研究简报

SCAR-PCR技术鉴定日本血吸虫尾蚴性别的应用

  • 莫筱瑾 ,
  • 吴群峰 ,
  • 冯正 ,
  • 徐斌 ,
  • 张颋 ,
  • 陈绅波 ,
  • 胡薇
展开
  • 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
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

收稿日期: 2020-05-20

  网络出版日期: 2021-01-12

基金资助

国家自然科学基金(31572513)

Sexing Schistosoma japonicum cercariae by sequence characterized amplified region-PCR

  • Xiao-jin MO ,
  • Qun-feng WU ,
  • Zheng FENG ,
  • Bin XU ,
  • Ting ZHANG ,
  • Shen-bo CHEN ,
  • Wei HU
Expand
  • 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 date: 2020-05-20

  Online published: 2021-01-12

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。

本文引用格式

莫筱瑾 , 吴群峰 , 冯正 , 徐斌 , 张颋 , 陈绅波 , 胡薇 . SCAR-PCR技术鉴定日本血吸虫尾蚴性别的应用[J]. 中国寄生虫学与寄生虫病杂志, 2020 , 38(6) : 777 -780 . DOI: 10.12140/j.issn.1000-7423.2020.06.017

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.

参考文献

[1] Sun J, Wang SW, Li C, et al. Transcriptome profilings of female Schistosoma japonicum reveal significant differential expression of genes after pairing[J]. Parasitol Res, 2014,113(3):881-892.
[2] Weerakoon KG, Gobert GN, Cai PF, et al. Advances in the diagnosis of human schistosomiasis[J]. Clin Microbiol Rev, 2015,28(4): 939-967.
[3] Luo F, Yin MB, Mo XJ, et al. An improved genome assembly of the fluke Schistosoma japonicum[J]. PLoS Negl Trop Dis, 2019,13(8):e0007612.
[4] Barnett R. Schistosomiasis[J]. Lancet, 2018,392(10163):2431.
[5] Short RB. Presidential address: sex and the single schistosome[J]. J Parasitol, 1983,69(1):3-22.
[6] Webster P, Mansour TE, Bieber D. Isolation of a female-specific, highly repeared Schistosoma mansoni DNA probe and its use in an assay of cercarial sex[J]. Mol Biochem Parasitol, 1989,36(3):217-222.
[7] Shaw JR, Marshall I, Erasmus DA. Schistosoma mansoni: in vitro stimulation of vitelline cell development by extracts of male worms[J]. Exp Parasitol, 1977,42(1):14-20.
[8] Liu FC, Ding H, Tian JM, et al. Differential gene expression, including Sjfs800, in Schistosoma japonicum females at pre-pairing, initial pairing and oviposition[J]. Parasit Vectors, 2019,12(1):414.
[9] Xin Y, Zhao N, Li Q, et al. Expression and detection of lentivirus-mediated green fluorescent protein in Schistosoma japonicum[J]. Chin J Parasitol Parasit Dis, 2016,34(6):517-521. (in Chinese)
[9] ( 辛玥, 赵楠, 李青, 等. 慢病毒介导的绿色荧光蛋白基因在日本血吸虫体内的表达和检测[J]. 中国寄生虫病学与寄生虫病杂志, 2016,34(6):517-521.)
[10] Zhao GH, Li J, Lin RQ, et al. An effective sequence characterized amplified region-PCR method derived from restriction site-amplified polymorphism for the identification of female Schistosoma japonicum of zoonotic significance[J]. Electrophoresis, 2010,31(4):641-647.
[11] Gasser RB, Morahan G, Mitchell GF. Sexing single larval stages of Schistosoma mansoni by polymerase chain reaction[J]. Mol Biochem Parasitol, 1991,47(2):255-258.
[12] Grevelding CG, Kampk?tter A, Kunz W. Schistosoma mansoni: sexing cercariae by PCR without DNA extraction[J]. Exp Parasitol, 1997,85(1):99-100.
[13] Boissier J, Durand P, Moné H. PCR effectiveness for sexing Schistosoma mansoni cercariae: application for sexing clonal cercarial populations[J]. Mol Biochem Parasitol, 2001,112(1):139-141.
[14] Xu J, Li CX, Duan ZL, et al. Multiplex PCR for sexing Schistosoma japonicum cercariae and its utility[J]. Parasitol Res, 2019,118(10):2885-2890.
文章导航

/

〈 〉