多联实时荧光PCR检测几种重要肠道致病菌的实验研究.docVIP

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(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【3q Vc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—04 Anexperimentonquantitativemethodofmulti—linkreal—timeluorescencePCRforde~cflonofSh/ge~,0l39Vibrio cholerae,Campylobacterjejuni.PENGYah—zhong,JIAHong,ZOUYuan,eta1.(1.TheShenzhenHospitalofBeijingUni. vemi~,Shengzhen518000,Guangdong,P.R.China;2.Shenzhenhighoccupationaltechnicalinstitute) Abstract:ObJectiveTofoundthequantitativemethodofmulti—linkrealtimeluorescencePCRforsimultaneousdetectionof Shigdla(SH),0139Vibriocholerae(0l39VC)andCampylobacter]ejuni(CJ).MethodsAccordingtotheopenreadingflame (ORF)ofsequenceofSh—ipaHgene,Ol39VC—wbfRgene,andCJ—Cgene,severalcouplesofspecificprimersandprobeswere designed,withutilizationofPrimerSelectsoftwareamongtheABIprimerexperess2.0andDNAStarrespectively.Optimalcombina— tionofeveryprimerandprobewasselected.Optimizationofconcen~ationofprimerandprobe, M,Taqenzymedosage,response conditionwerooptimized.Reactionsystemanddeterminationapproachofmulti—linkrealtimefluorescencePCR(MLRTL—PCR) wereestablishedfordetectionofSH,O139VCandCJfromclinicalspecimens.ResultsTheresultsshowedthatMLRTL—PCR possessedhighSpecificityandsensitivityfordetectionofSH,Ol39VCandCJandachieved100copies/m1.ConclusionThe methodofMLRTL—PCRcandetectSH,0139VCandCJfromclinicalspecimensimultaneouslyandquantitatively.Themethodis fast,convenient,accuratefavorableforthepurposesofearlydiagnosisandtreatment. Keywords:ShigeUa(SH);0139Vibriocholerae(oI39VC);Campylobacterjejuni(cJ);M