PCR-RFLP鉴定我国黑热病流行区6种常见蛉种

Typing 6common sandflies species from endemic areas of leishmaniasis in China by PCR-RFLP method

目的建立鉴定我国黑热病流行区6种常见蛉种的PCR-RFLP(PCR-restriction fragment length polymorphisms)方法。方法使用一对通用引物扩增线粒体COI基因,比较中华白蛉、长管白蛉、吴氏白蛉、亚历山大白蛉和歌乐山司蛉线粒体COI基因序列,寻找合适的限制性内切酶酶切位点,使用TaqI、PstI内切酶双酶切,通过电泳片段大小鉴别上述6种蛉种。结果长管白蛉、中华白蛉、吴氏白蛉、亚历山大白蛉、歌乐山司蛉和鳞喙司蛉酶切片段呈现长度不同的种特异条带,可以将上述我国常见的6种蛉种区分开来。结论基于COI基因序列差异建立的PCR-RFLP方法简便易行可靠,具有较高的灵敏度和特异性,可用于我国黑热病流行区6种常见蛉种的分类鉴定。

A PCR-RFLP method typing the prevalent sandflies from leishmaniasis endemic areas in China was established in our study.We designed a pair of universal primer for 6 species of sandflies.Species-specific differences encountered in the nucleotide sequences of the COI gene were used to design a PCR RFLP assay for the identification of Phlebotomus chinensis,Phlebotomus longiductus,Phlebotomus alexandri,Phlebotomus wui,Sergentomyia koloshanensis and Sergentomyia squamirostris.The specific patterns were developed by double digesting amplified COI gene with TaqI and PstI.The PCR-RFLP assay based on COI gene diversity to distinguish 6 species of sandflies was found to be highly specific,sensitive and simple.