甘南牦牛三种血液原虫多重PCR检测方法建立及流行病学调查

Establishment of Multiplex PCR Detection Method for Three Blood Protozoa in Gannan Yak and Epidemiological Investigation

为建立一种能同时快速检测弓形虫、环形泰勒虫、新孢子虫3种甘南牦牛血液原虫的多重PCR检测方法,掌握3种血液原虫在甘南牦牛中的流行情况,本研究以弓形虫hypothetical protein基因、环形泰勒虫Tams1基因、新孢子虫Nc-p43基因为靶标设计3对特异性引物,通过优化多重PCR反应条件,建立检测3种血液原虫的多重PCR方法,并检测甘南地区629份牦牛血清样品,进行流行病学分析。结果显示,建立的牦牛3种血液原虫多重PCR检测方法,具有特异性强、敏感性高和重复性好的优点,检测弓形虫、环形泰勒虫和新孢子虫DNA的最低浓度分别为0.01、0.02、0.01 ng·μL^(-1);流行病学调查结果显示,弓形虫、环形泰勒虫和新孢子虫的阳性率分别为4.29%、3.66%、5.88%,并存在混合感染现象,整体混合感染率为13.68%;季节性流行病学分析显示,3种原虫单一感染率均为冬季最高、春季最低(P>0.05),整体感染率为冬季最高、夏季最低(P>0.05)。按不同年龄分析,0~3岁牦牛3种原虫的整体感染率为27.83%,大于3岁的整体感染率27.46%(P>0.05)。分析风险因素发现,季节和年龄均不是牦牛感染3种原虫的风险因素(P>0.05)。本研究成功建立的多重PCR检测方法,可用于牦牛弓形虫、环形泰勒虫和新孢子虫的鉴别检测和流行病学调查,并检测了甘南地区牦牛三种血液原虫的流行情况,为该病的预防提供了重要的数据支撑。

To grasp the prevalence of three bloodborne parasites in Gannan yaks,a multiplex PCR assay for simultaneous detection of Toxoplasma gondii,Theileria annulare and Neospora caninum in Gannan yaks was established.In this study,three pairs of specific primers were designed using Toxoplasma gondii hypothetical protein gene,Tams1 gene of T.gondii and Nc-p43 gene of Neospora as the targets.Through optimizing the multiplex PCR reaction conditions,a multiplex PCR method detecting the three blood protozoa was established,and 629 yak sera from Gannan were tested for epidemiological analysis.The results showed that the established multiplex PCR method for detecting the three blood protozoa in yaks was high specificity,high sensitivity and good reproducibility,and the minimum concentrations of DNA for detecting Toxoplasma gondii,Theileria annulare and Neospora caninum were 0.01,0.02 and 0.01 ng·μL^(-1),respectively.The epidemiological investigation results showed that the positive rates were 4.29%,3.66%,and 5.88%for Toxoplasma gondii,Taylormycetes annulare and Neospora caninum respectively.Meantime,there were mixed infections among the three bloodborne parasites,and the overall mixed infection rate was 13.68%;Seasonal epidemiological analysis showed that the single infection rate of all three protozoa was the highest in winter and lowest in spring(P>0.05),and the overall infection rate was highest in winter and lowest in summer(P>0.05).According to different age analysis,the overall infection rate of the three protozoa in yaks of 0-3 years was 27.83%,and the overall infection rate for yaks older than 3 years was 27.46%(P>0.05).Analysis of risk factors revealed that neither season nor age was the risk factor for infection with the 3 protozoa in yaks(P>0.05).In this study,a multiplex PCR detection method was successfully established,which can be used for the differential detection and epidemiological investigation of Toxoplasma gondii,Theileria annulare and Neospora caninum in yaks,and the detected prevalence of three blood protozoa in yaks in Gannan region can provide important data support for the prevention of this disease.