牛无浆体PCR检测方法的建立及应用

Establishment and application of PCR assay for Anaplasma bovis

为建立快速、特异、敏感的牛无浆体(Anaplasma bovis)PCR检测方法及了解新疆部分地区牛无浆体感染情况,根据牛无浆体16S rRNA基因序列(MH255941.1)设计1对引物,建立了检测牛无浆体PCR方法,并进行特异性、敏感性、重复性试验、临床样品检测及系统进化分析。结果显示,所建立的方法能特异性地鉴别牛无浆体,与环形泰勒虫(Theileria annulata)、绵羊无浆体(A.ovis)、牛巴贝斯虫(Babesia bovis)、双芽巴贝斯虫(B.bigemina)基因组均无交叉反应,该方法检测下限可达到1.02×10^(-18)g/μL,比文献报道的PCR敏感10倍(8.9×10^(-18)g/μL);具有良好的重复性;对94份牛临床血液样品检测结果表明,牛无浆体的阳性率为54.3%(51/94),高于文献报道的PCR方法的检测结果37.2%(35/94);系统进化树显示,三个地区的牛无浆体菌株都不在同一分支上。结论,成功构建了牛无浆体PCR快速检测方法,这为牛无浆体的快速诊断及流行病学调查奠定基础。

The aim of this study was to establish a fast,specific and sensitive PCR method for the detection of Anaplasma bovis infection in Xinjiang.According to the sequence of 16S rRNA gene(MH255941.1),a pair of primers was designed and a PCR method for detection of A.bovis was established,and the specificity,sensitivity,reproducibility test,clinical sample detection and phylogenetic analysis were carried out.The results showed that the method can be used to specifically identify A.bovis,with no cross reaction with Theileria annulata,A.ovis,Babesia bovis,B.bigemina.The sensitivity reached 1.02×10^(-18) g/μL,which was 10 times as sensitive as the PCR reported in the literature(8.9×10^(-18) g/μL).This method had good repeatability.The 54.3%(51/94)positive rate in 94 clinical samples by the PCR assay was higher than that of the PCR test results(37.2%,35/94)reported in the literature.The phylogenetic tree showed that the A.bovis from the three regions were not on the same clades.In conclusion,a PCR method for the rapid detection of A.bovis in cattle was successfully constructed,which laid a foundation for the rapid diagnosis and epidemiological investigation of A.bovis.