聚合酶链反应检测杜克雷嗜血杆菌实验研究

Investigation on detection of Haemophilus ducreyi by polymerase chain reaction

目的 : 近年来 ,国内不断有软下疳病例报告 ,但无 1例有可靠的实验室依据。因此 ,探讨聚合酶链反应 (PCR)检测杜克雷嗜血杆菌 (H .ducreyi)的方法将其应用于临床实践 ,是目前我国性病防治工作的迫切要求。方法 : 根据杜克雷嗜血杆菌的特异性 16srRNA基因序列设计上下游引物对两株参考菌株进行PCR扩增 ,其扩增产物以 1.5 %琼脂糖凝胶电泳进行检测 ,并测序证实。并用其它的同源或相关病原微生物进行PCR扩增 ,证实其特异性。再以不同浓度菌悬液扩增检测其敏感性。结果 : 所试两株不同来源的杜克雷嗜血杆菌的扩增呈现单一扩增区带 ,电泳片段位置与预期结果 438bp相符 ,测序结果与基因库序列一致 ,并用具有同源及相关的微生物共 19种在相同条件下同时扩增 ,除杜克雷嗜血杆菌外 ,均未扩增出预期片断 ,测试的最高敏感度可达 10 - 6 ng μl。 结论 : 应用PCR技术检测杜克雷嗜血杆菌具有迅速、特异、敏感、简便的特点 ,在严格控制实验条件的情况下 ,PCR具有很大的实用价值 ,是诊断杜克雷嗜血杆菌感染的实验诊断方法。

Objective:To find out a more sensitive test for diagnosis of chancroid. Methods:Investigation on the necleotides sequences of 16srRNA gene of H.ducreyi was used to develop primer sets for diagnosis of chancroid by polymerase chain reaction (PCR) DNA amplification. Results:Two 16 base probes internal to this sequence were species specific for H.ducreyi when tested with 19 species of becteria, virus and fungus, thd reaction results showed 100% specificity. The two probes in combination with the specificity primers were 100% sensitivity with two strains of H.ducreyi. The highest sensitivity concentriation of the necleotides extracted form H.ducreyi was 10 -6 ng/μl. Conclusions :This is the preliminary research on PCR detecting H.ducreyi, and the further investigation will be conducted before putting it into used for confirmating diagnosis of chancroid. [