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 the construction of divalent gene probe for detection of heat-labile and 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EcoRI酶切，回收片段，重新构建LTh－STIb二价基因探针的克隆株。用辣报过氧化物防复合物（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。该探针与三公探针及LTh单价探针联合使用，能间接将LTh、STIa和STIb三种肠毒素分型。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 912bp polynucleotide divalent probe for heat-labile and heat-stable enterotoxins (LThSTIb) was constructed from the LTh-STIa-STIb 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 hybridizations demonstrated that this probe specifically identified bacterial colonies of human original ETEC that produced LTh, STIb and LTh-STIb. with the help of the trivalent probe and the LTh probe, the divalent probe was able to identify the LTh, STIb and STIa genes. These results suggest that the HRP-conjugated divalent probe is suitable for clinical detection and molecular epidemiolo gical studies of the ETEC diarrheal disease,