中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (6): 777-780.doi: 10.12140/j.issn.1000-7423.2020.06.017

• 研究简报 • 上一篇    下一篇

SCAR-PCR技术鉴定日本血吸虫尾蚴性别的应用

莫筱瑾1(), 吴群峰2, 冯正1, 徐斌1, 张颋1, 陈绅波1, 胡薇1,2,*()   

  1. 1 中国疾病预防控制中心寄生虫病预防控制所,国家热带病研究中心,世界卫生组织热带病合作中心,科技部国家级热带病国际联合研究中心,卫生部寄生虫病原与媒介生物学重点实验室,上海 200025
    2 复旦大学生命科学学院,上海 200433
  • 收稿日期:2020-05-20 出版日期:2020-12-30 发布日期:2021-01-12
  • 通讯作者: 胡薇
  • 作者简介:莫筱瑾(1981-),女,硕士,助理研究员,从事寄生虫病防控研究。E-mail: moxj@nipd.chinacdc.cn
  • 基金资助:
    国家自然科学基金(31572513)

Sexing Schistosoma japonicum cercariae by sequence characterized amplified region-PCR

MO Xiao-jin1(), WU Qun-feng2, FENG Zheng1, XU Bin1, ZHANG Ting1, CHEN Shen-bo1, HU Wei1,2,*()   

  1. 1 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention;Chinese Center for Tropical Diseases Research;WHO Collaborating Center of Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology;Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai 200025, China
    2 Department of Microbiology and Microbial Engineering, School of Life Science, Fudan University, Shanghai 200433, China
  • Received:2020-05-20 Online:2020-12-30 Published:2021-01-12
  • Contact: HU Wei
  • Supported by:
    National Natural Science Foundation of China(31572513)

摘要:

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。

关键词: 日本血吸虫, SCAR-PCR, 性别鉴定, 尾蚴, 单性感染

Abstract:

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.

Key words: Schistosoma japonicum, SCAR-PCR amplification, Sex identification, Cercariae, Monoinfection

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