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AnInexpensiveRT-PCREndpointDiagnosticAssayforSARS-CoV-2
UsingNestedPCR:DirectAssessmentofDetectionEfficiencyofRT-
qPCRTestsandSuitabilityforSurveillance
JayeshkumarNarsibhaiDavda#1,KeithFrank#1,SivakumarPrakash#1,2, GunjanPurohit1,Devi
PrasadVijayashankar1,2,DhiviyaVedagiri1,2,KarthikBharadwajTallapaka1,Krishnan
HarinivasHarshan1,2,ArchanaBharadwajSiva1,RakeshKumarMishra1,,JyotsnaDhawan1,
ImranSiddiqi1,2*
#Theseauthorscontributedequallytothiswork
Affiliation:
1CSIR-CentreforCellularandMolecularBiology(CSIR-CCMB),Hyderabad-500007.India
2AcademyofScientificandInnovativeResearch,CSIR-HRDCCampus,Sector19,Kamla
NehruNagar,Ghaziabad,UttarPradesh-201002.India
Authorforcorrespondence:imran@ccmb.res.in
Keywords:Covid-19,nestedPCR,endpointassay,pooledtesting,falsenegatives,
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Abstract
WithaviewtoextendingtestingcapabilitiesfortheongoingSARS-CoV-2pandemicwehave
developed a test that lowers cost and does not require real time quantitative reverse
transcription polymerase chain reaction (RT-qPCR).We developed a reverse transcription
nestedPCRendpointassay(RT-nPCR)andshowedthatRT-nPCRhascomparableperformance
tothestandardRT-qPCRtest.Inthecourseofcomparingtheresultsofbothtests,wefound
thatthestandardRT-qPCRtestcanhave lowdetectionefficiency(lessthan50%) inareal
testing scenariowhichmay be only partly explained by low viral representation inmany
samples.Thisfindingpointstotheimportanceofdirectlymonitoringdetectionefficiencyin
testenvironments.Wealsosuggestmeasuresthatwouldimprovedetectionefficiency.
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Introduction
ThecontinuingCovid-19pandemichascreatedanurgentneedforincreaseddiagnostictests
worldwide.Therequirementoftestshasexceededthenormaltestingcapacitiesavailablein
public and private hospitals and clinical research laboratories and also strained financial
resources. Themostwidelydeployed typeof test for identifying individuals infectedwith
SARS-CoV-2, based on recommendations of the World Health Organization (WHO) and
national health centres such as theUS Centers for Disease Control and Prevention (CDC)
detects the presence of viral RNA. The method employs real time quantitative reverse
transcriptionpolymerase chain reaction (RT-qPCR)ofRNAextracted fromnasopharyngeal
(NP)swabsamples,tomeasureamplificationofashortsegmentofaviralgeneinthecourse
ofaPCRreactionfollowingreversetranscriptionofviralRNA.PerformingtheRT-qPCRtest
requiresarealtimethermalcyclerwhichisanexpensiveinstrument.Mostmajorresearch
laboratoriesareequippedwithonlyalimitednumberandsmallerlaboratoriesmayhavenone
whichhasplacedconstraintsonthenumberoftestsaswellasplacesforconductingthem.
Further, the need for fluorescent oligonucleotide probes adds to the cost of the tests. A
numberofnewdiagnostictestingmethodsaimedatreducingthedependenceonexpensive
equipmentandkitsthatareinshortsupplyhavebeenproposedandareunderdevelopment
fordeployment(Broughtonetal.,2020;Rauchetal.,2020;Yanetal.,2020).However,given
theenormousandwidespreadcurrentneedfordiagnostictestingindiverseenvironmentsit
is equally important to increase the utilization of existing research capabilities that have
potentialbutarecurrentlynotbeingusedfortesting,throughtheuseofteststhatemploy
morewidelyavailableequipmentandreagentsusingsimpleestablishedmethods.
In order to extend the scope of diagnostic testing for SARS-CoV-2we explored a reverse
transcription nested PCR (RT-nPCR) approach that does not dependonRT-qPCRbut uses
standardRT-PCRaspartofanendpointassay.WedevelopedandtestedaRT-nPCRprotocol
comprisingamultiplexprimaryRT-PCRforamplificationoffourSARS-CoV-2ampliconsanda
controlhumanRPP30ampliconfollowedbyasecondarynestedPCRforindividualamplicons
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andvisualizationbyagarosegelelectrophoresis.WealsoexaminedtheuseofRT-nPCR in
pooledtestingandindirectamplificationwithoutRNAisolation.
RNAisolatedfromNPswabsamplesthathadbeenpreviouslytestedusingoneoftwoRT-
qPCRtestswasexaminedusingRT-nPCRandtheresultscompared.Wefoundthattakingboth
standard RT-qPCR tests together, the RT-nPCR test was able to correctly identify 90% of
samplesdetectedaspositivebyRT-qPCRandalsodetected13%samplesaspositiveamong
samplesthatwerenegativebythestandardRT-qPCRtest(likelyfalsenegatives).Basedon
the experimentally measured false negative rate by RT-nPCR tests from this study we
estimatedthatasmanyas50%ofpositivesamplesmayescapedetectioninsinglepasstesting
byRT-qPCRinanactualtestingscenario.
Results
NestedRT-PCRtestdevelopment
Wedesigned40setsofnestedoligonucleotideprimersforspecificallyamplifyingdifferent
regionsofSARS-CoV-2andnotSARS-CoVbasedonavailablesequenceinformation(Genbank
IDNC_004718.3forSARS-CoVandMT050493.1forSARS-CoV-2).Theseprimersweretested
foramplificationbyaprimaryPCR,followedbyasecondroundofnestedPCR.Thestarting
templatewascDNApreparedfromapoolofRNAisolatedfromtwoNPswabsamplesthat
hadpreviouslybeenidentifiedaspositiveusingaRT-qPCRdiagnostickit.Fromasetof40
candidateampliconsweselected4ampliconsthatgavevisibleamplificationintheprimary
PCR and strong bands in the secondary PCR reaction as visualized by agarose gel
electrophoresis.Theprimersequencesfortheseampliconswerecheckedagainst78SARS-
CoV-2sequencesfromIndianisolates.Exceptforasinglemismatchwithtwosequencesin
the middle for two primers, all primers were 100% identical to all 78 sequences. These
ampliconscomprisedportionsofOrf1ab,M,andNgenes(Figure1A;Table1).Theamplified
bandswereexcisedfromtheagarosegel,theDNAwasextracted,andidentityofeachband
wasconfirmedbysequencing.Similarlyasetofnestedprimerswasdevelopedforthehuman
RPP30 gene as a control. To test efficiency of amplification we used a dilution series of
ampliconsastemplatesinprimaryandsecondaryPCR.1-10moleculesofDNAindilutionsof
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isolatedviralampliconscouldbedetectedbynestedPCR(Figure1B).Todetectthepresence
ofSARS-CoV-2inRNAisolatedfromNPswabsweperformedamultiplexone-stepRT-PCRon
RNAfrompositiveandnegativesamplesusingpooledprimersforthefourviralamplicons
togetherwithhumanRPP30control.Theproductoftheprimaryone-stepRT-PCRwasused
astemplateinseparatenestedsecondaryPCRreactionsforeachoftheampliconsfollowed
bydetectionusingagarosegelelectrophoresis.UsingthenestedRT-PCRassaywewereable
to detect amplification of the four viral amplicons in RNA from positive samples and no
amplificationwasdetectedinnegativesamples(Figure2).
Pooledanddirecttesting
PooledtestinghasbeenusedforSARS-Co-V2detection(Yelinetal.,2020).Inordertoassess
thesuitabilityoftheRT-nPCRtestforanalysisofpooledsamples,weperformedtheteston
threesetsofpooledsamplescomprisingRNAisolatedfromdifferentdilutionsofapositive
sampleinapooloftennegativesamples.Forasamplehavingacyclethreshold(Ct)valueof
38.7fortheEgene,theRT-nPCRtestgaverobustdetectioninRNAfromundilutedsample
(positivefor3outof4viralamplicons)andlessrobustlyatadilutionof1:5(positivefor1out
of4viralamplicons;Figure3).FortwosampleshavingaCtvalueof28.7and34.5,theRT-
nPCRtestsuccessfullydetectedpresenceatadilutionof1:20.HencetheRT-nPCRtestwas
abletodetectpresenceofallthreesamplesinapoolof1:5.
Direct testingof samplesbyRT-qPCRwithoutRNA isolationhasbeendescribed in recent
reports(Bruceetal.,2020;Merindoletal.,2020;Smyrlakietal.,2020).Weperformeddirect
testingofheatinactivatedpositivesamplesbytheRT-nPCRtest.Using3µlofswabsample
directly,whichcorrespondsto1/5oftheamountusedintestingofisolatedRNA,theRT-nPCR
testdetectedapositivesamplehavinganEgeneCtvalueof27.9butnotasamplehavinga
higherCtof32.7(Figure4).Usingahighervolumeofswabsamplegaveweakeramplification
suggesting thepresenceof an inhibitor in theVTM.Weobserved improved amplification
whenpolyvinylpyrrolidonewasincludedinthePCRreaction.Passageofthesamplethrough
aSephadex-G50spincolumnalsorelievedthe inhibitionandgave improvedamplification.
OveralltheRT-nPCRtestiscapableofdetectingpositivesdirectlyinswabsamplesbutata
reducedsensitivitycomparedtoisolatedRNA.
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ComparisonofRT-nPCRwithRT-qPCR
InordertocomparetheperformanceofRT-nPCRwiththatofstandardRT-qPCRwetested
RNAsamplesthathadtestedpositivebyRT-qPCR.Samplesthatwerepositiveforatleasttwo
ampliconsoutoffourbyRT-nPCRwerecalledpositive,samplesthatwerepositiveforone
ampliconwereconsideredambiguousandnotincludedinthecomparison,andsamplesthat
werenegativeforallfourampliconswerecallednegative.WecomparedRT-nPCRtestresults
tosetsofpositiveandnegativeRNAsamplesthathadbeentestedbyeitheroftwoRT-qPCR
protocols,oneprovidedbyNationalInstituteofVirology(NIV),India(derivedfromCharite,
Berlin and CDC, USA protocols), and the other a commercial LabGun RT-qPCR kit
(LabGenomics,Korea).TheresultsindicatedthattheagreementbetweentheRT-nPCRand
NIVtestscouldberatedasmoderate(Cohen’sKappa=0.612,95%CI:0.42-0.81)(McHugh,
2012)whilethatbetweentheRT-nPCRandLabGuntestscouldberatedasalmostperfect
(Cohen’sKappa=0.905,95%CI:0.80-1.0;)(Figure5).TheloweragreementwiththeNIVset
wasdueto10samplesthatwerenegativebytheNIVprotocolbutpositiveintheRT-nPCR
test.Themeannumberofpositiveampliconsforthese10sampleswas3.3 indicatingthat
theywere likely to be true positives (i.e. false negatives). Based on the number of RNA
samples thatwere negative in the RT-qPCR tests but positive by RT-nPCRwe derived an
estimate of the proportion of positive samples thatwere being detected by theNIV and
LabGunRT-qPCRtests(Table2).TheNIVsetcovered400samplesandhadaprevalencerate
(% positives) of 15%. The detection efficiency for this setwas calculated to be 0.47. The
LabGun set comprised 1186 samples and had a prevalence rate of 3.7%. The detection
efficiencyforthesecondsetwasestimatedtobe0.44.Thereforeourestimatesuggeststhat
ahighproportionofpositivesamples(upto50%ormore)canbemissedbythestandardRT-
qPCRtestinarealtestingscenario.
Discussion
TheRT-nPCRtestdescribedabovedoesnotrequireareal timethermalcyclerandcanbe
performedinalaboratorythathasbasicmolecularbiologyequipment,athermalcycler,and
a BSL2 roomwith Class II laminar flow hood. It useswell-establishedmethodologies and
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reagentsthatarewidelyavailableandcanserveasabasisforbroadeningthescopeoftesting
forSARS-CoV-2.TheperformanceoftheRT-nPCRtestwascomparabletoRT-qPCRandthe
costofconsumablesforthetestbasedonlistpricesisaroundUS$7pertestabouthalfof
whichisthecostofRNAisolation.ThetestusestworoundsofPCRamplificationwhichdoes
increasepotentialforcontamination.Howeverwefoundthatbyfollowingasetofpractices
describedinadetailedprotocol(Supplementaryfile1),contaminationcouldbeavoided.
InthecourseofcomparingtheRT-nPCRandstandardRT-qPCRtestsweestimatedfromthe
rate of experimentally determined false negatives, that a high proportion (about 50%) of
positive samples were beingmissed by the RT-qPCR test applied in a single pass testing
protocol.Thisisahighescaperatewhichposesaconcerninindividualdiagnosisandmerits
monitoringandgreatercomparisonofresultsacrosstestingscenarios.Wealsosuggestthat
detectioncanbeimprovedbyrepeattestingofisolatedRNAandbyincreasingthenumberof
ampliconstested.
TheexistenceoffalsenegativesintheRT-qPCRtesthasbeeninferredinanumberofstudies
thatcomparedclinicaltestandsymptomaticdatawithRT-qPCRtestinginformation(Kucirka
etal.,2020;Lietal.,2020;Xiaoetal.,2020).Severalstudieshavecomparedtheperformance
ofdifferentRT-qPCRkitsonRNAfromclinicalsamplesprimarilytoassesstheperformanceof
thekits,andhavefoundagreementaswellasdifferencesthatalsopointtotheexistenceof
falsenegativetestresults(Hoganetal.,2020;Pujadasetal.,2020;vanKasterenetal.,2020;
Xiongetal.,2020).However,directexperimentalanalysisofRT-qPCRnegativeRNAsamples
at testing centres using different but related RT-PCR based tests as a way of estimating
detection efficiency has received limited attention likely because of the high demand for
diagnostictests,shortageoftestingkits,andcostoftesting.Thelowdetectionefficiencythat
wehaveestimatedmaynotbeentirelyexplainedbyalowrepresentationofthevirusinmany
of the samples as the mean number of positive amplicons by RT-nPCR for the 12 false
negatives (10 NIV + 2 LabGun) was 3.25 (Figure 5). Possible alternative explanations are
variabilityintestperformanceorinrepresentationofdifferentportionsoftheviralRNA.Low
detection efficiency could be a possible concern in other tests including those under
development that are based on reverse transcription as a first step followed by DNA
amplificationanddetection(Broughtonetal.,2020;Rauchetal.,2020;Yanetal.,2020).Many
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RT-qPCRtestsuseRNAcorrespondingtoabout1%ofswabsample.Increasingtheamountof
sampleforRNAisolationandconcentrationofthesamplearepossibleoptionsforimproving
detection,howeverthesewouldalsoincreasethenumberofoperationsandexpense,andit
would need to be assessed whether this would be compatible with medium to high
throughputprotocols.Protocolsbasedoninitialdetectionofasingleampliconfollowedbya
confirmatory test fora secondampliconmayalsocontribute to falsenegativesand lower
detection.
In conclusion the RT-nPCR endpoint assay for SARS-CoV-2 described above uses widely
availablereagents,lowerscosts,andobviatestheneedforarealtimetimethermalcycler.
Theassaycanthereforebeperformedinalargenumberofclinicalanddiagnosticlaboratories
lackingthisexpensivepieceofequipmentessentialforRT-qPCRtesting.Theperformanceof
RT-nPCRiscomparabletoRT-qPCRandanalysisofRT-qPCRtestedsamplesbyRT-nPCRshows
a high escape rate in detection of positives, highlighting a need for directly monitoring
detectionefficiencythroughassessmentoffalsenegativesinrealtestingenvironments.As
moreregionsoftheworldmoveintocommunitytransmissionphase,thereisalsoagreater
need for surveillance testing. Pooled testing by RT-nPCR can contribute to meeting this
requirement.
MaterialsandMethods
SampleCollection:
NPswabsampleswerecollectedfrompatientssuspectedofbeinginfectedwithSARS-CoV-2
andtheircontactsatdifferenthospitalsintheHyderabadvicinitybasedonIndianCouncilof
Medical Research (ICMR) guidelines (http://www.nie.gov.in/images/leftcontent_attach/COVID-
SARI_Sample_collection_SOP_255.pdf) and in accordancewith Institutional Ethics Committee
guidelines.Sampleswerecodedandanonymizedbeforeprocessinganddatacollection.
RNAisolation:
140µlofNPswabsampleinViralTransportMedium(VTM)wasusedforRNAisolationbythe
QIAamp®Viral RNA (cat# 52906) or equivalent kit as permanufacturer’s instructions. NP
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samplewaslysedinBSL3facility.RNAisolationstepswerecarriedoutinBSL2facility.RNA
waselutedin50µlofwater.ForpooledsampleRNAisolation,positivesamplewaspooled
withnegativesamplesin1:5,1:10and1:20ratioscomprising140µlofNPsampleandisolated
asabove.AllsafetyprecautionswerefollowedasperICMR-NIVguidelines.
PrimerdesignandPCR:
SARS-CoV-2fulllengthgenomicsequenceofanisolatefromKerala-Indiawasdownloaded
fromNCBI(GenbankIDMT050493.1).PrimersweredesignedforregionsspecificforSARS-
CoV-2,butnotSARS-CoV(GenbankIDNC_004718.ForthehumanRPP30gene(GenbankID
U77665.1) primers were designed on exon-exon junctions to avoid genomic DNA
amplification.OligonucleotideswereobtainedfromBioserve,Hyderabad.PooledRNAfrom
twopreviouslyidentifiedpositiveNPswabsampleswasusedforfirststrandcDNAsynthesis
(Takaraprimescriptkitcat#6110A).20µlofthecDNAreactionwasdilutedten-foldandused
forprimaryPCR.PrimaryPCRwasperformedusingEmeraldAmp®GTPCRMasterMix(Takara
cat#RR310A)10ul,forwardandreverseprimers(5µM)1µleach,dilutedcDNA3µl,and
water5µl.Thermalcyclingconditionswere1)95°C-2minutes2)95°C-15seconds3)60°C
-15seconds4)72°C-30seconds5)Repeatsteps2-4for4cycles6)95°C-15seconds7)55
°C-15seconds8)72°C-30seconds9)Repeatsteps6-8for39cycles10)72°C-2minutes
11) 15 °C – hold. The primary PCR product was diluted fifty-fold and 1 µl was used for
secondaryPCRusinganestedprimerpair.Thermalcyclingconditionswerethesameasfor
primaryPCR.PCRproductswere separatedbyelectrophoresisona1.5%agarosegel and
visualizedonaUVgeldocumentationsystem.Foramplicondilutionexperiments,bandswere
excisedfromthegel,DNAwasextracted(Macherey-NagelNucleospinkit,Cat#740609.250)
andsubclonedintopGEM-Tvector(Promega).TheinsertwasPCRamplifiedfromaplasmid
DNAclone,followedbygelpurificationandextraction.DNAwasquantifiedbyaQubitdsDNA
HSkit(ThermofisherCat#Q32851).
RT-nPCR:
PrimaryRT-PCRandsecondaryPCRwasperformedusingthePrimescriptIII1-stepRT-PCRkit
andEmeraldAmpGTPCRMix(Takara;detailedprotocolprovidedinSupplementaryFile1).
FordirecttestingofsamplewithoutRNAisolationNPsamplewasheatinactivatedat95°C
for10minutesinBSL3.1µlof5%PVP40(Sigmacat#PVP40-500G)wasincludedinPrimary
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PCRandother conditionswere kept same. For SephadexG50 (Sigma cat#GE17-0042-01)
experiment,aspincolumnwaspreparedasmentioned inSambrookandManiatismanual
andotherconditionswerekeptsame.ThespincolumnwasusedoutsidethePCRareato
avoidcontaminationfromaerosols.ImageswereeditedusingAdobePhotoshopandincluded
removal of intervening lanes in the gel between samples andDNAmarker indicatedby a
verticalline.
RT-qPCR:
NIVmethod:RT-qPCRforEgeneandRPP30genewasdonewith5µlofRNAtemplateby
followingFirstlinescreeningassayaccordingtoNationalInsitituteofVirology(ICMR-NIV)
https://www.icmr.gov.in/pdf/covid/labs/1_SOP_for_First_Line_Screening_Assay_for_2019_
nCoV.pdf.BasedonEgeneresultRdRpandOrf1bweretestedwith5µlofRNAsampleby
followingConfirmatoryassaygivenbyICMR-NIV
https://main.icmr.nic.in/sites/default/files/upload_documents/2_SOP_for_Confirmatory_As
say_for_2019_nCoV.pdf.
LabGunmethod:RT-qPCRforEgeneandRdRpwasperformedalongwithinternalcontrolas
per manufacturer’s instructions (LabGenomics - LabGunTM COVID-19 RT-PCR Kit cat#
CV9032B).4µlofRNAtemplatewasusedforthereaction.
Ethicalstatement:
The work described in this study was carried out in accordance with institutional ethics
committeeguidelines.
Authorcontributions:
JND, KF, SP, and IS designed the experiments. JND, KF, SP, GP, DPV, and DV performed
experiments.KBT,KHH,RKM,andABSsupervisedprocessingandprovisionofsamplesand
data.JND,KF,SP,ISandJDinterpretedtheexperimentalresults.IS,JND,SP,andKFwrotethe
paperwithinputsfromJD.
Acknowledgements:
TheTelenganastategovernmentandDirectorateofMedicalEducationareacknowledgedfor
providingtheswabsamplesusedinthisstudy.WethanktheCOVID-19teamofvolunteersat
.CC-BY-NC-ND 4.0 International licensemade available under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
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CCMBforprocessingthesamples.ThefulllistofvolunteersisprovidedasSupplementaryfile
2.ThisworkwassupportedbytheCouncilofScientificandIndustrialResearch(CSIR,India).
Competinginterests:
Theauthorsdeclarenocompetinginterests
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FigureLegends:
Figure1:NestedRT-PCRstrategyforSARS-CoV-2detection
A) SchematicofnestedPCRapproach
B) Detectionlimitbyserialdilutionofampliconsfrom1000–0.1molecules.
Figure2:RepresentativegelpictureofpositiveandnegativeRNAsamples.
Lanes1,2,and4:positivesamples;Lanes3,7,and8:negativesamples;Lane5:10
NTC;Lane6:20NTC.
Figure3:DetectionefficiencyofRT-nPCRtestinpooledsamples.
Samples with different Ct values for E gene pooled at different dilutions with
negative samples. Each pool was tested for amplification of 4 amplicons. UD:
undiluted.
Figure4:DetectionofSARS-CoV-2byRT-nPCRwithoutRNAisolation.
A) Reliefofinhibitionathighersamplevolumesbyadding0.5%ofPVP.Lanes1-4:E
geneCt=27.9;Lanes5-8:EgeneCt=32.7.Lanes1,5:1µl;2,6:3µl;3,7:5µl;4,8:
5µl+0.5%PVP.
B) Relief of inhibition at higher volume of NP swab sample by passing through
SephadexG-50spincolumn.Lane1,2:3µland9µlofNPswabsample.Lane3,4:
3µland9µlofNPswabsampleafterpassingthroughSephadexG-50column.
Figure5:ComparisonofRT-nPCRandRT-qPCRtests
A) ContingencytableofRT-nPCRwithRT-qPCRtests.Samplesthatwerepositivefor
onlyoneampliconbyRT-nPCRwere consideredambiguousandexcluded from
analysis. Thenumberof ambiguous sampleswas: fromNIV set: Positives0/20,
Negatives3/50;LabGunset:Positives4/30,Negatives2/41.
B) GraphshowingnumberofampliconsamplifiedbyRT-nPCRofsampleshavinga
rangeofCtvaluesforEgene.Ct25-31(n=21),Mean=4.0;Ct31-37(n=18),Mean
=3.61;Ct37-40(n=11),Mean=2.18.
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Table1:Listofprimers
Name Purpose Target
Gene
Sequence(5’-3’) Coordinatesin
MT050493.1
N1F1 PrimaryRT-PCR N AGGAAATTTTGGGGACCAGGAA 29102-29123
N1R1 PrimaryRT-PCR N CTGCTCATGGATTGTTGCAA 29491-29472
N1F2 SecondaryPCR N GATTACAAACATTGGCCGCAAATTG 29142-29166
N1R2 SecondaryPCR N GAAATCATCCAAATCTGCAGCAG 29462-29440
Orf1abF1 PrimaryRT-PCR ORF1AB GGGAAATTGTTAAATTTATCTCAACCTG 2182-2209
Orf1abR1 PrimaryRT-PCR ORF1AB CTGGTGTACCAACCAATGGAGC 2592-2571
Orf1abF2 SecondaryPCR ORF1AB AATTGTCGGTGGACAAATTGTC 2219-2240
Orf1abR2 SecondaryPCR ORF1AB ACTAGTAGGTTGTTCTAATGGTTG 2558-2535
MF1 PrimaryRT-PCR M TGGCAGATTCCAACGG 26504-26519
MR1 PrimaryRT-PCR M ACAGCGTCCTAGATGGTG 26979-26962
MF2 SecondaryPCR M CCTTGAACAATGGAACCTAG 26550-26569
MR2 SecondaryPCR M AGCAATACGAAGATGTCCAC 26958-26939
N2F1 PrimaryRT-PCR N GCCTCGGCAAAAACGTAC 29024-29041
N2R1 PrimaryRT-PCR N CTTGTGTGGTCTGCATGAG 29533-29515
N2F2 SecondaryPCR N GTAACACAAGCTTTCGGC 29061-29078
N2R2 SecondaryPCR N TGAGTTGAGTCAGCACTG 29506-29489
RPPF1 PrimaryRT-PCR RPP30 GCATGGCGGTGTTTGCAG
RPPR1 PrimaryRT-PCR RPP30 CTCCTTCTGATGGCCGAGG
RPPF2 SecondaryPCR RPP30 CGGACTTGTGGAGACAGCC
RPPR2 SecondaryPCR RPP30 CAAGCCTAGATTTGCCACGTC
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Table2:EstimateofDetectionEfficiencyofPositivesbyRT-qPCR
TestKit No.of
samples
(S)
RT-qPCR
Positives(P)
False
Negative
Rate(FNR)
EstimatedFalse
Negatives
FNE=(FNR)(S-P)
Detection
Efficiency
DE=P/(P+FNE)
NIV 400 60 10/50
=0.200
68 0.47
LabGun 1186 44 2/41
=0.049
56 0.44
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Fig1
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Fig2
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Fig4
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Fig5
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