羊吕氏泰勒虫PCR检测方法的建立与应用

摘要

为建立一种准确、敏感的羊吕氏泰勒虫检测方法,根据GenBank上发表的羊吕氏泰勒虫18S rRNA基因序列(登录号:KC735166.1),设计1对特异性引物,建立了PCR检测方法.筛选该方法的最佳反应条件及体系,并进行了敏感性、特异性、重复性试验及临床血液样品检测.结果表明,该方法能扩增出大小为962 bp的羊吕氏泰勒虫特异性片段,与GenBank收录的相关参考序列同源性高达99.4％～99.9％;与绵羊泰勒虫、尤氏泰勒虫、环形泰勒虫、莫氏巴贝斯虫和弓形虫基因组DNA均无交叉反应;DNA最低检测量为29.13 fg·μL-1;具有良好的重复性;对76份羊临床血液样品检测结果表明,羊吕氏泰勒虫感染率为77.63％,高于已报道的常规PCR检测结果.%To establish a PCR assay to detect Theileria luwenshuni,according to the published 18S rRNA gene sequence of T.luwenshuni in GenBank (Accession number:KC735166.1),a pair of primers were designed to detect the disease caused by T.luwenshuni.The optimum reaction conditions were screened,and studies were carried out to test the specificity,sensitivity and reproducibility of the clinical samples.The results of verification experiments showed that the targeted gene fragment was 962 bp in length and homology of PCR product was 99.4％～99.9％,and this detection method had no cross reaction with T.ovis,T.uilenbergi,T.annulata,Babesia motasi and Toxoplasma gondii.The sensitivity reached 29.13 fg·μL-1.This method had good repeatability.The 77.63％ positive rate in 76 clinical blood samples of sheep by the PCR assay was higher than that of the conventional PCR test results.