结合重组酶介导的核酸等温扩增和荧光探针快速检测日本血吸虫基因片段

doi:10.12140/j.issn.1000-7423.2019.01.005

中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (1): 23-27.doi: 10.12140/j.issn.1000-7423.2019.01.005

结合重组酶介导的核酸等温扩增和荧光探针快速检测日本血吸虫基因片段

赵松¹(), 刘燕红², 李婷¹, 李伟¹, 张键锋¹, 郭利川², 应清界², 羊海涛¹, 杨坤^1,^*()

1 国家卫生和计划生育委员会寄生虫病预防与控制技术重点实验室,江苏省寄生虫与媒介控制技术重点实验室,江苏省血吸虫病防治研究所,无锡 214064
2 江苏省奇天基因生物科技有限公司,无锡 214064

收稿日期:2018-11-22 出版日期:2019-02-28 发布日期:2019-03-18
通讯作者: 杨坤
作者简介:
作者简介：赵松（1978-）,男,硕士,助理研究员,从事寄生虫分子生物学研究。E-mail：shanechn@163.com
基金资助:
江苏省“科教强卫工程”医学重点人才（No. ZDRCA2016056）;江苏省卫生计生委科研课题（No. X201802）

Rapid detection of Schistosoma japonicum specific gene fragment by recombinase aided isothermal amplification combined with fluorescent probe

Song ZHAO¹(), Yan-hong LIU², Ting LI¹, Wei LI¹, Jian-feng ZHANG¹, Li-chuan GUO², Qing-jie YING², Hai-tao YANG¹, Kun YANG^1,^*()

1 Key Laboratory of National Health and Family Planning Commission on Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Jiangsu Province, Wuxi 214064, China
2 Jiangsu Qitian Gene Technology Co. Ltd, Wuxi 214064, China

Received:2018-11-22 Online:2019-02-28 Published:2019-03-18
Contact: Kun YANG
Supported by:
Supported by the Project of Invigorating Health Care through Science，Technology and Education, Jiangsu Provincial Medical Talent (No. ZDRCA2016056) and the Fundamental Research Funds of Jiangsu Commission of Health(No. X201802)

摘要/Abstract

摘要：

目的结合重组酶介导的核酸等温扩增（recombinase aided amplification,RAA）和荧光探针方法建立日本血吸虫特异性基因片段的快速检测方法。方法以日本血吸虫G28（SjG28）基因片段作为靶序列,应用DNAMAN 7.0软件设计合成引物及荧光探针,建立荧光RAA反应体系。扩增不同拷贝数的SjG28重组质粒,评价荧光RAA检测的敏感性;分别以日本血吸虫、曼氏血吸虫、细粒棘球绦虫、十二指肠钩口线虫、似蚓蛔线虫、华支睾吸虫基因组为模板进行荧光RAA检测,以评价该方法的特异性。采用荧光RAA分别检测500、200和50条日本血吸虫尾蚴感染的兔粪中虫卵DNA。分别在50只阴性钉螺中混入1、2、3、4、5、10只阳性钉螺,并提取DNA进行荧光RAA检测。结果以不同拷贝数的SjG28重组质粒为模板进行荧光RAA扩增,在5 min时即可观察到明显的荧光信号,随着拷贝数的不断降低,检测到荧光信号的时间也随之延长,不同拷贝数的扩增均在10 min内完成,最低可检出的质粒浓度为10拷贝/μl。以曼氏血吸虫、细粒棘球绦虫、十二指肠钩口线虫、似蚓蛔线虫及华支睾吸虫基因组DNA为模板的荧光RAA扩增结果均为阴性。不同数量尾蚴感染的兔粪中提取的DNA样品经荧光RAA检测,均能得到有效的扩增,检测可在15 min内完成。含不同数量阳性钉螺的50只阴性钉螺提取的日本血吸虫DNA经荧光RAA检测,均能得到有效的扩增,检测可在10 min内完成。结论本研究建立的可检测日本血吸虫核酸片段的荧光RAA方法,反应快捷,敏感性和特异性均较高。

关键词: 日本血吸虫, 基因检测, 核酸等温扩增, 荧光探针, 重组酶

Abstract:

Objective To establish a novel and rapid method to detect Schistosoma japonicum specific gene fragment using a real-time recombinase aided isothermal amplification (RAA) combined with a fluorescent probe. Methods S. japonicum G28（SjG28） was selected as the target gene to be detected, and the primers and fluorescent probes were designed accordingly. A real-time fluorescent RAA using RAA combined with fluorescent probe was established to detect SjG28. The sensitivity and specificity of the real-time fluorescent RAA was determined and optimized by using gradient dilutions of SjG28 recombinant plasmid as template and the genomic DNAs from other helminths of S. mansoni, Echinococcus granulosus, Ancylostoma duodenale, Ascaris lumbricoides and Clonorchis sinensis as controls. Then this method was applied to detect SjG28 in genomic DNA extracted from snails and feces of rabbits infected with S. japonicum. Results As low as 10 copies of SjG28 plasmid DNA could be detected by the developed real-time fluorescent RAA within 10 min. This method was able to detect SjG28 in the fecal samples from rabbit infected with different dose of cercarias (20, 200 and 500) within 15 min and snail pool (50) mixed with 1, 2, 3, 4, 5 and 10 cercaria-positive snails within 10 min. There was no product amplified from genomic DNAs extracted from other helminths: S. mansoni, E. granulosus, A. duodenale, A. lumbricoides and C. sinensis. Conclusion A novel and rapid florescent real-time fluorescent RAA method was established to detect S. japonicum with high sensitivity and specificity.

Key words: Schistosoma japonicum, Gene detection, Isothermal amplification of nucleic acid, Fluorescent probe, Recombinase

中图分类号:

R383.24

赵松, 刘燕红, 李婷, 李伟, 张键锋, 郭利川, 应清界, 羊海涛, 杨坤. 结合重组酶介导的核酸等温扩增和荧光探针快速检测日本血吸虫基因片段[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(1): 23-27.

Song ZHAO, Yan-hong LIU, Ting LI, Wei LI, Jian-feng ZHANG, Li-chuan GUO, Qing-jie YING, Hai-tao YANG, Kun YANG. Rapid detection of Schistosoma japonicum specific gene fragment by recombinase aided isothermal amplification combined with fluorescent probe[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(1): 23-27.

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参考文献 18

[1]	吴观陵. 我国血吸虫病免疫诊断发展的回顾与展望[J]. 中国寄生虫学与寄生虫病杂志, 2005, 23(s1): 323-328.
[2]	Zhou XN, Xu J, Chen HG, et al. Tools to support policy decisions related to treatment strategies and surveillance of schistosomiasis japonica towards elimination[J]. PLoS Negl Trop Dis, 2011, 5(12): e1408.
[3]	Xu J, Peeling RW, Chen JX, et al. Evaluation of immunoassays for the diagnosis of Schistosoma japonicum infection using archived sera[J]. PLoS Negl Trop Dis, 2011, 5(1): e949.
[4]	刘茜, 余传信. 血吸虫现症感染诊断方法的研究进展[J]. 中国血吸虫病防治杂志, 2016, 28(2): 220-224.
[5]	许静, 杨坤, 李石柱, 等. 我国血吸虫病传播控制后的监测体系[J]. 中国血吸虫病防治杂志, 2014, 26(1): 1-5.
[6]	周晓农. 开展精准防治实现消除血吸虫病的目标[J]. 中国血吸虫病防治杂志, 2016, 28(1): 1-4.
[7]	Spear RC, Seto EY, Carlton EJ, et al. The challenge of effective surveillance in moving from low transmission to elimination of schistosomiasis in China[J]. Int J Parasitol, 2011, 41(12): 1243-1247.
[8]	赵松, 李婷, 杨坤, 等. 重组酶介导的日本血吸虫特异性基因片段核酸等温扩增检测方法的建立[J]. 中国血吸虫病防治杂志, 2018, 30(3): 273-277, 306.
[9]	Zhu YC.Immunodiagnosis and its role in schistosomiasis control in China: a review[J]. Acta Trop, 2005, 96(2/3): 130-136.
[10]	You H, Mcmanus DP.Vaccines and diagnostics for zoonotic schistosomiasis japonica[J]. Parasitology, 2015, 142(2): 271-289.
[11]	Xia CM, Rong R, Lu ZX, et al. Schistosoma japonicum: a PCR assay for the early detection and evaluation of treatment in a rabbit model[J]. Exp Parasitol, 2009, 121(2): 175-179.
[12]	Utzinger J, Becker SL, Van Lieshout L, et al. New diagnostic tools in schistosomiasis[J]. Clin Microbiol Infect, 2015, 21(6): 529-542.
[13]	Xu J, Guan ZX, Zhao B, et al. DNA detection of Schistosoma japonicum: diagnostic validity of a LAMP assay for low-intensity infection and effects of chemotherapy in humans[J]. PLoS Negl Trop Dis, 2015, 9(4): e0003668.
[14]	Notomi T, Mori Y, Tomita N, et al. Loop-mediated isothermal amplification(LAMP): principle, features, and future prospects[J]. J Microbiol, 2015, 53(1): 1-5.
[15]	He P, Song LG, Xie H, et al. Nucleic acid detection in the diagnosis and prevention of schistosomiasis[J]. Infect Dis Poverty, 2016, 5: 25.
[16]	Li J, Macdonald J.Advances in isothermal amplification: novel strategies inspired by biological processes[J]. Biosens Bioelectronics, 2015, 64(64C): 196-211.
[17]	Colley DG, Bustinduy AL, Secor WE, et al. Human schistosomiasis[J]. Lancet, 2014, 383(9936): 2253-2264.
[18]	吴观陵. 人体寄生虫学[M]. 4版. 北京: 人民卫生出版社, 2013: 287.

结合重组酶介导的核酸等温扩增和荧光探针快速检测日本血吸虫基因片段

Rapid detection of Schistosoma japonicum specific gene fragment by recombinase aided isothermal amplification combined with fluorescent probe

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