肠毒素大肠杆菌LTh－STIb二价基因探针的克隆及其应用研究

Study on Cloning and Application of LTh-STIb Divalent Gene Probe for Detection of Heat-Stable Toxin in Enterotoxigenic E. coli

在肠毒素大肠杆菌（ＥＴＥＣ）ＬＴｈ－ＳＴＩａ－ＳＴＩｂ三价基因探针的基础上，用限制性内切酶ＥｃｏＲｌ酶切，回收片段．重新构建ＬＴｈ－ＳＴＩｂ二价基因探针的克隆株。用辣根过氧化物酶复合物（ＨＲＰ）直接标记二价基因探针，建立了增强型化学发光反应（ＥＣＬ）检测ＥＴＥＣ的方法．其敏感性可测到０．１ｐｇ的质粒ＤＮＡ或由１０个菌体培养而来的ＥＴＥＣ细胞，与同位素标记杂交方法的敏感性一致．且此探针仅与产ＳＴＩｂ、ＬＴｈ肠毒素的人源性ＥＴＥＣ菌株杂交，与ＳＴＩａ肠毒素菌株及其它非ＥＴＥＣ菌株均无杂交信号．该探针与三价探针及ＬＴｈ单价探针联合使用，能间接将ＬＴｈ、ＳＴＩａ和ＳＴＩｂ３种肠毒素分型。结果表明该方法简便快速、特异敏感，无放射性污染，是ＥＴＥＣ实验室诊断及分子流行病学调查的有效工具。

A 912 bp polynucleotide divalent probe for heat-labile and heat-stable enterotoxins(LTh, ST I b ) was constructed from the LTh-STI a-STI b trivalent gene probe. The divalent probe was directly conjugated with horseradish peroxidase(HRP) .then a procedure for detection of ETEC by enhanced chemiluminescence(ECL) was established. Slot-blot and colony hybridizations showed the HRP-conjugated probe was able to detect 0. 1pg plasmids DNA and as little as 10 original ETEC cells without stool. The hybridzations demonstrated that this probe specifically identified bacterial colonies of human original ETEC that produce LTh. STI b and LTh-STI b. With the help of the trivalent probe and the LTh probe .the bivalent probe was able to identify the LTh.STI b and STI a genes. These tesults suggest that the HRP-conjugated divalent probe is suitable for clinical detection and molecular epidemiological studies of the ETEC,diarrheal disease.