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1、多联实时荧光PCR检测几种重要肠道致病菌的实验研究2l06CHINATROPICALMEDICINEVo1.6No.12December2006中国热带医学2006年第6卷第l2期多联实时荧光PCR检测几种重要肠道致病菌的实验研究彭雁忠,贾宏,邹原,肖性龙,黄胜中,林镜中.[论着]摘要:目的建立一种能同时检测志贺氏茵(Sh),0L39霍乱弧茵(0l39Vc)和空肠弯曲茵(CJ)的多联实时荧光PCR定量方法.方法根据Sh的ipaH基因序列,0l39VC的wbfR基因序列,CJ的c基因序列的开放读码框(
2、orf),分别利用ABIprimerexperess2.0和BNAStar中的PrimerSelect软件设计出数对特异性的引物和探针,筛选出最佳的各组引物和探针组合,对引物,探针的浓度,Md浓度,Taq酶的用量,反应条件等进行优化,从而建立了捡测临床标本中Sh,O【3qVc和cJ的多联荧光PCR反应体系和检测方法.结果实验显示该方法具有特异性强,灵敏度高,均达到100copies/mlo结论多联实时荧光PCR方法可同时定量地检测临床标本中Sh,0mVC,CJ,结果显示快速简便,准确高效,有利于上述
3、病原茵所致疾病的早期诊断和治疗.关键词:志贺氏茵;0t39霍乱弧茵;多联实时荧光PCR空肠弯曲茵中图分类号:R378文献标识码:A文章编号:1009—9727(2006)12—2106—04Anexperimentonquantitativemethodofmulti—linkreal—timeluorescencePCRforde~cflonofSh/ge~,0l39Vibriocholerae,Campylobacterjejuni.PENGYah—zhong,JIAHong,ZOUYuan,e
4、ta1.(1.TheShenzhenHospitalofBeijingUni.vemi~,Shengzhen518000,Guangdong,P.R.China;2.Shenzhenhighoccupationaltechnicalinstitute)Abstract:ObJectiveTofoundthequantitativemethodofmulti—linkrealtimeluorescencePCRforsimultaneousdetectionofShigdla(SH),0139Vibr
5、iocholerae(0l39VC)andCampylobacter]ejuni(CJ).MethodsAccordingtotheopenreadingflame(ORF)ofsequenceofSh—ipaHgene,Ol39VC—wbfRgene,andCJ—Cgene,severalcouplesofspecificprimersandprobesweredesigned,withutilizationofPrimerSelectsoftwareamongtheABIprimerexpere
6、ss2.0andDNAStarrespectively.Optimalcombina—tionofeveryprimerandprobewasselected.Optimizationofconcen~ationofprimerandprobe,M',Taqenzymedosage,responseconditionwerooptimized.Reactionsystemanddeterminationapproachofmulti—linkrealtimefluorescencePCR(MLRTL
7、—PCR)wereestablishedfordetectionofSH,O139VCandCJfromclinicalspecimens.ResultsTheresultsshowedthatMLRTL—PCRpossessedhighSpecificityandsensitivityfordetectionofSH,Ol39VCandCJandachieved100copies/m1.ConclusionThemethodofMLRTL—PCRcandetectSH,0139VCandCJfro
8、mclinicalspecimensimultaneouslyandquantitatively.Themethodisfast,convenient,accuratefavorableforthepurposesofearlydiagnosisandtreatment.Keywords:ShigeUa(SH);0139Vibriocholerae(oI39VC);Campylobacterjejuni(cJ);Multi—linkreal—timeluoreseen
