CPA-核酸试纸条快速检测霍乱弧菌方法的建立与优化

生物技术通报 ›› 2013, Vol. 0 ›› Issue (7): 167-171.

CPA-核酸试纸条快速检测霍乱弧菌方法的建立与优化

秦强,朱金玲

（佳木斯大学基础医学院生物教研室，佳木斯 154007）

收稿日期:2012-12-24 修回日期:2013-07-19 出版日期:2013-07-19 发布日期:2013-09-02
作者简介:秦强，硕士研究生，研究方向：遗传病的分子诊断及治疗；E-mail：qinqiang1234567@126.com

Establishment and Optimization of CPA-nucleic Acid Test Strip for Rapid Detection of Vibrio cholerae

Qin Qiang, Zhu Jinling

（Biology Department，School of Basic Medical Sciences，Jiamusi University，Jiamusi 154007）

Received:2012-12-24 Revised:2013-07-19 Published:2013-07-19 Online:2013-09-02

摘要/Abstract

摘要： 用交叉引物恒温扩增技术（Cross priming amplification，CPA）和核酸试纸条检测方法（Nucleic acid test strip detection methord）快速检测霍乱弧菌。根据霍乱弧菌的MDH基因序列设计特异性的引物和探针，通过对反应体系与反应条件进行优化，确立最佳反应体系，并将CPA方法与PCR法进行比较与评价。对霍乱弧菌的检测具有高度特异性，对霍乱弧菌的检出极限达到了6×10 ² CFU/mL，高于PCR，而且具有较好的稳定性。交叉引物恒温扩增技术（Cross priming amplification，CPA）-核酸试纸条检测方法（Nucleic acid test strip detection methord）是一种特异、灵敏、快速、简便的霍乱弧菌的检测方法。

关键词: 霍乱弧菌, 交叉引物恒温扩增技术, 核酸试纸条检测方法

Abstract: It was to estabilish a cross priming amplification and nucleic acid test strip based rapid detection method of Vibrio cholerae. Specific primers and probes according to the the MDH gene sequence of the Vibrio cholerae were designed, and via optimizing the reaction system and the reaction conditions to establish the optimal reaction system, the CPA method with the PCR method were compared and evaluated. Result showed that the method was highly specific for the detection of Vibrio cholerae, the detection limit reached 6 × 10 ² CFU/mL, higher than that of the PCR, and has satisfactorg stability. The CPA-nucleic acid test strip detection method established in this study is a specific, sensitive, rapid and easy detection method of Vibrio cholerae.

Key words: Vibrio cholerae, Gross priming amplification（CPA）, Nucleic acid test strip

秦强,朱金玲. CPA-核酸试纸条快速检测霍乱弧菌方法的建立与优化[J]. 生物技术通报, 2013, 0(7): 167-171.

Qin Qiang, Zhu Jinling. Establishment and Optimization of CPA-nucleic Acid Test Strip for Rapid Detection of Vibrio cholerae[J]. Biotechnology Bulletin, 2013, 0(7): 167-171.

参考文献

[1] Colwell RR. Global climate and infectious diseases：the cholera paradigm[J]. Science, 1996, 274：2025-2031.
[2] Bhattacharya SK, Bhattacharya MK, Nair GB, et al. Clinical profile of acute diarrhoeal cases infected with thenew epidemic strain of V. cholerae O139：designation of the disease as cholera[J]. J Infect, 1993, 27：11-15.
[3] Varela P, Rivas M, Binsztein N, et al. Identification of toxigenic Vibrio cholerae from the Argentine outbreak by PCR for ctxA1 and ctxA2-B[J]. FEBS Lett, 1993, 315（1）：74-76.
[4] Goel AK, Ponmariappan S, Kamboj DV, et al. Single multiplex polymerase chain reaction for environmental surveillance of toxigenic pathogenic O1 and non-O1 Vibrio cholerae[J]. Folia Microbiol（Praha）, 2007, 52（1）：81-85.
[5] Albert MJ, Islam D, Nahar S, et al. Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR[J]. J Clin Microbiol, 1997, 35（6）：1633 1635.
[6] Dong ZH, Hu SX, Zeng YX, et al. Evaluation of clinical effect for rapid detection test-paper of Vibrio cholerae O139[J]. Appl Prev Med, 2004, 11（5）：973 -974.
[7] Xu GL, Hu L, Zhong HY, et al. Cross priming amplification：mechanism and optimization for isothermal DNA amplification[J]. Scientific Reports, 2012, | 2：246. doi：10.1038/srep00246
[8] 芮勇宇, 阚飙, 高守一, 等.不同群型霍乱弧菌和recA、dnaE和mdh管家基因序列分析[J].南方医科大学学报, 2006, 26（12）：1720-1724.
[9] 徐义刚, 李苏龙, 李丹丹, 等. DNA 环介导恒温扩增技术快速检测霍乱弧菌[J].生物工程学报, 2010, 26（3）：398-403.
[10] Fang RD, Li X, Hu L, et al. Cross-priming amplification for rapid detection of Mycobacterium tuberculosis in sputum specimens[J].Journal of Clinical Microbiology, 2009, 47（3）：847-847.