猪源肉孢子虫双重套式PCR检测方法的建立  

Establishment of a duplex nested PCR method for the detection of the Sarcocystis species from pigs

作　　者：刘婕 闫文朝[1] 钱伟锋[1] 韩利方[1] 薛瑞[1] LIU Jie;YANWen-chao;QIANWei-feng;HAN Li-fang;XUE Rui(College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471023,China)

机构地区：[1]河南科技大学动物科技学院,河南洛阳471023

出　　处：《中国预防兽医学报》2020年第7期680-684,共5页Chinese Journal of Preventive Veterinary Medicine

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

摘　　要：为了准确快速检测猪源肉孢子虫的种类,本研究根据米氏肉孢子虫和猪人肉孢子虫的18S rRNA基因序列设计双重套式PCR引物,优化并建立能够同时检测这两种猪源肉孢子虫的双重套式PCR方法。结果显示,双重套式PCR对米氏肉孢子虫和猪人肉孢子虫扩增产物的大小分别为415 bp和591 bp,而对枯氏肉孢子虫、刚地弓形虫、犬新孢子虫的检测结果均为阴性,对两种猪源肉孢子虫重组质粒标准品的检测下限均为5拷贝/μL,表明该双重套式PCR具有较强的特异性和较高的敏感性。利用本研究建立的方法随机对22份猪肉样品的检测结果显示,本研究建立的方法与肌肉压片镜检法的阳性符合率为100%,阴性符合率为89.5%,总符合率为90.9%,本研究建立的双重套式PCR方法可以实现对猪肉样品中米氏肉孢子虫和猪人肉孢子虫的快速检测。A duplex nested PCR assay was developed to detect the Sarcocystis species from pig specimens in this study.The primers were designed according to the 18S rRNA sequences of S.miescheriana(Sm18S rRNA)and S.suihominis(Ss18S rRNA)in this assay.The results showed that the amplified fragments of the PCR assay were 415-bp(Sm18S rRNA)and 591-bp(Ss18S rRNA)respectively.And the PCR assay was negative for the samples of S.cruzi,Toxoplasma gondii,and Neospora caninum.The detetion limit of the PCR assay was 5 copies/μL for the two plasmid standards of S.miescheriana and S.suihominis.These data indicated that this method had high specificity and sensitivity.Twenty-two random pork samples were then examined by using the PCR assay.Comparing with direct observation of squash slides under microscope,this method has 100%and 89.5%coincidence rate for the positive and negative samples and the total coincidence rate is 90.9%.These findings indicated that the PCR method established in this study could be used to rapidly detect S.miescheriana and S.suihominis in pork samples.

关 键 词：猪 米氏肉孢子虫 猪人肉孢子虫 双重套式PCR 18S rRNA 

分 类 号：S855.9[农业科学—临床兽医学]