from the denaturation temperature (TD), which refers to the TM as it applies to the melting of dsDNA. The rate of temperature change or ramp rate, the length of the incubation at each temperature and the number of times each cycle of temperatures is repeated are controlled by a programmable thermal cycler. Current technologies have significantly shortened the ramp rates, and therefore assay time, through the use of electronically controlled heating blocks or fan-forced heated air flows.
      The traditional diagnostic microbiological assays include microscopy, microbial culture, antigenaemia and serology. These can be limited by poor sensitivity, slow-growing or poorly viable organisms, narrow detection windows, complex interpretation, immunosuppression, antimicrobial therapy, high levels of background and non-specific cross-reactions [
      • Whelan AC
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      ]. Nonetheless, microbial culture produces valuable epidemiological data, revealing new, uncharacterised or atypical microbes and yielding intact or infectious organisms for further study [
      • Ogilvie M
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      ]. It is therefore clear that the role of the traditional assay continues to be an important one [
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      ,
      • Sintchenko V
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      ,
      • Ellis JS
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      Molecular diagnosis of influenza.
      ,
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      Laboratory confirmation of meningococcal disease in Scotland, 1993–9.
      ,
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      Development of polymerase chain reaction‐based assays for bacterial gene detection.
      ]. Additionally, PCR has some significant limitations. Our ability to design oligonucleotide primers only extends to our knowledge of a microorganism's genome as well as the ability of publicly available sequence databases to suitably represent all variants of that microbe. It is common for microbial genomes to contain unexpected mutations, which reduce or abrogate the function of a PCR. Traditionally, false-positives due to carryover contamination have caused considerable problems in the routine implementation of PCR in the diagnostic laboratory and have led to strict guidelines for the design of laboratories dedicated to performing PCR. Additionally, PCR may be too sensitive for some applications, detecting a microbe that is present at non-pathogenic levels. Thus, care is required when designing a PCR assay and interpreting its results.
      Existing combinations of PCR and amplicon detection assays will be called ‘conventional PCR’ throughout this review. The detection components include agarose gel electrophoresis [
      • Kidd IM
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      A non‐radioisotopic quantitative competitive polymerase chain reaction method: application in measurement of human herpesvirus 7 load.
      ], Southern blot [
      • Holland PM
      • Abramson RD
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      • Gelfand DH
      Detection of specific polymerase chain reaction product by utilizing the 5′−3′ exonuclease activity of Thermus aquaticus.
      ] and ELISA-like systems [
      • Van Der Vliet GME
      • Hermans CJ
      • Klatser PR
      Simple colorimetric microtiter plate hybridization assay for detection of amplified Mycobacterium leprae DNA.
      ]. Conventional PCR has been used to obtain quantitative data, with promising results [
      • Mackay IM
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      • Wei MQ
      Quantitative PCR‐ELAHA for the determination of retroviral vector transduction efficiency.
      ]. However, these approaches have suffered from the laborious post-PCR handling steps required to evaluate the amplicon [
      • Guatelli JC
      • Gingeras TR
      • Richman DD
      Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infection.
      ].
      The possibility that, in contrast to conventional PCR, the detection of amplicon could be visualised as the amplification progressed was a welcome one. This expanded the role of PCR from that of a pure research tool to that of a versatile technology permitting the development of routine diagnostic applications for the high-and low-throughput clinical microbiology laboratory [
      • Lomeli H
      • Tyagi S
      • Pritchard CG
      • Lizardi PM
      • Kramer FR
      Quantitative assays based on the use of replicatable hybridization probes.
      ,
      • Cockerill III, FR
      • Smith TF
      Rapid‐cycle real‐time PCR: a revolution for clinical microbiology.
      ]. Along the way, real-time assays have provided insight into the kinetics of the PCR as well as the efficiency of different nucleic acid extraction methods and the role that some compounds play in the inhibition of amplification [
      • Holland PM
      • Abramson RD
      • Watson R
      • Gelfand DH
      Detection of specific polymerase chain reaction product by utilizing the 5′−3′ exonuclease activity of Thermus aquaticus.
      ,
      • Lee LG
      • Connell CR
      • Bloch W
      Allelic discrimination by nick‐translation PCR with fluorogenic probes.
      ,
      • Livak KJ
      • Flood SJA
      • Marmaro J
      • Giusti W
      • Deetz K
      Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.
      ,
      • Heid CA
      • Stevens J
      • Livak KJ
      • Williams PM
      Real time quantitative PCR.
      ,
      • Gibson UEM
      • Heid CA
      • Williams PM
      A novel method for real time quantitative RT‐PCR.
      ,
      • Niesters HG
      • Van Esser J
      • Fries E
      • Wolthers KC
      • Cornelissen J
      • Osterhaus AD
      Development of a real‐time quantitative assay for detection of Epstein–Barr virus.
      ,
      • Read SJ
      Recovery efficiencies of nucleic acid extraction kits as measured by quantitative LightCycler™ PCR.
      ,
      • Biel SS
      • Held TK
      • Landt O
      • et al.
      Rapid quantification and differentiation of human polyomavirus DNA in undiluted urine from patients after bone marrow transplantation.
      ,
      • Petrik J
      • Pearson GJM
      • Allain J‐P
      High throughput PCR detection of HCV based on semiautomated multisample RNA capture.
      ]. Real-time PCR has made many more scientists familiar with the crucial factors contributing to successful amplification of nucleic acids. Today, real-time PCR is used to detect nucleic acids from food, vectors used in gene therapy protocols, genetically modified organisms, and areas of human and veterinary microbiology and oncology [
      • Klein D
      Quantification using real‐time PCR technology: applications and limitations.
      ,
      • Ahmed FE
      Detection of genetically modified organisms in foods.
      ,
      • Mhlanga MM
      • Malmberg L
      Using molecular beacons to detect single‐nucleotide polymorphisms with real‐time PCR.
      ].
      The monitoring of accumulating amplicon in real time has been made possible by the labelling of primers, oligonucleotide probes (oligoprobes) or amplicons with molecules capable of fluorescing. These labels produce a change in signal following direct interaction with, or hybridisation to, the amplicon. The signal is related to the amount of amplicon present during each cycle and will increase as the amount of specific amplicon increases. These chemistries have clear benefits over earlier radiogenic labels, including an absence of radioactive emissions, easy disposal and an extended shelf-life [
      • Matthews JA
      • Kricka LJ
      Analytical strategies for the use of DNA probes.
      ].
      A significant improvement introduced by real-time PCR is the increased speed with which it can produce results. This is largely due to the reduced cycle times, removal of separate post-PCR detection procedures, and the use of sensitive fluorescence detection equipment, allowing earlier amplicon detection [
      • Wittwer CT
      • Fillmore GC
      • Garling DJ
      Minimizing the time required for DNA amplification by efficient heat transfer to small samples.
      ,
      • Wittwer CT
      • Ririe KM
      • Andrew RV
      • David DA
      • Gundry RA
      • Balis UJ
      The LightCyclerTM: a microvolume multisample fluorimeter with rapid temperature control.
      ]. A reduced amplicon size may also play a role in this speed; however, it has been shown that decreased product size does not strictly correlate with improved PCR efficiency, and that the distance between the primers and the oligoprobe may play a more significant role [
      • Nitsche A
      • Steuer N
      • Schmidt CA
      • et al.
      Detection of human cytomegalovirus DNA by real‐time quantitative PCR.
      ,
      • Lunge VR
      • Miller BJ
      • Livak KJ
      • Batt CA
      Factors affecting the performance of 5′ nuclease PCR assays for Listeria monocytogenes detection.
      ].
      The technical disadvantages of using real-time PCR instead of conventional PCR include the need to break the seal of an otherwise closed system in order to monitor amplicon size, the incompatibility of certain platforms with some fluorescent chemistries, and the relatively restricted multiplex capabilities of current systems. Additionally, the start-up expense of real-time PCR may be prohibitive for low-throughput laboratories.
      Because most of the popular real-time PCR chemistries involve hybridisation of an oligoprobe(s) to a complementary sequence on one of the amplicon strands, the inclusion of more of the primer that creates that strand is beneficial to the generation of an increased fluorescent signal [
      • Gyllensten UB
      • Erlich HA
      Generation of single‐stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA‐DQA locus.
      ]. We have found that this asymmetric PCR approach improves the signal from both our conventional and real-time oligoprobe-hybridisation assays.
      Although some of the fluorescent labels have been given an associated nomenclature by their developer, the term ‘fluorophore’ will generally be used to describe these moieties, while their inclusion as labels on an oligonucleotide will be described as rendering it ‘fluorogenic’. The most commonly used fluorogenic oligoprobes rely upon fluorescence resonance energy transfer (FRET) between fluorogenic labels or between one fluorophore and a dark or black-hole non-fluorescent quencher (NFQ), which disperses energy as heat rather than fluorescence [
      • Didenko VV
      DNA probes using fluorescence resonance energy transfer (FRET): designs and applications.
      ]. FRET is a spectroscopic process by which energy is passed between molecules separated by 10-100 Å that have overlapping emission and absorption spectra [
      • Stryer L
      • Haugland RP
      Energy transfer: a spectroscopic ruler.
      ,
      • Heller MJ
      • Morrison LE
      ,
      • Clegg RM
      Fluorescence resonance energy transfer and nucleic acids.
      ]. Förster primarily developed the theory behind this process, which is a non-radiative induced dipole interaction [
      • Didenko VV
      DNA probes using fluorescence resonance energy transfer (FRET): designs and applications.
      ,
      • Förster T
      Zwischenmolekulare energiewanderung und fluoreszenz.
      ,
      • Selvin P
      Fluorescence resonance energy transfer.
      ].
      As alluded to earlier, post-amplification manipulation of the amplicon is not required for real-time PCR, because the fluorescent signals are directly measured as they pass out of the reaction vessel, so real-time PCR is often described as a ‘closed’ or homogeneous system. Apart from the time saved by amplifying and detecting template in a single tube, there is minimal potential for carryover contamination, and the assay's performance can be closely scrutinised without introducing errors due to handling of the amplicon [
      • Higuchi R
      • Fockler C
      • Dollinger G
      • Watson R
      Kinetic PCR analysis: real‐time monitoring of DNA amplification reactions.
      ]. In addition, real-time PCR has proven to be cost-effective on a per-run basis, when implemented in a high-throughput laboratory [
      • Martell M
      • Gómez J
      • Esteban JI
      • et al.
      High‐throughput real‐time reverse transcription‐PCR quantitation of hepatitis C virus RNA.
      ], particularly when replacing conventional, culture-based approaches to microbial detection.
      In the remainder of this review, the theory behind real-time PCR will be discussed. Additionally, its rapidly expanding use in the study of human infectious disease will provide an example of its acceptance and effectiveness in the diagnostic microbiology laboratory.

      AMPLICON DETECTION

      It is the detection process that discriminates real-time PCR from conventional PCR assays. There is a range of chemistries currently in use which can be broadly categorised as specific or non-specific for the amplicon's sequence [
      • Whitcombe D
      • Theaker J
      • Guy SP
      • Brown T
      • Little S
      Detection of PCR products using self‐probing amplicons and fluorescence.
      ]. These have recently been reviewed in detail [
      • Mackay IM
      • Arden KE
      • Nitsche A
      Real‐time PCR in virology.
      ]. Several additional reporter systems have since been described, and these will be discussed below; however, few applications have been described for the specific detection and genotyping of microbes.
      While the most common oligoprobes are based on traditional nucleic acid chemistry, the peptide nucleic acid (PNA) is becoming a more popular choice for oligonucleotide backbones. The PNA is a DNA analogue that is formed of neutral repeated N-(2-aminoethyl) glycine units instead of negatively charged sugar phosphates [
      • Egholm M
      • Buchardt O
      • Christensen L
      • et al.
      PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen bonding rules.
      ]. However, the PNA retains the same sequence recognition properties as DNA.
      In general, however, the specific and non-specific fluorogenic chemistries detect amplicon with the same sensitivity [
      • Wittwer CT
      • Ririe KM
      • Andrew RV
      • David DA
      • Gundry RA
      • Balis UJ
      The LightCyclerTM: a microvolume multisample fluorimeter with rapid temperature control.
      ].

      LINEAR OLIGOPROBES

      The use of a pair of adjacent, fluorogenic hybridisation oligoprobes was first described in the late 1980s [
      • Heller MJ
      • Morrison LE
      ,
      • Cardullo RA
      • Agrawai S
      • Flores C
      • Zamecnik PC
      • Wolf DE
      Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer.
      ], and, now known as ‘HybProbes’, they have become the manufacturer's chemistry of choice for the LightCycler (Roche Molecular Biochemicals, Mannheim, Germany), a capillary-based, microvolume fluorimeter and thermocycler with rapid temperature control [
      • Wittwer CT
      • Ririe KM
      • Andrew RV
      • David DA
      • Gundry RA
      • Balis UJ
      The LightCyclerTM: a microvolume multisample fluorimeter with rapid temperature control.
      ,
      • Wittwer CT
      • Herrmann MG
      • Moss AA
      • Rasmussen RP
      Continuous fluorescence monitoring of rapid cycle DNA amplification.
      ]. The upstream oligoprobe is labeled with a 3′ donor fluorophore (fluorescein isothiocyanate, FITC), and the downstream probe is commonly labelled with either a LightCycler Red 640 or Red 705 acceptor fluorophore at the 5′-terminus, so that when both oligoprobes are hybridised, the two fluorophores are located within 10 nucleotides of each other.
      Most recently described fluorogenic oligoprobes fall into the linear class of oligoprobe. The recently described double-stranded oligoprobes function by displacement hybridisation (Fig. 1a) [
      • Li Q
      • Luan G
      • Guo Q
      • Liang J
      A new class of homogeneous nucleic acid probes based on specific displacement hybridization.
      ]. In this process, a 5′ fluorophore-labelled oligonucleotide is, in its resting state, hybridised with a complementary, but shorter, quenching DNA strand that is 3′ end-labeled with an NFQ. When the full-length complementary sequence in the form of an amplicon is present, the reporter strand will preferentially hybridise to the longer amplicon, disrupting the quenched oligoprobe duplex and permitting the fluorophore to emit its excitation energy directly.
      Figure thumbnail gr1
      Fig. 1Oligoprobe chemistries. (a) Displacement probes. The shorter NFQ-labeled strand (Q; filled pentagon) is displaced when the fluorophore-labelled (F; open circle) strand hybridises to the specific and longer amplicon. (b) Q-PNA primers. Quenching is achieved in the absence of specific template by a short NFQ-labelled PNA molecule designed to hybridise with the fluorophore-labelled primer. (c) Light-up probes. These PNA probes fluoresce in the presence of a hybridised DNA strand due to their asymmetric thiazole orange fluorophore (T; open triangle). (d) HyBeacons. In close proximity to DNA, as occurs upon hybridisation with the specific amplicon, the fluorophore emits fluorescence. (e) DzyNA primers. When the primer is duplicated by the complementary strand (dashed line), a DNAzyme is created. In the presence of a complementary, dual-labelled oligonucleotide substrate, the continuously amplified DNAzyme will specifically cleave the template between the fluorophore and quencher (Q; open pentagon), releasing the labels and allowing fluorescence to occur. (f) Tripartite molecular beacons. The fluorophore is removed from the NFQ's influence upon opening of the hairpin because of hybridisation to specific amplicon, permitting fluorescence.
      This technique can also be used with a fluorophore-labelled primer, and, due to the added stringency of the complementary strand, the system acts as its own ‘hot-start’, as was shown using an NFQ-labeled PNA [
      • Stender H
      • Fiandaca M
      • Hyldig‐Nielsen JJ
      • Coull J
      PNA for rapid microbiology.
      ] strand (Q-PNA) (Fig. 1b) [
      • Fiandaca MJ
      • Hyldig‐Nielsen JJ
      • Gildea BD
      • Coull JM
      Self‐reporting PNA/DNA primers for PCR analysis.
      ]. In this system, the quenching probe is bound to unincorporated fluorogenic primer such that the NFQ and fluorophore are adjacent, resulting in a quenched system. Once the dsDNA amplicon is created by primer extension, however, the Q-PNA is displaced, and the fluorophore can fluoresce. The PNA backbones cannot be extended or hydrolysed by a DNA polymerase.
      The light-up probe is also a PNA to which the asymmetric cyanine fluorophore thiazole orange is attached (Fig. 1c) [
      • Svanvik N
      • Westman G
      • Wang D
      • Kubista M
      Light‐up probes. Thiazole orange‐conjugated peptide nucleic acid for the detection of target nucleic acid in homogeneous solution.
      ]. When hybridised with a nucleic acid target, as either a duplex or triplex, the fluorophore becomes strongly fluorescent. These oligoprobes do not interfere with the PCR or require conformational change, they are sensitive to single nucleotide mismatches and, because a single reporter is used, they allow the direct measurement of fluorescence instead of the measurement of a change in fluorescence between two fluorophores [
      • Svanvik N
      • Westman G
      • Wang D
      • Kubista M
      Light‐up probes. Thiazole orange‐conjugated peptide nucleic acid for the detection of target nucleic acid in homogeneous solution.
      ,
      • Isacsson J
      • Cao H
      • Ohlsson L
      • et al.
      Rapid and specific detection of PCR products using light‐up probes.
      ]. However, non-specific fluorescence has been reported during extended cycling [
      • Svanvik N
      • Sehlstedt U
      • Sjöback R
      • Kubista M
      Detection of PCR products in real time using light‐up probes.
      ].
      The HyBeacon is a single linear oligonucleotide internally labelled with a fluorophore that emits an increased signal upon formation of a duplex with the target DNA strand (Fig. 1d) [
      • French DJ
      • Archard CL
      • Brown T
      • McDowell DG
      HyBeacon™ probes: a new tool for DNA sequence detection and allele discrimination.
      ,
      • French DJ
      • Archard CL
      • Andersen MT
      • McDowell DG
      Ultra‐rapid analysis using HyBeacon™ probes and direct PCR amplification from saliva.
      ]. The HyBeacon is labelled at the 3′-terminus with a phosphate or octanediol molecule to prevent Taq-mediated extension. This technique is used with all the non-incorporating nucleotide-based oligoprobe chemistries used in real-time PCR to ensure that they do not function as a primer. This chemistry does not require destruction, interaction with a second oligoprobe or secondary structure changes to produce a signal, and it is relatively cheap and simple to design.

      DUAL-LABELLED OLIGOPROBES

      In the early 1990s, an innovative approach involved nick-translation PCR in combination with dual-fluorophore-labelled oligoprobes was introduced [
      • Lee LG
      • Connell CR
      • Bloch W
      Allelic discrimination by nick‐translation PCR with fluorogenic probes.
      ]. In the first truly homogeneous assay of its kind, a fluorophore was added to the 5′-terminus and another to the middle of a sequence-specific oligoprobe. When in such close proximity, the 5′ reporter fluorophore (6-carboxy-fluoroscein; FAM) transferred laser-induced excitation energy by FRET to the 3′ quencher fluorophore (6-carboxy-tetramethyl-rhodamine; TAMRA). The oligoprobe hybridised to its template prior to the extension step, and the fluorophores were subsequently released during the primer extension step as a result of the 5′ to 3′ endonuclease activity of a suitable DNA polymerase. Once the labels were separated, the reporter's emissions were no longer quenched, and the instrument monitored the resulting fluorescence. Today, these oligoprobes are labelled at each terminus and are called 5′ nuclease, hydrolysis or TaqMan oligoprobes. The nuclease oligoprobe is the manufacturer's chemistry of choice for the ABI Prism sequence detection systems.
      A modification of the 5′ nuclease chemistry has resulted in the minor groove binding (MGB) oligoprobes [
      • Afonina IA
      • Reed MW
      • Lusby E
      • Shishkina IG
      • Belousov YS
      Minor groove binder‐conjugated DNA probes for quantitative DNA detection by hybridization‐triggered fluorescence.
      ]. This chemistry, commercially called the Eclipse oligoprobes, replaces the TaqMan oligoprobe's standard TAMRA quencher with a proprietary NFQ and incorporates a molecule that hyperstabilises the oligoprobe-target duplex by folding into the minor groove of the dsDNA [
      • Kutyavin IV
      • Afonina IA
      • Mills A
      • et al.
      3′‐Minor groove binder‐DNA probes increase sequence specificity at PCR extension temperatures.
      ,
      • Afonina IA
      • Sanders S
      • Walburger D
      • Belousov YS
      Accurate SNP typing by real‐time PCR.
      ]. A fluorophore is attached to the 3′ end, and in the unbound state the oligoprobe assumes a random coil configuration that is efficiently quenched. This chemistry allows the use of very short (12-17-nucleotide) oligoprobes because of a 15–30 °C rise in their TM resulting from the interaction of the MGB with the DNA helix. These short oligoprobes are ideal for detecting single-nucleotide polymorphisms (SNPs), because they are more significantly destabilised by nucleotide changes within the hybridisation site than are larger oligoprobes.
      Another dual-labelled oligonucleotide sequence has been used as the signal-generating portion of the DzyNA-PCR system (Fig. 1e) [
      • Todd AV
      • Fuery CJ
      • Impey HL
      • Applegate TL
      • Haughton MA
      DzyNA‐PCR. Use of DNAzymes to detect and quantify nucleic acid sequences in a real‐time fluorescent format.
      ]. Here, the reporter and quencher molecules are separated following specific cleavage of the oligonucleotides holding them in close proximity. This cleavage is performed by a DNAzyme, which is created during the PCR as the complement of an antisense DNAzyme sequence included in the 5′ tail of one of the primers. Upon cleavage, the fluorophores are released, allowing the production of fluorescence in an identical manner to a hydrolysed TaqMan oligoprobe.

      HAIRPIN OLIGONUCLEOTIDES

      Molecular beacons were the first hairpin oligoprobes to be used in real-time PCR. The molecular beacon's fluorogenic labels are positioned at the termini of the oligoprobe. The labels are held in close proximity by distal stem regions of homologous base pairing deliberately designed to create a hairpin structure. The closed hairpin is quenched due either to FRET or direct collision transfer of energy occurring at the molecular level as a consequence of the intimate proximity of the labels [
      • Tyagi S
      • Bratu DP
      • Kramer FR
      Multicolor molecular beacons for allele discrimination.
      ]. In the presence of a complementary sequence, designed to occur within the bounds of the primer binding sites, the oligoprobe will hybridise, shifting into an open configuration. The fluorophore is now spatially removed from the quencher's influence, allowing fluorescent emissions to be monitored [
      • Tyagi S
      • Kramer FR
      Molecular beacons: probes that fluoresce upon hybridization.
      ]. This structural change occurs in each cycle, increasing in cumulative intensity as the amount of specific amplicon increases. The quencher, DABCYL (4-(4′-dimeth-ylamino-phenylazo)-benzene), differs from that described for the nuclease oligoprobes because it is an NFQ.
      Recently, tripartite molecular beacons have been added to this class of fluorogenic chemistry (Fig. 1f) [
      • Nutiu R
      • Li Y
      Tripartite molecular beacons.
      ]. These oligoprobes have been designed to fulfill a need for suitably high-throughput chemistries and they combine a molecular beacon's hairpin with long or unlabelled single-stranded arms, each designed to hybridise to an oligonucleotide labelled with either a fluorophore or an NFQ. The system is quenched in the hairpin state due to the close proximity of the labels, but fluorescent when hybridised to the specific amplicon strand. Because the function of these oligoprobes depends upon correct hybridisation of the stem and two oligoprobes, their accurate design is crucial [
      • Bustin SA
      Absolute quantification of mRNA using real‐time reverse transcription polymerase chain reaction assays.
      ].
      Finally, a self-quenching hairpin primer has recently been described which is commercially entitled the light upon extension (LUX) fluorogenic primer [
      • Nazarenko I
      • Lowe B
      • Darfler M
      • Ikonomi P
      • Schuster D
      • Raschtchian A
      Multiplex quantitative PCR using self‐quenched primers labeled with a single fluorophore.
      ]. This chemistry is dark in the absence of specific amplicon, through the natural quenching ability of a carefully placed guanosine nucleotide. The natural quencher is brought into close proximity with the FAM or JOE 5′ 2,7-dimethoxy-4,5-dichloro-6-carboxy-fluoroscein fluorophore via a stretch of 5′ and 3′ complementary sequences. In the presence of specific target, the primer hybridises, opening the hairpin and permitting fluorescence from the fluorophore.

      MICROBIAL QUANTITATION

      Although the terminology is often confused, real-time PCR does not inherently imply quantitative PCR. To quantify the amount of template present in a sample, thought must be given to the type and number of controls required. Standards are used to allow calculation of the amount of template present in a patient sample, while internal controls (ICs) are mostly used to determine the occurrence of false-negative reactions, examine the ability to amplify from a preparation of nucleic acids, and, more rarely in real-time PCR, as a standard for quantitation. Certainly, the reliability of quantitative PCR methods is intimately associated with the choice and quality of the assay controls [
      • Celi FS
      • Mentuccia D
      • Proietti‐Pannunzi L
      • Di Gioia CRT
      • Andreoli M
      Preparing poly‐A‐containing RNA internal standards for multiplex competitive RT‐PCR.
      ,
      • Alexandre I
      • Zammatteo N
      • Ernest I
      • et al.
      Quantitative determination of CMV DNA using a combination of competitive PCR amplification and sandwich hybridization.
      ].
      No matter what control is chosen, it is imperative to accurately determine its concentration [
      • Zimmermann K
      • Manhalter JW
      Technical aspects of quantitative competitive PCR.
      ] and to ensure that ICs are added at suitable levels in order to prevent extreme competition with the wild-type template for reagents [
      • Brightwell G
      • Pearce M
      • Leslie D
      Development of internal controls for PCR detection of Bacillus anthracis.
      ]. The use of a spectrometer is inadequate for quantitating a control molecule [
      • Glasel JA
      Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios.
      ]; however, in combination with an experimental and statistical analysis, the reliability of the values is greatly enhanced [
      • Bagnarelli P
      • Menzo S
      • Valenza A
      • et al.
      Quantitative molecular monitoring of human immunodeficiency virus type 1 activity during therapy with specific antiretroviral compounds.
      ,
      • Wang Z
      • Spadoro J
      Determination of target copy number of quantitative standards used in PCR based diagnostic assays.
      ,
      • Rodrigo AG
      • Goracke PC
      • Rowhanian K
      • Mullins JI
      Quantitation of target molecules from polymerase chain reaction‐based limiting dilution assays.
      ,
      • Taswell C
      Limiting dilution assays for the determination of immunocompetent cell frequencies.
      ,
      • Sykes PJ
      • Brisco MJ
      • Snell LE
      • et al.
      Minimal residual disease in childhood acute lymphoblastic leukaemia quantified by aspirate and trephine: is the disease multifocal?.
      ]. Finally, one must remember that the results of quantitation using a molecular control need to be expressed relative to a suitable biological marker, e.g., in terms of the volume of plasma, the number of cells or the mass of tissue or genomic nucleic acid, thus allowing comparability between assay results and testing sites [
      • Niesters HGM
      Quantitation of viral load using real‐time amplification techniques.
      ].

       Standards for quantitation

      Most commonly, an exogenous control is created using a cloned amplicon, a portion of the target organism's genome, or simply the purified amplicon itself [
      • Borson ND
      • Strausbauch MA
      • Wettstein PJ
      • Oda RP
      • Johnston SL
      • Landers JP
      Direct quantitation of RNA transcripts by competitive single‐tube RT‐PCR and capillary electrophoresis.
      ]. This control forms the basis of an external standard curve created from the data produced by the individual amplification of a dilution series of exogenous control. The concentration of an unknown, which is amplified in the same reaction, but in a separate vessel, can then be found from the standard curve. While the external standard curve is the more commonly described quantitative approach, it frequently suffers from uncontrolled and unmonitored inter-vessel variations. Some platforms have overcome this issue by including a capacity to detect and correct for variation in the emissions of a non-participating, or ‘passive’, internal reference fluorophore (6-carboxy-N,N,N′,N′-tetramethylrhod-amine; ROX). The corrected values, obtained from a ratio of the emission intensity of the fluorophore and ROX, are called RQ+. To further control amplification fluctuations, the fluorescence from a ‘no-template’ control reaction (RQ) is subtracted from RQ+, resulting in the δRQ value that indicates the magnitude of the signal generated for the given PCR [
      • Gelmini S
      • Orlando C
      • Sestini R
      • et al.
      Quantitative polymerase chain reaction‐based homogeneous assay with fluorogenic probes to measure c‐cerB‐2 oncogene amplification.
      ]. Assays that lack this capacity are more appropriately described as semiquantitative.

       Internal controls (ICs)

      The use of an IC was described in the earliest of PCR experiments as an important quality control [
      • Chehab FF
      • Doherty M
      • Cai S
      • Kan YW
      • Cooper S
      • Rubin EM
      Detection of sickle cell anaemia and thalassaemias.
      ,
      • Reiss RA
      • Rutz B
      Quality control PCR: a method for detecting inhibitors of Taq DNA polymerase.
      ], particularly when performing competitive quantitation. When such a control is added before template purification (extraction control) or amplification (amplification control), it is called an exogenous IC, since it does not occur naturally within the nucleic acid preparation, but is co-amplified within the same reaction. Ideally, the IC should hybridise to the same primers, have an identical amplification efficiency [
      • Zimmermann K
      • Manhalter JW
      Technical aspects of quantitative competitive PCR.
      ,
      • Orlando C
      • Pinzani P
      • Pazzagli M
      Developments in quantitative PCR.
      ], and contain a discriminating feature such as a change in its length [
      • Celi FS
      • Mentuccia D
      • Proietti‐Pannunzi L
      • Di Gioia CRT
      • Andreoli M
      Preparing poly‐A‐containing RNA internal standards for multiplex competitive RT‐PCR.
      ,
      • Celi FS
      • Mentuccia D
      • Proietti‐Pannunzi L
      • Di Gioia CRT
      • Andreoli M
      Preparing poly‐A‐containing RNA internal standards for multiplex competitive RT‐PCR.
      ,
      • Brightwell G
      • Pearce M
      • Leslie D
      Development of internal controls for PCR detection of Bacillus anthracis.
      ,
      • Möller A
      • Jansson JK
      Quantification of genetically tagged cyanobacteria in Baltic sea sediment by competitive PCR.
      ,
      • Hall LL
      • Bicknell GR
      • Primrose L
      • Pringle JH
      • Shaw JA
      • Furness PN
      Reproducibility in the quantification of mRNA levels by RT‐PCR‐ELISA and RT competitive‐PCR‐ELISA.
      ] or, more commonly in today's oligoprobe-based methods, a change in the sequence [
      • Alexandre I
      • Zammatteo N
      • Ernest I
      • et al.
      Quantitative determination of CMV DNA using a combination of competitive PCR amplification and sandwich hybridization.
      ,
      • Aberham C
      • Pendl C
      • Gross P
      • Zerlauth G
      • Gessner M
      A quantitative, internally controlled real‐time PCR assay for the detection of parvovirus B19 DNA.
      ] of the wild-type target [
      • Kearns AM
      • Guiver M
      • James V
      • King J
      Development and evaluation of a real‐time quantitative PCR for the detection of human cytomegalovirus.
      ,
      • Gruber F
      • Falkner FG
      • Dorner F
      • Hämmerle T
      Quantitation of viral DNA by real‐time PCR applying duplex amplification, internal standardization, and two‐colour fluorescence detection.
      ].
      However, IC templates that bind different primers or have different amplification efficiencies can still prove useful as standards for semiquantitative PCR or relative quantitation.
      An endogenous control is a template that occurs naturally within the specimen being examined. Housekeeping genes often fulfill this role, and they have been successfully used to quantitate gene expression by RT-PCR and monitor the integrity of a template after its purification [
      • Chehab FF
      • Doherty M
      • Cai S
      • Kan YW
      • Cooper S
      • Rubin EM
      Detection of sickle cell anaemia and thalassaemias.
      ]. When endogenous controls are used for the quantitation of RNA, it is essential that the housekeeping gene is minimally regulated and exhibits a constant and cell cycle-independent basal level of transcription [
      • Selvey S
      • Thompson EW
      • Matthaei K
      • Lea RA
      • Irving MG
      • Griffiths LR
      β‐Actin—an unsuitable internal control for RT‐PCR.
      ]. This is not the case for some commonly used genes such as β-actin, whereas studies have shown that an 18S rRNA target meets the desired criteria [
      • Selvey S
      • Thompson EW
      • Matthaei K
      • Lea RA
      • Irving MG
      • Griffiths LR
      β‐Actin—an unsuitable internal control for RT‐PCR.
      ,
      • Thellin O
      • Zorzi W
      • Lakaye B
      • et al.
      Housekeeping genes as internal standards: use and limits.
      ].

       Relative vs. absolute quantitation

      The amount of template in a sample can be described either relatively or absolutely. Relative quantitation is the simpler approach, and describes changes in the amount of a target sequence compared to its level in a related matrix or within the same matrix by comparison to the signal from an endogenous or other reference control. Absolute quantitation is more demanding but states the exact number of nucleic acid targets present in the sample in relation to a specific unit, making it easier to compare data from different assays and laboratories [
      • Pfaffl MW
      • Horgan GW
      • Dempfle L
      Relative expression software tool (REST©) for group‐wise comparison and statistical analysis of relative expression results in real‐time PCR.
      ,
      • Freeman WM
      • Walker SJ
      • Vrana KE
      Quantitative RT‐PCR: pitfalls and potential.
      ]. Absolute quantitation may be necessary when there is a lack of sequential specimens to demonstrate a relative change in microbial load, or when no suitably standardised reference reagent is available.
      A highly accurate approach used for absolute quantitation by conventional PCR utilises competitive coamplification of one or a series of ICs of known concentration with a wild-type target nucleic acid of unknown concentration [
      • Becker‐Andre M
      • Hahlbrock K
      Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY).
      ,
      • Clementi M
      • Menzo S
      • Manzin A
      • Bagnarelli P
      Quantitative molecular methods in virology.
      ,
      • Gilliland G
      • Perrin S
      • Bunn HF
      ,
      • Siebert PD
      • Larrick JW
      Competitive PCR.
      ]. However, conventional competitive quantitation is technically demanding, requiring significant development and optimisation compared to quantitation by real-time PCR, which is better suited to the quick decision-making required in a clinical environment [
      • Locatelli G
      • Santoro F
      • Veglia F
      • Gobbi A
      • Lusso P
      • Malnati MS
      Real‐time quantitative PCR for human herpesvirus 6 DNA.
      ,
      • Wall SJ
      • Edwards DR
      Quantitative reverse transcription‐polymerase chain reaction (RT‐PCR): a comparison of primer‐dropping, competitive, and real‐time RT‐PCRs.
      ,
      • Tanaka N
      • Kimura H
      • Iida K
      • et al.
      Quantitative analysis of cytomegalovirus load using a real‐time PCR assay.
      ]. Software with the ability to calculate the concentration of an unknown by comparing real-time PCR signals generated by a coamplified target and IC is rare but emerging [
      • Pfaffl MW
      • Horgan GW
      • Dempfle L
      Relative expression software tool (REST©) for group‐wise comparison and statistical analysis of relative expression results in real‐time PCR.
      ]. In addition, new or improved formulae are appearing which aim to make quantitation more reliable and simpler [
      • Liu W
      • Saint DA
      A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics.
      ].

       Acquisition of fluorescence data

      Fluorescence data generated by real-time PCR assays are generally collected from PCR cycles that occur within the linear amplification portion of the reaction, where conditions are optimal and the fluorescence accumulates in proportion to the amplicon [
      • Mackay IM
      • Arden KE
      • Nitsche A
      Real‐time PCR in virology.
      ] (Fig. 2). This is in contrast to signal detection from the endpoint of the reaction, where the final amount of amplicon may have been affected by inhibitors, poorly optimised reaction conditions or saturation effects due to the presence of excess double-stranded amplicon. In fact, at the endpoint there may be no relationship between the initial template and final amplicon concentrations. Because the emissions from fluorescent chemistries are temperature-dependent, data are generally acquired only once/cycle, at the same temperature [
      • Wittwer CT
      • Herrmann MG
      • Moss AA
      • Rasmussen RP
      Continuous fluorescence monitoring of rapid cycle DNA amplification.
      ].
      Figure thumbnail gr2
      Fig. 2Kinetic analysis. The ideal amplification curve of a real-time PCR (solid), when plotted as fluorescence intensity against the cycle number, is a sigmoidal curve. Early amplification cannot be viewed because the emissions are masked by the background noise. However, when enough amplicon is present, the assay's exponential progress can be monitored as the rate of amplification enters a linear phase (LP). Under ideal conditions, the amount of amplicon increases at a rate of one log10 every 3.32 cycles. As primers and enzyme become limiting, and products inhibitory to the PCR and overly competitive to oligoprobe hybridisation accumulate, the reaction slows, entering a transition phase (TP) and eventually reaching a plateau phase (PP) where there is little or no increase in fluorescence. The point at which the fluorescence surpasses the noise threshold (dashed horizontal line) is called the threshold cycle or crossing point (CT or CP; indicated by an arrow), and this value is used in the calculation of template quantity during quantitative real-time PCR. Also shown are curves representing a titration of template (dashed curves), consisting of decreasing starting template concentrations, which produce higher CT or CP values, respectively. Data for the construction of a standard curve are taken from the LP.
      The fractional cycle number at which the real-time fluorescence signal mirrors progression of the reaction above the background noise is used as an indicator of successful target amplification [
      • Wilhelm J
      • Hahn M
      • Pingoud A
      Influence of DNA target melting behavior on real‐time PCR quantification.
      ]. Most commonly, this is called the threshold cycle (CT), but a similar value is described for the LightCycler, and the fractional cycle is called the crossing point (CP). The CT is defined as the PCR cycle in which the gain in fluorescence generated by the accumulating amplicon exceeds ten standard deviations of the mean baseline fluorescence, using data taken from cycles 3–15 [
      • Jung R
      • Soondrum K
      • Neumaier M
      Quantitative PCR.
      ]. The CT and CP are proportional to the number of target copies present in the sample [
      • Gibson UEM
      • Heid CA
      • Williams PM
      A novel method for real time quantitative RT‐PCR.
      ] and are assumed to represent equal amounts of amplicon present in each tube or capillary, since the CT and CP values represent the fractional cycle number for each sample at a single fluorescence intensity value. In practice, the CT and CP are calculated after the definition of a noise band that excludes data from early PCR cycles that cannot be distinguished from background noise. The final CT and CP values are the fractional cycles at which a single fluorescence value (usually at or close to the noise band) intersects each sample's plotted PCR curve [
      • Wilhelm J
      • Hahn M
      • Pingoud A
      Influence of DNA target melting behavior on real‐time PCR quantification.
      ] (Fig. 2). The accuracy of the CT or CP depends upon the concentration and nature of the fluorescence-generating component, the amount of template initially present, the sensitivity of the platform, and the platform's ability to discriminate specific fluorescence from background noise.

       Improved quantitation using real-time PCR

      Significant improvements in the quantitation of microbial load by real-time PCR result from the detection system's enormous dynamic range, which can accommodate at least eight log10 copies of nucleic acid template [
      • Gruber F
      • Falkner FG
      • Dorner F
      • Hämmerle T
      Quantitation of viral DNA by real‐time PCR applying duplex amplification, internal standardization, and two‐colour fluorescence detection.
      ,
      • Locatelli G
      • Santoro F
      • Veglia F
      • Gobbi A
      • Lusso P
      • Malnati MS
      Real‐time quantitative PCR for human herpesvirus 6 DNA.
      ,
      • Ishiguro T
      • Saitoh J
      • Yawata H
      • Yamagishi H
      • Iwasaki S
      • Mitoma Y
      Homogeneous quantitative assay of hepatitis C virus RNA by polymerase chain reaction in the presence of a fluorescent intercalater.
      ,
      • Kimura H
      • Morita M
      • Yabuta Y
      • et al.
      Quantitative analysis of Epstein–Barr virus load by using a real‐time PCR assay.
      ,
      • Najioullah F
      • Thouvenot D
      • Lina B
      Development of a real‐time PCR procedure including an internal control for the measurement of HCMV viral load.
      ,
      • Ryncarz AJ
      • Goddard J
      • Wald A
      • Huang M‐L
      • Roizman B
      • Corey L
      Development of a high‐throughput quantitative assay for detecting herpes simplex virus DNA in clinical samples.
      ,
      • Monopoeho S
      • Mignotte B
      • Schwartzbrod L
      • et al.
      Quantification of enterovirus RNA in sludge samples using single tube real‐time RT‐PCR.
      ,
      • Alexandersen S
      • Oleksiewicz MB
      • Donaldson AI
      The early pathogenesis of foot‐and‐mouth disease in pigs infected by contact: a quantitative time‐course study using TaqMan RT‐PCR.
      ,
      • Abe A
      • Inoue K
      • Tanaka T
      • et al.
      Quantitation of hepatitis B virus genomic DNA by real‐time detection PCR.
      ,
      • Brechtbuehl K
      • Whalley SA
      • Dusheiko GM
      • Saunders NA
      A rapid real‐time quantitative polymerase chain reaction for hepatitis B virus.
      ,
      • Moody A
      • Sellers S
      • Bumstead N
      Measuring infectious bursal disease virus RNA in blood by multiplex real‐time quantitative RT‐PCR.
      ]. The broad dynamic range avoids the need for pre-dilution of an amplicon before detection, or the need to repeat an assay using a diluted sample because a preliminary result falls outside the limits of the assay. Both of these problems occur commonly when using conventional endpoint PCR assays for quantitation, as their detection systems are unable to encompass the products of high template loads while maintaining adequate sensitivity [
      • Brechtbuehl K
      • Whalley SA
      • Dusheiko GM
      • Saunders NA
      A rapid real‐time quantitative polymerase chain reaction for hepatitis B virus.
      ,
      • Weinberger KM
      • Wiedenmann E
      • Böhm S
      • Jilg W
      Sensitive and accurate detection of hepatitis B virus DNA using a kinetic fluorescence detection system (TaqMan PCR).
      ,
      • Schaade L
      • Kockelkorn P
      • Ritter K
      • Kleines M
      Detection of cytomegalovirus DNA in human specimens by LightCycler PCR.
      ,
      • Kawai S
      • Yokosuka O
      • Kanda T
      • Imazeki F
      • Maru Y
      • Saisho H
      Quantification of hepatitis C virus by TaqMan PCR: comparison with HCV amplicor monitor assay.
      ]. The flexibility of real-time PCR is further demonstrated by its ability to detect one target in the presence of a vast excess of another target during duplexed assays [
      • Ryncarz AJ
      • Goddard J
      • Wald A
      • Huang M‐L
      • Roizman B
      • Corey L
      Development of a high‐throughput quantitative assay for detecting herpes simplex virus DNA in clinical samples.
      ].
      Real-time PCR is also a particularly attractive alternative to conventional PCR for the study of microbial load because of its low inter-assay and intra-assay variability [
      • Locatelli G
      • Santoro F
      • Veglia F
      • Gobbi A
      • Lusso P
      • Malnati MS
      Real‐time quantitative PCR for human herpesvirus 6 DNA.
      ,
      • Abe A
      • Inoue K
      • Tanaka T
      • et al.
      Quantitation of hepatitis B virus genomic DNA by real‐time detection PCR.
      ,
      • Schutten M
      • Van Den Hoogen B
      • Van Der Ende ME
      • Gruters RA
      • Osterhaus ADME
      • Niesters HGM
      Development of a real‐time quantitative RT‐PCR for the detection of HIV‐2 RNA in plasma.
      ] and its equivalent or improved sensitivity compared to microbial culture, or conventional single-round and nested PCR [
      • Clarke SC
      • Reid J
      • Thom L
      • Denham BC
      • Edwards GFS
      Laboratory confirmation of meningococcal disease in Scotland, 1993–9.
      ,
      • Locatelli G
      • Santoro F
      • Veglia F
      • Gobbi A
      • Lusso P
      • Malnati MS
      Real‐time quantitative PCR for human herpesvirus 6 DNA.
      ,
      • Monopoeho S
      • Mignotte B
      • Schwartzbrod L
      • et al.
      Quantification of enterovirus RNA in sludge samples using single tube real‐time RT‐PCR.
      ,
      • Kearns AM
      • Turner AJL
      • Taylor CE
      • George PW
      • Freeman R
      • Gennery AR
      LightCycler‐based quantitative PCR for rapid detection of human herpesvirus 6 DNA in clinical material.
      ,
      • Kupferschmidt O
      • Krüger D
      • Held TK
      • Ellerbrok H
      • Siegert W
      • Janitschke K
      Quantitative detection of Toxoplasma gondii DNA in human body fluids by TaqMan polymerase chain reaction.
      ,
      • Kennedy MM
      • Lucas SB
      • Russell‐Jones R
      • et al.
      HHV8 and female Kaposi's sarcoma.
      ,
      • Capone RB
      • Pai SI
      • Koch WM
      • Gillison ML
      Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV‐associated head and neck squamous cell carcinoma.
      ,
      • Leutenegger CM
      • Klein D
      • Hofmann‐Lehmann R
      • et al.
      Rapid feline immunodeficiency virus provirus quantitation by polymerase chain reaction using the TaqMan fluorogenic real‐time detection system.
      ,
      • Smith IL
      • Halpin K
      • Warrilow D
      • Smith GA
      Development of a fluorogenic RT‐PCR assay (TaqMan) for the detection of Hendra virus.
      ,
      • Van Elden LJR
      • Nijhuis M
      • Schipper P
      • Schuurman R
      • Van Loon AM
      Simultaneous detection of influenza viruses A and B using real‐time quantitative PCR.
      ,
      • Lanciotti RS
      • Kerst AJ
      • Nasci RS
      • et al.
      Rapid detection of west nile virus from human clinical specimens, field‐collected mosquitoes, and avian samples by a TaqMan reverse transcriptase‐PCR assay.
      ]. Real-time PCR has been reported to be at least as sensitive as Southern blot, still considered by some as the gold standard for probe-based hybridisation assays [
      • Capone RB
      • Pai SI
      • Koch WM
      • Gillison ML
      Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV‐associated head and neck squamous cell carcinoma.
      ].

      MICROBIAL GENOTYPING

      Although nucleotide sequencing is still the gold standard for characterising unknown nucleic acids, it is a relatively lengthy process. The development of real-time PCR has partially addressed this failing by providing a tool capable of routine detection of characterised mutations, insertions or deletions.
      Most fluorescent chemistries used for real-time PCR do not rely upon a destructive process to generate a signal. Therefore, they may be able to perform a genotyping role at the completion of the PCR. The SYBR green and HybProbe chemistries are most commonly used to perform these analyses; however, the double-stranded and light-up oligoprobes and HyBeacons should also function in this role. Other chemistries, such as the TaqMan and Eclipse oligoprobes and hairpin oligonucleotides, discriminate these nucleotide changes using two sets of oligoprobes to differentiate the wild-type from the altered sequences. While this is a perfectly legitimate and functional approach to genotyping by real-time PCR, the extra fluorogenic oligonucleotides increase the overall cost of the assay. Additionally, the number of different microbes that can be discriminated during multiplex real-time PCR is reduced, since two fluorophores must be assigned to analyse each microbe. The occurrence of a mismatch between a hairpin oligonucleotide and its target has a greater destabilising effect on the duplex than the introduction of an equivalent mismatch between the target and a linear oligoprobe. This is because the hairpin structure provides a highly stable alternative configuration. Therefore, hairpin oligonucleotides are more specific than the more common linear oligoprobes, making them ideal candidates for detecting SNPs [
      • Tyagi S
      • Bratu DP
      • Kramer FR
      Multicolor molecular beacons for allele discrimination.
      ].
      Genotyping data are obtained after the completion of the PCR, and therefore represent an endpoint analysis. The amplicon is denatured and rapidly cooled to encourage the formation of fluorophore and target strand complexes. The temperature is then gradually raised, and the fluorescence from each vessel is continuously recorded. The detection of sequence variation using fluorescent chemistries relies upon the destabilisation incurred as a result of the change(s). The non-specific chemistries reflect these changes in the context of the entire dsDNA amplicon, requiring the dissociation of fluorogenic molecules from the dsDNA, which only occurs upon melting of the duplex. The sequence changes have a different impact upon the specific fluorogenic chemistries, altering the expected TM in a manner that reflects the particular nucleotide change. The resulting rapid decrease in fluorescence using either approach can be presented as a ‘melt peak’ using software capable of calculating the negative derivative of the fluorescence change with temperature (Fig. 3).
      Figure thumbnail gr3
      Fig. 3Fluorescence melting curve analysis. At the completion of a real-time PCR using a fluorogenic chemistry, the reaction can be cooled to a temperature below the expected TM of the oligoprobes and then heated to above 90°C at a fraction of a degree/second (a). During heating, the emissions of the reporter or acceptor fluorophore can be constantly acquired (b). Software calculates the negative derivative of the fluorescence with temperature, producing a clear melt peak that indicates the TM of the oligoprobe-target melting transition (black peak; c) or the TD of melting dsDNA. When one or more nucleotide changes are present, the TM or TD is shifted (grey peak). This shift is reproducible and can be used diagnostically to genotype microbial templates.
      Importantly, different nucleotide changes destabilise hybridisation to different degrees, and this can be incorporated into the design of genotyping assays to ensure maximum discrimination between melt peaks. The least destabilising mismatches include G (G:T, G:A and G:G), whereas the most destabilising include C (C:C, C:A and C:T) [
      • Bernard PS
      • Lay MJ
      • Wittwer CT
      Integrated amplification and detection of the C677T point mutation in the methyltetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves.
      ].

      MULTIPLEXREAL-TIME PCR

      Multiplex PCR uses one or more primer sets to potentially amplify multiple templates within a single reaction [
      • Chamberlain JS
      • Gibbs RA
      • Ranier JE
      • Nguyen PN
      • Caskey CT
      Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification.
      ,
      • Elnifro EM
      • Ashshi AM
      • Cooper RJ
      • Klapper PE
      Multiplex PCR: optimization and application in diagnostic virology.
      ]. However, its use in real-time PCR has led to confusion in the traditional terminology. Multiplex real-time PCR more commonly refers to the use of multiple fluorogenic oligoprobes for the discrimination of amplicons that may have been produced by one or several primer pairs. The development of multiplex real-time PCR has proven problematic because of the limited number of fluorophores available [
      • Lee LG
      • Connell CR
      • Bloch W
      Allelic discrimination by nick‐translation PCR with fluorogenic probes.
      ] and the frequent use of monochromatic energising light sources. Although excitation by a single wavelength produces bright emissions from a suitably receptive fluorophore, the number of fluorophores that can be excited by that wavelength is limited [
      • Tyagi S
      • Marras SAE
      • Kramer FR
      Wavelength‐shifting molecular beacons.
      ].
      The discovery and application of the non-fluorescent quenchers has made available some wavelengths that were previously occupied by the emissions from the early quenchers themselves. This should permit the future inclusion of a greater number of spectrally discernable oligoprobes/reaction, and highlights the need for a single non-fluorescent quencher that can quench a broad range of emission wavelengths (e.g., 400–600 nm). The impressive electron-donating properties of guanosine make it an ideal natural quencher, and its use has contributed to the growing number of assays that only require a single fluorophore/target [
      • Nazarenko I
      • Pires R
      • Lowe B
      • Obaidy M
      • Raschtchian A
      Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes.
      ].
      Early real-time PCR systems contained optimised filter sets to minimise overlap of the emission spectra from the fluorophores. Despite this, the number of fluorophores that could be combined and clearly distinguished was limited. More recent real-time PCR platforms have incorporated either multiple light-emitting diodes, or a tungsten light source that emits over a wide range of wavelengths. When these platforms also incorporate high-quality optical filters, it is possible to use many of the current real-time PCR detection chemistries on the one machine. Unfortunately some platforms are not suitably constructed. Even if these improvements are included, the platform can still only perform four-colour oligoprobe multiplexing, and one colour is ideally set aside for use as an IC. Some real-time PCR designs have made use of conserved single or multiple nucleotide changes among similar templates to allow their differentiation by concurrent changes to the oligoprobe's TM or the amplicon's TD [
      • Espy MJ
      • Uhl JR
      • Mitchell PS
      • et al.
      Diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR.
      ,
      • Nicolas L
      • Milon G
      • Prina E
      Rapid differentiation of old world Leishmania species by LightCycler polymerase chain reaction and melting curve analysis.
      ]. Combining the use of multiple fluorophores with the discrimination of additional targets by temperature allows the identification of a significantly larger number of amplicon targets [
      • Wittwer CT
      • Herrmann MG
      • Gundry CN
      • Elenitoba‐Johnson KSJ
      Real‐time multiplex PCR assays.
      ]; however, this combined approach has not been applied to the diagnosis of infectious disease on a significant scale [
      • Espy MJ
      • Ross TK
      • Teo R
      • et al.
      Evaluation of LightCycler PCR for implementation of laboratory diagnosis of herpes simplex virus infections.
      ], possibly because of the sequence variation among many microbial genes [
      • Kearns AM
      • Turner AJL
      • Taylor CE
      • George PW
      • Freeman R
      • Gennery AR
      LightCycler‐based quantitative PCR for rapid detection of human herpesvirus 6 DNA in clinical material.
      ,
      • Schalasta G
      • Arents A
      • Schmid M
      • Braun RW
      • Enders G
      Fast and type‐specific analysis of herpes simplex virus types 1 and 2 by rapid PCR and fluorescence melting‐curve‐analysis.
      ,
      • Loparev VN
      • McCaustland K
      • Holloway BP
      • Krause PR
      • Takayama M
      • Schmid DS
      Rapid genotyping of varicella‐zoster virus vaccine and wild‐type strains with fluorophore‐labeled hybridization probes.
      ,
      • Read SJ
      • Mitchell JL
      • Fink CG
      LightCycler multiplex PCR for the laboratory diagnosis of common viral infections of the central nervous system.
      ,
      • Whiley DM
      • Mackay IM
      • Sloots TP
      Detection and differentiation of human polyomaviruses JC and BK by LightCycler PCR.
      ]. Far more commonly, this approach has been used for the detection of human genetic diseases, where as many as 27 possible nucleotide substitutions have been detected using only one or two fluorophores [
      • Schütz E
      • Von Ashen N
      • Oellerich M
      Genotyping of eight thiopurine methyltransferase mutations: three‐color multiplexing, ‘two‐color/shared’ anchor, and fluorescence‐quenching hybridization probe assays based on thermodynamic nearest‐neighbour probe design.
      ,
      • Bohling SD
      • King TC
      • Wittwer CT
      • Elenitoba‐Johnson KSJ
      Rapid simultaneous amplification and detection of the MBR/JH chromosomal translocation by fluorescence melting curve analysis.
      ,
      • Lay MJ
      • Wittwer CT
      Real‐time fluorescence genotyping of factor V Leiden during rapid‐cycle PCR.
      ,
      • Herrmann MG
      • Dobrowolski SF
      • Wittwer CT
      Rapid β‐globin genotyping by multiplexing probe melting temperature and color.
      ,
      • Lee LG
      • Livak KJ
      • Mullah B
      • Graham RJ
      • Vinayak RS
      • Woudenberg TM
      Seven‐color, homogeneous detection of six PCR products.
      ,
      • Gundry CN
      • Bernard PS
      • Herrmann MG
      • Reed GH
      • Wittwer CT
      Rapid F508del and F508C assay using fluorescent hybridization probes.
      ,
      • Bernard PS
      • Wittwer CT
      Homogeneous amplification and variant detection by fluorescent hybridization probes.
      ,
      • Elenitoba KSJ
      • Bohling SD
      • Wittwer CT
      • King TC
      Multiplex PCR by multicolor fluorimetry and fluorescence melting curve analysis.
      ].
      To date, there have been only a handful of diagnostic microbial assays that can truly co-amplify and discriminate more than two fluorophores. An impressive multiplex, real-time PCR protocol discriminated between four retroviral target sequences [
      • Vet JAM
      • Majithia AR
      • Marras SAE
      • et al.
      Multiplex detection of four pathogenic retroviruses using molecular beacons.
      ]; however, conventional multiplex PCR using endpoint detection has easily discriminated between more than five different amplified sequences, indicating a greater degree of flexibility [
      • Quereda C
      • Corral I
      • Laguna F
      • et al.
      Diagnostic utility of a multiplex herpesvirus PCR assay performed with cerebrospinal fluid from human immunodeficiency virus‐infected patients with neurological disorders.
      ,
      • Weigl JAI
      • Puppe W
      • Gröndahl B
      • Schmitt H‐J
      Epidemiological investigation of nine respiratory pathogens in hospitalized children in Germany using multiplex reverse‐transcriptase polymerase chain reaction.
      ,
      • Stockton J
      • Ellis JS
      • Saville M
      • Clewley JP
      • Zambon MC
      Multiplex PCR for typing and subtyping influenza and respiratory syncytial viruses.
      ,
      • Kehl SC
      • Henrickson KJ
      • Hua W
      • Fan J
      Evaluation of the hexaplex assay for detection of respiratory viruses in children.
      ,
      • Echevarria JE
      • Erdman DD
      • Swierkosz EM
      • Holloway BP
      • Anderson LJ
      Simultaneous detection and identification of human parainfluenza viruses 1, 2, and 3 from clinical samples by multiplex PCR.
      ,
      • Henegariu O
      • Heerema NA
      • Dloughy SR
      • Vance GH
      • Vogt PH
      Multiplex PCR: critical parameters and step‐by‐step protocol.
      ].
      Future development of novel chemistries and improved real-time instrumentation and software should significantly improve the ability to multiplex fluorophores for enhanced real-time PCR assays. Perhaps a chimera of real-time PCR and microarray technology, in combination with microfluidic devices, may advance all three technologies to a point where the desired number of templates could be easily amplified and discriminated.

      SPECIFIC APPLICATIONS FOR MICROBIOLOGY

      Real-time PCR assays have been extremely useful for studying microbial agents of infectious disease, where they have helped to clarify many disease processes. Most of the assays presented in the literature have increased the frequency of microbial detection compared to non-PCR techniques, making the implementation of real-time PCR attractive to many.
      Of course, real-time PCR has also proven valuable for basic microbiological research, where its ability to amplify template from a wide array of sample types (Table 1) has made it an ideal system for application across the various microbiological disciplines [
      • Schalasta G
      • Eggers M
      • Schmid M
      • Enders G
      Analysis of human cytomegalovirus DNA in urines of newborns and infants by means of a new ultrarapid real‐time PCR‐system.
      ]. Increasingly, these applications are difficult to review, due to their use as a tool within, rather than the focus of, a published study.
      Table 1An incomplete list indicating the extraordinary variety of sample types from which nucleic acids can be successfully prepared, amplified and detected using real-time PCR assays
      Nucleic acid originsReferences
      Plants[
      • Skaf JS
      • Schultz MH
      • Hirata H
      • De Zoeten GA
      Mutational evidence that VPg is involved in the replication and not the movement of Pea enation mosaic virus‐1.
      ]
      Animals[
      • Alexandersen S
      • Oleksiewicz MB
      • Donaldson AI
      The early pathogenesis of foot‐and‐mouth disease in pigs infected by contact: a quantitative time‐course study using TaqMan RT‐PCR.
      ,
      • Taylor MJ
      • Hughes MS
      • Skuce RA
      • Neill SD
      Detection of Mycobacterium bovis in bovine clinical specimens using real‐time fluorescence and fluorescence resonance energy transfer probe rapid‐cycle PCR.
      ]
      Urban sludge[
      • Monopoeho S
      • Mignotte B
      • Schwartzbrod L
      • et al.
      Quantification of enterovirus RNA in sludge samples using single tube real‐time RT‐PCR.
      ]
      Microbial culture[
      • Cane PA
      • Cook P
      • Ratcliffe D
      • Mutimer D
      • Pillay D
      Use of real‐time PCR and fluorimetry to detect lamivudine resistance‐associated mutations in hepatitis B virus.
      ,
      • Woo THS
      • Patel BKC
      • Smythe LD
      • Symonds ML
      • Norris MA
      • Dohnt MF
      Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid‐cycle PCR.
      ,
      • De Viedma DG
      • Diaz Infanntes M
      • Lasala F
      • Chaves F
      • Alcalá L
      • Bouza E
      New real‐time PCR able to detect in a single tube multiple rifamin resistance mutations and high‐level isoniazid resistance mutations in Mycobacterium tuberculosis.
      ,
      • Shrestha NK
      • Tuohy MJ
      • Hall GS
      • Isada CM
      • Procop GW
      Rapid identification of Staphylococcus aureus and the mecA gene from BacT/ALERT blood culture bottles by using the LightCycler system.
      ,
      • Reischl U
      • Linde H‐J
      • Metz M
      • Leppmeier B
      • Lehn N
      Rapid identification of methicillin‐resistant Staphylococcus aureus and simultaneous species confirmation using real‐time fluorescence PCR.
      ,
      • Rauter C
      • Oehme R
      • Diterich I
      • Engele M
      • Hartung T
      Distribution of clinically relevant Borrelia genospecies in ticks assessed by a novel, single‐run, real‐time PCR.
      ,
      • Pietilä J
      • He Q
      • Oksi J
      • Viljanen MK
      Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene.
      ,
      • Semighini CP
      • Marins M
      • Goldman MHS
      • Goldman GH
      Quantitative analysis of the relative transcript levels of ABC transporter Atr genes in Aspergillus nidulans by real‐time reverse transcription‐PCR assay.
      ,
      • Tanriverdi S
      • Tanyeli A
      • Baslamisli F
      • et al.
      Detection and genotyping of oocysts of Cryptosporidium parvum by real‐time PCR and melting curve analysis.
      ,
      • Blair PL
      • Witney A
      • Haynes JD
      • Moch JK
      • Carucci DJ
      • Adams JH
      Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real‐time RT‐PCR.
      ,
      • Garin D
      • Peyrefitte C
      • Crance J‐M
      • Le Faou A
      • Jouan A
      • Bouloy M
      Highly sensitive Taqman® PCR detection of Puumala hantavirus.
      ]
      Solid tissues[
      • White PA
      • Pan Y
      • Freeman AJ
      • et al.
      Quantification of hepatitis C virus in human liver and serum samples by using LightCycler reverse transcriptase PCR.
      ,
      • Argaw T
      • Ritzhaupt A
      • Wilson CA
      Development of a real time quantitative PCR assay for detection of porcine endogenous retrovirus.
      ,
      • Eishi Y
      • Suga M
      • Ishige I
      • et al.
      Quantitative analysis of mycobacterial and propionibacterial DNA in lymph nodes of Japanese and European patients with sarcoidosis.
      ,
      • Matsumura M
      • Hikiba Y
      • Ogura K
      • et al.
      Rapid detection of mutations in the 23S rRNA gene of Helicobacter pylori that confers resistance to clarithromycin treatment to the bacterium.
      ,
      • Gibson JR
      • Saunders NA
      • Owen RJ
      Novel method for rapid determination of clarithromycin sensitivity in Helicobacter pylori.
      ,
      • Chisholm SA
      • Owen RJ
      • Teare EL
      • Saverymuttu S
      PCR‐based diagnosis of Helicobacter pylori infection and real‐time determination of clarithromycin resistance directly from human gastric biopsy samples.
      ,
      • Fenollar F
      • Fournier P‐E
      • Raoult D
      • Gérolami R
      • Lepidi H
      • Poyart C
      Quantitative detection of Tropheryma whipplei DNA by real‐time PCR.
      ,
      • Bruña‐Romero O
      • Hafalla JCR
      • González‐Aseguinolaza G
      • Sano G
      • Tsuji M
      • Zavala F
      Detection of malaria liver‐stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real‐time PCR.
      ,
      • Pevenstein SR
      • Williams RK
      • McChesney D
      • Mont EK
      • Smialek JE
      • Straus SE
      Quantitation of latent varicella‐zoster virus and herpes simplex virus genomes in human trigeminal ganglia.
      ]
      Cerebrospinal fluid[
      • Schalasta G
      • Arents A
      • Schmid M
      • Braun RW
      • Enders G
      Fast and type‐specific analysis of herpes simplex virus types 1 and 2 by rapid PCR and fluorescence melting‐curve‐analysis.
      ,
      • Monpoeho S
      • Coste‐Burel M
      • Costa‐Mattioli M
      • et al.
      Application of a real‐time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid.
      ,
      • Corless CE
      • Guiver M
      • Borrow R
      • et al.
      Development and evaluation of a ‘real‐time’ RT‐PCR for the detection of enterovirus and parechovirus RNA in CSF and throat swab samples.
      ,
      • Verstrepen WA
      • Kuhn S
      • Kockx MM
      • Van De Vyvere ME
      Rapid detection of enterovirus RNA in cerebrospinal fluid specimens with a novel single‐tube real‐time reverse transcription‐PCR assay.
      ,
      • Probert WS
      • Bystrom SL
      • Khashe S
      • Schrader KN
      • Wong JD
      5′‐Exonuclease assay for detection of serogroup Y Neisseria meningitidis.
      ]
      Peripheral blood mononuclear cells[
      • Ohyashiki JK
      • Suzuki A
      • Aritaki K
      • et al.
      Use of real‐time PCR to monitor human herpesvirus 6 reactivation after allogeneic bone marrow transplantation.
      ]
      Bone marrow[
      • Kupferschmidt O
      • Krüger D
      • Held TK
      • Ellerbrok H
      • Siegert W
      • Janitschke K
      Quantitative detection of Toxoplasma gondii DNA in human body fluids by TaqMan polymerase chain reaction.
      ]
      Whole blood[
      • Stevens SJC
      • Verkuijlen SAWM
      • Van Den Brule AJC
      • Middeldorp JM
      Comparison of quantitative competitive PCR with LightCycler‐based PCR for measuring Epstein–Barr virus DNA load in clinical specimens.
      ,
      • Lee M‐A
      • Tan C‐H
      • Aw L‐T
      • et al.
      Real‐time fluorescence‐based PCR for detection of malaria parasites.
      ]
      Plasma[
      • Schutten M
      • Van Den Hoogen B
      • Van Der Ende ME
      • Gruters RA
      • Osterhaus ADME
      • Niesters HGM
      Development of a real‐time quantitative RT‐PCR for the detection of HIV‐2 RNA in plasma.
      ]
      Serum[
      • Laue T
      • Emmerich P
      • Schmitz H
      Detection of dengue virus RNA inpatients after primary or secondary dengue infection by using the TaqMan automated amplification system.
      ,
      • White PA
      • Pan Y
      • Freeman AJ
      • et al.
      Quantification of hepatitis C virus in human liver and serum samples by using LightCycler reverse transcriptase PCR.
      ,
      • Gallagher A
      • Armstrong AA
      • MacKenzie J
      • et al.
      Detection of Epstein–Barr virus (EBV) genomes in the serum of patients with EBV‐associated Hodgkin's disease.
      ,
      • Costa C
      • Costa J‐M
      • Desterke C
      • Botterel F
      • Cordonnier C
      • Bretagne S
      Real‐time PCR coupled with automated DNA extraction and detection of galactomannan antigen in serum by enzyme‐linked immunosorbent assay for diagnosis of invasive aspergillosis.
      ,
      • Costa J‐M
      • Pautas C
      • Ernault P
      • Foulet F
      • Cordonnier C
      • Bretagne S
      Real‐time PCR for diagnosis and follow‐up of Toxoplasma reactivation after allogeneic stem cell transplantation using fluorescence resonance energy transfer hybridization probes.
      ]
      Swabs[
      • Corless CE
      • Guiver M
      • Borrow R
      • et al.
      Development and evaluation of a ‘real‐time’ RT‐PCR for the detection of enterovirus and parechovirus RNA in CSF and throat swab samples.
      ,
      • Ke D
      • Ménard C
      • Picard FJ
      • et al.
      Development of conventional and real‐time PCR assays for the rapid detection of group B streptococci.
      ,
      • Schweiger B
      • Zadow I
      • Heckler R
      • Timm H
      • Pauli G
      Application of a fluorogenic PCR assay for typing and subtyping influenza viruses in respiratory samples.
      ]
      Bronchoalveolar lavage[
      • Hayden RT
      • Uhl JR
      • Qian X
      • et al.
      Direct detection of Legionella species from bronchoalveolar lavage and open lung biopsy specimens: comparison of LightCycler PCR, in situ hybridization, direct fluorescence antigen detection, and culture.
      ,
      • Reischl U
      • Linde H‐J
      • Lehn N
      • Landt O
      • Barratt K
      • Wellinghausen N
      Direct detection and differentiation of Legionella spp. & Legionella pneumophila in clinical specimens by dual‐colour real‐time PCR and melting curve analysis.
      ]
      Amniotic fluid[
      • Costa J‐M
      • Ernault P
      • Gautier E
      • Bretagne S
      Prenatal diagnosis of congenital toxoplasmosis by duplex real‐time PCR using fluorescence resonance energy transfer hybridization probes.
      ]
      Saliva and sputum[
      • Furuta Y
      • Ohtani F
      • Sawa H
      • Fukuda S
      • Inuyama Y
      Quantitation of varicella‐zoster virus DNA in patients with Ramsay Hunt syndrome and zoster sine herpete.
      ,
      • Klaschik S
      • Lehmann LE
      • Raadts A
      • Book M
      • Hoeft A
      • Stuber F
      Real‐time PCR for the detection and differentiation of Gram‐positive and Gram‐negative bacteria.
      ]
      Faeces[
      • Ibekwe AM
      • Watt PM
      • Grieve CM
      • Sharma VK
      • Lyons SR
      Multiplex fluorogenic real‐time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands.
      ,
      • Wolk DM
      • Schneider SK
      • Wengenack NL
      • Sloan LM
      • Rosenblatt JE
      Real‐time PCR method for detection of Encephalitozoon intestinalis from stool specimens.
      ]
      Urine[
      • Carman WF
      • Wallace LA
      • Walker J
      • et al.
      Rapid virological surveillance of community influenza infection in general practice.
      ,
      • Schalasta G
      • Eggers M
      • Schmid M
      • Enders G
      Analysis of human cytomegalovirus DNA in urines of newborns and infants by means of a new ultrarapid real‐time PCR‐system.
      ,
      • Kearns AM
      • Draper B
      • Wipat W
      • et al.
      LightCycler‐based quantitative PCR for detection of cytomegalovirus in blood, urine and respiratory samples.
      ]

       Viruses

      Within microbiology, the application of real-time PCR has had the biggest impact upon the field of virology, where studies have qualitatively investigated the role of viruses in a range of human diseases [
      • Kato T
      • Mizokami M
      • Mukaide M
      • et al.
      Development of a TT virus DNA quantification system using real‐time detection PCR.
      ]. Also, epidemiological studies of co-infections have been improved by these molecular techniques, which can reliably measure the amount of two nucleic acid targets present within a single sample [
      • Kearns AM
      • Turner AJL
      • Taylor CE
      • George PW
      • Freeman R
      • Gennery AR
      LightCycler‐based quantitative PCR for rapid detection of human herpesvirus 6 DNA in clinical material.
      ,
      • Zerr DM
      • Huang M‐L
      • Corey L
      • Erickson M
      • Parker HL
      • Frenkel LM
      Sensitive method for detection of human herpesvirus 6 and 7 in saliva collected in field studies.
      ,
      • Furuta Y
      • Ohtani F
      • Sawa H
      • Fukuda S
      • Inuyama Y
      Quantitation of varicella‐zoster virus DNA in patients with Ramsay Hunt syndrome and zoster sine herpete.
      ]. Real-time PCR has also improved the discrimination of multiple viral genotypes within a single reaction vessel [
      • Jordens JZ
      • Lanham S
      • Pickett MA
      • Amarasekara S
      • Aberywickrema I
      • Watt PJ
      Amplification with molecular beacon primers and reverse line blotting for the detection and typing of human papillomaviruses.
      ] and provided an alternative to morbidity and mortality assays for virus detection. An example is Newcastle disease virus, which exists as two radically different pathogenic phenotypes caused by small nucleotide changes that can be easily detected using fluorescence melting curve analysis to reveal the genetic pathotype of the strain [
      • Aldous EW
      • Collins MS
      • McGoldrick A
      • Alexander DJ
      Rapid pathotyping of Newcastle disease virus (NDV) using fluorogenic probes in a PCR assay.
      ].
      Direct and indirect links between viral infection and chronic conditions such as sarcoma [
      • Kennedy MM
      • Lucas SB
      • Russell‐Jones R
      • et al.
      HHV8 and female Kaposi's sarcoma.
      ,
      • Kennedy MM
      • Lucas SB
      • Jones RR
      • et al.
      HHV8 and Kaposi's sarcoma: a time cohort study.
      ,
      • Kennedy MM
      • Cooper K
      • Howells DD
      • et al.
      Identification of HHV8 in early Kaposi's sarcoma: implications for Kaposi's sarcoma pathogenesis.
      ,
      • O'Leary JJ
      • Kennedy M
      • Luttich K
      • et al.
      Localisation of HHV‐8 in AIDS related lymphadenopathy.
      ,
      • O'Leary J
      • Kennedy M
      • Howells D
      • et al.
      Cellular localisation of HHV‐8 in Castleman's disease: is there a link with lymphnode vascularity?.
      ], carcinoma [
      • Capone RB
      • Pai SI
      • Koch WM
      • Gillison ML
      Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV‐associated head and neck squamous cell carcinoma.
      ,
      • Lo YMD
      • Chan LYS
      • Lo K‐W
      • et al.
      Quantitative analysis of cell‐free Epstein–Barr virus DNA in plasma of patients with nasopharyngeal carcinoma.
      ], cervical intra-epithelial neoplasia [
      • Josefsson A
      • Livak K
      • Gyllensten U
      Detection and quantitation of human papillomavirus by using the fluorescent 5′ exonuclease assay.
      ,
      • Swan DC
      • Tucker RA
      • Holloway BP
      • Icenogle JP
      A sensitive, type‐specific, fluorogenic probe assay for detection of human papillomavirus DNA.
      ,
      • Lanham S
      • Herbert A
      • Watt P
      HPV detection and measurement of HPV‐16, telomerase, and surviving transcripts in colposcopy clinic patients.
      ] and lymphoproliferative disorders [
      • MacKenzie J
      • Gallagher A
      • Clayton RA
      • et al.
      Screening for herpesvirus genomes in common acute lymphoblastic leukemia.
      ,
      • Jabs WJ
      • Hennig H
      • Kittel M
      • et al.
      Normalized quantification by real‐time PCR of Epstein–Barr virus load in patients at risk for posttransplant lymphoproliferative disorders.
      ] can be relatively easily studied using real-time PCR. Other studies have described the presence of flaviviruses [
      • Ishiguro T
      • Saitoh J
      • Yawata H
      • Yamagishi H
      • Iwasaki S
      • Mitoma Y
      Homogeneous quantitative assay of hepatitis C virus RNA by polymerase chain reaction in the presence of a fluorescent intercalater.
      ,
      • Lanciotti RS
      • Kerst AJ
      • Nasci RS
      • et al.
      Rapid detection of west nile virus from human clinical specimens, field‐collected mosquitoes, and avian samples by a TaqMan reverse transcriptase‐PCR assay.
      ,
      • Laue T
      • Emmerich P
      • Schmitz H
      Detection of dengue virus RNA inpatients after primary or secondary dengue infection by using the TaqMan automated amplification system.
      ,
      • White PA
      • Pan Y
      • Freeman AJ
      • et al.
      Quantification of hepatitis C virus in human liver and serum samples by using LightCycler reverse transcriptase PCR.
      ,
      • Callahan JD
      • Wu S‐JL
      • Dion‐Schultz A
      • et al.
      Development and evaluation of serotype‐ and group‐specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus.
      ,
      • Ratge D
      • Scheiblhuber B
      • Landt O
      • Berg J
      • Knabbe C
      Two‐round rapid‐cycle RT‐PCR in single closed capillaries increases the sensitivity of HCV RNA detection and avoids amplicon carry‐over.
      ,
      • Komurian‐Pradel F
      • Paranhos‐Baccalà G
      • Sodoyer M
      • et al.
      Quantitation of HCV RNA using real‐time PCR and fluorimetry.
      ,
      • Beames B
      • Chavez D
      • Guerra B
      • Notvall L
      • Brasky KM
      • Lanford RE
      Development of a primary tamarin hepatocyte culture system for GB virus‐B: a surrogate model for hepatitis C virus.
      ], hepadnaviruses [
      • Brechtbuehl K
      • Whalley SA
      • Dusheiko GM
      • Saunders NA
      A rapid real‐time quantitative polymerase chain reaction for hepatitis B virus.
      ,
      • Weinberger KM
      • Wiedenmann E
      • Böhm S
      • Jilg W
      Sensitive and accurate detection of hepatitis B virus DNA using a kinetic fluorescence detection system (TaqMan PCR).
      ,
      • Cane PA
      • Cook P
      • Ratcliffe D
      • Mutimer D
      • Pillay D
      Use of real‐time PCR and fluorimetry to detect lamivudine resistance‐associated mutations in hepatitis B virus.
      ], herpesviruses [
      • Niesters HG
      • Van Esser J
      • Fries E
      • Wolthers KC
      • Cornelissen J
      • Osterhaus AD
      Development of a real‐time quantitative assay for detection of Epstein–Barr virus.
      ,
      • Nitsche A
      • Steuer N
      • Schmidt CA
      • et al.
      Detection of human cytomegalovirus DNA by real‐time quantitative PCR.
      ,
      • Locatelli G
      • Santoro F
      • Veglia F
      • Gobbi A
      • Lusso P
      • Malnati MS
      Real‐time quantitative PCR for human herpesvirus 6 DNA.
      ,
      • Tanaka N
      • Kimura H
      • Iida K
      • et al.
      Quantitative analysis of cytomegalovirus load using a real‐time PCR assay.
      ,
      • Kimura H
      • Morita M
      • Yabuta Y
      • et al.
      Quantitative analysis of Epstein–Barr virus load by using a real‐time PCR assay.
      ,
      • Najioullah F
      • Thouvenot D
      • Lina B
      Development of a real‐time PCR procedure including an internal control for the measurement of HCMV viral load.
      ,
      • Ryncarz AJ
      • Goddard J
      • Wald A
      • Huang M‐L
      • Roizman B
      • Corey L
      Development of a high‐throughput quantitative assay for detecting herpes simplex virus DNA in clinical samples.
      ,
      • Schaade L
      • Kockelkorn P
      • Ritter K
      • Kleines M
      Detection of cytomegalovirus DNA in human specimens by LightCycler PCR.
      ,
      • Kearns AM
      • Turner AJL
      • Taylor CE
      • George PW
      • Freeman R
      • Gennery AR
      LightCycler‐based quantitative PCR for rapid detection of human herpesvirus 6 DNA in clinical material.
      ,
      • Kennedy MM
      • Lucas SB
      • Russell‐Jones R
      • et al.
      HHV8 and female Kaposi's sarcoma.
      ,
      • Capone RB
      • Pai SI
      • Koch WM
      • Gillison ML
      Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV‐associated head and neck squamous cell carcinoma.
      ,
      • Loparev VN
      • McCaustland K
      • Holloway BP
      • Krause PR
      • Takayama M
      • Schmid DS
      Rapid genotyping of varicella‐zoster virus vaccine and wild‐type strains with fluorophore‐labeled hybridization probes.
      ,
      • Schalasta G
      • Eggers M
      • Schmid M
      • Enders G
      Analysis of human cytomegalovirus DNA in urines of newborns and infants by means of a new ultrarapid real‐time PCR‐system.
      ,
      • Furuta Y
      • Ohtani F
      • Sawa H
      • Fukuda S
      • Inuyama Y
      Quantitation of varicella‐zoster virus DNA in patients with Ramsay Hunt syndrome and zoster sine herpete.
      ,
      • Lo YMD
      • Chan LYS
      • Lo K‐W
      • et al.
      Quantitative analysis of cell‐free Epstein–Barr virus DNA in plasma of patients with nasopharyngeal carcinoma.
      ,
      • Kearns AM
      • Draper B
      • Wipat W
      • et al.
      LightCycler‐based quantitative PCR for detection of cytomegalovirus in blood, urine and respiratory samples.
      ,
      • Stevens SJC
      • Verkuijlen SAWM
      • Van Den Brule AJC
      • Middeldorp JM
      Comparison of quantitative competitive PCR with LightCycler‐based PCR for measuring Epstein–Barr virus DNA load in clinical specimens.
      ,
      • Gallagher A
      • Armstrong AA
      • MacKenzie J
      • et al.
      Detection of Epstein–Barr virus (EBV) genomes in the serum of patients with EBV‐associated Hodgkin's disease.
      ,
      • Fernandez C
      • Boutolleau D
      • Manichanh C
      • Mangeney N
      • Agut H
      • Gautheret‐Dejean A
      Quantitation of HHV‐7 genome by real‐time polymerase chain reaction assay using MGB probe technology.
      ,
      • Biggar RJ
      • Whitby D
      • Marshall V
      • Linhares AC
      • Black F
      Human herpesvirus 8 in Brazilian Amerindians: a hyperendemic population with a new subtype.
      ,
      • Ohyashiki JK
      • Suzuki A
      • Aritaki K
      • et al.
      Use of real‐time PCR to monitor human herpesvirus 6 reactivation after allogeneic bone marrow transplantation.
      ,
      • Lallemand F
      • Desire N
      • Rozenbaum W
      • Nicolas J‐C
      • Marechal V
      Quantitative analysis of human herpesvirus 8 viral load using a real‐time PCR assay.
      ,
      • White I
      • Campbell TC
      Quantitation of cell‐free cell‐associated Kaposi's sarcoma associated herpesvirus DNA by real‐time PCR.
      ,
      • Peter JB
      • Sevall JS
      Review of 3200 serially received CSF samples submitted for type‐specific HSV detection by PCR in the reference laboratory setting.
      ,
      • Hawrami K
      • Breur J
      Development of a fluorogenic polymerase chain reaction assay (TaqMan) for the detection and quantitation of varicella zoster virus.
      ], orthomyxoviruses [
      • Van Elden LJR
      • Nijhuis M
      • Schipper P
      • Schuurman R
      • Van Loon AM
      Simultaneous detection of influenza viruses A and B using real‐time quantitative PCR.
      ], parvoviruses [
      • Gruber F
      • Falkner FG
      • Dorner F
      • Hämmerle T
      Quantitation of viral DNA by real‐time PCR applying duplex amplification, internal standardization, and two‐colour fluorescence detection.
      ], papovaviruses [
      • Biel SS
      • Held TK
      • Landt O
      • et al.
      Rapid quantification and differentiation of human polyomavirus DNA in undiluted urine from patients after bone marrow transplantation.
      ,
      • Whiley DM
      • Mackay IM
      • Sloots TP
      Detection and differentiation of human polyomaviruses JC and BK by LightCycler PCR.
      ,
      • Jordens JZ
      • Lanham S
      • Pickett MA
      • Amarasekara S
      • Aberywickrema I
      • Watt PJ
      Amplification with molecular beacon primers and reverse line blotting for the detection and typing of human papillomaviruses.
      ], paramyxoviruses [
      • Smith IL
      • Halpin K
      • Warrilow D
      • Smith GA
      Development of a fluorogenic RT‐PCR assay (TaqMan) for the detection of Hendra virus.
      ,
      • Aldous EW
      • Collins MS
      • McGoldrick A
      • Alexander DJ
      Rapid pathotyping of Newcastle disease virus (NDV) using fluorogenic probes in a PCR assay.
      ,
      • Whiley DM
      • Syrmis MW
      • Mackay IM
      • Sloots TP
      Detection of human respiratory syncytial virus in respiratory samples by LightCycler reverse transcriptase PCR.
      ], pestiviruses [
      • Vilcek Š
      • Paton DJ
      A RT‐PCR assay for the rapid recognition of border disease virus.
      ], picornaviruses [
      • Monopoeho S
      • Mignotte B
      • Schwartzbrod L
      • et al.
      Quantification of enterovirus RNA in sludge samples using single tube real‐time RT‐PCR.
      ,
      • Alexandersen S
      • Oleksiewicz MB
      • Donaldson AI
      The early pathogenesis of foot‐and‐mouth disease in pigs infected by contact: a quantitative time‐course study using TaqMan RT‐PCR.
      ,
      • Monpoeho S
      • Coste‐Burel M
      • Costa‐Mattioli M
      • et al.
      Application of a real‐time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid.
      ,
      • Nijhuis M
      • Van Maarseveen N
      • Schuurman R
      • et al.
      Rapid and sensitive routine detection of all members of the genus Enterovirus in different clinical specimens by real‐time PCR.
      ,
      • Corless CE
      • Guiver M
      • Borrow R
      • et al.
      Development and evaluation of a ‘real‐time’ RT‐PCR for the detection of enterovirus and parechovirus RNA in CSF and throat swab samples.
      ,
      • Watkins‐Reidel T
      • Woegerbauer M
      • Hollemann D
      • Hufnagl P
      Rapid diagnosis of enterovirus infections by real‐time PCR on the LightCycler using the TaqMan format.
      ,
      • Verstrepen WA
      • Kuhn S
      • Kockx MM
      • Van De Vyvere ME
      Rapid detection of enterovirus RNA in cerebrospinal fluid specimens with a novel single‐tube real‐time reverse transcription‐PCR assay.
      ,
      • Verstrepen WA
      • Bruynseels P
      • Mertens AH
      Evaluation of a rapid real‐time RT‐PCR assay for the detection of enterovirus RNA in cerebrospinal fluid specimens.
      ], poxviruses [
      • Espy MJ
      • Cockerill III, FR
      • Meyer RF
      • et al.
      Detection of smallpox virus DNA by LightCycler PCR.
      ], retroviruses [
      • Schutten M
      • Van Den Hoogen B
      • Van Der Ende ME
      • Gruters RA
      • Osterhaus ADME
      • Niesters HGM
      Development of a real‐time quantitative RT‐PCR for the detection of HIV‐2 RNA in plasma.
      ,
      • Leutenegger CM
      • Klein D
      • Hofmann‐Lehmann R
      • et al.
      Rapid feline immunodeficiency virus provirus quantitation by polymerase chain reaction using the TaqMan fluorogenic real‐time detection system.
      ,
      • Lewin SR
      • Vesanen M
      • Kostrikis L
      • et al.
      Use of real‐time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1‐infected individuals on prolonged effective antiretroviral therapy.
      ,
      • Argaw T
      • Ritzhaupt A
      • Wilson CA
      Development of a real time quantitative PCR assay for detection of porcine endogenous retrovirus.
      ,
      • Choo CK
      • Ling MT
      • Suen CKM
      • Chan KW
      • Kwong YL
      Retrovirus‐mediated delivery of HPV16 E7 antisense RNA inhibited tumorigenicity of CaSki cells.
      ,
      • Klein D
      • Janda P
      • Steinborn R
      • Salmons B
      • Günzburg WH
      Proviral load determination of different feline immunodeficiency virus isolates using real‐time polymerase chain reaction: influence of mismatches on quantification.
      ], rhabdoviruses [
      • Smith IL
      • Northill JA
      • Harrower BJ
      • Smith GA
      Detection of Australian bat lyssavirus using a fluorogenic probe.
      ] and TT virus [
      • Iriyama M
      • Kimura H
      • Nishikawa K
      • et al.
      The prevalence of TT virus (TTV) infection and its relationship to hepatitis in children.
      ].
      A significant number of studies have used PCR to detect viral load, and have proved its usefulness as an indicator of the extent of active infection, interactions between virus and host, and the changes in viral load as a result of antiviral therapies, all of which can play a role in the treatment regimen selected [
      • Nitsche A
      • Steuer N
      • Schmidt CA
      • Landt O
      • Siegert W
      Different real‐time PCR formats compared for the quantitative detection of human cytomegalovirus DNA.
      ,
      • Clementi M
      Quantitative molecular analysis of virus expression and replication.
      ,
      • Limaye AP
      • Jerome KR
      • Kuhr CS
      • et al.
      Quantitation of BK virus load in serum for the diagnosis of BK virus‐associated nephropathy in renal transplant recipients.
      ]. Conventional quantitative PCR has already proven that the application of nucleic acid amplification to the monitoring of viral load provides a useful marker of disease progression and the efficacy of antiviral compounds [
      • Clementi M
      • Menzo S
      • Manzin A
      • Bagnarelli P
      Quantitative molecular methods in virology.
      ,
      • Clementi M
      Quantitative molecular analysis of virus expression and replication.
      ,
      • Holodniy M
      • Katzenstein D
      • Sengupta S
      • et al.
      Detection and quantification of human immunodeficiency virus RNA in patient serum by use of the polymerase chain reaction.
      ,
      • Roberts TC
      • Brennan DC
      • Buller RS
      • et al.
      Quantitative polymerase chain reaction to predict occurrence of symptomatic cytomegalovirus infection and assess response to ganciclovir therapy in renal transplant recipients.
      ,
      • Rollag H
      • Sagedal S
      • Holter E
      • Degre M
      • Ariansen S
      • Nordal KP
      Diagnosis of cytomegalovirus infection in kidney transplant recipients by a quantitative RNA–DNA hybrid capture assay for cytomegalovirus DNA in leukocytes.
      ,
      • Kaneko S
      • Murakami S
      • Unoura M
      • Kobayashi K
      Quantitation of hepatitis C virus RNA by competitive polymerase chain reaction.
      ,
      • Menzo S
      • Bagnarelli P
      • Giacca M
      • Manzin A
      • Varaldo PE
      • Clementi M
      Absolute quantitation of viremia in human immunodeficiency virus infection by competitive reverse transcription and polymerase chain reaction.
      ]. Because disease severity and viral load are linked, the use of real-time PCR quantitation has proven beneficial when studying the role of viral reactivation or persistence in the progression of disease [
      • Nitsche A
      • Steuer N
      • Schmidt CA
      • et al.
      Detection of human cytomegalovirus DNA by real‐time quantitative PCR.
      ,
      • Tanaka N
      • Kimura H
      • Iida K
      • et al.
      Quantitative analysis of cytomegalovirus load using a real‐time PCR assay.
      ,
      • Kimura H
      • Morita M
      • Yabuta Y
      • et al.
      Quantitative analysis of Epstein–Barr virus load by using a real‐time PCR assay.
      ,
      • Najioullah F
      • Thouvenot D
      • Lina B
      Development of a real‐time PCR procedure including an internal control for the measurement of HCMV viral load.
      ,
      • Kearns AM
      • Turner AJL
      • Taylor CE
      • George PW
      • Freeman R
      • Gennery AR
      LightCycler‐based quantitative PCR for rapid detection of human herpesvirus 6 DNA in clinical material.
      ,
      • Furuta Y
      • Ohtani F
      • Sawa H
      • Fukuda S
      • Inuyama Y
      Quantitation of varicella‐zoster virus DNA in patients with Ramsay Hunt syndrome and zoster sine herpete.
      ,
      • Lo YMD
      • Chan LYS
      • Lo K‐W
      • et al.
      Quantitative analysis of cell‐free Epstein–Barr virus DNA in plasma of patients with nasopharyngeal carcinoma.
      ,
      • Laue T
      • Emmerich P
      • Schmitz H
      Detection of dengue virus RNA inpatients after primary or secondary dengue infection by using the TaqMan automated amplification system.
      ,
      • Ohyashiki JK
      • Suzuki A
      • Aritaki K
      • et al.
      Use of real‐time PCR to monitor human herpesvirus 6 reactivation after allogeneic bone marrow transplantation.
      ,
      • Lallemand F
      • Desire N
      • Rozenbaum W
      • Nicolas J‐C
      • Marechal V
      Quantitative analysis of human herpesvirus 8 viral load using a real‐time PCR assay.
      ,
      • Hawrami K
      • Breur J
      Development of a fluorogenic polymerase chain reaction assay (TaqMan) for the detection and quantitation of varicella zoster virus.
      ,
      • Limaye AP
      • Huang M‐L
      • Leisenring W
      • Stensland L
      • Corey L
      • Boeckh M
      Cytomegalovirus (CMV) DNA load in plasma for the diagnosis of CMV disease before engraftment in hematopoietic stem‐cell transplant recipients.
      ,
      • Limaye AP
      • Huang M‐L
      • Leisenring W
      • Stensland L
      • Corey L
      • Boeckh M
      Cytomegalovirus (CMV) DNA load in plasma for the diagnosis of CMV disease before engraftment in hematopoietic stem‐cell transplant recipients.
      ,
      • Chang L‐J
      • Urlacher V
      • Iwakuma T
      • Cui Y
      • Zucali J
      Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system.
      ,
      • Hoshino Y
      • Kimura H
      • Kuzushima K
      • et al.
      Early intervention in post‐transplant lymphoproliferative disorders based on Epstein–Barr viral load.
      ,
      • Machida U
      • Kami M
      • Fukui T
      • et al.
      Real‐time automated PCR for early diagnosis and monitoring of cytomegalovirus infection after bone marrow transplantation.
      ,
      • Gault E
      • Michel Y
      • Nicolas J‐C
      • Belabani C
      • Nicolas J‐C
      • Garbarg‐Chenon A
      Quantification of human cytomegalovirus DNA by real‐time PCR.
      ,
      • Tanaka N
      • Kimura H
      • Hoshino Y
      • et al.
      Monitoring four herpesviruses in unrelated cord blood transplantation.
      ]. Alterations to a microbe's tropism or its replication, and the effects that these changes have on a host cell, can also be followed using real-time PCR [
      • Kennedy MM
      • O'Leary JJ
      • Oates JL
      • et al.
      Human herpes virus 8 (HHV‐8) in Kaposi's sarcoma: lack of association with Bcl‐2 and p53 protein expression.
      ,
      • Kennedy MM
      • Biddolph S
      • Lucas SB
      • et al.
      CD40 upregulation is independent of HHV‐8 in the pathogenesis of Kaposi's sarcoma.
      ,
      • Kennedy MM
      • Biddolph S
      • Lucas SB
      • et al.
      Cyclin D1 expression and HHV‐8 in Kaposi sarcoma.
      ].
      The role of highly sensitive and rapid real-time PCR assays in the thorough assessment of viral gene therapy vectors before their use in clinical trials has become an important one. Nuclease oligoprobes have been most commonly used for these studies, which assess the biodistribution, function and purity of the novel ‘drug’ preparations [
      • Choo CK
      • Ling MT
      • Suen CKM
      • Chan KW
      • Kwong YL
      Retrovirus‐mediated delivery of HPV16 E7 antisense RNA inhibited tumorigenicity of CaSki cells.
      ,
      • Klein D
      • Janda P
      • Steinborn R
      • Salmons B
      • Günzburg WH
      Proviral load determination of different feline immunodeficiency virus isolates using real‐time polymerase chain reaction: influence of mismatches on quantification.
      ,
      • Smith IL
      • Northill JA
      • Harrower BJ
      • Smith GA
      Detection of Australian bat lyssavirus using a fluorogenic probe.
      ,
      • Iriyama M
      • Kimura H
      • Nishikawa K
      • et al.
      The prevalence of TT virus (TTV) infection and its relationship to hepatitis in children.
      ,
      • Nitsche A
      • Steuer N
      • Schmidt CA
      • Landt O
      • Siegert W
      Different real‐time PCR formats compared for the quantitative detection of human cytomegalovirus DNA.
      ,
      • Clementi M
      Quantitative molecular analysis of virus expression and replication.
      ,
      • Limaye AP
      • Jerome KR
      • Kuhr CS
      • et al.
      Quantitation of BK virus load in serum for the diagnosis of BK virus‐associated nephropathy in renal transplant recipients.
      ,
      • Holodniy M
      • Katzenstein D
      • Sengupta S
      • et al.
      Detection and quantification of human immunodeficiency virus RNA in patient serum by use of the polymerase chain reaction.
      ,
      • Roberts TC
      • Brennan DC
      • Buller RS
      • et al.
      Quantitative polymerase chain reaction to predict occurrence of symptomatic cytomegalovirus infection and assess response to ganciclovir therapy in renal transplant recipients.
      ,
      • Rollag H
      • Sagedal S
      • Holter E
      • Degre M
      • Ariansen S
      • Nordal KP
      Diagnosis of cytomegalovirus infection in kidney transplant recipients by a quantitative RNA–DNA hybrid capture assay for cytomegalovirus DNA in leukocytes.
      ,
      • Kaneko S
      • Murakami S
      • Unoura M
      • Kobayashi K
      Quantitation of hepatitis C virus RNA by competitive polymerase chain reaction.
      ,
      • Menzo S
      • Bagnarelli P
      • Giacca M
      • Manzin A
      • Varaldo PE
      • Clementi M
      Absolute quantitation of viremia in human immunodeficiency virus infection by competitive reverse transcription and polymerase chain reaction.
      ,
      • Limaye AP
      • Huang M‐L
      • Leisenring W
      • Stensland L
      • Corey L
      • Boeckh M
      Cytomegalovirus (CMV) DNA load in plasma for the diagnosis of CMV disease before engraftment in hematopoietic stem‐cell transplant recipients.
      ,
      • Chang L‐J
      • Urlacher V
      • Iwakuma T
      • Cui Y
      • Zucali J
      Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system.
      ,
      • Hoshino Y
      • Kimura H
      • Kuzushima K
      • et al.
      Early intervention in post‐transplant lymphoproliferative disorders based on Epstein–Barr viral load.
      ,
      • Machida U
      • Kami M
      • Fukui T
      • et al.
      Real‐time automated PCR for early diagnosis and monitoring of cytomegalovirus infection after bone marrow transplantation.
      ,
      • Gault E
      • Michel Y
      • Nicolas J‐C
      • Belabani C
      • Nicolas J‐C
      • Garbarg‐Chenon A
      Quantification of human cytomegalovirus DNA by real‐time PCR.
      ,
      • Tanaka N
      • Kimura H
      • Hoshino Y
      • et al.
      Monitoring four herpesviruses in unrelated cord blood transplantation.
      ,
      • Kennedy MM
      • O'Leary JJ
      • Oates JL
      • et al.
      Human herpes virus 8 (HHV‐8) in Kaposi's sarcoma: lack of association with Bcl‐2 and p53 protein expression.
      ,
      • Kennedy MM
      • Biddolph S
      • Lucas SB
      • et al.
      CD40 upregulation is independent of HHV‐8 in the pathogenesis of Kaposi's sarcoma.
      ,
      • Kennedy MM
      • Biddolph S
      • Lucas SB
      • et al.
      Cyclin D1 expression and HHV‐8 in Kaposi sarcoma.
      ,
      • Gerard CJ
      • Arboleda MJ
      • Solar G
      • Mule JJ
      • Kerr WG
      A rapid and quantitative assay to estimate gene transfer into retrovirally transduced hematopoietic stem/progenitor cells using a 96‐well format PCR and fluorescent detection system universal for MMLV‐based proviruses.
      ,
      • Rohr U‐P
      • Wulf M‐A
      • Stahn S
      • Steidl U
      • Haas R
      • Kronenwett R
      Fast and reliable titration of recombinant adeno‐associated virus type‐2 using quantitative real‐time PCR.
      ,
      • Josefsson AM
      • Magnusson PKE
      • Ylitalo N
      • et al.
      Viral load of human papilloma virus 16 as a determinant for development of cervical carcinoma in situ: a nested case–control study.
      ,
      • Hackett NR
      • El Sawy T
      • Lee LY
      • et al.
      Use of quantitative TaqMan real‐time PCR to track the time‐dependent distribution of gene transfer vectors in vivo.
      ,
      • Sanburn N
      • Cornetta K
      Rapid titer determination using quantitative real‐time PCR.
      ,
      • Scherr M
      • Battmer K
      • Blömer U
      • Ganser A
      • Grez M
      Quantitative determination of lentiviral vector particle numbers by real‐time PCR.
      ].
      Likewise, the study of new and emerging viruses has been ideally complemented by the use of homogeneous real-time PCR assays as tools to demonstrate and strengthen epidemiological links between unique viral sequences and the clinical signs and symptoms experienced by patients [
      • Smith IL
      • Halpin K
      • Warrilow D
      • Smith GA
      Development of a fluorogenic RT‐PCR assay (TaqMan) for the detection of Hendra virus.
      ,
      • Lanciotti RS
      • Kerst AJ
      • Nasci RS
      • et al.
      Rapid detection of west nile virus from human clinical specimens, field‐collected mosquitoes, and avian samples by a TaqMan reverse transcriptase‐PCR assay.
      ,
      • Smith IL
      • Northill JA
      • Harrower BJ
      • Smith GA
      Detection of Australian bat lyssavirus using a fluorogenic probe.
      ,
      • Lanciotti RS
      • Kerst AJ
      Nucleic acid sequence‐based amplification assays for rapid detection of West Nile and St Louis encephalitis.
      ,
      • Mackay IM
      • Jacob KC
      • Woolhouse D
      • et al.
      Molecular assays for the detection of human metapneumovirus.
      ,
      • Halpin K
      • Young PL
      • Field HE
      • Mackenzie JS
      Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus.
      ,
      • Gibb TR
      • Norwood DA
      • Woollen N
      • Henchal EA
      Development and evaluation of a fluorogenic 5′‐nuclease assay to identify Marburg virus.
      ,
      • Gibb TR
      • Norwood DA
      • Woollen N
      • Henchal EA
      Development and evaluation of a fluorogenic 5′ nuclease assay to detect and differentiate between Ebola virus subtypes Zaire and Sudan.
      ].
      The speed and flexibility of real-time PCR has also proven useful for commercial interests who require exquisite sensitivity to screen for microbial contamination within large-scale reagent preparations produced from eukaryotic expression systems [
      • Brorson K
      • Swann PG
      • Lizzio E
      • Maudru T
      • Peden K
      • Stein KE
      Use of a quantitative product‐enhanced reverse transcriptase assay to monitor retrovirus levels in mAb cell‐culture and downstream processing.
      ,
      • De Wit C
      • Fautz C
      • Xu Y
      Real‐time quantitative PCR for retrovirus‐like particle formation in CHO cell culture.
      ].

       Bacteria

      The benefits to the patient from rapid real-time PCR assays are most notable when applied to the detection of bacteria. The results can quickly inform the clinician as to the infection status of the patient, allowing a more specific and timely application of antibiotics. This can limit the potential for toxicity due to shotgun treatment regimens, reduce the duration of a hospital stay and prevent the improper use of antibiotics, thus minimising the potential for resistant strains to emerge.
      Broad applications of real-time PCR can augment or replace traditional culture or histochemical assays, as was seen with the creation of a molecular assay capable of classifying bacteria in the same way as a Gram stain [
      • Klaschik S
      • Lehmann LE
      • Raadts A
      • Book M
      • Hoeft A
      • Stuber F
      Real‐time PCR for the detection and differentiation of Gram‐positive and Gram‐negative bacteria.
      ]. However, specific bacterial species are more frequently the focus for real-time PCR assays, especially when long culture times can be replaced by rapid and specific gene detection. Leptospira genospecies, Mycobacterium and Propionibacterium spp., Chlamydia spp., Legionella pneumophila and Listeria monocytogenes have all been detected and in some cases quantitated with the use of real-time PCR assays [
      • Lunge VR
      • Miller BJ
      • Livak KJ
      • Batt CA
      Factors affecting the performance of 5′ nuclease PCR assays for Listeria monocytogenes detection.
      ,
      • Woo THS
      • Patel BKC
      • Smythe LD
      • Symonds ML
      • Norris MA
      • Dohnt MF
      Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid‐cycle PCR.
      ,
      • Miller N
      • Cleary T
      • Kraus G
      • Young AK
      • Spruill G
      • Hnatyszyn HJ
      Rapid and specific detection of Mycobacterium tuberculosis from acid‐fast bacillus smear‐positive respiratory specimens and BacT/ALERT MP culture bottles by using fluorogenic probes and real time‐PCR.
      ,
      • O'Mahony J
      • Hill C
      A real time PCR assay for the detection and quantitation of Mycobacterium avium subsp. paratuberculosis using SYBR green and the Light Cycler.
      ,
      • Torres MJ
      • Criado A
      • Palomares JC
      • Aznar J
      Use of real‐time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance‐associated mutations in Mycobacterium tuberculosis.
      ,
      • Eishi Y
      • Suga M
      • Ishige I
      • et al.
      Quantitative analysis of mycobacterial and propionibacterial DNA in lymph nodes of Japanese and European patients with sarcoidosis.
      ,
      • Kraus G
      • Cleary T
      • Miller N
      • et al.
      Rapid and specific detection of the Mycobacterium tuberculosis complex using fluorogenic probes and real‐time PCR.
      ,
      • De Viedma DG
      • Diaz Infanntes M
      • Lasala F
      • Chaves F
      • Alcalá L
      • Bouza E
      New real‐time PCR able to detect in a single tube multiple rifamin resistance mutations and high‐level isoniazid resistance mutations in Mycobacterium tuberculosis.
      ,
      • Li Q‐G
      • Liang J‐X
      • Luan G‐Y
      • Zhang Y
      • Wang K
      Molecular beacon‐based homogeneous fluorescence PCR assay for the diagnosis of infectious diseases.
      ,
      • Creelan JL
      • McCullough SJ
      Evaluation of strain‐specific primer sequences from an abortifacient strain of ovine Chlamydophila abortus (Chlamydia psittaci) for detection of EAE by PCR.
      ,
      • Hayden RT
      • Uhl JR
      • Qian X
      • et al.
      Direct detection of Legionella species from bronchoalveolar lavage and open lung biopsy specimens: comparison of LightCycler PCR, in situ hybridization, direct fluorescence antigen detection, and culture.
      ,
      • Ballard AL
      • Fry NK
      • Chan L
      • et al.
      Detection of Legionella pneumophila using real‐time PCR hybridisation assay.
      ,
      • Wellinghausen N
      • Frost C
      • Marre E
      Detection of Legionellae in hospital water samples by quantitative real‐time LightCycler PCR.
      ,
      • Reischl U
      • Linde H‐J
      • Lehn N
      • Landt O
      • Barratt K
      • Wellinghausen N
      Direct detection and differentiation of Legionella spp. & Legionella pneumophila in clinical specimens by dual‐colour real‐time PCR and melting curve analysis.
      ].
      The detection of Neisseria gonorrhoeae has benefited from real-time PCR, particularly in the role of a confirmatory test when the specificity of commercial assays fails [
      • Whiley DM
      • LeCornec GM
      • Mackay IM
      • Siebert DJ
      • Sloots TP
      A real‐time PCR assay for the detection of Neisseria gonorrhoeae by LightCycler.
      ]. This example highlights the need for care when choosing a bacterial PCR target, especially when that target exists on a plasmid that is exchanged among other bacteria, providing potentially confusing diagnostic results. Neisseria meningitidis causes meningococcal disease, and real-time PCR has proven to be a powerful tool that can be quickly developed for the rapid discrimination of currently circulating pathogens [
      • Probert WS
      • Bystrom SL
      • Khashe S
      • Schrader KN
      • Wong JD
      5′‐Exonuclease assay for detection of serogroup Y Neisseria meningitidis.
      ].
      The detection and monitoring of antibiotic resistance among clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, Helicobacter pylori, Enterococcus faecalis and Enterococcus faecium has also benefited from real-time applications [
      • Randegger CC
      • Hachler H
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