基于不同靶基因的3种住肉孢子虫PCR检测方法比较  

Comparison of Three Sarcocystis PCR Detection Methods Based on Different Target Genes

作　　者：孙滔 米荣升[2,3,4] 张烨华 张世杰 张晓丽 贾海燕 曾江勇[5] 黄燕 龚海燕[2,3,4] 韩先干[2,3,4] 陈兆国 赵权[1] SUN Tao;MI Rong-sheng;ZHANG Ye-hua;ZHANG Shi-jie;ZHANG Xiao-li;JIA Hai-yan;ZENG Jiang-yong;HUANG Yan;GONG Hai-yan;HAN Xian-gan;CHEN Zhao-guo;ZHAO Quan(College of Animal Science and Technology,Jilin Agricultural University,Changchun Jilin,130118,China;Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai,200241,China;Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards (Shanghai) of Ministry of Agriculture,Shanghai,200241,China;Key Laboratory of Animal Parasitology of Ministry of Agriculture,Shanghai,200241,China;Tibet Livestock Research Institute, Tibet Academy of Agriculture and Animal Science,Lhasa Tibet,850009,China)

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]中国农业科学院上海兽医研究所,上海200241 [3]农业农村部动物产品质量安全生物性危害因子风险评估实验室(上海),上海200241 [4]农业农村部动物寄生虫学重点实验室,上海200241 [5]西藏自治区农牧科学院畜牧兽医研究所,西藏拉萨850009

出　　处：《动物医学进展》2020年第12期1-6,共6页Progress In Veterinary Medicine

基　　金：国家重点研发计划项目(2018YFD0502305);上海市科技兴农项目(2019-02-08-00-08-F01151);国家农产品质量安全风险评估项目(GJFP2019022);中央级公益性科研院所基本科研业务费项目(2019JB13)。

摘　　要：为筛选敏感、特异的住肉孢子虫(Sarcocystis spp.)PCR检测方法,对以住肉孢子虫18S rRNA、28S rRNA和线粒体细胞色素C氧化酶I基因(cox 1)的部分片段为靶基因设计的3套引物建立的PCR方法进行敏感性和特异性试验,并对田间采集的100份牛源和猪源肌肉样品进行检测。结果显示,以18S rRNA和28S rRNA为靶基因的PCR检测方法的敏感性相似,DNA最小检出量均为1×10-2 ng/μL;而以cox 1为靶基因的PCR检测方法敏感性相对较低,DNA的最低检出量为1 ng/μL。对7种不同寄生虫DNA进行特异性试验,结果以cox 1基因为靶基因的PCR方法特异性较好,仅能够扩增出枯氏住肉孢子虫(Sarcocystis cruzi)DNA;以18S rRNA为靶基因的PCR法,除枯氏住肉孢子虫外,刚地弓形虫和微小隐孢子虫也有相似扩增条带;以28S rRNA为靶基因的PCR法,除枯氏住肉孢子虫外,刚地弓形虫、微小隐孢子虫和欧猥迭宫绦虫DNA也能扩增出杂带,但大小不符。利用3种方法对100份牦牛和猪肉样品进行扩增,发现以18S rRNA、28S rRNA和cox 1为靶基因的PCR法的阳性率分别为36.00%(36/100)、43.00%(43/100)和38.00%(38/100)。结果表明,以cox 1为靶基因的PCR法有良好的特异性,适合用于田间动物肌肉样品中住肉孢子虫的检测,为开展动物住肉孢子虫病分子流行病学及防控研究提供了依据。In order to select a sensitive and specific PCR detection method for Sarcocystis spp.,three PCR detection methods based on 18S rRNA,28S rRNA and mitochondrial cytochrome C oxidase I gene(cox 1)of Sarcocystis,respectively,were compared for sensitivity and specificity.These methods were also used for detection of 100 field yak muscle and pork samples.The results showed that the sensitivities of PCR detection based on 18S rRNA and 28S rRNA were similar,and can amply 1×10-2 ng/μL DNA successfully,and the minimum detection DNA amount of PCR based on cox 1 was 1 ng/μL.The results of specificity test on seven different parasite species DNAs showed that the PCR method based on cox 1 gene could only amplify Sarcocystis cruzi.Besides Sarcocystis,PCR method based on 18S rRNA could also amplified Toxoplasma gondii and Cryptosporidium parvum samples.PCR method based on 28S rRNA could also amplified Toxoplasma gondii,Cryptosporidium parvum and Spirometra erinaceieuropaei samples,however,the amplicon sizes were different with Sarcocystis cruzi.Detection results of field yak muscle and pork showed that the Sarcocystis positive rate using PCR methods based on 18S rRNA,28S rRNA and cox 1 gene were 36.00%(36/100),43.00%(43/100),38.00%(38/100),respectively.These results indicated that the PCR method based on cox 1 gene had good specificity and suited for Sarcocystis detection in field animal muscles.The work provided basis for the research on the molecular epidemiology and prevention and control of sarcocystosis.

关 键 词：18S rRNA基因 28S rRNA基因 cox 1基因 PCR方法 住肉孢子虫 

分 类 号：S852.723[农业科学—基础兽医学] S851.34[农业科学—兽医学]