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Abstract

Polymerasechainreactionbasedassaysproviderapid,simple,andsensitivedetectionofbacterialgenes,butarenot
withouttheirdrawbacks.ThisreviewsummarizestheprincipaladvantagesanddisadvantagesofPCRbasedbacterialgene
detection,providesguidelinesforthedevelopmentandvalidationofnewPCRassays,anddescribespotentialpitfallsthat
maybeencounteredandhowthesecanbeavoided.2000PublishedbyElsevierScienceB.V.
Keywords:Polymerasechainreaction(PCR);Primerdesign;Review

1.DiagnosticPCRinbacteriologicalresearch
Thepolymerasechainreaction(PCR)has been agreat boonto bacteriologicalresearch.Its many uses
includethedetectionofspecicDNAorRNAsequences(JohnsonandBrown,1996;Persing,1996),DNA
sequencing, amplication ngerprinting (Welsh and McClelland, 1990;CaetanoAnolles,1993; Versalovic
and Schneid, 1994), detection and identication ofnonculturablemicroorganisms (Fredricks and Relman,
1999), andsitedirectedmutagenesis (Link and Phillips, 1997). The most familiar of these is simple gene
detection for molecu lar epidemiological purposes. In this role PCR can substitute for DNAprobe
hybridizationassays (John son and Stell, 2000a,b), or can provide a genetic surrogate for traditional
phenotypictests,whichin

*Tel.:116127252000,ext.4185;fax:116127252273.Emailaddress:johns007@tc.umn.edu(J.R.Johnson).

someinstancesmaybetechnicallydemanding,insensitive,orunreliable(JohnsonandStell,2000a,b).As
comparedwithprobehybridizationassays,PCRcansavetime,particularlyifonlysmallnumbersofsamples
are being tested or if primers can be multiplexed such that multiple genes are detected simultaneously
(HenegariuandHeerema,1997;FredricksandRelman,1999;JohnsonandStell,2000a,b).Thetremendous
sensitivityofPCRallowstargetgenestobedetectedwhenpresentinextremelylowconcentrations,thereby
permittingthedetectionofaminorbacterialsubpopulationwithinacomplexmixedorawithouttheneedfor
isolationoftheorganismsofinterest(Lo,1994;JohnsonandBrown,1998).TheexquisitespecicityofPCR
derivesfromthereactionsstrictrequirementforprecisenucleotidesequencecomplementarityateachprimer
bindingsite,particularlyatthe39 endoftheprimers(DieffenbachandDveksler,1995;Lisby,1999;Williams
andWilson,1999).Thisspecicityallowsverynediscriminationbetweenclosely
01677012/00/$seefrontmatter2000PublishedbyElsevierScienceB.V.PII:S01677012(00)001603
202J.R.Johnson/JournalofMicrobiologicalMethods41(2000)201209

relatedvariantsofthesamegenetoadegreenotpossiblewithprobehybridization,whichisconsiderably
moretolerantoflowlevelsequencedegeneracy(JohnsonandBrown,1996;JohnsonandStell,2000a,b).
PCRalsohastheintangibleappealofrepresentingmoderntechnology.
2.IsPCRreallythebestapproach?
However,beforedevelopinganewdiagnosticPCRassayoradoptinganestablishedone,itbehoovesthe
investigatortoconsiderthepotentialdisadvantagesofPCR.Reagentsanddisposablematerials(e.g.,special
PCRtubesandpipettortips)arefairlyexpensive,particularlyincomparisonwiththoserequiredformany
traditionalphenotypictestsorprobehybridization,whichtodaycanbedonewithoutradioactivityortheuseof
toxicchemicalssuchasphenol(FredricksandRelman,1999).Althoughthermalcyclersandthenecessary
gelelectrophoresisequipmententailonlymodeststartupcosts,ifmultipledifferentPCRreactionsandgels
aretoberunsimultaneously,multiplecyclersandelectrophoresissetupsmaybeneeded,whichincreases
costsandconsumeslaboratoryspace.

TheexquisitesensitivityofPCRisadoubleedgedswordwhichmakesfalsepositiveresultsfromeventhe
mostminutedegreeofcontaminationaseriousthreat(KwokandHiguchi,1989;FredricksandRelman,1999).
Consequently,contaminationmustbevigilantlyguardedagainstbyobservanceoffastidioussteriletechnique
andtheuseofDNAfreereagents,specialpipettorsand/orpipettetips,plusaclean(noampliedDNA)
PCRsetuparea(KwokandHiguchi,1989;FredricksandRelman,1999).Foralaboraborythatisnotalready
equippedforPCR,theneedforseparatepreandpostPCRareasaddstothespacerequirements.Evenwith
theuseoftheseextensivepreventivemeasures,contaminationstillmustbemonitoredforbytheinclusionin
everyamplicationrunofnegativecontrolswhichwereprocessedinparallelwiththeunknownsamples,
hence had an equal opportunity for contamination (Fredricks and Relman, 1999). Separate analysis of
duplicateindependentlypreparedtemplateDNAsamplesfromeachspecimenprovidesaddedprotection
againstfalsepositiveresultsfromcontamination.
TheintoleranceofPCRtobasepairmismatchesnearthe39 endoftheprimer,althoughenhancingassay
specicity,canalsoreducethesensitivyofPCRfordetectinggeneswhichexhibitsequencedegeneracy,ifthe
primerrecognitionsitehappenstoincludeavariableregionorposition(JohnsonandStell,2000).Evena
singlebasemismatchatthevery39 terminusoftheprimercanpreventeffectiveamplicationandrenderthe
reactionnegative.Ironically,foratraitthatishighlyexpressed(e.g.,Escherichiacolialphahemolysin)butis
encodedbyagenewhichexhibitssubstantialsequencedegeneracy(e.g.,hlyA),thiscanmakePCRactually
lesssensitivethanasimple,inexpensive,traditionalphenotypicassay,e.g.,overnightgrowthonbloodagar
(unpublisheddata).
Finally, economy of scale can make blotting more efcient than PCR for analyzing large numbers of
samples,particularlyifforonlyasinglegene.Forblotting,over100samplescanbedottedontoastandard
size(10315cm)nylonmembrane.Duplicatemembranescanbyhybridizedwithprobe,washed,anddetected
inonecontainerwithlittlemoreeffortthanrequiredtosimilarlyprocessanindividualsample.Incontrast,with
PCRtheworkofprocessingincreasesapproximatelyinproportiontothenumberofsamplesprocessed.
IffortheintendedapplicationPCRdoesseemworththetroubleandexpense,asearchforanexistingassay
maysavethepotentialuserthetimeandcostassociatedwithdevelopingandvalidatinganewone.Ofnote,
evenifpublishedprimersareavailable,investigatorsareadvisedtocheckthesecarefullyagainstthepublished
sequenceforaccuracyandtoevaluatethemforotherperformancecharacteristics,asdetailedbelowfornew
primerselection.Inseveralinstanceswehavebeenledastraybypublishedprimersequencesthatwerefrankly
inerrororthatweresuboptimalforuseinPCR.However,ifnosatisfactoryalternativeisavailable,theinves
tigatorusuallycanreadilydevelopaPCRbasedassaytomeetthespecicneed,asdescribedbelow.
3.Sequence
FundamentaltothedevelopmentofPCRbasedgenedetectionassaysistheknowledgeofthenucleotide
sequenceofthegeneofinterest.The
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appropriateDNAsequencesusuallycanbelocatedbysearchingtheGenBankdatabases(http://www.ncbi.
nlm.nih.gov/Entrez/nucleotide.html)withkeywordsthatincludethegenenameandspecies.Inmany
instances,sequencewillbeavailableformultipleversionsofthegenefromdifferentstrains,inwhichcaseit
is advisable to retrieve and compare the different versions, either manually or digitally (e.g.,
usingCLUSTAL(HigginsandThompson,1996)or BLAST:http://www.ncbi.nlm.nih.gov/blast/blast.cgi).The
possibilitythatsequencedifferencesbetweendifferentversionsofthesamegenemaybeartifactualrather
thanrealmustbeconsidered(Pennisi,1999).Occasionally,genesequencesarenotavailableinGenBankand
mustbeobtaineddirectlyfromtheinvestigator(ifunpublished)orextractedfromapublication.
4.Primerdesign

Manycomputerapplicationsareavailabletoassistwithinitialprimerselection,includingPrimer3(Rozen
andSkaletsky,1998),PrimerDesigner(version4,1994,ScienticandEducationalSoftware,Durham,NC:
see http: / / www.scied.com / ses]pd4.htm), and others (see http: / / biochem.boehringermannheim.com
/prod]inf/manuals/pcr] man/Chapter02/CHAP01Seite12.htm).Primerdesignprogramstypicallyallow
theusertospecifyvariousparameters,afterwhichthecomputersearchesthedesignatedtargetsequencefor
suitableprimerregionsandeitherselectsthebestoftheseorsuggestsarangeofalternativeswhichtheuser
canfurtherevaluatemanually.However,wehavefoundthatdirectinspectionofgenesequences,followed
bytrialanderrorevaluationofcandidateprimersasderivedfrom thosesequencesinsimulated PCR,e.g.,
usingtheapplicationAmplify(version1.2or2.0,forMacintoshonly:EngelsB.UniversityofWisconsin,
Madison:seehttp://www.wisc.edu/genetics/CATG/amplify/index.html)(Engels,1993),isalsousually
quiteeffective.
Itisgenerallyrecommendedthatprimersshouldincludefrom17to28basestoprovideadequatespecicity
fortheregionofinterest(InnisandGelfand,1990;Rybicki,1996).Theyshouldhaveapurine(GC)content
of4060%toprovideameltingtemperature(Tm)of55808,whichallowsforan
nealingtemperaturesinthedesiredrangeof55688.PairedprimersshouldhaveasimilarTm.Primersideally
shouldincludeoneormorepurinesatthe39terminustoprovideaddedstabilitytothetemplateprimerduplex
atthecritical39 polymerizationinitiationsite(i.e.,asocalled39 GCclamp).However,runsofthreeof
moreCsorGsatthe39 endsofprimersmaypromotemisprimingatGorCrichsequencesandshouldbe
avoided,asshouldlongruns,i.e.,morethansixrepeats,ofanybase.Finally,complementaritybetweenthe
39 endsofpairedprimers,orinternalcomplementaritywithinanindividualprimer(whichcangiveriseto
undesirablesecondarystructuresuchashairpins)shouldbeavoided(InnisandGelfand,1990).
Insomeinstances,primerlocationswillbeconstrainedbyaneedtoankaparticularrestrictionsiteor
anotherspeciclandmarkwithinagene,ortostaywithinadenedvariable,conserved,orepitopeencoding
region(JohnsonandBrown,1996;JohnsonandStell,2000a,b).Withthelackofsuchconstraints,candidate
primerscanbe selectedfromanysuitableregionofthe geneof interest.The desiredampliconsizemay
inuencewhichgeneregionsarescrutinizedforselectionofcandidateforwardandreverseprimers,since
PCRproductsoffrom150to700bpinlengtharemostefcientlyampliedbyconventionalPCRandare
mosteasilydetectedbygelelectrophoresis.Differencesinlengthbetweenforwardandreverseprimerswhich
willbepairedwithoneanotherarenotimportantsolongastheprimersexhibitsimilarpredictedannealing
temperatures orstabilityof theprimertemplateduplex, asestimated byprimerdesignorPCR simulation
programs.
Eachcandidateforwardandreverseprimershouldbecheckedagainstitselfandagainstothercandidate
primers to exclude potentialprimerdimeror hairpin formation, which can be done most effectively
usingprimerdesignorsimulatedPCRprograms.Candidateprimersalsoshouldbecomparedwiththewhole
thesequenceofinteresttoevaluateforpotentialsecondarybindingsiteswithinthegene,whichifsufciently
complementaryandorientedappropriatelycangiverisetospuriousPCRproductsandinterferewiththe
formationofthedesiredamplicon.Toconrmspecicityforthegeneofinterest,candidateprimersalso
should be compared against the sequence databanks using aBLASTsearch. Al though homology with
sequencesfromirrelevant
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organismsisunlikelytocauseaproblem,homologywithothersequencesfromtheorganismsofinterest,or
withsequencesfromdifferentorganismsthatmaybeencounteredinthesamplestobeanalyzed,couldleadto
nonspecic primer annealing and possibly amplication of spurious PCR products, and hence should be
avoided.
5.Assaydevelopmentandoptmization

Thesinglebestcandidateforwardandreverseprimersarenextsynthesized.Apostsynthesiscleanupstep
isdesirabletoremoveresidualsaltswhichmightinteferewiththeamplicationreaction(CaveandBingen,
1994).Thiscanbeaccomplishedbysepharosecolumnpurication,whichisoftenincludedinthepurchase
priceofcommerciallysynthesizedprimers.HPLCpurication,whicheliminatesmutantprimersthatmight
interferewithstructuralanalyses,isavailableforanextracharge.Ifthislevelofpuricationisdesiredfor
primerslongerthanapproximately60nucleotides,polyacrylamidegelelectrophoresismustbedoneinsteadof
HPLC.
FortheinitialdevelopmentandsubsequentvalidationofaPCRassayitisessentialtohaveknownpositive
andnegativecontrolDNAsamples.Itismostconvenientifappropriateviableorganismsareavailablefrom
whichtemplateDNAcanbepreparedasneeded,usingthesamemethodaseventuallywillbeusedforthetest
bacteriatobeanalyzedusingtheassay.Recombinantstrains,althoughseeminglyidealaspositivecontrols
becauseoftheirspeciccontentofthegeneofinterest,havethetheoreticaldisadvantageofnotreproducing
thephysicalandgeneticcharacteristicsofthewildtypestrainsthateventuallywillbeprocessedintheassay(if
theassayistobeusedwithwildtypeunknownstrains),hencemaybemisleading.Formanybacteria,includ
ingevenhardygrampositivessuchasEnterococcusspp.,DNAofsufcientquantityandqualityfordiagnostic
PCRcanbeobtainedbyboilingtheorganismsinwater,followedbycentrifugationtoremovebacterialdebris
(WoodsandVersalovic,1993;JohnsonandStell,2000a,b).Forlesshardybacteria,thesimpleadditionofan
aliquotofintactorganismsdirectlytotheamplicationmixmaysufce,sincetheinitialdenaturationstepof
the
thermalcyclingprotocolservestolysethebacteria(JoshiandBaichwal,1991),althoughinourexperience
this approach yields more variability in results than does the use of boiled, centrifuged template DNA
preparations.
The initial test amplications can be set up by using generic default concentrations for the various
componentsoftheamplicationmixasrecommendedbythemanufactureroftheparticularpolymerase
used,orbyusingcommercialPCRbeads,inwhichcasetheconcentrationsofallingredientsexceptprimerand
templateDNA arepredetermined bythemanufacturer. Inour experience, theuseofaninitial hotstart
routine,whichpreventsnonspecicprimingandpolymerizationfromoccurringasthereactionmixtureis
heatedfromroomtemperatureto
.908CduringtheinitialPCRcycle,hasbeenextremelyhelpfulineliminatingspuriousPCRproducts
(noise)andenhancingtheyieldofthedesiredproduct(ChouandRussell,1992).Hotstartcanbe
accomplishedmanuallybywitholdingonecomponentofthereactionmixturefromthetubesduringthe
initialsetup,andaddingitduringtherstPCRcycleonlyafterthetemperaturehasexceeded708C(Chouand
Russell,1992).However,thisisinconvenientandintroducesariskofirreproducibilityandcarry
overcontamination.Hotstartcanbedoneusingacommercialthermallyactivatedpolymeraseforonly
slightlygreatercostbutwithconsiderablelaborsavingsandreducedriskofcontamination(http:/
/biochem.boehringermannheim.com/prod]inf/manuals/pcr]manChapter03/CHAP03Seite53.htm).
Numerousaspectsoftheamplicationmix(e.g.,pHandconcentrationofmagnesium,primers,anddNTPs)
can be manipulated,andvarious genericor proprietaryPCRenhancerscanbe added,to achieverobust
amplication ofonlythe bandofinterest(InnisandGelfand, 1990;Rybicki, 1996). Commercial kits are
available to assist with rapid assay optimization. However, in our experience the single most important
determinantofassayperformance,otherthantheprimersthemselves,istheannealingtemperature,whichcan
beraisedorloweredasneededtoeliminatespuriousbandsandtobringoutthebandofinterest(Rychlikand
Spencer,1990).Alternatively,orinaddition,aninitialtouchdownroutinecanbeused.Intouchdown
cycling,
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during the rst few PCR cycles the annealing temperature is set higher than the planned nal annealing
temperature,thenisdecreasedincrementallywitheachroundofPCRuntiltheplannedannealingtemperature

isreached(DonandCox,1991;GallegoandMartinez,1997).Onlyrarelyshoulditbenecessarytoabandona
primerbecauseofpoorperformanceandtosynthesizeanalternateprimer.

6.Assayvalidation
Althoughthedemonstrationthatanewlydesignedprimerpaircangenerateanampliconoftheexpectedsize
frompositivecontrolDNAandfailtoreactwithanegativecontrolsampleisencouraging,thisreallyisonly
therststepin the validationof anew PCR assay.Itis important tomore extensivelyconrmboththe
specicityandthesensitivityoftheassay(FredricksandRelman,1999).Specicitycanbefurtherconrmed
bydemonstrating(1)thattheampliedbandactuallyrepresentsthegenesequenceofinterest,and(2)that
amplicationisabsentwithnegativecontrolsamplesotherthanjustthenegativecontrolusedduringinitial
assay development. Sen sitivity can be further conrmed by demonstrating that the assay can detect all
membersofalargerpopulationofknownpositivecontrols.Theinadequacyofrelianceonasinglepositiveand
negativecontrolsampleforassayvalidationisapparentfromthe95%condenceintervalsaroundestimated
sensitivityorspecicityvaluesof100%asderivedfromanalysisofeventworeferencesamples,sincethese
includevaluesaslowas16%(LentnerandDiem,1982).Therealsoisanundesirablecircularityinusingasa
positivecontrolonlythestrainthatservedasthesourceforthegenesequenceonwhichtheparticularprimers
werebased,sincethisapproachessentiallytestsonlythemechanicsofthePCRreaction,notwhethertheassay
willbeusefulintherealworldapplicationofevaluatingunknownwildtypestrainsthatmayhaveslightly
differentversionsofthegeneofinterestthandoesthesourcestrain.
Conrmation that a PCR product actually repre sentstheexpected gene sequencecan be obtained by
Southernhybridizationusingagenespecicprobe,
byrestrictionendonucleaseanalysis(incomparisonwithfragmentlengthsaspredictedbasedontheknown
gene sequence), bysinglestrandedconforma tional polymorphism analysis, or by direct DNA sequence
determination(FredricksandRelman,1999).Thisstepmightseemsuperuous,inviewoftheseeminglylow
probabilitythatanampliconofpreciselythepredictedsizecouldbegeneratedbyspuriousbindingofboth
primerstoheterologoustemplateDNAsequences.However,wehaveobservedexactlythisphenomoninone
instanceinwhichprimersspecicforaputativeEnterococcusvirulencegene(espA)yieldedanampliconofthe
expectedsizewithE.coliDNAwhichhadbeenincludedasanegativecontrol(Fig.1),despitetheabsenceof
the predicted homology between the primer sequences and theE. coligenome. Sequenc ing of theE.
coliespAPCRproductrevealedittoactuallyrepresentaportionoflacZ,theE.colibetagalactosidasegene,
whichexhibitsnonucleotidesequencehomologywithespA.
ConrmationofanewPCRassayssensitivityandspecicityinalargerpopulationrequirestheavaila
bilityofknownpositiveandnegativecontrolstrains.Thestatusofcontrolstrainswithrespecttothetraitof
interest canhave been determined previously, or can be dened as part of the validation process. Direct
validationoftheampliconitself(asdiscussedabove)becomeslesscriticalifalargecontrolpopulationis
availableinwhichthegenerationofanampliconoftheexpectedsizecanbedemonstratedwith(andonly
with)strainsthatareactuallypositiveforthetraitofinterest,accordingtosomecomparisonstandardmethod.
Probehybridizationistheusualmethodusedtodenestrainsaspositiveornegativeforthegeneofinterest
(LeBouguenecandArchambaud,1992;YamamotoandTerai,1995;JohnsonandRusso,1998;Johnsonand
Stell,2000a,b).However,particularlyforPCRassaysdesignedtodiscriminatebetweenhighlyhomologous
allelesofaparticulargene,probehybridization(whichislikelytobeinsensitivetosuchsubtledifferences)
maybeinadequateasacomparisonstandard.Inthiscircumstance,eithercomparisonwithaphenotypespecic
forthedifferentalleles(ifareliablephenotypeisavailable),ordirectconrmationofamplicons,maybe
necessary(JohnsonandBrown,1996;JohnsonandStell,2000a,b).

206J.R.Johnson/JournalofMicrobiologicalMethods41(2000)201209

Fig.1.SpuriousamplicationofaputativeEnterococcusspecicPCRproductfromE.coli.MultiplexPCRwasdoneusingve
primerpairsostensiblyspecicforEnterococcusgenescylA(cytolysin:430bpproduct),asa1(aggregationsubstance:380bp
product),espA(enterococcal surface protein A: 315 bp product),ebsA(enterococcal binding substance: 240 bp product),
andgel(gelatinase:200bpproduct).FourdifferentpositivecontrolstrainsofEnterococcus(lanes24and6)demonstratethe
expectedcombinationsofbands.TheE.colistrain(lane5),whichwasusedasanegativecontrol,demonstratesaband(white
arrow)atapproximatelythepositionoftheespAproduct.SequencingoftheE.colibandshowedittorepresentlacZ,anE.
coligenewithouthomologytoespA.M,100bpmolecularweightladder(lanes1and7).

When comparing PCR with probe results, it is important to recognize that PCR assays and probe
hybridizationanswersomewhatdifferentquestions.Forapositiveproberesult,DNAregionsofsufcient
homology and length must be present to allow retention of the labeled probe during washings. These
homologousregionscanbeconnedtoonlyportionsoftheprobe,andcanbediscontinuouslydistributed
along the probe. Consequently, probe hybridization is fairly tolerant of insertions, dele tions, and point
mutations, so long as a sufcient length of homologous sequence remains to support probe binding. In
contrast,forapositivePCRresult,bothprimerrecognitionsitesmustbepresent,conserved(particularlyat
the 39 end), and oriented as in the original version of the gene. However, the actual sequence of the
interveningDNAbetweentheprimerbindingsitesislargelyirrelevant,exceptinthatitslengthdeterminesthe
sizeoftheampliedPCRproduct.

OneconsequenceofthesedifferencebetweenprobehybridizationandPCRisthatprobehybridizationis
less likely than is PCR to be affected by deletions or mutations that involve one or both primer sites,
particularlyatthe39 end.Suchaltera
tionscanrenderaPCRassaynegativedespitethepresenceofsomeorallofthegeneofinterest,without
interferingwithprobehybridization.Wehaveencounteredwhatwepresumetobeexamplesofthissituationin
strainsthatareweaklyprobepositivebutPCRnegativeforagene,asituationwhichislikelyexplainedby
partial deletion of the gene, with loss of one primer site (Johnson and Stell, 2000a,b). We also have
encounteredstrainsthatareconcordantlyPCRandprobepositiveforageneatoneextremeofanoperon
(e.g.,papA),concordantlyPCRandprobenegativeforageneattheotherendofthesameoperon(e.g.,papG),
anddiscordantlyprobepositivebutPCRnegativeforaninterveninggene(e.g.,papEF),aphenomenonwhich
islikelyexplainedbyabsenceofoneendoftheoperon,includingthereverseprimersiteforthemiddlegene
(unpublished).
However,insomecircumstancesPCRcanactuallybemoresensitivetothepresenceofagene(orremnants
thereof)thanisprobehybridization.Ifprimersitesareconserved,butinterveningsequencesaresufciently
degenerate, a generic probe may not hybridize. Alternatively, probes may be too long for the available
template.Wehaveencounteredwhat
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probablyrepresentsthelattersituationinE.colistrainsthatappeartobedeletedformostofthepapoperonbut
whichyieldapositivePCRresultwithprimersthatarespecicforsiteswithintheresidual5 9 fragmentofthe
upstreamgenepapA.Suchstrainsgiveaweakornegativesignalwhenhybridizedwithalonger(papAH)
DNAprobe,eventhoughtheprobeincludestheremnantpapAregion,presumablybecausetheportionofthe
probethatndsacomplementarysequencetowhichitcanhybridizeisnotlongenough(relativetothetotal
lengthoftheprobe)toallowproberetentionduringwashings(JohnsonandStell,2000a,b).Theseshadesof
graygenotypesasrevealedbydiscrepenciesbetweenPCRandprobehybridizationraisethedenitionalissue
ofwhatismeantbypositivityforthegeneofinterest,orwhatdegreeofpositivityisrelevantfortheanalytical
purposesathand.
ArelatedissuethatsometimesarisesduringcomparisonofPCRwithprobe(orofonePCRassaywith
another)pertainstoselectivedetectionofdifferentregionsofageneoroperon.Asarstapproximation,all
portionsofageneoroperoncanbeconsideredequivalentaspotentialtargetsformoleculardetectionofthe
traitofinterest.Accordingtothisassumption,resultsfromthedetectionofonetargetregionbyoneassay
couldlegitimatelybecomparedwithresultsfromdetectionofanearbytargetregionfromthesamegeneor
operonbyadifferentassay,asawaytodeterminewhichassayismoresensitiveorspecicfortheparticular
trait.However,thisissomewhatofanoversimplication,sinceinrealitymanywildtypestrainshaveincom
pletecopiesofcertaingenesoroperons.
An example of this problem is provided by a recent study in whichpapCPCR (Le Bouguenec and
Archambaud, 1992) was compared withpapEFPCR to determine which assay was best for assessing
thepapstatus(asdenedbypapEFGprobehybridization)ofwildtypeE.colistrains(YamamotoandTerai,
1995).ThendingthatpapEFPCRcorrespondedbestwithpapEFGprobehybridizationwasinterpretedas
indicating the superiority ofpapEFPCR overpapCPCR, i.e., thatpapCPCR lacked sensitivity. We
subsequentlyusedprimersandprobesformultiplepapregions(JohnsonandStell,2000a,b)tofurtheranalyze
thesixstrainsthatintheabovestudyhadgiventhediscrepantpapCandpapEF
PCRresults.TheexplanationforthepreviouslynoteddiscrepancybetweenpapEFandpapCPCRwasfound
tobethatthesixstrainswerelargelydevoidofpapsequencesotherthanpapEF,includingpapG.Thetwo
PCRassaysthuswereboth100%accuratewithrespecttothestrainspapstatusintheirrespectiveregionsof
thepapoperon. Ironically, that these strains contained only a remnant ofpapwas inapparent from their
positivepapEFGprobehybridizationresults,whichinthepreviousstudyhadcausedthemtobeclassied
aspappositive,asiftheycontainedacompletecopyofpap(YamamotoandTerai,1995).

7.Multiplexing
An increasingly popular maneuver that increases the efciency of diagnostic PCR (although possibly
interferingwithsensitivityinsomecircumstances)istocombinemultipleprimersinonePCRreactionsoasto
simultaneouslyamplifymultipledifferentgeneregions, aprocesssometimestermedmultiplyprimed or
multiplexPCR(LeBouguenecandArchambaud,1992;YamamotoandTerai,1995;JohnsonandBrown,
1996; Karkkainen and Kaup pinin, 1998; Mitsumori and Terai, 1998; Johnson and Stell, 2000a,b).
Multiplexing imposes additional constraints on primer selection. The distribution of amplicon sizes to be
generatedbyeachprimerpoolshouldbesuchthattheproductscanbereadilydiscriminatedbyinspectionin
agarosegels,forwhichaminimumincrementalsizedifferenceof20%betweenadjacentbandsisdesirable
(e.g.,200bp,240bp,290bp,etc.).Allprimerstobecombinedinthesamepoolmustbecompatiblewithone
another(whichcanbeevaluatedduringprimerselectionusingPCRsimulationsoftware),shouldnothave
overlappingtargetregions,andideallyshouldhavesimilarannealingtemperatures.Whenmultipleallelesofa
givengenearetobedetectedusingamultiplexprimerpoolitsometimesispossibletodeviseauniversal
(consensus)primerforoneorientation(i.e.,senseorantisense)(KarkkainenandKauppinin,1998;Johnsonand
Stell,2000a,b).Allelespecicityisthendeterminedbyindividualprimersintheoppositeorientationwhichare
complimentarytoallelespecicregionslocatedatvaryingdistances
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from the consensus primer recognition site, such that in combination with the consensus forward primer
eachallelespecicprimeryieldsanampliconofdistinctivesize(KarkkainenandKauppinin,1998;Johnson
andStell,2000a,b).
AchievementofbalancedsimultaneousamplicationofallpossibleproductsfromamultiplexPCRmix
oftenrequiresempiricaladjustmentofassayconditions(HenegariuandHeerema,1997).Inourexperience,the
mostusefulparametertovaryistheprimerconcentration,sincebandintensityvariesroughlyinparallelwith
(althoughnotstrictlyinproportionto)theconcentrationofthecorrespondingprimer(s).Surprisinglylarge
reductionsintheconcentrationsofprimersspecicforother(competing)bandsasgeneratedbythesame
primer pool may be needed to permit a comparatively weak band to emerge with sufcient intensity.
AlthoughvariouscommercialproductsareavailablethatostensiblyassistinoptimizingmultiplexPCRassays,
inourexperiencewithE.coli(JohnsonandBrown,1996;JohnsonandStell,2000a,b)andEnterococcus(un
published)itusuallyispossibletoachieveanacceptableresultbyusingstandardreagentsandempirically
adjusting the annealing temperature and primer concentrations, so long as the primers in cluded in the
multiplexpoolareindividuallywelldesignedandarecompatiblewithoneanother.

8.Summary
Diagnostic PCR is a powerful tool for bac teriological research. Although in some circum stances
alternative methods may be preferable, PCR assays do offer many potential advantages over traditional
methodologies. New assays can be de veloped readily even by investigators with only a modicum of
experiencewithPCR,solongasestablishedprinciplesareobserved.Scrupulousassayvalidationandcareful
attentiontoqualitycontrolduringassayuseareessential.

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