聚合酶链反应结合探针杂交检测幽门螺杆菌

PolymerasechainreactiontestandhybridizationfordetectionofHelicobacterpylori

目的建立特异、灵敏的聚合酶链反应（ＰＣＲ）结合长臂光敏生物素探针杂交检测幽门螺杆菌（ＨＰ）的方法。方法用ＰＣＲ产物直接克隆并测序，以含ＨＰＤＮＡ序列的重组质粒外源片段为探针。用细菌培养、单纯ＰＣＲ和ＰＣＲ结合杂交（ＰＣＲ－Ｓｂ）检测经胃镜诊断为慢性胃炎、胃溃疡、十二指肠球部炎症、十二指肠溃疡的胃活检组织、唾液和粪便标本中的ＨＰ。结果检测胃活检标本７９份，ＰＣＲ－Ｓｂ阳性率为９９％，单纯ＰＣＲ及细菌培养的阳性率分别为９５％和６５％，ＰＣＲ－Ｓｂ的灵敏度达到１ｆｇ。唾液标本的检测阳性率为３５％，粪便标本的检测结果均为阴性。结论测序证实了ＰＣＲ扩增的正确性。ＰＣＲ结合长臂光敏生物素探针杂交是特异、灵敏、简便和对人体无害的检测ＨＰ的方法。

ObjectiveTodevelopasensitiveandspecifictechniqueforthedetectionofHeli-cobacterpylori(HP)withpolymerasechainreaction(PCR)andhybridization.MethodsConstructarecombinantplasmidwithproductofPCRandvectorplasmidpUC18.DNAfragmentsdigestedfromthisrecombinantplasmidwereusedasprobes.Pepticbiopsy,salivaandfecesspecimensweredetectedbyculture,directPCRandPCRplushybridization.ResultsSequencingshowthePCRproductshadthesamesequencewithHPureADNA.PCRplushybridizationcouldimprovethesen-sitivityupto1fg.It′spositiverateinthedetectionofpepticbiopsysampleswasthehighest99%,andthepositiveratesofdirectPCRandculturewere95%and65%respectively.Salivasampleshadmuchlesspositiveratethanpepticbiopsyspecimens,asof35%,thoughHPcouldnotbedetectedfromfecessamples.ConclusionsDetectingHPwithPCRplushybridizationwasveryspecific,sen-sitive,simpleandharmless.Itisusefulindiagnosisofinfection,epidemiologicalresearchandsoon.