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2003,AmericanSocietyforMicrobiology.AllRightsReserved.

IntraspeciesPolymorphismofCryptosporidiumparvumRevealedbyPCR-RestrictionFragmentLengthPolymorphism（RFLP）andRFLP-Single-StrandConformationalPolymorphismAnalyses

ZhiliangWu,IsaoNagano,ThidarutBoonmars,TakumiNakada,andYuzoTakahashi*

DepartmentofParasitology,GifuUniversitySchoolofMedicine,Tsukasa40,Gifu500-8705,Japan

Received27January2003/Accepted26May2003

ABSTRACT

Top

Abstract

Introduction

MaterialsandMethods

Results

Discussion

References

Aglycoprotein（Cpgp40/15）-encodinggeneofCryptosporidiumparvumwasanalyzedtorevealintraspeciespolymorphismwithinC.parvumisolates.Forty-oneisolateswerecollectedfromdifferentgeographicalorigins（Japan,Italy,andNepal）andhosts（humans,calves,andagoat）.TheseisolateswerecharacterizedbymeansofDNAsequencing,PCR-restrictionfragmentlengthpolymorphism（PCR-RFLP）,andRFLP-single-strandconformationalpolymorphism（RFLP-SSCP）analysesofthegeneforCpgp40/15.ThesequenceanalysisindicatedthattherewasDNApolymorphismbetweengenotypeIandII,aswellaswithingenotypeI,isolates.TheDNAandaminoacidsequenceidentitiesbetweengenotypesIandIIdiffered,dependingontheisolates,rangingfrom73.3to82.9%and62.4to80.1%,respectively.ThoseamonggenotypeIisolatesdiffered,dependingontheisolates,rangingfrom69.0to85.4%and54.8to79.2%,respectively.BecauseofthehighresolutiongeneratedbyPCR-RFLPandRFLP-SSCP,theisolatesofgenotypeIcouldbesubtypedasgenotypesIa1,Ia2,Ib,andIe.TheisolatesofgenotypeIIcouldbesubtypedasgenotypesIIa,IIb,andIIc.Theisolatesfromcalves,agoat,andoneJapanesehumanwereidentifiedasgenotypeII.WithingenotypeII,theisolatesfromJapanwereidentifiedasgenotypeIIa,thosefromcalvesinItalywereidentifiedasgenotypeIIb,andthegoatisolatewasidentifiedasgenotypeIIc.AllofthegenotypeIisolateswerefromhumans.TheJapaneseisolate（codeno.HJ3）andalloftheNepaleseisolateswereidentifiedasgenotypesIa1andIa2,respectively.TheItalianisolateswereidentifiedasgenotypeIb,andtheJapaneseisolate（codeno.HJ2）wasidentifiedasgenotypeIe.Thus,thePCR-RFLP-SSCPanalysisofthisglycoproteinCpgp40/15genegeneratedahighresolutionthathasnotbeenachievedbypreviousmethodsofgenotypicdifferentiationofC.parvum.

INTRODUCTION

AmongknownCryptosporidiumspp.（seethereviewbyXiaoetal.[19]）,Cryptosporidiumparvumissupposedlythemainspeciesthatinfectshumans（7）.PreviousstudieshaveshownthatC.parvumismainlycomposedoftwogenotypes,IandII.Theformerhasbeenfoundinhumanpatientsandisthereforereferredtoasthehumantype（5,7）.GenotypeIIwasfirstfoundincattleandisthereforereferredtoasthecattletype.Sincethen,however,thistypehasbeenfoundinawiderangeofmammals,includinghumans（4,8）.Thus,bothgenotypesareresponsibleforoutbreaksofhumancryptosporidiosisandsporadicinfectionsinimmunocompromisedindividuals（6）.

Despiteextensiveearlierstudies,itisstillunclearwhetherthepathogenesis,virulence,infectivity,ordrugsensitivityofCryptosporidiumisrelatedtosomespecificgenotypeornot.Also,genotypicinformationisneededtodetectCryptosporidiumbymeansofPCRfortapwatermonitoringand/ortotracetheinfectionrouteoftheparasite.

Genefingerprintingisapromisingmethodwithwhichtofulfillsuchrequirements.Recently,onegeneencodingaglycoprotein（namedCpgp40/15）ofC.parvumwaslistedasacandidatetargetgeneforgeneticanalysisofC.parvum.SequenceanalysisoftheCpgp40/15geneindicatedthepresenceofpolymorphicvariantswithingenotypeIisolates,whichhavebeendividedintofivesubcategoriessofar（2,11）.ThisstudywasundertakentosearchformoreadvantagesoftheCpgp40/15genefortheidentificationandgenefingerprintingofC.parvumisolatesadaptingmoresophisticatedmethods,namely,restrictionfragmentlengthpolymorphism（RFLP）andsingle-strandconformationalpolymorphism（SSCP）.

MATERIALSANDMETHODS

Parasiteisolates.

Atotalof41isolatesofC.parvumwereused.SixisolateswerefromcalvesinGifu,Japan（codeno.CGJ1toCGJ6）;

11werefromcalvesinKobe,Japan（codeno.CKJ1toCKJ11）;

1wasfromacalfinNagoya,Japan（codeno.CNJ1）;

8werefromcalvesinItaly（codeno.CI1toCI8）;

1wasfromagoatinItaly（codeno.GI1）;

3werefromhumansinJapan（codeno.HJ1toHJ3）;

5werefromhumansinItaly（codeno.HI1toHI5）;

and6werefromhumansinNepal（codeno.HN1toHN6）.

Allfecalsampleswerepreservedin2%K2Cr2O7,andoocystswereisolatedbythesucroseflotationmethod.Thesamplesweresubjectedtofurtherpurificationwithanimmunomagneticseparationkit（Dynabeadsanti-Cryptosporidium;

DynalAS,Oslo,Norway）.

TemplateDNAforPCRwaspreparedasdescribedpreviously（15）.Inbrief,oocystswerefrozenandthawedfivetimesandtreatedat100°

Cfor20min.ThesampleswerethendigestedwithproteinaseKatafinalconcentrationof200µ

g/mlat55°

Cfor3handheatedat95°

Cfor5mintostopthedigestion.Thus-treatedsamplesweredirectlyusedforPCRundertheconditionsdescribedinthefollowingparagraph.

DevelopmentofPCRprimerandPCRconditions.

Apairofprimers（CCGTTATAGTCTCCGCTGTAandAAAGCAGAGGAACCGGCAT）weredevelopedforamplificationofthegeneforCpgy40/15onthebasisofthepublishedsequencedata（GenBankaccessionnumberAF022929）（11）.

ThePCRconditionsusedwereasfollows:

1initialdenaturationcycleof94°

Cfor3min;

35cyclesof94°

Cfor30s,54°

Cfor30s,and72°

Cfor1min;

and1finalextensioncycleof72°

Cfor10min.

DNAsequencing.

TheCpgy40/15-encodinggenesfrom16isolates（including8calfisolatesfromJapanandItaly,1goatisolatefromItaly,and7humanisolatesfromJapan,Italy,andNepal）weresequenced.PCRproductswerepurifiedwithaGeneCleanIIKit（Bio101,Carlsbad,Calif.）.DNAfragmentswereligatedintoapT7BlueT-Vector（Novagen,Inc.,Madison,Wis.）.TherecombinantplasmidswereintroducedintocompetentcellsofEscherichiacoliJM109.TheplasmidDNAwasisolatedfromE.coliwithaFlexiPrepKit（AmershamPharmaciaBiotechInc.,Piscataway,N.J.）.

TheDNAwassequencedwithaThermoSequenasecyclesequencingkit（USBCorporation,Cleveland,Ohio）andanautomaticsequencer（LIC-4200;

AlokaCo.,Ltd.）.ThesequencedatawereanalyzedwithDNASISsoftware（HitachiSoftwareEngineering,Tokyo,Japan）.HomologysearchingofthenucleotideandproteinsequencedatabasewascarriedoutwiththeBLASTprogramattheNationalCenterforBiotechnologyInformation（Bethesda,Md.）.PairwisesequencealignmentsandproteinidentitydeterminationswereperformedwithClustalW1.81andPHYLIPDNADISTsoftware.

RFLPandSSCP.

PCR-RFLPanalysiswasperformedaspreviouslydescribed（14,16）.PCRproductswerepurifiedbyethanolprecipitationandthendigestedwiththeappropriaterestrictionendonucleases（AluIandRsaI）inaccordancewiththemanufacturer'

sinstructions.

PCR-RFLP-SSCPisahighlysensitivemethoddevelopedbyWuetal.（16）.Inbrief,thePCR-RFLPproductwasmixedata1:

1ratiowithdenaturingsolution（95%[vol/vol]formamide,0.02%[wt/vol]xylenecyanol,0.02%[wt/vol]bromophenolblue,20mMEDTA[pH8.0]）andheatedat95°

Cfor5min.Thedenaturedsampleswerecoolediniceimmediately.Seven-microlitersampleswereappliedtothegel,andtheDNAsampleswererunat600V,50mA,30W,and10°

Cfor110min.Afterelectrophoresis,thegelwasstainedwithaPlusOneDNAsilverstainingkit（AmershamPharmaciaBiotechAB,Uppsala,Sweden）.

RESULTS

AnalysisoftheCpgp40/15gene.

Ofthe41isolates,16fromdifferentgeographicaloriginsandhostswerechosenforDNAsequencing.ThelengthsoftheampliconsproducedbytheprimerdevelopedforCpgy40/15werevariableamongisolates,rangingfrom883to961bp,andtheDNAsequencesoftheampliconswerealsodiverse,asshowninTable1andFig.1.

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TABLE1.Sequence,PCR-RFLP,andRFLP-SSCPanalysesoftheCpgp40/15geneforgenotypingofC.parvumisolates

Viewlargerversion（85K）:

FIG.1.DNAsequencediversitiesamongC.parvumisolatesintheCpgp40/15gene.Thegenesfrom16isolatesweresequencedandtentativelygroupedintoseventypesthatareindicatedontheleftasIIa,IIb,IIc,Ia1,Ia2,Ib,andIe（fortheisolatenames,seeMaterialsandMethods）.Dotsindicateidenticalbasepairs,andhyphensindicategaps.ThedifferencesbetweenIIaandIIbandbetweenIa1andIa2areindicatedbyshading.TheGenBankaccessionnumbersareAY167589（CKJ7）,AY167590（CI2）,AY167591（GI1）,AY167594（HJ3）,AY167595（HN6）,AY167596（HI2）,andAY167593（HJ2）.

TheDNAsequencesofisolatesfromcalvesfromdifferentgeographicalareasofJapan（codeno.CGJ2,CGJ5,CKJ1,CKJ3,CKJ7,andCNJ1）andonefromahumanpatientinJapan（codeno.HJ1）werequitesimilar,withmorethan99%identity,andwereidentifiedasgenotypeII（Fig.1）.

IsolatesfromItaliancalves（codeno.CI2andCI8）showedmorethan98%DNAseq