Real-Time PCR in the Microbiology Laboratory

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R EVIEW 10.1111/j.1198-743X.2004.00722.x

Real-time PCR in the microbiology laboratoryI. M. Mackay1,2

1Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre and 2Department of

Paediatrics, Royal Childrens Hospital, Brisbane, Queensland, Australia

A B S T R A C T

Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted asthe 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 hascatalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk ofcarryover contamination is minimised. There is an increasing number of chemistries which are used todetect PCR products as they accumulate within a closed reaction vessel during real-time PCR. Theseinclude the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucle-otide 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 traditionalterminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted thedevelopment of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation andgenotyping of the microbial causes of infectious disease, will also be discussed. Because theamplification hardware and the fluorogenic detection chemistries have evolved rapidly, this reviewaims to update the scientist on the current state of the art. Additionally, the advantages, limitations andgeneral background of real-time PCR technology will be reviewed in the context of the microbiologylaboratory.

Keywords Real-time PCR, quantitation, molecular, diagnostics

Accepted: 26 December 20021

Clin Microbiol Infect 2004; 10: 190212

B A C K G R O U N D

Diagnostic microbiology is in the midst of a newera. Rapid nucleic acid amplification and detec-tion technologies are quickly displacing the tra-ditional assays based on pathogen phenotyperather than genotype. The polymerase chainreaction (PCR) [1,2] has increasingly been des-cribed as the latest gold standard for detectingsome microbes, but such claims can only be takenseriously when each newly described assay issuitably compared to its characterised predeces-sors [36]. PCR is the most commonly usednucleic acid amplification technique for the

diagnosis of infectious disease, surpassing theprobe and signal amplification methods. The PCRcan amplify DNA or, when preceded by a reversetranscription (RT) incubation at 4255 C, RNA.RT-PCR is the most sensitive method for thedetection and quantitation of mRNA, especiallyfor low-abundance templates [710]. The PCRprocess can be divided into three steps. First,double-stranded DNA (dsDNA) is separated attemperatures above 90 C. Second, oligonucleo-tide primers generally anneal at 5060 C, and,finally, optimal primer extension occurs at7078 C. The temperature at which the primeranneals is usually referred to as the TM. This is thetemperature at which 50% of the oligonucleotidetarget duplexes have formed. In the case of real-time PCR, the oligonucleotide could represent aprimer or a labelled probe. The TM differs from thedenaturation temperature (TD), which refers to theTM as it applies to the melting of dsDNA. The rate

Corresponding author and reprint requests: I. M. Mackay,CVRU, SASVRC, Royal Childrens Hospital, Herston Road,Herston, Queensland 4029, AustraliaE-mail: [email protected]

2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases


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of temperature change or ramp rate, the length ofthe incubation at each temperature and the num-ber of times each cycle of temperatures is repeatedare controlled by a programmable thermal cycler.Current technologies have significantly shortened

the ramp rates, and therefore assay time, throughthe use of electronically controlled heating blocksor fan-forced heated air flows.

The traditional diagnostic microbiologicalassays include microscopy, microbial culture,antigenaemia and serology. These can be limitedby poor sensitivity, slow-growing or poorly viableorganisms, narrow detection windows, complexinterpretation, immunosuppression, antimicrobialtherapy, high levels of background and non-specific cross-reactions [11,12]. Nonetheless,microbial culture produces valuable epidemio-

logical data, revealing new, uncharacterised oratypical microbes and yielding intact or infectiousorganisms for further study [13]. It is thereforeclear that the role of the traditional assay contin-ues to be an important one [1418]. Additionally,PCR has some significant limitations. Our abilityto design oligonucleotide primers only extends toour knowledge of a microorganisms genome aswell as the ability of publicly available sequencedatabases to suitably represent all variants of thatmicrobe. It is common for microbial genomes tocontain unexpected mutations, which reduce or

abrogate the function of a PCR. Traditionally,false-positives due to carryover contaminationhave caused considerable problems in the routineimplementation of PCR in the diagnostic laborat-ory and have led to strict guidelines for the designof laboratories dedicated to performing PCR.Additionally, PCR may be too sensitive for someapplications, detecting a microbe that is present atnon-pathogenic levels. Thus, care is requiredwhen designing a PCR assay and interpreting itsresults.

Existing combinations of PCR and amplicondetection assays will be called conventional PCRthroughout this review. The detection compo-nents include agarose gel electrophoresis [19],Southern blot [20] and ELISA-like systems [21].Conventional PCR has been used to obtain quan-titative data, with promising results [22]. How-ever, these approaches have suffered from thelaborious post-PCR handling steps required toevaluate the amplicon [23].

The possibility that, in contrast to conventionalPCR, the detection of amplicon could be visual-

ised as the amplification progressed was awelcome one. This expanded the role of PCRfrom that of a pure research tool to that of aversatile technology permitting the developmentof routine diagnostic applications for the high-

and low-throughput clinical microbiology labor-atory [24,25]. Along the way, real-time assayshave provided insight into the kinetics of the PCRas well as the efficiency of different nucleic acidextraction methods and the role that some com-pounds play in the inhibition of amplification[20,2633]. Real-time PCR has made many morescientists familiar with the crucial factors contri-buting to successful amplification of nucleic acids.Today, real-time PCR is used to detect nucleicacids from food, vectors used in gene therapyprotocols, genetically modified organisms, and

areas of human and veterinary microbiology andoncology [3436].The monitoring of accumulating amplicon in

real time has been made possible by the labellingof primers, oligonucleotide probes (oligoprobes)or amplicons with molecules capable of fluores-cing. These labels produce a change in signalfollowing direct interaction with, or hybridisationto, the amplicon. The signal is related to theamount of amplicon present during each cycleand will increase as the amount of specificamplicon increases. These chemistries have clear

benefits over earlier radiogenic labels, includingan absence of radioactive emissions, easy disposaland an extended shelf-life [37].

A significant improvement introduced by real-time PCR is the increased speed with which it canproduce results. This is largely due to the reducedcycle times, removal of separate post-PCR detec-tion procedures, and the use of sensitive fluores-cence detection equipment, allowing earlieramplicon detection [38,39]. A reduced ampliconsize may also play a role in this speed; however, ithas been shown that decreased product size doesnot strictly correlate with improved PCR effi-ciency, and that the distance between the primersand the oligoprobe may play a more significantrole [40,41].

The technical disadvantages of using real-timePCR instead of conventional PCR include the needto break the seal of an otherwise closed system inorder to monitor amplicon size, the incompatibil-ity of certain platforms with some fluorescentchemistries, and the relatively restricted multiplexcapabilities of current systems. Additionally, the

Mackay Real-time PCR in the microbiology laboratory 191

2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 190212


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start-up expense of real-time PCR may be prohib-itive for low-throughput laboratories.

Because most of the popular real-time PCRchemistries involve hybridisation of an oligo-probe(s) to a complementary sequence on one of

the amplicon strands, the inclusion of more of theprimer that creates that strand is beneficial to thegeneration of an increased fluorescent signal [42].We have found that this asymmetric PCRapproach improves the signal from both ourconventional and real-time oligoprobe-hybridisa-tion assays.

Although some of the fluorescent labels havebeen given an associated nomenclature by theirdeveloper, the term fluorophore will generally beused to describe these moieties, while their inclu-sion as labels on an oligonucleotide will be

described as rendering it fluorogenic. The mostcommonly used fluorogenic oligoprobes relyupon fluorescence resonance energy transfer(FRET) between fluorogenic labels or betweenone fluorophore and a dark or black-hole non-fluorescent quencher (NFQ), which dispersesenergy as heat rather than fluorescence [43]. FRETis a spectroscopic process by which energy ispassed between molecules separated by 10100 A

that have overlapping emission and absorptionspectra [4446]. Forster primarily developed thetheory behind this process, which is a non-radia-

tive induced dipole interaction [43,47,48].As alluded to earlier, post-amplification mani-pulation of the amplicon is not required for real-time PCR, because the fluorescent signals aredirectly measured as they pass out of the reactionvessel, so real-time PCR is often described as aclosed or homogeneous system. Apart from thetime saved by amplifying and detecting templatein a single tube, there is minimal potential forcarryover contamination, and the assays per-formance can be closely scrutinised withoutintroducing errors due to handling of the ampl-icon [49]. In addition, real-time PCR has proven tobe cost-effective on a per-run basis, when imple-mented in a high-throughput laboratory [50],particularly when replacing conventional, cul-ture-based approaches to microbial detection.

In the remainder of this review, the theorybehind real-time PCR will be discussed. Addi-tionally, its rapidly expanding use in the study ofhuman infectious disease will provide an exampleof its acceptance and effectiveness in the diagnos-tic microbiology laboratory.

A M P L I C O N D E T E C T I O N

It is the detection process that discriminates real-time PCR from conventional PCR assays. There isa range of chemistries currently in use which can

be broadly categorised as specific or non-specificfor the amplicons sequence [51]. These haverecently been reviewed in detail [52]. Severaladditional reporter systems have since been des-cribed, and these will be discussed below; how-ever, few applications have been described for thespecific detection and genotyping of microbes.

While the most common oligoprobes are basedon traditional nucleic acid chemistry, the peptidenucleic acid (PNA) is becoming a more popularchoice for oligonucleotide backbones. The PNA isa DNA analogue that is formed of neutral repea-

ted N-(2-aminoethyl) glycine units instead ofnegatively charged sugar phosphates [53].However, the PNA retains the same sequencerecognition properties as DNA.

In general, however, the specific and non-specific fluorogenic chemistries detect ampliconwith the same sensitivity [39].

L I N E A R O L I G O P R O B E S

The use of a pair of adjacent, fluorogenic hybrid-isation oligoprobes was first described in the late

1980s [45,54], and, now known as HybProbes,they have become the manufacturers chemistryof choice for the LightCycler (Roche MolecularBiochemicals, Mannheim, Germany), a capillary-based, microvolume fluorimeter and thermocy-cler with rapid temperature control [39,55]. Theupstream oligoprobe is labeled with a 3 donorfluorophore (fluorescein isothiocyanate, FITC),and the downstream probe is commonly labelledwith either a LightCycler Red 640 or Red 705acceptor fluorophore at the 5-terminus, so thatwhen both oligoprobes are hybridised, the twofluorophores are located within 10 nucleotides ofeach other.

Most recently described fluorogenic oligo-probes fall into the linear class of oligoprobe.The recently described double-stranded oligo-probes function by displacement hybridisation(Fig. 1a) [56]. 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 withan NFQ. When the full-length complementary

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Fig. 1. Oligoprobe 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 hybridisewith the fluorophore-labelled primer. (c) Light-up probes. These PNA probes fluoresce in the presence of a hybridisedDNA strand due to their asymmetric thiazole orange fluorophore (T; open triangle). (d) HyBeacons. In close proximity toDNA, 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 acomplementary, dual-labelled oligonucleotide substrate, the continuously amplified DNAzyme will specifically cleave thetemplate between the fluorophore and quencher (Q; open pentagon), releasing the labels and allowing fluorescence tooccur. (f) Tripartite molecular beacons. The fluorophore is removed from the NFQs influence upon opening of the hairpinbecause of hybridisation to specific amplicon, permitting fluorescence.




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sequence in the form of an amplicon is present,the reporter strand will preferentially hybridise tothe longer amplicon, disrupting the quenchedoligoprobe duplex and permitting the fluoro-phore to emit its excitation energy directly.

This technique can also be used with a fluor-ophore-labelled primer, and, due to the addedstringency of the complementary strand, thesystem acts as its own hot-start, as was shownusing an NFQ-labeled PNA [57] strand (Q-PNA)(Fig. 1b) [58]. In this system, the quenching probeis bound to unincorporated fluorogenic primersuch that the NFQ and fluorophore are adjacent,resulting in a quenched system. Once the dsDNAamplicon is created by primer extension, how-ever, the Q-PNA is displaced, and the fluorophorecan fluoresce. The PNA backbones cannot be

extended or hydrolysed by a DNA polymerase.The light-up probe is also a PNA to which theasymmetric cyanine fluorophore thiazole orangeis attached (Fig. 1c) [59]. When hybridised with anucleic acid target, as either a duplex or triplex,the fluorophore becomes strongly fluorescent.These oligoprobes do not interfere with the PCRor require conformational change, they are sensi-tive to single nucleotide mismatches and, becausea single reporter is used, they allow the directmeasurement of fluorescence instead of the meas-urement of a change in fluorescence between two

fluorophores [59,60]. However, non-specific fluor-escence has been reported during extended cyc-ling [61].

The HyBeacon is a single linear oligonucleotideinternally labelled with a fluorophore that emitsan increased signal upon formation of a duplexwith the target DNA strand (Fig. 1d) [62,63]. TheHyBeacon is labelled at the 3-terminus with aphosphate or octanediol molecule to prevent Taq-mediated extension. This technique is used withall the non-incorporating nucleotide-based oligo-probe chemistries used in real-time PCR to ensurethat they do not function as a primer. Thischemistry does not require destruction, interac-tion with a second oligoprobe or secondarystructure changes to produce a signal, and it isrelatively cheap and simple to design.

D U A L - L A B E L L E D O L I G O P R O B E S

In the early 1990s, an innovative approachinvolved nick-translation PCR in combinationwith dual-fluorophore-labelled oligoprobes was

introduced [26]. In the first truly homogeneousassay of its kind, a fluorophore was added to the5-terminus and another to the middle of asequence-specific oligoprobe. When in such closeproximity, the 5 reporter fluorophore (6-carboxy-

fluoroscein; FAM) transferred laser-inducedexcitation energy by FRET to the 3 quencherfluorophore (6-carboxy-tetramethyl-rhodamine;TAMRA). The oligoprobe hybridised to its tem-plate prior to the extension step, and the fluor-ophores were subsequently released during theprimer extension step as a result of the 5 to 3endonuclease activity of a suitable DNA polym-erase. Once the labels were separated, the repor-ters emissions were no longer quenched, and theinstrument monitored the resulting fluorescence.Today, these oligoprobes are labelled at each

terminus and are called 5

nuclease, hydrolysis orTaqMan oligoprobes. The nuclease oligoprobe isthe manufacturers chemistry of choice for theABI Prism sequence detection systems.

A modification of the 5 nuclease chemistry hasresulted in the minor groove binding (MGB)oligoprobes [64]. This chemistry, commerciallycalled the Eclipse oligoprobes, replaces the Taq-Man oligoprobes standard TAMRA quencherwith a proprietary NFQ and incorporates amolecule that hyperstabilises the oligoprobetar-get duplex by folding into the minor groove of the

dsDNA [65,66]. A fluorophore is attached to the3 end, and in the unbound state the oligoprobeassumes a random coil configuration that isefficiently quenched. This chemistry allows theuse of very short (1217-nucleotide) oligoprobesbecause of a 1530 C rise in their TM resultingfrom the interaction of the MGB with the DNAhelix. These short oligoprobes are ideal for detect-ing single-nucleotide polymorphisms (SNPs),because they are more significantly destabilisedby nucleotide changes within the hybridisationsite than are larger oligoprobes.

Another dual-labelled oligonucleotide sequ-ence has been used as the signal-generatingportion of the DzyNA-PCR system (Fig. 1e) [67].Here, the reporter and quencher molecules areseparated following specific cleavage of the oligo-nucleotides holding them in close proximity. Thiscleavage is performed by a DNAzyme, which iscreated during the PCR as the complement ofan antisense DNAzyme sequence included inthe 5 tail of one of the primers. Upon clea-vage, the fluorophores are released, allowing




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the production of fluorescence in an identicalmanner to a hydrolysed TaqMan oligoprobe.

H A I R P I N O L I G O N U C L E O T I D E S

Molecular beacons were the first hairpin oligo-probes to be used in real-time PCR. The molecularbeacons fluorogenic labels are positioned at thetermini of the oligoprobe. The labels are held inclose proximity by distal stem regions of homol-ogous base pairing deliberately designed to createa hairpin structure. The closed hairpin isquenched due either to FRET or direct collisiontransfer of energy occurring at the molecular levelas a consequence of the intimate proximity of thelabels [68]. In the presence of a complementarysequence, designed to occur within the bounds of

the primer binding sites, the oligoprobe willhybridise, shifting into an open configuration.The fluorophore is now spatially removed fromthe quenchers influence, allowing fluorescentemissions to be monitored [69]. This structuralchange occurs in each cycle, increasing in cumu-lative intensity as the amount of specific ampliconincreases. The quencher, DABCYL (4-(4-dimeth-ylamino-phenylazo)-benzene), differs from thatdescribed for the nuclease oligoprobes because itis an NFQ.

Recently, tripartite molecular beacons have

been added to this class of fluorogenic chemistry(Fig. 1f) [70]. These oligoprobes have beendesigned to fulfill a need for suitably high-throughput chemistries and they combine amolecular beacons hairpin with long or unla-belled single-stranded arms, each designed tohybridise to an oligonucleotide labelled witheither a fluorophore or an NFQ. The system isquenched in the hairpin state due to the closeproximity of the labels, but fluorescent whenhybridised to the specific amplicon strand.Because the function of these oligoprobes dependsupon correct hybridisation of the stem and twooligoprobes, their accurate design is crucial [8].

Finally, a self-quenching hairpin primer hasrecently been described which is commerciallyentitled the light upon extension (LUX) fluoro-genic primer [71]. This chemistry is dark in theabsence of specific amplicon, through the naturalquenching ability of a carefully placed guanosinenucleotide. The natural quencher is broughtinto close proximity with the FAM or JOE5 2,7-dimethoxy-4,5-dichloro-6-carboxy-fluoroscein

fluorophore via a stretch of 5 and 3 comple-mentary sequences. In the presence of specifictarget, the primer hybridises, opening the hairpinand permitting fluorescence from the fluoro-phore.

M I C R O B I A L Q U A N T I T A T I O N

Although the terminology is often confused, real-time PCR does not inherently imply quantitativePCR. To quantify the amount of template presentin a sample, thought must be given to the typeand number of controls required. Standards areused to allow calculation of the amount oftemplate present in a patient sample, whileinternal controls (ICs) are mostly used to deter-mine the occurrence of false-negative reactions,

examine the ability to amplify from a preparationof nucleic acids, and, more rarely in real-timePCR, as a standard for quantitation. Certainly, thereliability of quantitative PCR methods is inti-mately associated with the choice and quality ofthe assay controls [72,73].

No matter what control is chosen, it is imper-ative to accurately determine its concentration[74] and to ensure that ICs are added at suitablelevels in order to prevent extreme competitionwith the wild-type template for reagents [75]. Theuse of a spectrometer is inadequate for quantitat-

ing a control molecule [76]; however, in combi-nation with an experimental and statisticalanalysis, the reliability of the values is greatlyenhanced [7781]. Finally, one must rememberthat the results of quantitation using a molecularcontrol need to be expressed relative to a suitablebiological marker, e.g., in terms of the volume ofplasma, the number of cells or the mass of tissueor genomic nucleic acid, thus allowing compar-ability between assay results and testing sites [82].

Standards for quantitation

Most commonly, an exogenous control is createdusing a cloned amplicon, a portion of the targetorganisms genome, or simply the purified ampl-icon itself [83]. This control forms the basis of anexternal standard curve created from the dataproduced by the individual amplification of adilution series of exogenous control. The concen-tration of an unknown, which is amplified in thesame reaction, but in a separate vessel, can thenbe found from the standard curve. While the




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external standard curve is the more commonlydescribed quantitative approach, it frequentlysuffers from uncontrolled and unmonitored inter-vessel variations. Some platforms have overcomethis issue by including a capacity to detect and

correct for variation in the emissions of a non-participating, or passive, internal referencefluorophore (6-carboxy-N,N,N,N-tetramethylrhod-amine; ROX). The corrected values, obtained froma ratio of the emission intensity of the fluorophoreand ROX, are called RQ+. To further controlamplification fluctuations, the fluorescence from ano-template control reaction (RQ) is subtractedfrom RQ+, resulting in the DRQ value thatindicates the magnitude of the signal generatedfor the given PCR [84]. Assays that lack thiscapacity are more appropriately described as

semiquantitative.

Internal controls (ICs)

The use of an IC was described in the earliest ofPCR experiments as an important quality con-trol [85,86], particularly when performing com-petitive quantitation. When such a control isadded before template purification (extractioncontrol) or amplification (amplification control),it is called an exogenous IC, since it does notoccur naturally within the nucleic acid pre-

paration, but is co-amplified within the samereaction. Ideally, the IC should hybridise to thesame primers, have an identical amplificationefficiency [74,87], and contain a discrimin-ating feature such as a change in its length[72,72,75,88,89] or, more commonly in todaysoligoprobe-based methods, a change in the seq-uence [73,90] of the wild-type target [91,92].However, IC templates that bind different prim-ers or have different amplification efficienciescan still prove useful as standards for semi-quantitative PCR or relative quantitation.

An endogenous control is a template thatoccurs naturally within the specimen being exam-ined. Housekeeping genes often fulfill this role,and they have been successfully used to quanti-tate gene expression by RT-PCR and monitor theintegrity of a template after its purification [85].When endogenous controls are used for thequantitation of RNA, it is essential that thehousekeeping gene is minimally regulated andexhibits a constant and cell cycle-independentbasal level of transcription [93]. This is not the

case for some commonly used genes such asb-actin, whereas studies have shown that an 18SrRNA target meets the desired criteria [93,94].

Relative vs. absolute quantitation

The amount of template in a sample can bedescribed either relatively or absolutely. Relativequantitation is the simpler approach, and des-cribes changes in the amount of a target sequencecompared to its level in a related matrix or withinthe same matrix by comparison to the signal froman endogenous or other reference control. Abso-lute quantitation is more demanding but statesthe exact number of nucleic acid targets present inthe sample in relation to a specific unit, making iteasier to compare data from different assays and

laboratories [7,95]. Absolute quantitation may benecessary when there is a lack of sequentialspecimens to demonstrate a relative change inmicrobial load, or when no suitably standardisedreference reagent is available.

A highly accurate approach used for absolutequantitation by conventional PCR utilises com-petitive coamplification of one or a series of ICs ofknown concentration with a wild-type targetnucleic acid of unknown concentration [9699].However, conventional competitive quantitationis technically demanding, requiring significant

development and optimisation compared toquantitation by real-time PCR, which is bettersuited to the quick decision-making required in aclinical environment [100102]. Software with theability to calculate the concentration of anunknown by comparing real-time PCR signalsgenerated by a coamplified target and IC is rarebut emerging [7]. In addition, new or improvedformulae are appearing which aim to makequantitation more reliable and simpler [103].

Acquisition of fluorescence data

Fluorescence data generated by real-time PCRassays are generally collected from PCR cyclesthat occur within the linear amplification portionof the reaction, where conditions are optimal andthe fluorescence accumulates in proportion to theamplicon [52] (Fig. 2). This is in contrast to signaldetection from the endpoint of the reaction, wherethe final amount of amplicon may have beenaffected by inhibitors, poorly optimised reactionconditions or saturation effects due to the




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presence of excess double-stranded amplicon. Infact, at the endpoint there may be no relationshipbetween the initial template and final ampliconconcentrations. Because the emissions from fluor-escent chemistries are temperature-dependent,data are generally acquired only oncecycle, atthe same temperature [55].

The fractional cycle number at which the real-time fluorescence signal mirrors progression ofthe reaction above the background noise is usedas an indicator of successful target amplification[104]. Most commonly, this is called the thresholdcycle (CT), but a similar value is described for theLightCycler, and the fractional cycle is called thecrossing point (CP). The CT is defined as the PCR

cycle in which the gain in fluorescence generatedby the accumulating amplicon exceeds ten stand-ard deviations of the mean baseline fluorescence,using data taken from cycles 315 [105]. The CTand CP are proportional to the number of target

copies present in the sample [29] and are assumedto represent equal amounts of amplicon present ineach tube or capillary, since the CT and CP valuesrepresent the fractional cycle number for eachsample at a single fluorescence intensity value. Inpractice, the CT and CP are calculated after thedefinition of a noise band that excludes data fromearly PCR cycles that cannot be distinguishedfrom background noise. The final CT and CPvalues are the fractional cycles at which a singlefluorescence value (usually at or close to the noiseband) intersects each samples plotted PCR curve

[104] (Fig. 2). The accuracy of the CT or CPdepends upon the concentration and nature of thefluorescence-generating component, the amountof template initially present, the sensitivity of theplatform, and the platforms ability to discrimin-ate specific fluorescence from background noise.

Improved quantitation using real-time PCR

Significant improvements in the quantitation ofmicrobial load by real-time PCR result from thedetection systems enormous dynamic range,

which can accommodate at least eight log10

copiesof nucleic acid template [92,100,106114]. Thebroad dynamic range avoids the need for pre-dilution of an amplicon before detection, or theneed to repeat an assay using a diluted samplebecause a preliminary result falls outside thelimits of the assay. Both of these problems occurcommonly when using conventional endpointPCR assays for quantitation, as their detectionsystems are unable to encompass the products ofhigh template loads while maintaining adequatesensitivity [113,115117]. The flexibility of real-time PCR is further demonstrated by its ability todetect one target in the presence of a vast excessof another target during duplexed assays [109].

Real-time PCR is also a particularly attractivealternative to conventional PCR for the study ofmicrobial load because of its low inter-assay andintra-assay variability [100,112,118] and its equiv-alent or improved sensitivity compared to micro-bial culture, or conventional single-round andnested PCR [17,100,110,119126]. Real-time PCRhas been reported to be at least as sensitive as

Fig. 2. Kinetic analysis. The ideal amplification curve of areal-time PCR (solid), when plotted as fluorescence inten-sity against the cycle number, is a sigmoidal curve. Earlyamplification cannot be viewed because the emissions aremasked by the background noise. However, when enoughamplicon is present, the assays exponential progress canbe monitored as the rate of amplification enters a linearphase (LP). Under ideal conditions, the amount of ampl-icon increases at a rate of one log10 every 3.32 cycles. Asprimers and enzyme become limiting, and productsinhibitory to the PCR and overly competitive to oligoprobehybridisation accumulate, the reaction slows, entering atransition phase (TP) and eventually reaching a plateauphase (PP) where there is little or no increase in fluores-cence. The point at which the fluorescence surpasses thenoise threshold (dashed horizontal line) is called the

threshold cycle or crossing point (CT

or CP

; indicated byan arrow), and this value is used in the calculation oftemplate quantity during quantitative real-time PCR. Alsoshown are curves representing a titration of template(dashed curves), consisting of decreasing starting templateconcentrations, which produce higher CT or CP values,respectively. Data for the construction of a standard curveare taken from the LP.




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Southern blot, still considered by some as the goldstandard for probe-based hybridisation assays[122].

M I C R O B I A L G E N O T Y P I N G

Although nucleotide sequencing is still the goldstandard for characterising unknown nucleicacids, it is a relatively lengthy process. Thedevelopment of real-time PCR has partiallyaddressed this failing by providing a tool capableof routine detection of characterised mutations,insertions or deletions.

Most fluorescent chemistries used for real-timePCR do not rely upon a destructive process togenerate a signal. Therefore, they may be able toperform a genotyping role at the completion of

the PCR. The SYBR green and HybProbe chem-istries are most commonly used to perform theseanalyses; however, the double-stranded and light-up oligoprobes and HyBeacons should also func-tion in this role. Other chemistries, such as theTaqMan and Eclipse oligoprobes and hairpinoligonucleotides, discriminate these nucleotidechanges using two sets of oligoprobes to differ-entiate the wild-type from the altered sequences.While this is a perfectly legitimate and functionalapproach to genotyping by real-time PCR, theextra fluorogenic oligonucleotides increase the

overall cost of the assay. Additionally, the num-ber of different microbes that can be discrimin-ated during multiplex real-time PCR is reduced,since two fluorophores must be assigned toanalyse each microbe. The occurrence of a mis-match between a hairpin oligonucleotide and itstarget has a greater destabilising effect on theduplex than the introduction of an equivalentmismatch between the target and a linear oligo-probe. This is because the hairpin structureprovides a highly stable alternative configuration.Therefore, hairpin oligonucleotides are more spe-cific than the more common linear oligoprobes,making them ideal candidates for detecting SNPs[68].

Genotyping data are obtained after the com-pletion of the PCR, and therefore represent anendpoint analysis. The amplicon is denatured andrapidly cooled to encourage the formation offluorophore and target strand complexes. Thetemperature is then gradually raised, and thefluorescence from each vessel is continuouslyrecorded. The detection of sequence variation

using fluorescent chemistries relies upon thedestabilisation incurred as a result of the chan-ge(s). The non-specific chemistries reflect thesechanges in the context of the entire dsDNAamplicon, requiring the dissociation of fluorogen-

ic molecules from the dsDNA, which only occursupon melting of the duplex. The sequence chan-ges have a different impact upon the specificfluorogenic chemistries, altering the expected TMin a manner that reflects the particular nucleotidechange. The resulting rapid decrease in fluores-cence using either approach can be presented as amelt peak using software capable of calculatingthe negative derivative of the fluorescence changewith temperature (Fig. 3).

Importantly, different nucleotide changesdestabilise hybridisation to different degrees,

and this can be incorporated into the design ofgenotyping assays to ensure maximum discrim-ination between melt peaks. The least destabilis-ing mismatches include G (G:T, G:A and G:G),whereas the most destabilising include C (C:C,C:A and C:T) [127].

M U L T I P L E X R E A L - T I M E P C R

Multiplex PCR uses one or more primer sets topotentially amplify multiple templates within asingle reaction [128,129]. However, its use in real-

time PCR has led to confusion in the traditionalterminology. Multiplex real-time PCR more com-monly refers to the use of multiple fluorogenicoligoprobes for the discrimination of ampliconsthat may have been produced by one or severalprimer pairs. The development of multiplex real-time PCR has proven problematic because of thelimited number of fluorophores available [26] andthe frequent use of monochromatic energisinglight sources. Although excitation by a singlewavelength produces bright emissions from asuitably receptive fluorophore, the number offluorophores that can be excited by that wave-length is limited [130].

The discovery and application of the non-fluorescent quenchers has made available somewavelengths that were previously occupied bythe emissions from the early quenchers them-selves. This should permit the future inclusion ofa greater number of spectrally discernable oligo-probes/reaction, and highlights the need for asingle non-fluorescent quencher that can quencha broad range of emission wavelengths (e.g.,




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400600 nm). The impressive electron-donatingproperties of guanosine make it an ideal naturalquencher, and its use has contributed to thegrowing number of assays that only require asingle fluorophoretarget [131].

Early real-time PCR systems contained optim-ised filter sets to minimise overlap of the emissionspectra from the fluorophores. Despite this, thenumber of fluorophores that could be combinedand clearly distinguished was limited. Morerecent real-time PCR platforms have incorporatedeither multiple light-emitting diodes, or a tung-sten light source that emits over a wide range ofwavelengths. When these platforms also incor-

porate high-quality optical filters, it is possible touse many of the current real-time PCR detectionchemistries on the one machine. Unfortunatelysome platforms are not suitably constructed. Evenif these improvements are included, the platform

can still only perform four-colour oligoprobemultiplexing, and one colour is ideally set asidefor use as an IC. Some real-time PCR designs havemade use of conserved single or multiple nucleo-tide changes among similar templates to allowtheir differentiation by concurrent changes to theoligoprobes TM or the amplicons TD [132,133].Combining the use of multiple fluorophores withthe discrimination of additional targets by tem-perature allows the identification of a significantlylarger number of amplicon targets [134]; however,this combined approach has not been applied to

the diagnosis of infectious disease on a significantscale [135], possibly because of the sequencevariation among many microbial genes [119,136139]. Far more commonly, this approach has beenused for the detection of human genetic diseases,where as many as 27 possible nucleotide substi-tutions have been detected using only one or twofluorophores [140147].

To date, there have been only a handful ofdiagnostic microbial assays that can trulyco-amplify and discriminate more than two fluor-ophores. An impressive multiplex, real-time PCR

protocol discriminated between four retroviraltarget sequences [148]; however, conventionalmultiplex PCR using endpoint detection haseasily discriminated between more than fivedifferent amplified sequences, indicating a greaterdegree of flexibility [149154].

Future development of novel chemistries andimproved real-time instrumentation and softwareshould significantly improve the ability to multi-plex fluorophores for enhanced real-time PCRassays. Perhaps a chimera of real-time PCR andmicroarray technology, in combination withmicrofluidic devices, may advance all three tech-nologies to a point where the desired number oftemplates could be easily amplified and discrim-inated.

S P E C I F I C A P P L I C A T I O N S F O RM I C R O B I O L O G Y

Real-time PCR assays have been extremely usefulfor studying microbial agents of infectious dis-ease, where they have helped to clarify many

Fig. 3. Fluorescence melting curve analysis. At the com-pletion of a real-time PCR using a fluorogenic chemistry,the reaction can be cooled to a temperature below theexpected TM of the oligoprobes and then heated to above90C at a fraction of a degree/second (a). During heating,the emissions of the reporter or acceptor fluorophore canbe constantly acquired (b). Software calculates the negativederivative of the fluorescence with temperature, producinga clear melt peak that indicates the TM of the oligoprobetarget melting transition (black peak; c) or the TD ofmelting dsDNA. When one or more nucleotide changes arepresent, the TM or TD is shifted (grey peak). This shift isreproducible and can be used diagnostically to genotypemicrobial templates.




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disease processes. Most of the assays presented inthe literature have increased the frequency ofmicrobial detection compared to non-PCR tech-niques, making the implementation of real-timePCR attractive to many.

Of course, real-time PCR has also provenvaluable for basic microbiological research, whereits ability to amplify template from a wide arrayof sample types (Table 1) has made it an idealsystem for application across the various micro-biological disciplines [155]. Increasingly, theseapplications are difficult to review, due to theiruse as a tool within, rather than the focus of, apublished study.

Viruses

Within microbiology, the application of real-timePCR has had the biggest impact upon the field ofvirology, where studies have qualitatively inves-tigated the role of viruses in a range of humandiseases [156]. Also, epidemiological studies ofco-infections have been improved by thesemolecular techniques, which can reliably measurethe amount of two nucleic acid targets presentwithin a single sample [119,157,158]. Real-timePCR has also improved the discrimination ofmultiple viral genotypes within a single reactionvessel [159] and provided an alternative to mor-

bidity and mortality assays for virus detection. Anexample is Newcastle disease virus, which existsas two radically different pathogenic phenotypescaused by small nucleotide changes that can be

easily detected using fluorescence melting curveanalysis to reveal the genetic pathotype of thestrain [160].

Direct and indirect links between viral infectionand chronic conditions such as sarcoma [121,161

164], carcinoma [122,165], cervical intra-epithelialneoplasia [166168] and lymphoproliferativedisorders [169,170] can be relatively easilystudied using real-time PCR. Other studies havedescribed the presence of flaviviruses [106,126,171176], hepadnaviruses [113,115,177], herpesvi-ruses [30,40,100,102,107109,116,119,121,122,137,155,158,165,178187], orthomyxoviruses [125],parvoviruses [92], papovaviruses [32,139,159],paramyxoviruses [124,160,188], pestiviruses [189],picornaviruses [110,111,190195], poxviruses [196],retroviruses [118,123,197200], rhabdoviruses

[201] and TT virus [202].A significant number of studies have used PCRto detect viral load, and have proved its useful-ness as an indicator of the extent of activeinfection, interactions between virus and host,and the changes in viral load as a result ofantiviral therapies, all of which can play a role inthe treatment regimen selected [203205]. Con-ventional quantitative PCR has already proventhat the application of nucleic acid amplificationto the monitoring of viral load provides a usefulmarker of disease progression and the efficacy of

antiviral compounds [97,204,206210]. Becausedisease severity and viral load are linked, theuse of real-time PCR quantitation has provenbeneficial when studying the role of viral reacti-vation or persistence in the progression of disease[40,102,107,108,119,158,165,171,183,184,187,211,211216]. Alterations to a microbes tropism or itsreplication, and the effects that these changeshave on a host cell, can also be followed usingreal-time PCR [217219].

The role of highly sensitive and rapid real-timePCR assays in the thorough assessment of viralgene therapy vectors before their use in clinicaltrials has become an important one. Nucleaseoligoprobes have been most commonly used forthese studies, which assess the biodistribution,function and purity of the novel drug prepara-tions [199,220225].

Likewise, the study of new and emergingviruses has been ideally complemented by theuse of homogeneous real-time PCR assays as toolsto demonstrate and strengthen epidemiologicallinks between unique viral sequences and the

Table 1. An incomplete list indicating the extraordinaryvariety of sample types from which nucleic acids can besuccessfully prepared, amplified and detected using real-time PCR assays

Nucleic acid origins References

Plants [293]Animals [111,260]Urban sludge [110]Microbial culture [177,234,240,253,254,266,267,

280,284,292,294]Solid tissues [172,198,238,256258,271,288,295]

Cerebrospinal fluid [136,190,192,194,248]Peripheral blood

mononuclear cells[183]

Bone marrow [120]Whole blood [179,291]Plasma [118]Serum [171,172,180,279,287]Swabs [192,259,296]Bronchoalveolar lavage [243,246]Amniotic fluid [286]Saliva and sputum [158,233]Faeces [264,285]Urine [12,155,178]




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clinical signs and symptoms experienced bypatients [124,126,201,226230].

The speed and flexibility of real-time PCR hasalso proven useful for commercial interests whorequire exquisite sensitivity to screen for micro-

bial contamination within large-scale reagentpreparations produced from eukaryotic expres-sion systems [231,232].

Bacteria

The benefits to the patient from rapid real-timePCR assays are most notable when applied to thedetection of bacteria. The results can quicklyinform the clinician as to the infection status ofthe patient, allowing a more specific and timelyapplication of antibiotics. This can limit the

potential for toxicity due to shotgun treatmentregimens, reduce the duration of a hospital stayand prevent the improper use of antibiotics, thusminimising the potential for resistant strains toemerge.

Broad applications of real-time PCR can aug-ment or replace traditional culture or histochem-ical assays, as was seen with the creation of amolecular assay capable of classifying bacteria inthe same way as a Gram stain [233]. However,specific bacterial species are more frequently thefocus for real-time PCR assays, especially when

long culture times can be replaced by rapid andspecific gene detection. Leptospira genospecies,Mycobacterium and Propionibacterium spp., Chla-mydia spp., Legionella pneumophila and Listeriamonocytogenes have all been detected and in somecases quantitated with the use of real-time PCRassays [41,234246].

The detection of Neisseria gonorrhoeae has bene-fited from real-time PCR, particularly in the roleof a confirmatory test when the specificity ofcommercial assays fails [247]. This example high-lights the need for care when choosing a bacterialPCR target, especially when that target exists on aplasmid that is exchanged among other bacteria,providing potentially confusing diagnosticresults. Neisseria meningitidis causes meningococ-cal disease, and real-time PCR has proven to be apowerful tool that can be quickly developed forthe rapid discrimination of currently circulatingpathogens [248].

The detection and monitoring of antibioticresistance among clinical isolates ofStaphylococcusaureus, Staphylococcus epidermidis, Helicobacter

pylori, Enterococcus faecalis and Enterococcus fae-cium has also benefited from real-time applica-tions [249258]. Additionally, the understandingand treatment of fulminant diseases such asmeningitis, sepsis, inflammatory bowel disease

and food-poisoning caused by characterised bac-teria such as the group B streptococci and Myco-bacterium spp., Escherichia coli and Bacteroidesvulgatus [259265] have been enhanced by thespeedy return of results, which also aids trackingof microbial outbreaks to their source.

Real-time PCR has made possible the rapidquantitation and differentiation of some of themore exotic pathogenic bacteria, such as the tick-borne spirochete Borrelia burgdorferi [266268] andthe methanotropic bioremediating Methylocystisspp. [269].

The involvement of treponemes in the devel-opment of periodontal disease has been studiedusing 5 nuclease chemistry, revealing a microbialrole in every stage [270]. In addition, measure-ment of the bacterial load of Tropheryma whippleihas allowed the discrimination of environmentalcontamination and low-level colonisation fromactive infection [271].

More recently, there has been an explosion ofliterature indicating that real-time PCR is the toolof choice for the rapid detection of microbes usedas agents of biological warfare. In some cases, the

assays have allowed rapid discrimination ofweaponised pathogens from the harmless labor-atory-adapted or vaccine-related strains. At theforefront of the available literature are assays todetect Bacillus anthracis spores and the bacteriumsvirulence-encoding plasmids or chromosomalmarkers [272,273,273277]. Conventional assaysmay take 48 h to complete, and, for obviousreasons, this is an unacceptable lag period.

Fungi, parasites and protozoans

The smallest number of applications have beenrelated to the study of fungal, parasitic andprotozoan pathogens of humans. Nonetheless,real-time PCR assays have significantly contribu-ted to the general diagnosis of invasive diseasecaused by Aspergillus fumigatus and Aspergillus

flavus [278,279]. Also, monitoring the transcriptionlevels of certain Aspergillus nidulans transportergenes has provided important information abouttheir role in multiresistance [280]. In addition,real-time assays have been used when investi-




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gating buildings for the presence of potentiallyharmful levels of toxigenic fungal spores, orconidia, such as those produced by Stachybotryschartarum [281283].

Cryptosporidium parvum oocysts and the spores

from Encephalitozoon spp. have been successfullygenotyped or speciated using real-time PCR,which has significantly improved on laboratorydiagnosis using microscopy and histochemicalstaining, especially for low concentrations ofexcreted material [284,285].

Rapid serological detection ofToxoplasma gondiiis often hampered by the presence of the parasitein patients who are immunocompromised. Addi-tionally, the length of time required for traditionalculture or mouse inoculation is excessive. There-fore, rapid molecular methods have vastly

improved the detection of this microbe [120,286].Additionally, this technology is useful for thestudy of T. gondii responses to antimicrobialtherapies [287].

Detection of malarial parasites using a mousemodel in combination with real-time PCR hasimproved result turnaround time and meant thatparasite load data can be generated [288,289].Real-time PCR has also proven useful for directin-vivo detection and quantitation of malarialparasites with a high level of sensitivity [290,291],in addition to monitoring the stage-specific mat-

uration ofPlasmodium falciparum via the transcrip-tion of specific genes [292].

C O N C L U S I O N S A N D S U M M A R Y

Microbiology is ideally suited for the benefits ofsensitivity and rapidity that PCR has brought tothe research laboratory. However, the advent ofreal-time PCR has further improved the role ofPCR in the high-throughput environment byadding a detection system capable of enormousdynamic range, homogeneity of amplification anddetection, and the ability to genotype an ampli-fied nucleic acid without the need for additionalsteps. Unfortunately, many of the genotypingapplications have been trialled first in the field ofhuman genetics, where there is frequently a moreabundant source of template, and the geneticchanges, once characterised, remain constant.Nonetheless, this review has highlighted thegeneral acceptance of real-time PCR in theresearch and diagnostic microbiology laboratory,and its popularity is continuing to expand.

Advances in the development of fluorophores,nucleotide labelling and the novel application ofoligoprobe hybridisation have provided real-timePCR technology with a broad enough commercialbase to promote its usefulness to the wider non-

research scientific community. Robotic nucleicacid extraction and liquid-handling systems, com-bined with rapid thermal cyclers and instrumen-tation capable of detecting and differentiatingmultiple amplicons using many of the chemistriesdescribed in this and other reviews, make real-time PCR an attractive and viable proposition forthe routine diagnostic laboratory. Many laborat-ories rely upon tissue culture to isolate microbialagents of infectious disease, in combination withserological methods to further confirm the iden-tity of the isolates or to monitor a patients

immune response to an infectious agent. Suchmethods, while providing an important source ofinformation about unknown and emerging path-ogens, may take a prolonged and clinically signi-ficant amount of time to complete.

According to the literature, the most widelyused fluorogenic probe format is the 5 nucleaseoligoprobe, although that is most likely due to itscommercial maturity. The rate of publicationsdescribing other methods, especially those utilis-ing the LightCycler in combination with a pair ofHybProbes, is significant and changing the bal-

ance rapidly, especially in the area of microbialdetection and genotyping. There are also morevirus-detecting real-time PCR applications des-cribed in the literature than for any other microbe.The more recently developed oligoprobe chemis-tries have been used in only a few innovativeapplications, but they will be better understood astheir benefits and limitations are more widelydescribed, and hopefully they will allow a greatervariety of options for microbial genotyping.

Recent developments in multiplex real-timePCR have suggested a future in which easyidentification, genotyping and quantitation ofmicrobial targets in single, rapid reactions willbe commonplace. Of course, real-time PCR is byno means restricted to microbiology, as significantachievements have already been made in the areaof human genetic diagnostics, applying all thebenefits of real-time PCR to enhance the detectionof genetic disease. However, the technology isonly as reliable as the accompanying controls andassociated quality assurance programmes. Thisincludes the quality of standards, the use of




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suitably controlled standard curves, and the needto fully optimise, validate and evaluate each andevery new assay against previously standardisedassays. Without such care, real-time PCR willprovide fast but inaccurate data to the clinician,

who will surely come to rely upon such assays,as they represent a growing proportion of theresult-generating tests within the diagnosticmicrobiology environment. In addition, commer-cial interests will undoubtedly play an expandingrole in determining which technologies enter intothe mainstream.

Perhaps in combination with micro- andmacroarray technology and emerging microflui-dic devices, real-time PCR assays that can dis-criminate as many targets as desired, whileproducing quantitative data at a greatly increased

speed, will consolidate fluorogenic nucleic acidamplification as a routine and incredibly power-ful tool for the laboratory of tomorrow.

A C K N O W L E D G M E N T S

This work was supported by a Royal Childrens HospitalFoundation Grant (922-034), which was sponsored by theWoolworths Care for Kids campaign. The author wishes tothank Dr Katherine Arden for critical review of this manu-script.

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