聚合酶链式反应及限制性片段长度多态性技术检测乙型肝炎病毒P基因区YMDD基因序列变异

Detection of the YMDD motif mutation of P gene of hepatitis B virus using polymerase chain reaction-restriction fragment length polymorphism

目的建立检测基因变异的聚合酶链反应(PCR)限制性片段长度多态性(Restrictionfragmentlengthpolymophism,RFLP)法。方法从服用拉米夫定1年以上的慢性乙肝病例中选取10例抽取血清,分别采用错配套式PCR-RFLP技术、PCR产物直接测序和重组克隆测序,检测P基因YMDD变异。结果在10个病例中,PCR-RFLP检测到7例为野毒株、1例为混合株感染、2例为变异株;PCR产物直接测序,9例为野株、1例为变异株;对2份用PCR-RFLP法检测为非野株,而PCR测序为阴性的标本采用克隆后测序,发现均为混合株感染。结论通过对几种检测方法的对比,认为克隆后测序的结果最精确;而从实用性和经济角度来看,采用PCR-RFLP进行初步筛检的方法具有更大的意义,值得推广。

Objective To establisha convenientapproachto implementpolymerasechainreaction-restrictionfragmentlength polymorphism(PCR-RFLP)fordetectingof genemutation.Methods Serumsampleswerecollectedfrom10patientswith chronichepatitisB who hadreceivedorallamivudineforat least1year,and3methodsincludingmismatchedPCR-RFLP,directsequenceanalysisof thePCRproduct andcloningpriorto secondarysequenceanalysiswereemployedrespectivelyto examinetheYMDDmotifmutationof P geneof hepatitisB virus(HBV).Results PCR-RFLPrevealedwild-typeHBV infectionin7cases, mixedinfectionin1caseandHBVmutantinfectionin2cases.Withdirectsequenceanalysisof thePCR product,9caseswerefoundwithwild-typeHBVinfection and1withHBVmutantinfection.However,2non-wild-type HBV-infectedcasesas approvedby PCR-RFLP,oppositeto theresultsby directsequenceanalysis,werebothshownby sequenceanalysisaftercloningto suffermixedinfection.Conclusion Sequenceanalysisfollowingcloningisthebestwayto detectYMDDmotifmutationin HBV,butin termsof economyandpracticability,mismatchedPCR-RFLPis of greater significanceinmassscreeningof YMDDmotifmutantHBVthantheother2approaches.