瑟氏泰勒虫血液直接PCR检测方法的应用

Application of a direct PCR method for detection of Theileria Sergenti

为建立快速、灵敏、特异、低成本的瑟氏泰勒虫检测方法,根据GenBank上发表的瑟氏泰勒虫p33基因序列设计合成1对引物,通过PCR退火温度的筛选、特异性试验、敏感性试验和重复性试验,建立瑟氏泰勒虫血液直接PCR检测方法。该方法能扩增瑟氏泰勒虫p33基因的1段416 bp序列,而与弓形虫、新孢子虫、牛附红细胞体基因序列均无交叉反应。该方法检测出的1μL血液最大稀释倍数为40。应用血液直接PCR方法和常规PCR对吉林省8个地区某牛场采取的210份样本进行检测的结果表明:直接PCR阳性率为30.0%,而常规PCR阳性率为25.7%,差异不显著(P>0.05);两者的总符合率为94%,阳性符合率为80%,阴性符合率为92%;2种方法之间的Kappa值为0.85,说明该法更具有推广和应用价值。该试验成功应用了血液直接PCR检测方法,为瑟氏泰勒虫病的快速诊断和流行病学调查提供了一种新的方法。

The aim is to establish a rapid,sensitive,specific and low cost method for the detection of Theileria sergenti.A pair of primers were designed and synthesized according to the sequence of p33 gene published on GenBank.A direct PCR detection method for the blood of Theileria sergenti was established.This method could specifically amplify a 416 bp sequence of the p33 gene of Theileria sergenti.There was no cross reaction with Toxoplasma gondii,Neospora caninum,Bovine eperythrozoon.The maximum dilution concentration of 1μL blood detected by this method was 40 times and the sensitivity was good.The direct blood PCR method and routine PCR were used to detect 210 samples from a cattle farm in 8 areas of Jilin Province.The results displayed that the positive rate of direct PCR was 30.0%,while the positive rate of routine PCR was 25.7%,there was no significant difference(P>0.05).The total coincidence rate was 94%,the positive coincidence rate was 80%and the negative coincidence rate was 92%.The Kappa value between the two methods was 0.85.It shows that the method is worth popularizing and applying.In this experiment,the direct PCR method was successfully applied to detect the blood,it provides a new method for rapid diagnosis and epidemiological investigation of Theileria sergenti disease.