SCAR-PCR技术鉴定日本血吸虫尾蚴性别的应用

Sexing Schistosoma japonicum cercariae by sequence characterized amplified region-PCR

采用日本血吸虫单条毛蚴感染单个阴性钉螺的方法,培育获得可释放单一性别尾蚴的阳性钉螺。收集每只阳性钉螺释放的单一性别尾蚴,用序列特异扩增区域PCR(SCAR-PCR)鉴定尾蚴性别;用同一钉螺逸出的单一性别尾蚴感染小鼠后21 d,剖杀小鼠,收集成虫,判定成虫的性别以验证尾蚴性别(实验动物感染法)并评价SCAR-PCR鉴定单条尾蚴性别的正确性。结果显示,仅雌性尾蚴能扩增出153 bp的特异性条带,其鉴定结果与实验动物感染法的判定结果一致。SCAR-PCR检测DNA下限为16.5 ng,相当于1条尾蚴DNA量。采集经动物实验确认的雄性和雌性尾蚴各40条,应用SCAR-PCR检测出其中37条雌性尾蚴和40条雄性尾蚴。SCAR-PCR检测单条雌性尾蚴的正确性为92.5%(37/40),检测单条雄性尾蚴的正确性为100%(40/40)。应用SCAR-PCR可以鉴别日本血吸虫尾蚴的性别。

To identify the sex of Schistosoma japonicum cercaria,single sex cercariae were prepared.Monosexual miracidia were used to infect single parasite-negative snail for breeding positive snails being able to shed single sex cercaria.The single-sex cercariae released from each positive snail were collected for sex identification using the sequence characterized amplified region-PCR(SCAR-PCR)method.To confirm the sex of cercariae obtained,single-sex cercariae from the same snail were used to infect mice,and the adult worms were harvested on 21 d post-infection for verifying the worm gender morphologically,and examining the accuracy of the PCR method for detecting DNA of one single cercaria.The PCR results indicated that the female cercariae presented a specific band at 153 bp,showing a concordance with the findings of infected mouse experiment.The minimum DNA amount detectable in the PCR assay was 16.5 ng,equivalent to the DNA amount of one cercaria.To verify the accuracy of the PCR method,40 female and 40 male sex-known cercariae verified morphologically on their derived adult worms were examined using the PCR method,and found 37 females and 40 males respectively from the sex-known samples,indicative of a sex-discriminating accuracy of 92.5%(37/40)for female and 100%(40/40)for male cercariae of S.japonicum.The results demonstrated that the cercaria sex could be differentiated using the SCAR-PCR method.