An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV … · 2020. 6. 8. · 6 Comparison of...

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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:[email protected]

Keywords:Covid-19,nestedPCR,endpointassay,pooledtesting,falsenegatives,

.CC-BY-NC-ND 4.0 International licensemade available under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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

The copyright holder for this preprintthis version posted June 9, 2020. ; https://doi.org/10.1101/2020.06.08.139477doi: bioRxiv preprint

https://doi.org/10.1101/2020.06.08.139477http://creativecommons.org/licenses/by-nc-nd/4.0/

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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




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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.




15

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




16

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




Fig1




Fig2




Fig4




Fig5



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