猪囊等孢球虫和十二指肠贾第虫双重巢式PCR检测方法的建立与应用  

Establishment and application of a duplex nested PCR method for detection of Cystoisospora suis and Giardia duodenalis

作　　者：李静[1] 刘珍珍 陈凯丽 王朋林 张龙现[1] 梁楠[1,2] 菅复春[1] LI Jing;LIU Zhen-zhen;CHEN Kai-li;WANG Peng-lin;ZHANG Long-xian;LIANG Nan;JIAN Fu-chun(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;College of Animal Husbandry Engineering,Henan Vocational College of Agriculture,Zhengzhou 451450,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450002 [2]河南农业职业学院牧业工程学院,河南郑州451450

出　　处：《中国兽医科学》2022年第5期595-602,共8页Chinese Veterinary Science

基　　金：国家现代农业产业技术体系项目(CARS-35);国家重点研发计划项目(2016YFD0500707)。

摘　　要：为建立同步检测猪囊等孢球虫和十二指肠贾第虫的方法,根据猪囊等孢球虫SSU rRNA和十二指肠贾第虫gdh基因位点建立并优化了一套双重巢式PCR方法,同时对该方法的敏感性、特异性和重复性在临床样品检测中进行验证。结果显示,利用该双重巢式PCR扩增的SSU rRNA和gdh目的产物大小分别为530和432 bp,与单病原巢式PCR的检测结果一致。该方法对猪囊等孢球虫和十二指肠贾第虫的检测下限分别为3.85×10^(-4)和5.51×10^(-3)ng/μL;与毕氏肠微孢子虫、安氏隐孢子虫和大肠杆菌基因组DNA均无交叉反应;利用单巢式PCR和建立的双重巢式PCR方法分别对河南省部分地区的212份猪粪便样品进行特异性扩增,结果显示单巢式PCR与双重巢式PCR检测结果的符合率为100%。本研究首次成功建立了特异性强、灵敏度高的猪囊等孢球虫和十二指肠贾第虫双重巢式PCR检测方法,为猪肠道原虫分子流行病学调查提供了更高效的技术手段。In order to establish a duplex nested PCR method for simultaneous detection of Cystoisospora suis and Giardia duodenalis,a duplex nested PCR method was established and optimized according to C.suis SSU rRNA gene and G.duodenalis gdh gene locus.The results showed that the sizes of the target products amplified by the duplex nested PCR for SSU rRNA and gdh were 530 bp and 432 bp,respectively,which was consistent with the results of single pathogen nested PCR.The detection limits of this method for C.suis and G.duodenalis were 3.85×10^(-4)ng/μL and 5.51×10^(-3)ng/μL,respectively.There was no crossreaction with Enterocytozoon bieneusi,Cryptosporidium andersoni and Escherchia coli genomic DNA.A total of 212 pig feces samples collected from some areas of Henan Province were specifically amplified,which showed that the amplification results of single PCR were consistent with that of duplex PCR,and the coincidence rate was 100%.The duplex nested PCR with high specificity and sensitivity for simultaneous detection of C.suis and G.duodenalis was successfully established for the first time,which provided a more efficient technical means for molecular epidemiological investigation of intestinal protozoa in pigs.

关 键 词：猪囊等孢球虫 十二指肠贾第虫 SSU rRNA GDH 双重巢式PCR 

分 类 号：S852.72[农业科学—基础兽医学]