羊附红细胞体巢式PCR检测方法的建立与应用

Establishment and application of a nested-PCR assay for detection of Mycoplasma ovis

为了建立一种更加准确、敏感的羊附红细胞体检测方法,根据Gen Bank上发表的羊附红细胞体16S rRNA基因序列(登录号:AF338268),设计内、外2对特异性引物,建立了巢式PCR检测方法。筛选该方法的最佳反应条件,并进行了特异性、敏感性、重复性试验及临床样本检测。结果表明,该方法能扩增出大小为506 bp的羊附红细胞体特异性片段,与Gen Bank收录的相关参考序列同源性为97.8%~99.6%;与猪附红细胞体、嗜吞噬细胞无浆体、绵羊无浆体、莫氏巴贝斯虫、吕氏泰勒虫基因组DNA均无交叉反应;DNA最低检测量为0.654 fg·μL^(-1);具有良好的重复性;对77份羊临床血液样品检测结果表明,羊附红细胞体感染率为71.43%,高于常规PCR和已报道的巢式PCR检测结果。

To establish a nested PCR assay for detection of Mycoplasma ovis, according to the published Mycoplasma ovis 16S rRNA gene sequence in GenBank （Accession number： AF338268） , two pairs of primers were designed inside and outside to establish nested PCR assay for detecting the disease caused by Mycoplasma ovis. The optimum reaction conditions were screened, and the specificity, sensitivity and reproducibility of test and detection of clinical samples were carried out. The results of verification experiments showed that the targeted gene fragment was 506 bp in length and homology of nested PCR product was 97.8% to 99.6% , and this detection method had no cross reaction with Eperythrozoon suis , Anaplasma phagocytophilum , Anaplasma ovis , Babesia motasi , and Theileria luwenshuni. The sensiti positive rate in 77 vlty cli reached 0. 654 fg ·μL^-1. This method had good nical samples by the nested PCR assay was PCR and the reported nested PCR test resuhs repeatability. The 71.43% higher than that by the conventional