甘南牦牛三种消化道线虫多重PCR检测方法的建立及流行病学调查

Establishment of a multiplex PCR method for detection of three nematodes in digestive tract and its application in yak in Gannan Prefecture,China

为建立一种能同时检测奥氏奥斯特线虫、捻转血矛线虫和环纹背带线虫的多重PCR检测方法,掌握三种线虫在甘南牦牛中的流行情况,本研究以三种线虫的核糖体ITS序列为靶标设计3对特异性引物,建立了多重PCR检测方法,同时检测986份甘南牦牛粪便样品,对三种线虫进行流行病学分析。结果显示,本研究建立的多重PCR方法检测到奥氏奥斯特线虫、捻转血矛线虫、环纹背带线虫DNA的最低浓度分别为0.03、0.03、0.02 ng/μL,三种线虫的感染率分别为12.58%、14.30%、11.36%,混合感染率为12.98%。1—4月份3种消化道线虫的感染率显著高于5—8月份和9—12月份(P<0.01),3种消化道线虫的感染率在0~3岁与大于3岁的牦牛之间差异不显著。本研究成功建立了用于牦牛奥氏奥斯特线虫、捻转血矛线虫、环纹背带线虫的多重PCR检测方法;甘南牦牛三种线虫的流行率较高,提示甘南牦牛消化道线虫的防控不能忽视。

The objective of this study was to establish a multiplex PCR method that could be used to detect Ostertagia ostertagi,Haemonchus contortus and Teladorsagia circumcincta in digestive tract of livestock at one time,and using this method to investigated the prevalence of the three nematode species in yaks in Gannan Prefecture,China.Three pairs of specific primer were designed according to ribosomal ITS sequences of three kinds of nematodes,and the reaction conditions ware optimized based on the results of PCR,and then this method used to detect the three kinds of nematodes in feces of 986 yaks that collected from Gannan.In result,the minimum concentrations for the detection of DNA of Ostertagia ostertagi,Haemonchus contortus and Teladorsagia circumcincta were 0.03 ng/μL,0.03 ng/μL and 0.02 ng/μL,respectively.The prevalence rates of three kinds of nematodes were 12.58%,14.30%and 11.36%,respectively,and more the rate of polyinfection was 12.98%.In addition,the prevalence of three gastrointestinal nematodes from January to April was significantly higher than that from May to December(P<0.01),but there was no significant difference between the infection rate in yak aged 0—3 years and over 3 years.The results indicated that the multiplex PCR method could be used for Ostertagia ostertagi,Haemonchus contortus and Teladorsagia circumcincta detection in yak,and the prevalence rate of gastrointestinal nematodes in yak is high in Gannan Prefecture,China,which remind us that the combating this disease should not be ignored in yak.