蜱传反刍动物艾立希体巢式PCR检测方法的建立及应用

Establishment and application of a nested PCR method for tick-borne Ehrlichia ruminantium detection

为建立一种快速、敏感检测反刍动物艾立希体的方法,本研究根据GenBank中登录的反刍动物艾立希体pCS20基因保守区设计2对特异性引物,经各反应条件的优化,建立了反刍动物艾立希体巢式PCR检测方法。结果显示,该方法可以特异性检测反刍动物艾立希体DNA,而对牛巴贝斯虫、双芽巴贝斯虫、牛环形泰勒虫和弓形虫的检测均为阴性,具有良好的特异性;该方法灵敏度可达1.04×101拷贝/μL,是反刍动物艾立希体实时荧光PCR检测试剂盒的10倍,是常规PCR的1 000倍。对50只血蜱、花蜱和微小牛蜱DNA进行检测,巢式PCR、反刍动物艾立希体实时荧光PCR检测试剂盒和常规PCR的阳性检出率分别为26.0%,14.0%和0.0%。本试验建立的巢式PCR检测方法适用于反刍动物艾立希体病的早期诊断和分子流行病学调查,为蜱传反刍动物艾立希体病的防控提供技术支持。

A nested PCR assay was established for the detection of Ehrlichia ruminantium with two pairs of specific primers based on the E.ruminantium pCS20gene.The results showed that the assay was specific for detecting E.ruminantium,but not for B.bovis,Babesia bigemina,Theileria annulate and Toxoplasma gondii.The sensitivity of the method was 1.04×101copies/μL,which was 10times as long as the Ehrlichia ruminantiumreal-time PCR kit and 1 000times as long as the conventional PCR.Among 50ticks of Haemaphysalis,Amblyomma and Boophilus microplus,the positive rate was 26.0%detected by nested PCR,14.0%by Ehrlichia ruminantium real-time PCR kit and 0.0%by conventional PCR.The results showed that the nested PCR assay could be used for early detection and epidemiology investigation of Ehrlichia ruminantium,which will be a technical support for the tick-borne Ehrlichia ruminantium prevention and control.