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
王晓星,崔艳艳,菅复春,张忠义,宁建峰,张龙现,王荣军,宁长申
摘要(Abstract)：
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。
关键词(KeyWords)： 羊附红细胞体;16S rRNA;巢式PCR
基金项目(Foundation): 农业部国家现代肉羊产业技术体系建设专项资金项目(nycytx-39);; 河南省教育厅现代畜牧业河南省协同创新中心
作者(Author): 王晓星,崔艳艳,菅复春,张忠义,宁建峰,张龙现,王荣军,宁长申
DOI: 10.16445/j.cnki.1000-2340.2016.04.011
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