牛源肉孢子虫多重PCR检测方法的建立  

Establishment of a multiplex PCR method for the detection of the Sarcocystis species from cattle

作　　者：韩利方[1] 马超锋 钱伟锋[1] 张旻[1] 位治国[1] 闫文朝[1] HAN Li-fang;MA Chao-feng;QIAN Wei-feng;ZHANG Min;WEI Zhi-guo;YAN Wen-chao(College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471023,China;Xinyang Center for Animal Disease Control and Prevention,Xingyang 464000,China)

机构地区：[1]河南科技大学动物科技学院,河南洛阳471023 [2]信阳市动物疫病预防控制中心,河南信阳464000

出　　处：《中国预防兽医学报》2022年第2期152-156,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：河南省科技攻关计划项目(172102110017);河南省自然科学基金项目(182300410088)。

摘　　要：为了准确快速鉴别检测牛源肉孢子虫的种类,本研究根据毛样肉孢子虫、黑氏肉孢子虫、人肉孢子虫、枯氏肉孢子虫和罗氏肉孢子虫5种牛源肉孢子虫的18S rRNA或COX1基因序列设计多重PCR引物,优化并建立了能够同时检测这5种牛源肉孢子虫的多重PCR方法。结果显示,多重PCR对毛样肉孢子虫、黑氏肉孢子虫、人肉孢子虫、枯氏肉孢子虫和罗氏肉孢子虫扩增产物的大小依次为1007 bp、643 bp、535 bp、400 bp和287 bp。特异性试验结果显示,该方法除了对毛样肉孢子虫、黑氏肉孢子虫、人肉孢子虫、枯氏肉孢子虫和罗氏肉孢子虫可扩增出相应大小的目的条带外,对米氏肉孢子虫、刚地弓形虫、犬新孢子虫的检测结果均为阴性,特异性较强;敏感性试验结果显示,该方法对5种牛源肉孢子虫重组质粒标准品的检测下限均为6拷贝/μL,敏感性较高。利用本研究建立的多重PCR方法对34份随机采集的牛肉样品检测,结果显示,多重PCR与肌肉压片镜检法的阳性符合率为100%,阴性符合率为92%,总符合率为94.1%,表明本实验建立的该多重PCR方法可用于临床检测。本研究首次建立了牛源肉孢子虫的多重PCR鉴别检测方法,为牛和人肉孢子虫病诊断和分子流行病学研究提供更有效的技术手段。In order to detect the Sarcocystis species from cattle accurately and quickly,the multiplex PCR primers based on18 S rDNA or COX1 sequences of the five bovine-borne Sarcocystis species S.hirsuta,S.heydorni,S.hominis,S.cruzi and S.rommeli were designed in this study.The multiplex PCR method which can detect these five species of Sarcocystis was established and optimized.The result showed that specific,fragments were amplified by this multiplex PCR method in the samples containing S.hirsuta(1007 bp),S.heydorni(643 bp),S.hominis(535 bp),S.cruzi(400 bp)and S.rommeli(287 bp),respectively.The amplification results were negative for S.miescheriana,Toxoplasma gondii and Neospora caninum.The detection limit was6 copies/μL when plasmid standards were used as PCR templates.These data indicated that this method is highly specific and sensitive.Thirty-four beef samples selected randomly were examined by using the multiplex PCR method.The positive samples examined by the microscopic observation of squashing slides were 100%positive in the PCR.But two negative samples in the microscopic observation are positive in the PCR.The total coincidence rate was 94.1%.In conclusion,the multiplex PCR for detecting the five Sarcocystis species from cattle meat is an effective molecular tool for molecular epidemiology of cattle samples..

关 键 词：牛 肉孢子虫 多重PCR 特异性 敏感性 

分 类 号：S855.9[农业科学—临床兽医学]