用多重PCR技术检测小管福寿螺体内广州管圆线虫幼虫

Multiplex PCR Assay for the Detection of Angiostrongylus cantonensis Larvae in Pomacea canaliculata

目的建立一种多重PCR方法检测小管福寿螺体内的广州管圆线虫幼虫。方法根据广州管圆线虫核糖体小亚基rDNA基因序列(GenBank登录号为AY295804)设计特异性引物,并与小管福寿螺16srDNA的特异性引物组合,建立多重PCR检测方法。分别以阳性和阴性小管福寿螺DNA为模板进行多重PCR扩增,电泳鉴定并测序。用阴性小管福寿螺DNA倍比稀释200条广州管圆线虫Ⅲ期幼虫DNA模板,使之浓度分别为1200ng/μl、120ng/μl、12ng/μl、1200pg/μl、120pg/μl和12pg/μl,检测该方法的敏感性。野外采集小管福寿螺172只,肺检法检测后,进行多重PCR扩增,计算敏感性和特异性。结果电泳和测序结果证实该多重PCR检测方法能有效扩增出目的片段,小管福寿螺和广州管圆线虫的目的片段分别为550和405bp。该方法可检测出广州管圆线虫Ⅲ期幼虫DNA的最小浓度为120pg/μl。肺检法和多重PCR法检测均为阳性结果的45只,两法均为阴性的100只。肺检法为阴性、多重PCR法为阳性的24只,肺检法为阳性而多重PCR法检测为阴性的3只。多重PCR的敏感性和特异性分别为93.8%(45/48)和80.6%(100/124)。多重PCR法的阳性检出率为40.1%(69/172),肺检法阳性检出率为27.9%(48/172),差异具有统计学意义(χ2=14.8,P<0.01)。结论建立了检测小管福寿螺体内广州管圆线虫的多重PCR方法。

Objective To establish a multiplex PCR assay for detecting Angiostrongylus cantonensis larvae in Pomacea canaliculata. Methods A pair of specific primers was designed based on the sequences of the small subunit rDNA of A. cantonensis （GenBank jAY295804）, in combination with 16s rDNA specific primers of P. canaliculata, a multi- plex PCR was developed. The PCR was performed on positive and negative snails, and the amplified products were analyzed by agarose gel electrophoresis and DNA sequencing. DNA template of 200 Ⅲ stage larvae of A. cantonensis was diluted by negative snail DNA （1 200 ng/μl, 120 ng/μl, 12 ng/μl, 1 200 pg/μl, 120 pg/μl and 12 pg/μl）, to find the minimum detectable level. Single blind method was used to evaluate the accuracy. After being detected by lung microscopy, 172 snails from field were tested by the multiplex PCR to assess the sensitivity and specificity. Results Agarose gel electrophoresis and DNA sequencing analysis indicated that the target sequences were efficiently amplified by the PCR assay （550 bp for P. canaliculata, 405 bp for A. cantonensis）. The minimum detectable level was 120 pg/μl. The coincidence between the two methods stood for 84.3% （145/172）, including 45 positives and 100 negatives. 24 snails were PCR positive and microscopy negative, 3 snails were PCR negative and microscopy positive. The sensitivity and specificity of multiplex PCR was 93.8% and 80.6%, respectively. Its positive rate （40.1%, 69/172） was significant higher than that of lung-microscopy （27.9%, 48/172）（χ2=14.8, P0.01）. Conclusion A multiplex PCR method has been developed for the detection of A. cantonensis larvae in P. canaliculata.