PCR方法诊断家犬感染细粒棘球绦虫的特异性及在临床诊断中的应用价值

Evaluation of a repeated sequence of Echinococcus granulosus for copro-PCR diagnosis in dogs and its value in clinical diagnosis

目的验证PCR方法检测终宿主家犬感染细粒棘球绦虫的特异性及其在临床诊断中的应用价值。方法取感染细粒棘球绦虫家犬粪便,显微镜下计数细粒棘球绦虫虫卵,稀释后PCR检测其敏感性。同时取家犬小肠内水泡带绦虫、多头绦虫、羊绦虫,将其DNA与细粒棘球绦虫DNA一起进行PCR扩增,观察其特异性。对133 bp的目标条带进行序列测定,对比分析。结果PCR检测终宿主家犬感染细粒棘球绦虫具有高度的灵敏性,即使粪便中有1个虫卵(8pg的DNA),也能测出,但扩增后特异性较差,无法利用扩增带型进行诊断。4种寄生虫均具有相同的133 bp DNA重复序列,在400 bp均可检测出相同条带。结论PCR方法检测终宿主感染细粒棘球绦虫DNA重复序列尽管有优良的灵敏性,因其特异性差,不适合用于临床诊断和推广。

Objective To assess sensitivity and specificity of primers（Eg1121a/Eg1122a） used to perform coproPCR for Echinococcus granulosus infection in definitive hosts. Methods The number of eggs from samples taken in the feces of dogs was estimated under microscopy and their DNA was extracted, then PCR Egl121a/Egl122a was tested on different DNA concentrations to examine their sensitivity. To examine their specificity, the DNA of 3 parasites： T. hydatigena, T. multiceps and T. ovis, taken in the intestines of the same dog, and of Echinococcus granulosus, were tested. The targeted DNA 133 bp fragment was sequenced and analyzed. Results The method, able to detect one egg equivalent to 8 pg of DNA, was of high sensitivity, but of poor specificity after amplification. The 133 bp fragment was found for all the parasites studied; this amplicon presented the same sequence for all the parasites. A 400 bp band was also observed for all the parasites. Conclusions These primers, despite their good sensitivity, can not be used for diagnosis of Echinococcus granulosus infection in definitive host as a routine method because of its poor specificity, even after sequencing analysis.