环介导等温扩增技术快速检测沙眼衣原体

doi:10.3969/j.issn.1673-8640.2017.09.014

检验医学 ›› 2017, Vol. 32 ›› Issue (9): 809-812.DOI: 10.3969/j.issn.1673-8640.2017.09.014

• 技术研究与评价·论著 • 上一篇下一篇

环介导等温扩增技术快速检测沙眼衣原体

王雪亮¹, 刘芬², 蒋玲丽¹, 鲍芸¹, 杨依绡¹, 肖艳群¹, 王华梁¹

1.上海市临床检验中心分子生物学室,上海 200126
2.上海生物制品研究所有限责任公司研发部,上海 200052

收稿日期:2016-09-05 出版日期:2017-09-30 发布日期:2017-09-30
作者简介:null
作者简介：王雪亮,男,1981年生,硕士,主管技师,主要从事临床分子诊断质量管理与控制研究。
基金资助:
上海市卫生和计划生育委员会青年基金项目（20134Y010）

Rapid determination of Chlamydia trachomatis by loop-mediated isothermal amplification

WANG Xueliang¹, LIU Fen², JIANG Lingli¹, BAO Yun¹, YANG Yixiao¹, XIAO Yanqun¹, WANG Hualiang¹

1.Department of Molecular Biology， Shanghai Center for Clinical Laboratory，Shanghai 200126，China;
2.Research and Development Department， Shanghai Institute of Biological Products Co.， Ltd.， Shanghai 200052，China

Received:2016-09-05 Online:2017-09-30 Published:2017-09-30

摘要/Abstract

摘要：

目的建立一种新的简单、快速检测沙眼衣原体（CT）的环介导等温扩增（LAMP）技术。方法采用PrimerExplorer V4软件,针对CT隐蔽性质粒基因保守区域设计4重引物（2重内游引物,2重外游引物）,并对LAMP反应体系和反应条件进行优化,评价其敏感性和特异性,将其与实时荧光定量聚合酶链反应（PCR）对临床样本的检测结果进行比较。结果建立的LAMP技术特异性高,与其他常见菌株检测无交叉反应;敏感性高（100 拷贝/μL）。用建立的LAMP技术与实时荧光定量PCR对90例临床样本进行检测,检测结果为阳性40例、阴性50例,二者符合率为100%。结论成功建立了检测CT隐蔽性质粒基因的LAMP技术,为CT的快速检测提供了新的手段。

关键词: 环介导等温扩增, 核酸检测, 沙眼衣原体

Abstract:

Objective To establish a novel loop-mediated isothermal amplification（LAMP） for the simple and rapid determination of Chlamydia trachomatis（CT）.Methods Four primers（2 inner primers and 2 outer primers） for LAMP were designed by targeting the cryptic plasmid gene of CT using PrimerExplorer V4 software. The reaction system and conditions of LAMP were optimized,and the sensitivity and specificity were evaluated. Meanwhile,the results of LAMP were compared with those of real-time fluorescence quantitation polymerase chain reaction（PCR）. Results There was no cross reaction with other common bacteria,and the sensitivity was 100 copies/μL. A total of 90 clinical specimens were determined by LAMP. Compared with real-time fluorescence quantitation PCR,40 of 90 cases were positive,and 50 of 90 cases were negative by LAMP. There was a consistency of 100% between LAMP and real-time fluorescence quantitation PCR.Conclusions LAMP targeting the cryptic plasmid gene of CT is successfully established,and it can be as a novel tool for the rapid determination of CT.

Key words: Loop-mediated isothermal amplification, Nucleic acid determination, Chlamydia trachomatis

中图分类号:

R446.61

王雪亮, 刘芬, 蒋玲丽, 鲍芸, 杨依绡, 肖艳群, 王华梁. 环介导等温扩增技术快速检测沙眼衣原体[J]. 检验医学, 2017, 32(9): 809-812.

WANG Xueliang, LIU Fen, JIANG Lingli, BAO Yun, YANG Yixiao, XIAO Yanqun, WANG Hualiang. Rapid determination of Chlamydia trachomatis by loop-mediated isothermal amplification[J]. Laboratory Medicine, 2017, 32(9): 809-812.

图/表 5

参考文献 17

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引物	引物序列（5'~3'）	长度（bp）
F3	CCCATGCTTTCAGATTTGC	19
B3	TGGCAGTTACTATAATTTACCATAC	25
FIP	CACAATTAGAAGACGATTCCTTCCTAG- CGGTCAAATAGAGCAAG	44
BIP	TGTTGGGAAAAATAGACATGGATCGG- CGGAGAATTTACTAATTTTTGG	48
CT-F	AAGCTTAATTTAATCGCGAATCAGA	25
CT-R	TCTTTGGATAGCTGCTAATGCATGG	25

引物	引物序列（5'~3'）	长度（bp）
F3	CCCATGCTTTCAGATTTGC	19
B3	TGGCAGTTACTATAATTTACCATAC	25
FIP	CACAATTAGAAGACGATTCCTTCCTAG- CGGTCAAATAGAGCAAG	44
BIP	TGTTGGGAAAAATAGACATGGATCGG- CGGAGAATTTACTAATTTTTGG	48
CT-F	AAGCTTAATTTAATCGCGAATCAGA	25
CT-R	TCTTTGGATAGCTGCTAATGCATGG	25

环介导等温扩增技术快速检测沙眼衣原体

Rapid determination of Chlamydia trachomatis by loop-mediated isothermal amplification

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