伊氏锥虫与路氏锥虫二重PCR鉴别检测方法的建立

Development of a dual PCR assay for the detection of Trypanosoma evansi and Trypanosoma lewisi

目的建立伊氏锥虫和路氏锥虫二重PCR鉴别检测方法。方法根据路氏锥虫18S rRNA基因和伊氏锥虫RoTat 1.2VSG基因特异序列设计两对引物,通过优化PCR反应体系与反应条件,建立种特异二重PCR鉴别检测方法。结果用建立的二重PCR鉴别检测方法能特异扩增路氏锥虫和伊氏锥虫目的片段,大小分别为261bp和482bp;路氏锥虫和伊氏锥虫的最低检出量分别均达1个虫体,检测日本血吸虫、杜氏利氏曼原虫与瑟氏泰勒虫等其他血液寄生虫均为阴性。结论建立的二重PCR检测体系灵敏度高、特异性强,适用于路氏锥虫和伊氏锥虫的感染检测及流行病学调查。

Objective A dual PCR assay was developedto detect Trypanosoma evansi and Trypanosoma lewisi.Methods The assay targets the 18S rRNA of T.lewisi,and RoTat 1.2 VSG of T.evansi.Primers for the test were designed from sequence data.The assay was tested against Schistosoma japonicum,Leishmania donovani and Theileria sergenti. Results The assay was tested using blood from infected rats and was found to be sensitive,detecting less than one genomic equivalent of T.lewisi and T.evansi.All of the negative control samples tested negative.Conclusion The dual PCR assay was more sensitive than the Haematocrit centrifugation test（HCT） and faster than and the mouse inoculation（MI） assay.This assay has the ability to rapidly detect T.lewisi and T.evansi and distinguish organisms present in blood samples from infected animals.