5对引物检测犬棘球蚴病感染方法评价

Assessment of 5 pairs of primers molecularly diagnosing infection of Echinococcus spp. in dogs

目的 评估5对引物检测犬棘球蚴病感染的PCR方法并建立可将多房棘球绦虫及细粒棘球绦虫羊株（G1型）和骆驼株（G6型）区分的方法。方法 选用引物BG1／3、Bretange：1／2、Eg1f／1r、EM—H15／H17和Cabrera：1／2，扩增稀释的细粒棘球绦虫虫卵DNA，进行敏感度测定。对水泡带绦虫、多头绦虫、羊绦虫、多房棘球绦虫、细粒棘球绦虫G1型和G6型进行PCR扩增，测定引物的特异度。结果 5对引物在体外均能检测出1个虫卵的稀释度。Cabrera：1／2引物能在6种寄生虫样本中扩增出相同片段大小的条带：Bretange：1／2引物可以扩增出多房棘球绦虫、细粒棘球绦虫G1型和G6型；BG1／3引物可以在多房棘球绦虫和细粒棘球绦虫G1型中扩增出1条带，在细粒棘球绦虫G6型中扩增出2条带：Eg1f／1r可以在细粒棘球绦虫G1型和G6型中扩增出相同片段大小的条带；EM—H15／H17可以在多房棘球绦虫和细粒棘球绦虫G6型中扩增出相同的条带。结论 5对引物中BG1／3具有较强的特异性，其余4对引物特异性不强；利用BG1／3和Eg1f／1r、EM—H15／H17组合可以方便区分细粒棘球绦虫G1型、G6型和多房棘球绦虫。

Objective To assess polymerase chain reaction with 5 pair primers in identifying infection of Echinococcus granulosus and Echinococcus multilocularis in hosts （humans and dogs ） and find a way for discriminating Echinococcus multilocularis from Echinococcus granulosus and sheep strain G1 from camel stain G6. Methods After calculating the eggs under microcopy and diluting the egg, 5 PCR primers BG1/3, Bretange： 1/2, Eg1f/1r, EM-H15/H17 and Cabrera： 1/2 were chosen to performed the amplification. Parasites including T. hydatigena, T. muhiceps and T. ovis commonly existing in dog＇s intestine, Echinococcus granulosus （G1 and G6） and Echinococcus multilocularis were amplified, the specificity of the products was determined. Results All the methods could detect even at the content of one egg by PCR. Cabrera： 1/2 primer could amplify same size of DNA target among all the samples. The same size band could be inspected in Echinococcus multilocularis, G1 and G6 using Bretange： 1/2. Using primer BG1/3, Echinococcus multilocularis and G1 samples showed one band with the same size. And it showed two bands for G6 sample. The same size target fragment could be amplified in G1 and G6 using primer Eg1f/1r. The amplicon size of Echinococcus multilocularis and G6 were the same using primer EM-H15/H17. Conclusions Among 5 pairs of primers, BG1/3 has a good specificity, others don＇t. Combination of use of BG1/3 with Eg1f/1r and EM-H15/H17 facilitates the differential diagnosis of Echinococcus in China.