Yersinia pestis detection by loop-mediated isothermal amplification combined with magnetic bead capture of DNA

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Clinical

Microbiology

Yersinia

pestis

detection

by

loop-mediated

isothermal

amplification

combined

with

magnetic

bead

capture

of

DNA

Na

Feng

_,

_Yazhou

_Zhou

_,

_Yanxiao

_Fan

_,

_Yujing

_Bi

_,

_Ruifu

_Yang

_,

_Yusen

_Zhou

_,

Xiaoyi

Wang

a_{AnhuiMedicalUniversity,Anhui,People’sRepublicofChina}

b_{BeijingInstituteofMicrobiologyandEpidemiology,StateKeyLaboratoryofPathogenandBiosecurity,LaboratoryofAnalytical} Microbiology,Beijing,China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22June2016 Accepted17March2017 Availableonline26August2017 AssociateEditor:RoxanePiazza

Keywords: Plague Yersiniapestis Loop-mediatedisothermal amplification Magneticbeads

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b

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Wedevelopedaloop-mediatedisothermalamplification(LAMP)assayforthedetectionofY. pestisbytargetingthe3asequenceonchromosome.All11speciesofthegenusYersiniawere usedtoevaluatethespecificityofLAMPandPCR,demonstratingthattheprimershadahigh levelofspecificity.ThesensitivityofLAMPorPCRwas2.3or23CFUforpureculture,whereas 2.3×104_or2.3×106_{CFUforsimulatedspleenandlungsamples.Forsimulatedliversamples,}

thesensitivityofLAMPwas2.3×106_{CFU,butPCRwasnegativeatthelevelof2.3×10}7_CFU.

Aftersimulatedspleenandlungsamplesweretreatedwithmagneticbeads,thesensitivity ofLAMPorPCRwas2.3×103_or2.3×106_{CFU,whereas2.3×10}5_or2.3×107_{CFUformagnetic}

bead-treatedliversamples.Theseresultsindicatedthatsomecomponentsinthetissues couldinhibitLAMPandPCR,andlivertissuesampleshadastrongerinhibitiontoLAMP andPCRthanspleenandlungtissuesamples.LAMPhasahighersensitivitythanPCR,and magneticbeadcaptureofDNAscouldremarkablyincreasethesensitivityofLAMP.LAMPis asimple,rapidandsensitiveassaysuitableforapplicationinthefieldorpovertyareas.

©2017PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade Microbiologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Plague isazoonotic disease causedbyGram-negative bac-terium Yersinia pestis, which is occasionally transmitted to humansfromY.pestis-infectedrodentsviathebitesofinfected fleas.1 _{Y.pestisis thoughttohaveevolved from aserotype}

O:1bstrain ofY.pseudotuberculosisabout 6000–10,000years

∗ _{Correspondingauthors.}

E-mails:yszhou@bmi.ac.cn(Y.Zhou),wgenome@163.com(X.Wang).

ago although these two species cause remarkably differ-ent diseases.2–3 _{Y.pestisis ahighly virulent and infectious}

pathogen, and was classifiedas aCategory Apathogen by theU.S.CentersforDiseaseControlandPrevention.4

Histori-cally,Y.pestishasgivenrisetothreemajorplaguepandemics, leadingtomillionsofhumandeaths.Recently,theincreased outbreakofplaguearoundtheworldisreportedannuallyto

https://doi.org/10.1016/j.bjm.2017.03.014

1517-8382/©2017PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Table1–SequenceoftheprimersforLAMPandPCRreactions.

Primernames Type Sequence(5–3) Primerlength(bp)

3a-F3 Forwardouterprimer ACTACCATCCCCTCAAGGTT 20

3a-B3 Backwardouterprimer GAGGGCGTTTTGGTAGAGAA 20

3a-FIP Forwardinnerprimer CACCCGCGTTATCTCATCCCG-TTTTCGAGTAGGGTTAGGTGGGC 44

3a-BIP Backwardinnerprimer CATGGACGTATGGCGGGTCA-TTTTGTGATGCCGTCCAATGCA 42

3a-LF Forwardloopprimer ACCGCCATGAAATGGACAATG 21

theWorldHealthOrganization(WHO),andplaguewas clas-sifiedasare-emerginginfectiousdiseasebytheWHO.5–6_In

addition,plaguehasbeenattractingaconsiderableattention becauseitscausativeagenthasalwaysbeenrecognizedasone oftheclassicalbiologicalwarfareorbioterrorismagents.7–8_To

minimizethesethreats,developmentofarapidmethodfor thedetectionofY.pestisisessential.

Y. pestis was often detected by bacterial isolation and microscopyobservation,9_{phagelysisassay,}10–11_ELISAassays

basedonthedetectionofF1antigenandantibodiesagainst

Y.pestis,12–15 _{conventional PCR assays,}16–18 _real-time

quan-titative PCR assays,19–28 _{biosensors based on fiber-optic or}

upconvertingphosphortechnology,29–31_solid-phase

radioim-munoassaybasedon radiolabeled monoclonalantibodyfor thedetectionofplagueantigen.32_{Allthesemethodsare}

play-inganimportantrole inthediagnosis ofplague, butthese methodseitheraretime-consumingandlaborious,orrequire expensiveequipmentandpersonnelwithahighlevelof tech-nicalexpertise.However,permanentsurveillanceofthefociof plaguelocatedinthepoorregionstopredictfutureepizootics inrodentsandexposureriskforhumansorinvestigationof samples suspectedof bioterrorism on site requires a sim-ple,rapidand efficientdiagnostic method. Acolloidalgold particles-based lateral-flow (LF)strip detection method for

Y.pestishasbeen developedbasedonantibodies toF1and LcrVproteins.33–34_{Thisdipsticktestisalow-cost,easy-to-use}

andrapidscreeningmethodinthesurveillanceofplagueor investigationofsamplessuspectedofbioterrorismonsite,but nucleicacid-basedrapiddetectiontechnologycouldbeamore powerfulalternativefordetectingY.pestis.

Loop-mediatedisothermalamplification(LAMP) technol-ogy has received considerable attention because it allows efficientlyamplificationofDNAwithhighspecificityand sen-sitivityunder isothermalconditions of60–65◦C.TheLAMP reactioncanbeaccomplishedwithinlessthan1hbasedon asetoffourtosixprimersandtheBstDNApolymerasewith stranddisplacementactivity.35_{Themethodismoresuitable}

forfieldapplications,especiallyinpovertyareas,becauseit doesnotrequirespecializedorexpensiveequipment,andonly asimpleandinexpensivewaterbathorheatingblockcan sat-isfyLAMPassay.36–37Inaddition,LAMPresultscanbereadby thenakedeye,orthelateralflowdipstick(LFD)under natu-rallight,whichmakesthedetectionresultseasytobejudged inthefield.Currently,theLAMPtechniquehasbeenwidely usedinthediagnosisofinfectiousdiseases,38_anditismore

sensitiveindetectingbacteriacomparedtotheconventional polymerasechainreaction(PCR)method.39–40 _{Inthis study,}

weconstructasimpleandrapidLAMPmethodfordetection ofY.pestisbasedonthespecificsequence3a(GenBank acces-sionno.AF350075)thatisaspecificfragmentlocatedon Y. pestischromosome foundby using acomparative genomic

method.41 _{Thespecificity of the method wasevaluated by}

usingall11speciesofthegenusYersinia.Thesensitivitywas evaluatedbyusingY.pestispureculture,andsimulatedtissue samples, andmagneticbead-treatedsimulatedtissue sam-ples.

Materials

and

methods

Bacterialstrains,reagents,instrumentsandanimals Y.pestisEVvaccinestrain, Y.pseudotuberculosis,Y. enterocolit-ica,Y.frederiksenii,Y.intermedia,Y.kristensenii,Y.bercovieri,Y. mollaretii,Y.rohdei,Y.ruckeri,andY.aldovaewereusedto evalu-ate thespecificityofLAMPand PCR;10× thermopolbuffer, MgSO4 (100mM),and Bst DNAPolymerase, LargeFragment

(8000U/ml)were purchased from NEB(Beijing, China); Cal-ceinandreal-timeturbidity meterLA-320cwere purchased from EikenChinaCO., LTD.;TaqDNA polymerase(5U)and dNTPs(2.5mM)werepurchasedfromTaKaRa(Dalian,China); SM3-P100,amino-modifiedsilica-coatedmagneticbeads,was purchased formShanghai Allrun Nano Science & Technol-ogyCO.,LTD.BALB/cwereobtainedfromLaboratoryAnimal ResearchCenter,AcademyofMilitaryMedicalScience,China (licensed from the Ministry of Health in General Logistics DepartmentofChinesePeople’sLiberationArmy,PermitNo. SCXK-2007-004).TheprotocolswereapprovedbyCommittee oftheWelfareandEthicsofLaboratoryAnimals,Beijing Insti-tuteofMicrobiologyandEpidemiology.Theexperimentswere conductedstrictlyincompliancewiththeRegulationsofGood LaboratoryPracticefornonclinicallaboratorystudiesofdrug issuedbytheNationalScientificandTechnologicCommittee ofPeople’sRepublicofChina.

LAMPandPCRassays

ForLAMPreaction,asetoffiveprimerswasdesigned accord-ing to the published sequence of 3a in Y. pestis KIM D46 (GenBank accession no.: AF350075) using Primer Explorer version 4. A forward inner primer (FIP), a backward inner primer(BIP),twoouterprimers(F3and B3),andaloop for-ward primer (LF) were used for LAMP amplification. The sequencesoftheprimersareshowninTable1.LAMPreaction was performedinatotalvolumeof25␮Lmixture contain-ing 10× Thermopol buffer (2.5␮L), 100mM MgSO4 (1.5␮L),

2.5mMdNTPMix(14␮L),100mM3a-FIP(0.4␮L),100mM 3a-BIP(0.4␮L),10mM3a-F3(0.5␮L),10mM3a-B3(0.5␮L),10mM 3a-LF(0.2␮L),8000U/mLofBstDNAPolymerase,Large Frag-ment (1␮L), water (3␮L), template DNA (1␮L). The LAMP reactionwascarriedoutat65◦Cfor60minandinactivatedat 80◦Cfor5mininwaterbath.FordirectvisualdetectionofDNA

A

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G

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Time (min) Time (min)

T urbidity (400nm) T urbidity (400nm) 1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 6 7 7 7 7 8 8 8 8 9 9 9 10 10 1 2 3 5 5 6 6 7 7 8 8 9 9 10 10 1 2 3 4 1 2 3 4 5 6 7 8 9 10 4 10 11 11 11 11 11 12 12 12 12 13 13 14 5 6 7 8 9 10 1 2 3 4 11 12

Fig.1–Thesensitivity(A–D)oftheLAMPorPCRforthedetectionofY.pestisusingserialdilutionsofextractedDNA,andthe specificity(E–H)oftheLAMPorPCRforthedetectionofY.pestisusing20ngDNAfromeachbacteriumofthegenusYersinia.

(A)DetectionofLAMPproductswithareal-timeturbiditymeter.1–8:20ng–0.002pg;10:negativecontrol.(B)Visualizationof LAMPproductsstainedwithcalceinandinspectedundernaturallight.Tube1–9:20ng–0.0002pg;10:negativecontrol.(C) DetectionofLAMPproductswithagarosegelelectrophoresis.Lane1:marker;2–10:20ng–0.0002pg;11:negativecontrol.(D) ElectrophoreticanalysisofPCRproductswithagarosegel.Lane1:marker;2–10:20ng–0.0002pg;11:negativecontrol;12: marker.(E)DetectionoftheLAMPproductswithareal-timeturbiditymeter.1:Y.pestis;2:Y.pseudotuberculosis;3:Y. enterocolitica;4:Y.frederiksenii;5:Y.intermedia;6:Y.kristensenii;7:Y.bercovieri;8:Y.mollaretii;9:Y.rohdei;10:Y.ruckeri;11:Y. aldovae;12:negativecontrol.(F)VisualizationofLAMPproductsstainedwithcalceinandinspectedundernaturallight.Tube 1:Y.pestis;2:Y.pseudotuberculosis;3:Y.enterocolitica;4:Y.frederiksenii;5:Y.intermedia;6:Y.kristensenii;7:Y.bercovieri;8:Y. mollaretii;9:Y.rohdei;10:Y.ruckeri;11:Y.aldovae;12:negativecontrol.(G)DetectionofLAMPproductswithagarosegel electrophoresis.Lane1:marker;2:Y.pestis;3:Y.pseudotuberculosis;4:Y.enterocolitica;5:Y.frederiksenii;6:Y.intermedia;7:Y. kristensenii;8:Y.bercovieri;9:Y.mollaretii;10:Y.rohdei;11:Y.ruckeri;12:Y.aldovae;13:negativecontrol.(H)Electrophoretic analysisofPCRproductswith1.5%agarosegel.Lane1:marker;2:Y.pestis;3:Y.pseudotuberculosis;4:Y.enterocolitica;5:Y. frederiksenii;6:Y.intermedia;7:Y.kristensenii;8:Y.bercovieri;9:Y.mollaretii;10:Y.rohdei;11:Y.ruckeri;12:Y.aldovae;13: negativecontrol;14:marker.

amplification,1␮LofFluorescentDetectionReagent contain-ingcalcein(EIKENCHINACO.,LTD.)wasaddedtothereaction tubebeforetheLAMPreaction.Forapositivereaction,acolor changefromorangetogreenwasobservedthroughthenaked eyeundernaturallight,whereasanegativereactionmixture remained orange. Alternatively, specific DNA amplification wasmonitoredspectrophotometricallybyareal-time turbid-itymeter.Toconfirmthatcolorchangeinreactiontubesisa resultofDNAamplification,LAMPproductwasalsodetected by0.8% agarose gel electrophoresisand ethidiumbromide staining.

ConventionalPCRreactionwasperformedinatotal vol-ume of 25␮L mixture containing 10×buffer (3␮L), 2.5mM dNTPs(0.6␮L),10mM3a-F3(0.5␮L),10mM3a-B3(0.5␮L),5U

ofTaqDNApolymerase(0.1␮L),deionizedwater(15.3␮L)and template DNA (5␮L).F3 and B3 primers were usedfor the conventional PCR amplification(Table 1). Thereaction was subjectedtoaninitialdenaturationat95◦Cfor5min,followed by 35 cycles ofdenaturationat95◦C for30s, annealing at 56◦Cfor30s,extensionat72◦Cfor1minandafinal exten-sionat72◦Cfor5min.ThePCRproductswere analyzedby 1.5%agarosegelelectrophoresisandstainedwithethidium bromide.

ExtractionofbacterialgenomicDNA

Forpreparationofhigh-qualitygenomicDNA,Y.pestisEV vac-cinestrain andother bacteria werecultured inLuriabroth

1.2 1 0.8 0.6 0.4 0.2 0 -0.2 1.2 1.4 1.6 1 0.8 0.6 0.4 0.2 0 -0.2 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 1 2 ₁ 3 4 3 5 6 5 7 8 7 2 4 6 8 9 10 9 10 11 12

Time (min) _{Time (min)}

T urbidity (400nm) T urbidity (400nm)

A

E

B

F

C

D

Fig.2–Thesensitivity(A–C)oftheLAMPandPCRforthedetectionofY.pestisbyusingtheDNAextractedcrudelyfrom serialdilutionsofbacterialsolutions,andthespecificity(D–F)oftheLAMPandPCRforthedetectionofY.pestisusing crudelyextractedDNAfromeachbacterium(2.3×107_{CFU)ofthegenusYersinia.(A)DetectionoftheLAMPproductswitha}

real-timeturbiditymeter.1–9:2.3×107_–2.3×10−1_{CFUofY.pestis;10:negativecontrol.(B)VisualizationofLAMPproducts}

stainedwithcalceinandinspectedundernaturallight.Tube1–9:2.3×107_–2.3×10−1_{CFUofY.pestis;10:negativecontrol.(C)}

ElectrophoreticanalysisofPCRproductswithagarosegel.Lane1:marker;Lane2–10:2.3×107_–2.3×10−1_{CFUofY.pestis;11:}

negativecontrol;12:marker.(D)DetectionofLAMPproductswithareal-timeturbiditymeter.1:Y.pestis;2:Y.

pseudotuberculosis;3:Y.enterocolitica;4:Y.frederiksenii;5:Y.intermedia;6:Y.kristensenii;7:Y.bercovieri;8:Y.mollaretii;9:Y. rohdei;10:Y.ruckeri;11:Y.aldovae;12:negativecontrol.(E)VisualizationofLAMPproductsstainedwithcalceinand

inspectedundernaturallight.Tube1:Y.pestis;2:Y.pseudotuberculosis;3:Y.enterocolitica;4:Y.frederiksenii;5:Y.intermedia;6:

Y.kristensenii;7:Y.bercovieri;8:Y.mollaretii;9:Y.rohdei;10:Y.ruckeri;11:Y.aldovae;12:negativecontrol.(F)Electrophoretic analysisofPCRproductswithagarosegel.Lane1:marker;2:Y.pestis;3:Y.pseudotuberculosis;4:Y.enterocolitica;5:Y. frederiksenii;6:Y.intermedia;7:Y.kristensenii;8:Y.bercovieri;9:Y.mollaretii;10:Y.rohdei;11:Y.ruckeri;12:Y.aldovae;13: negativecontrol;14:marker.

(LB)at26◦Cand37◦C,respectively,andthencollectedby cen-trifugation.Thecollectedbacterialpelletwasresuspendedin SEbuffer(0.15MNaCl,0.1MEDTA-2Na·2H2O,adjustedtopH

8.0by10M HCl),addedtoafinal concentrationof2%SDS, andthenplacedinawaterbathat60◦Cfor10min.Then,an equalvolumeofphenol,chloroformandisoamylalcohol mix-ture(25:4:1)wasaddedandthesolutionwasmixedbyslowly inverting or rotatingto denature proteins, then to remove proteins.Theaqueousphase wasadded afinal concentra-tionof50–100␮g/mlofRNasetodegradeRNAsat37◦C for 30–60min.Then,anequalvolumeofchloroformwasadded, shakingvigorously.Theaqueousphasewasprecipitatedwith twotimesvolumeofethanol,andthentheDNAwasspooled outwithaglassrod,rinsedwith70%ethanol,anddriedinair. Finally,theDNAwasdissolvedintoTEbuffer(10mMTris–HCl, 1mM EDTA-2Na·2H2O, adjusted to pH 8.0 by 10M HCl)

foruse.

ForsimpleandrapidisolationofbacterialDNA,the bac-terialcells wereresuspended indeionized water,and then genomicDNAwasreleasedbyboilingthebacterial suspen-sionat100◦Cfor10min.Thesupernatantwascollectedand usedastheDNAtemplateinbothLAMPandconventionalPCR reactions.

CaptureofbacterialgenomicDNAbymagneticbeads

BacterialgenomicDNAwasreleasedbyboilingbacterial sus-pensionat100◦Cfor10min,300␮Lofthesupernatantwas mixed with the same volume of binding buffer [4M NaCl and20%polyethyleneglycol(PEG)8000],followedbyaddition of0.2mgmagneticnanoparticlesSM3-P100.Thesuspension wasvibratedvigorouslyfor15s,followedbystandingatroom temperaturefor5min.Themagneticbeadswerecollectedby usinganexternalmagnet,andthesupernatantwasdiscarded. Themagnetic pelletwas washed twotimes with200␮Lof 70% ethanol, and then driedcompletely atroom tempera-ture.Finally,themagneticpelletwasresuspendedin100␮L ofdeionizedwatertoeluteboundDNAatroomtemperature for5minwithcontinuousagitation.Thesupernatant contain-ingthegenomicDNAwasusedastheDNAtemplateinboth LAMPandconventionalPCRassays.

Preparationofsimulatedsamplesandmagneticbead captureofDNA

Thespleen,liverand lungswere collected asepticallyfrom pathogen-free BALB/c mice, and then blended with the

1.2 1 0.8 0.6 0.4 0.2 0 -0.2 1.2 1 0.8 0.6 0.4 0.2 0 -0.2 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 1 2 _{1 2} 3 4 _{3 4} 5 6 5 6 7 8 _{7 8} 9 10 _{9 10} 5 6 7 8 9 10 1 2 3 4 1 2 3 4 5 6 7 8 9 10 5 6 7 8 9 10 1 2 3 4 11 12 5 6 7 8 9 10 1 2 3 4 11 12

A

E

B

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Fig.3–DetectionofY.pestisinsimulatedspleensamplesbyLAMPandPCR.ThesensitivityofLAMPorPCRwas

determinedbyusingtheDNAextractedbyboilingsimulatedspleensamplefor10min(A–C)orbyusingtheDNAcaptured bymagneticbeads(D–F).(A)DetectionofLAMPproductswithareal-timeturbiditymeter.1–8:2.3×100_–2.3×107_CFUofY. pestis;9:positivecontrol;10:negativecontrol.(B)VisualizationofLAMPproductsstainedwithcalceinandinspectedunder naturallight.Tube1–8:2.3×100_–2.3×107_{CFUofY.pestis;9:positivecontrol;10:negativecontrol.(C)Electrophoretic}

analysisofPCRproductswithagarosegel.Lane1:marker;Lane2–9:2.3×107_–2.3×100_{CFUofY.pestis;10:positivecontrol;}

11:negativecontrol;12:marker.(D)DetectionofLAMPproductswithareal-timeturbiditymeter.1–8:2.3×100_–2.3×107_CFU

ofY.pestis;9:positivecontrol;10:negativecontrol.(E)VisualizationofLAMPproductsstainedwithcalceinandinspected undernaturallight.Tube1–8:2.3×100_–2.3×107_{CFUofY.pestis;9:positivecontrol;10:negativecontrol.(F)Electrophoretic}

analysisofPCRproductswithagarosegel.Lane1:marker;Lane2–9:2.3×107_–2.3×100_{CFUofY.pestis;10:positivecontrol}

(20ngDNA);11:negativecontrol;12:marker.

appropriate volume of PBS buffer to prepare tissue homogenates. Y. pestis was cultured in LB, collected by centrifugation,andthenresuspendedindeionizedwaterto prepareserial10-folddilutionsofbacterialsuspension.The spleen,liverandlungtissuehomogenateswererespectively inoculatedwiththesame volumeofserial10-folddilutions ofbacterialsuspensiontoobtaindifferentconcentrationsof artificially contaminated samples (2.3×107_–2.3×10−1_CFU).

Noninoculated tissue homogenates were mixed with the same volume ofdeionized water tobe used asa negative control. Artificially contaminated samples for each tissue were boiledat100◦C for10min,and then centrifugatedat 10,000×gfor2min.Thesupernatantscanbedirectlyusedas theDNAtemplateinbothLAMPandconventionalPCRassays. The supernatants can be further used to isolate bacterial DNAbymagneticbeadsaccordingtotheprotocoldescribed above, and thenused inboth LAMP and conventional PCR assays.

DeterminationofLAMPorPCRsensitivity

ThesensitivityofLAMPorPCRforthedetectionofY.pestiswas evaluatedwiththehigh-qualitygenomicDNA.Serial10-fold dilutionsofDNAweremadeinsteriledeionizedwater,and usedastheDNAtemplatestoperformLAMPandPCRassays.

ThesensitivityofLAMPorPCRwasevaluatedwithY.pestis

pureculture.Serial10-folddilutionsofY.pestiscellsweremade insteriledeionizedwater,boiledat100◦Cfor10min,andthe supernatantswereusedforLAMPandconventionalPCR reac-tions.ThesensitivityofLAMPorPCRwasevaluatedwiththe simulatedsamplespreparedbyusingmousespleen,liverand lungs. Serial10-fold dilutionsofY.pestiscells were respec-tively spikedinto thesame volumeoftissuehomogenates, andboiledat100◦Cfor10min.Thesupernatantsweredirectly used for LAMP and PCR assays. In addition, the bacterial DNAwascapturedfromthesupernatantsbymagneticbeads, andthenusedtodeterminethesensitivityofLAMPandPCR assays.

EvaluationofspecificityofLAMPorPCR

ThespecificityofLAMP orPCRforthedetectionofY.pestis

wasevaluatedbyusingthehigh-qualitybacterialDNAs(20ng) from11speciesofbacteriainthegenusYersinia.Thespecificity oftheLAMPorPCRwasalsoevaluatedwithpureculturesfrom 11speciesofbacteriainthegenusYersinia.Eachbacteriumwas dilutedto107_{CFUinsteriledeionizedwater,boiledat100}◦_C

for10min,andthesupernatantswereusedforLAMPandPCR reactions.

T

urbidity (400nm)

T

urbidity (400nm)

Time (min) Time (min)

1 3 5 7 9 2 4 6 8 10 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 1.2 1.2 1 0.8 0.6 0.4 0.2 0 -0.2 1 0.8 0.6 0.4 0.2 0 -0.2 1 3 5 7 9 2 4 6 8 10 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 11 12 5 6 7 8 9 10 1 2 3 4 11 12 5 6 7 8 9 10 1 2 3 4

A

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Fig.4–DetectionofY.pestisinsimulatedliversamplesbyLAMPandPCR.ThesensitivityofLAMPorPCRwasdetermined byusingtheDNAextractedbyboilingsimulatedliversamplefor10min(A-C)orbyusingtheDNAcapturedbymagnetic beads(D–F).(A)DetectionofLAMPproductswithareal-timeturbiditymeter.1–8:2.3×100_–2.3×107_{CFUofY.pestis;9:}

positivecontrol;10:negativecontrol.(B)VisualizationofLAMPproductsstainedwithcalceinandinspectedundernatural light.Tube1–8:2.3×100_–2.3×107_{CFUofY.pestis;9:positivecontrol;10:negativecontrol.(C)ElectrophoreticanalysisofPCR}

productswithagarosegel.Lane1:marker;Lane2–9:2.3×107_–2.3×100_{CFUofY.pestis;10:positivecontrol(20ngDNA);11:}

negativecontrol;12:marker.(D)DetectionofLAMPproductswithareal-timeturbiditymeter.1–8:2.3×100_–2.3×107_CFUof Y.pestis;9:positivecontrol;10:negativecontrol.(E)VisualizationofLAMPproductsstainedwithcalceinandinspected undernaturallight.Tube1–8:2.3×100_–2.3×107_{CFUofY.pestis;9:positivecontrol;10:negativecontrol.(F)Electrophoretic}

analysisofPCRproductswithagarosegel.Lane1:marker;Lane2–9:2.3×107_–2.3×100_{CFUofY.pestis;10:positivecontrol}

(20ngDNA);11:negativecontrol;12:marker.

Results

ThesensitivityofdetectionofY.pestisbyLAMPandPCR assays

To determine the sensitivity of LAMP or PCR assay, serial 10-folddilutions of high-purityY.pestis DNAs were tested. TheresultsshowedthatthelowestdetectionlimitforLAMP was0.02ngofY.pestisDNAwhentheproductwasdetected bythenakedeye,areal-timeturbiditymeteroragarosegel electrophoresisandethidiumbromidestaining,whereasthe sensitivityforPCRwas0.2ngofY.pestisDNA(Fig.1A–D).These resultsindicatedthatLAMPhadahighersensitivitythanPCR forthedetectionofY.pestisDNA,andtwo LAMPdetection methodshadthesamesensitivity.WhenY.pestiswas deter-minedbyusingserial10-folddilutionsofbacterialsolution, thesensitivityforLAMPwas2.3CFUofY.pestiswhenthe prod-uctwasdetectedbyareal-timeturbiditymeter,thenakedeye undernaturallightoragarosegelelectrophoresis,whereasthe sensitivityforPCRwas23CFUofY.pestis(Fig.2A–D).Three LAMPdetectionmethodsalsohadthesamesensitivity,and hadahighersensitivitythanPCR.

ThespecificityofdetectionofY.pestisbyLAMPandPCR assays

TodeterminethespecificityforthedetectionofY.pestisby LAMPorPCRassay,11speciesofthegenusYersiniaweretested.

Whenthehigh-purityDNAsextractedfrom11speciesofthe genusYersiniawereusedforthedetectionofY.pestis, respec-tively,theLAMPproductwasonlydetectedinthereactiontube ofY.pestis,andnoLAMPampliconsappearedinthereaction tubesofotherYersiniastrains.Similarly,whenthehigh-purity DNAs extractedfrom 11 speciesofthe genusYersiniawere respectivelyusedtodetectY.pestisbyPCR,specificbandwas onlyobservedinY.pestisEVvaccinestrain(Fig.1D–F).When

Y. pestis was determined by using 107_{CFU of pure culture}

from11speciesofthegenusYersinia,LAMPproductwasonly detectedinthereactiontubeofY.pestis,andnoLAMP ampli-consappearedinthereactiontubesofotherYersiniastrains. Similarly,when11speciesofthegenusYersiniawereusedto detectY.pestisbyPCR,specificbandwasonlyobservedinY. pestisEVvaccinestrain(Fig.2D–F).

DetectionofY.pestisinsimulatedsamplesbyLAMPand PCRassays

TodeterminethesensitivityforthedetectionofY.pestisin simulated spleen,liverand lung samplesbyLAMP or PCR. Artificiallycontaminatedspleen,liverandlungsamples con-tainingdifferentconcentrationsofY.pestiswerepreparedby mixing spleen,liverand lungtissuehomogenateswiththe samevolumeofserial10-folddilutionsofY.pestissuspension. Artificiallycontaminatedspleen,liverandlungsampleswere boiled for10min, and thenthe supernatants were directly

T

urbidity (400nm)

T

urbidity (400nm)

Time (min) Time (min)

1.2 1 0.8 0.6 0.4 0.2 0 -0.2 1.2 1 0.8 0.6 0.4 0.2 0 -0.2 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 11 12 5 6 7 8 9 10 1 2 3 4 11 12 2 4 6 8 10 2 4 6 8 10 1 3 5 7 9 1 3 5 7 9

A

E

B

F

C

D

Fig.5–DetectionofY.pestisinsimulatedlungsamplesbyLAMPandPCR.ThesensitivityofLAMPorPCRwasdetermined byusingtheDNAextractedbyboilingsimulatedlungsamplesfor10min(A–C)orbyusingtheDNAcapturedbymagnetic beads(D–F).(A)DetectionofLAMPproductswithareal-timeturbiditymeter.1–8:2.3×100_–2.3×107_{CFUofY.pestis;9:}

positivecontrol;10:negativecontrol.(B)VisualizationofLAMPproductsstainedwithcalceinandinspectedundernatural light.Tube1–8:2.3×100_–2.3×107_{CFUofY.pestis;9:positivecontrol;10:negativecontrol.(C)ElectrophoreticanalysisofPCR}

productswithagarosegel.Lane1:marker;Lane2–9:2.3×107_–2.3×100_{CFUofY.pestis;10:positivecontrol(20ngDNA);11:}

negativecontrol;12:marker.(D)DetectionofLAMPproductswithareal-timeturbiditymeter.1–8:2.3×100_–2.3×107_CFUof Y.pestis;9:positivecontrol;10:negativecontrol.(E)VisualizationofLAMPproductsstainedwithcalceinandinspected undernaturallight.Tube1–8:2.3×100_–2.3×107_{CFUofY.pestis;9:positivecontrol;10:negativecontrol.(F)Electrophoretic}

analysisofPCRproductswithagarosegel.Lane1:marker;Lane2–9:2.3×107_–2.3×100_{CFUofY.pestis;10:positivecontrol}

(20ngDNA);11:negativecontrol;12:marker.

determinedbyLAMPandPCRassays,respectively.The sensi-tivityfortheartificiallycontaminatedspleenbyLAMPorPCR assaywas2.3×104 _or2.3×106_{CFUofY.pestis,respectively.}

TheLAMPassaywas100-foldmoresensitivethanPCRassay (Fig.3A–C).Thelowestdetectionlimitfortheartificially con-taminatedliversamplesbyLAMPwas2.3×106_{CFUofY.pestis,}

butPCRassaydidn’tdetect2.3×107_{CFUofY.pestisinthe}

sim-ulatedliversamples(Fig.4A–C).Thelowestdetectionlimitfor theartificiallycontaminatedlungsamplesbyLAMPandPCR assaywas2.3×104_and2.3×106_{CFUofY.pestis,respectively.}

TheLAMPassaywas100-foldmoresensitivethanPCRassay (Fig.5A–C).ToincreasethesensitivityforthedetectionofY. pestisinthe simulatedsamples,the supernatantsobtained byboilingtheartificiallycontaminatedspleen,liverandlung sampleswererespectivelymixedwiththemagneticbeadsto capturetheDNAsforLAMPandPCRassays.AftertheDNAs inthesimulatedspleensampleswerecapturedbymagnetic beads,thesensitivityforLAMPorPCRassaywas103_or106_CFU

ofY.pestis,respectively.LAMPassaywas1000-foldmore sen-sitive than PCRassay (Fig.3Eand F). Thelowestdetection limitfortheartificiallycontaminatedliverbyLAMPorPCRwas 2.3×105_or2.3×107_{CFUofY.pestis.LAMPassaywas100-fold}

moresensitivethanPCRassay(Fig.4EandF).Thesensitivity fortheartificiallycontaminatedlungsbyLAMPorPCRassay was103or106CFUofY.pestis.LAMPassaywas1000-foldmore sensitivethanPCRassay(Fig.5EandF).

Discussion

Y.pestisstrainsusuallycontainthreevirulenceplasmidspCD1, pMT1andpPCP1,ofwhichpCD1wasinheritedfromits ances-torY.pseudotuberculosis.Theplasmid pCD1 mainlyencodes typethreesecretionsystem(T3SS),whichplaysanessential roleinthepathogenesisofY.pestis.42–43 _{Thenewlyacquired}

pPCP1 encodesasurfaceproteinase, plasminogenactivator (Pla), that isresponsible forthe increaseofthe pathogen’s invasive abilityinbubonic plague and thereplication ofY. pestisrapidlyinthelungs.44–45_{AnotherplasmidpMT1encodes}

F1antigen,oneofthemajorprotectiveantigensinY.pestis,

andthemurinetoxin(Ymt)requiredforsurvivalofY.pestis

inthemidgutofflea.46_{Immunologicaldiagnosis ofY.pestis}

was mainlybased onF1 antigenor its antibodies,but this methodcouldnotdetectthevirulentY.pestisstrainslacking F1antigen.47_{Inaddition,ourrecentstudiesfoundsome}

nat-uralisolatesofY.pestiswithoneortwoplasmids.Bycontrast, thetargetgenesonchromosomemightbemorestable.Inthis study,weconstructedaLAMPmethodtodetectY.pestisbased onthe3asequenceonchromosome,whichmightrecognizea greateramountofsamplesthanpreviousstudies.Currently, therehavebeentworeportsthatdescribedthedetectionof

Y.pestisbyLAMP.48–49 _{Comparedwiththesetworeports,our}

detectionofY.pestisDNA,butshowedahighersensitivitythan Dong’smethodforthedetectionofY.pestispureculture.All thesethreemethodshadahigherspecificity.However,thetwo previousreportsare basedonthetarget ofF1antigengene encodedonthepMT1forthedetectionofY.pestispureculture andhigh-qualitygenomicDNA.Evidently,thesetwomethods cannotdetectY.pestisstrainslackingF1antigenonthe plas-midpMT1,andaLAMPmethodisalsoneededtodetectY.pestis

inactualsamples.BasedontheLAMPmethodweconstructed inthisstudy,wedeterminedsimulatedspleen,liverandlung tissuesamplescombinedwithmagneticbeadcaptureofY. pestisDNA.

ThegenusYersiniacontains 11species,ofwhich Yersinia enterocolitica, Yersinia pseudotuberculosis and Y. pestis are pathogenic for humans and animals, whereas other eight species,formerlycalledY.enterocolitica-likeisolates, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia bercovieri,Yersiniamollaretii,Yersiniarohdei,Yersiniaruckeri,and

Yersiniaaldovaeareopportunisticpathogensmostlyfoundin theenvironment.1_{Toevaluatethespecificityoftheprimers}

basedon the 3asequence forthedetection ofY.pestis, all 11 speciesofthe genus Yersinia,closely related organisms, weretestedintheexperiments.Theresultsshowedthatthe primersdesignedinthisstudyhadahighlevelofspecificity forY.pestisbyLAMPorPCR.However,inourrecentstudy, 3a-negativenaturalisolatesofY.pestiswere foundinDingbian County,ShaanxiProvince,China,50_{indicatingthata3a-based}

LAMPorPCRmethodisalsonotreliableforthedetectionofY. pestis.Therefore,amulti-targetLAMPorPCRdetectionmethod shouldbedevelopedforthereliableidentificationofY.pestis

inthefuture.

TodeterminethesensitivityforthedetectionofY.pestis,we firstuseserial10-folddilutionsofhigh-qualityDNAtoperform LAMPorPCRassay.Theresultsshowedthatthelowest detec-tionlimitofLAMPorPCRassaywas0.02or0.2ngofY.pestis

DNA,indicatingthatLAMPassaywas10-foldmoresensitive thanconventionalPCRassay.Then,serial10-folddilutionsof

Y.pestispureculturewereusedtocrudelyextractDNAby boil-ing,andthendetectedbyLAMPandPCR.Theresultsshowed thatthesensitivityofLAMPorPCRassaywas2.3or23CFUofY. pestis,indicatingthatLAMPassaywas10-foldmoresensitive thanPCRassay.Inaddition,theresultsalsoshowedthatthe detectionofLAMPproductsbythenakedeyewasassensitive asthatbyareal-timeturbiditymeter.LAMPamplificationcan beaccomplishedwithin60minatanisothermaltemperature of65◦C.Therefore,LAMPassayisasimple,rapidand sensi-tivemethodsuitableforapplicationinthefield,especiallyin povertyareas.

ToevaluatethereliabilityforthedeterminationofY.pestis

byLAMPand PCRassaysinactualsamples,wedetermined

Y.pestisinthesimulatedspleen,liverandlungsamples.The resultsshowedthatthesensitivityofthreesimulated sam-plesweremuchlowerthanthatofY.pestispurecultureand DNA,indicatingthatsomecomponentsinthespleen,liverand lungtissuesmightinhibitLAMPandPCRreactions.Thisresult wassimilartothepreviousstudiesinwhichclinicalsamples, suchasserum,plasmaandurine,hadaninhibitiontoboth LAMPandPCRassay.51–52_{Inaddition,ourresultsalsoshowed}

thatthesimulatedliversamplehadalowersensitivitythan thesimulatedspleenandlungsamplesforthedetectionofY.

pestisbyLAMPandPCR.Thisresultindicatedthattheremight bemorebioactivecomponentsinlivertissuethatcouldinhibit LAMPandPCRreactions.ComparedwithPCRforthe detec-tion of threesimulated samples,LAMP assaywas 100-fold moresensitivethanPCRassay,indicatingthatLAMPwasless affected byinhibitorysubstances presentinmouse spleen, liverandlungtissuesamplesthanPCR.LAMPisnotonlymore simple,rapidandsensitivethanPCR,butalsomoresuitable todetectspleen,liverandlungtissuesamplesthanPCR.

To further enhance the sensitivity for the detection of actualsamples,magneticbeadswereusedtocaptureY.pestis

DNAs from three simulatedsamples. Afterthe DNAswere capturedfromthesimulatedspleenandlungsampleswith magneticbeads, the sensitivityofthese twosampleswere increasedforthedetectionofY.pestisbyLAMP,butthe sensi-tivityofthesetwosampleswereunchangedforthedetection ofY.pestisbyPCR, andLAMP was1000-foldmoresensitive thanPCR.AftertheDNAswerecapturedfromsimulatedliver sampleswith magneticbeads, thesensitivity ofLAMP and PCRwereequallyincreased,andthesensitivityofLAMPwas 100-foldmoresensitivethanthatofPCR.Theseresults indi-catedthatmagneticbeadcaptureofDNAscouldremarkably increasethesensitivityofLAMPforthedetectionofY.pestis

inspleen,liverandlungtissuesamples.

Conflicts

of

interest

Theauthorsdeclarethattheyhavenocompetinginterests.

Acknowledgement

FinancialsupportforthisstudycamefromtheNational Nat-uralScienceFoundationofChina(31430006,81171529).

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