分子信标荧光定量PCR技术检测结核分枝杆菌方法建立及其.pdf

四川大学学报(医学版)
2010;41(1):162165
JSichuanUniv(MedSciEdi)
分子信标荧光定量PCR技术检测结核分枝
杆菌方法建立及其临床应用3

方梅,陆巧荣,洪志强
昆山市疾病预防控制中心(昆山215300)

【摘要】目的建立结核分枝杆菌的分子信标荧光定量PCR检测方法,探讨该方法检测结核分枝杆菌的临
床应用价值。方法针对结核分枝杆菌Ag85BDNA特异保守序列设计引物和分子信标探针,并构建标准品。采
用分子信标荧光定量PCR法、抗酸染色法、集菌培养法检测158例肺结核患者痰液标本,比较3种方法的检出率。
结果所建立方法最低检测限度为2个基因拷贝/反应,在2×102～2×108基因拷贝/mL范围内,Ct值同DNA量
的对数呈线性关系。该方法检测5株非分枝杆菌和6株非结核分枝杆菌结果均为阴性,检测结核分枝杆菌H37Rv
出现75bp特异条带。分子信标荧光定量PCR法、抗酸染色法、集菌培养法阳性检出率分别为60.13%、16.46%、
31.01%,荧光定量PCR法检出率明显高于抗酸染色法和集菌培养法。结论分子信标荧光定量PCR法具有较高
的灵敏度和特异度,对结核病的诊断有一定的临床意义。
【关键词】结核分枝杆菌分子信标荧光定量PCR
【中图分类号】R372

EstablishmentandApplicationofMolecularBeaconReal2timePCRintheDetectionofMycobacteriumtuberculosis
FANGMei,LUQiao2rong,HONGZhi2qiang.KunshanCenterofDiseaseControlandPrevention,Kunshan
215300,China
【Abstract】ObjectiveToestablishamethodfordetectionofM.tuberculosisbymolecularbeaconreal2time
PCRandevaluateitsapplicationintheclinicaldetectionofM.tuberculosis.MethodsTheprimersandmolecular
probeweredesignedtoM.tuberculosisspecificDNAsequenceforAg85B,andconstituteastandards.158sputum
specimensfrompatientswithpulmonarytuberculosisweredetectedusingmolecularbeaconreal2timePCR,acid2fast
stainandcultivation,andthedetectionratesofthethreemethodswerecompared.ResultsThelowestdetection
limitofthedetectedmethodis2genomecopiesperreaction,thelinearstandardcurvewasobtainedbetween2×102
and2×108DNAcopies/mL.Themethoddetected5strainsofnon2mycobacterium,6strainsofnon2tuberculosis
mycobacteriumandM.tuberculosisH37Rv,onlyM.tuberculosisH37Rvhad75bpspecificband.Thepositiverates
ofdetectionusingmolecularbeaconreal2timePCR,acid2faststainandcultivationwere60.13%,16.46%,31.01%
respectively,thepositiveratesofdetectionofreal2timePCRwassignificantlyhigherthanthatofacid2faststainand
cultivation.ConclusionMolecularbeaconreal2timePCRhashighsensitivityandspecificityinthedetectionofM.
tuberculosis,whichisclinicalsignificanceforthediagnosisoftuberculosis.
【Keywords】MycobacteriumtuberculosisMolecularbeaconReal2timePCR

结核病已发展为世界性严重公共卫生问题,我化等优点,被应用于结核分枝杆菌的定量检测研
国是全球