作者：李若琳¹, 曹月¹, 梁宇晨¹, 李梦雪¹, 朱婷¹, 鞠丹², 张彪³, 谢轶⁴, 许文明⁵, 孙群¹

作者单位：1. 四川大学生命科学学院 生物资源与生态环境教育部重点实验室, 四川 成都 610064;
2. 天津红日药业股份有限公司, 天津 301700;
3. 成都市食品药品检验研究院, 四川 成都 610045;
4. 四川大学华西医院实验医学科, 四川 成都 610041;
5. 四川大学华西第二医院 四川大学-香港中文大学生殖医学联合实验室, 四川 成都 610041

关键词：环介导等温扩增技术;沙眼衣原体;gyrA基因;快速检测

摘要：

建立一种利用环介导等温扩增技术（loop-mediated isothermal amplification, LAMP）快速检测沙眼衣原体的方法。针对沙眼衣原体gyrA基因设计LAMP特异引物，并加入一条环引物LB提高反应效率，对反应的特异性、灵敏度和重复性进行评价，同时用沙眼衣原体阳性的临床样本进行LAMP检测验证。实时荧光LAMP结果表明，建立的LAMP方法特异性好，不与10种常见的病原微生物以及生殖道感染相关细菌发生非特异反应，灵敏度可以达到4.13×10¹ copies/μL；20次重复试验的Cq值变异系数为7.23%，反应稳定性良好；用LAMP方法对6份临床阳性样本进行检测，阳性符合率为100%。因此，该研究建立的沙眼衣原体LAMP检测方法快速、灵敏、稳定性好，能为感染筛查和早期诊断提供参考。

Development of a loop-mediated isothermal amplification method for the rapid detection of Chlamydia trachomatis
LI Ruolin¹, CAO Yue¹, LIANG Yuchen¹, LI Mengxue¹, ZHU Ting¹, JU Dan², ZHANG Biao³, XIE Yi⁴, XU Wenming⁵, SUN Qun¹
1. Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China;
2. Tianjin Chasesun Pharmaceutical Co., Ltd., Tianjin 301700, China;
3. Chengdu institute for Food and Drug Control, Chengdu 610045, China;
4. Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China;
5. Joint Laboratory for Reproductive Medicine, SCU-CUHK, West China Second University Hospital, Sichuan University, Chengdu 610041, China
Abstract: A method was developed for the rapid detection of Chlamydia trachomatis by using loop-mediated isothermal amplification (LAMP). The LAMP specific primers were designed for the gyrA gene of C.trachomatis, and a loop primer LB was added to improve the reaction efficiency. The specificity, sensitivity and repeatability of the reaction were evaluated, followed by the testing on the clinical positive samples of C. trachomatis by LAMP. Real-time fluorescent LAMP results showed that the LAMP method established had good specificity, without non-specific reaction with 10 common pathogenic bacteria associated with reproductive tract infections, and itssensitivity could reach 4.13×10¹ copies/μL. A total of 20 repeatingtests were performed, and the variation coefficient of Cq value was 7.23%, indicating that the stability of the LAMP methodwas satisfactory. Six clinically positive samples were used for validation of LAMP developed, and the positive coincidence rate was 100%. Accordingly the LAMP method established for detecting C.trachomatis is rapid, sensitive and stable, and it can provide a reference for screening and early diagnosis of C. trachomatis causing infection.
Keywords: loop-mediated isothermal amplification technique;Chlamydia trachomatis;gyrA gene;rapid detection
2020, 46(4):65-69  收稿日期: 2020-01-02;收到修改稿日期: 2020-03-02
基金项目: 国家重点研发计划（项目编号：SQ2019YFE010495）；成都市技术创新研发项目（2020-YF05-00306-SN）
作者简介: 李若琳(1996-),女,四川大英县人,硕士研究生,专业方向为病原微生物
参考文献
[1] MILLMAN K, BLACK C M, JOHNSON R E, et al. Population-based genetic and evolutionary analysis of Chlamydia trachomatis urogenital strain variation in the United States[J]. Journal of Bacteriology, 2004, 186(8):2457-2465
[2] WIESENFELD H C. Screening for Chlamydia trachomatis infections in women[J]. The New England Journal of Medicine, 2017, 376(8):765-773
[3] 刘原君, 王千秋. 沙眼衣原体感染的诊断与实验室检查现状[J]. 中国医学文摘:皮肤科学, 2016(3):316-321
[4] NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12):E63
[5] World Health Organization. The use of loop-mediated isothermal amplification (TB-LAMP) for the diagnosis of pulmonary tuberculosis:policy guidance.[EB/OL].Geneva:WHO, 2016-08-08[2019-12-24]. https://apps.who.int/iris/handle/10665/249154
[6] BY P, PAPP J, SCHACHTER J, et al. Recommendations for the laboratory-based detection of Chlamydia trachomatis and Neisseria gonorrhoeae-2014[J]. MMWR Recommendations and reports:Morbidity and mortality weekly report recommendations and reports/Centers for Disease Control, 2014, 63(1-19)
[7] NOTOMI T, MORI Y, TOMITA N, et al. Loop-mediated isothermal amplification (LAMP):principle, features, and future prospects[J]. Journal of Microbiology, 2015, 53(1):1-5
[8] 王斌. 沙眼衣原体感染后HeLa-229细胞的miRNA差异表达及其功能研究[D]. 天津:天津医科大学, 2007.
[9] NAGAMINE K, HASE T, NOTOMI T. Accelerated reaction by loop-mediated isothermal amplification using loop primers[J]. Molecular and Cellular Probes, 2002, 16(3):223-229
[10] 唐卫明, 薛耀华, 郑和平. 生殖道沙眼衣原体感染的挑战与应对[J]. 中华皮肤科杂志, 2017(5):313-315
[11] NIEMZ A, FERGUSON T M, BOYLE D S. Point-of-care nucleic acid testing for infectious diseases[J]. Trends in Biotechnology, 2011, 29(5):240-250
[12] HURTLE W, BODE E, KULESH D A, et al. Detection of the Bacillus anthracis gyrA gene by using a minor groove binder probe[J]. Journal of Clinical Microbiology, 2004, 42(1):179-185
[13] ROCHA D J P, SANTOS C S, PACHECO L G C. Bacterial reference genes for gene expression studies by RT-qPCR:survey and analysis[J]. Antonie van Leeuwenhoek, 2015, 108(3):685-693