Rapid, specific and sensitive detection of <i>Vibrio vulnificus</i> by loop-mediated isothermal amplification targeted to <i>vvhA </i>gene

ZHANG Lina; WANG Mingyi; CONG Dianxia; DING Shuyan; CONG Rinan; YUE Jinyong; GENG Jianli; HU Chengjin

doi:10.1007/s13131-018-1182-8

利用扩增vvhA基因的环介导等温扩增技术快速，特异而敏感地检测创伤弧菌

doi: 10.1007/s13131-018-1182-8

1.
济南军区总医院实验诊断科, 济南 250031, 中国
2.
济南军区总医院实验诊断科, 济南 250031, 中国;大连医科大学附属威海市立医院中心实验室, 威海 264200, 中国
3.
大连医科大学附属威海市立医院中心实验室, 威海 264200, 中国

基金项目: The Major PLA Research Project of "The 12th Five-Year Plan" for Medical Science Development under contract No. BWS12J014; the Primary Research & Development Plan of Shandong Province under contract No. 2016GSF121036.

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- 收稿日期: 2017-03-27

Rapid, specific and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification targeted to vvhA gene

1.
Department of Laboratory Medicine, General Hospital of Jinan Military Region of PLA, Jinan 250031, China
2.
Department of Laboratory Medicine, General Hospital of Jinan Military Region of PLA, Jinan 250031, China;Department of Central Laboratory, Weihai Municipal Hospital affiliated to Dalian Medical University, Weihai 264200, China
3.
Department of Central Laboratory, Weihai Municipal Hospital affiliated to Dalian Medical University, Weihai 264200, China

摘要

摘要: 创伤弧菌是一种可感染人类的河口病原菌。建立快速，特异而敏感的检测方法，有助于创伤弧菌感染的早期疾病诊断和及时治疗。本研究设计了针对vvhA基因的一系列引物（包括两对外部引物和两对内部引物），采用环介导等温扩增技术（LAMP）来检测创伤弧菌。结果显示，本方法的最适扩增温度是63℃，反应仅需35分钟。扩增产物不仅可以用含有DNA ladder的琼脂糖凝胶电泳检出，也可借助钙黄绿素直接肉眼观察。采用45株菌株检测该方法的特异性，其中所有创伤弧菌均被检出而其他菌株检测结果皆为阴性。本方法的敏感性是普通PCR扩增的100倍，同时利用该方法可以准确检测出所有的模拟样品、临床标本及环境样品。与其他已知方法比较，针对vvhA基因的介导等温扩增技术可以快速、简单、敏感及特异地鉴定创伤弧菌。
- 创伤弧菌 /
- 环介导等温扩增 /
- vvhA基因
Abstract: Vibrio vulnificus is an estuarine bacterial pathogen for human. The rapid, specific and sensitive detection of V. vulnificus is urgently needed for early disease diagnosis and timely treatment of V. vulnificus infection. In the study, a loop-mediated isothermal amplification (LAMP) technique was developed for V. vulnificus detection with a set of primers, composed of two out primers and two inner primers targeted to vvhA gene. The optimal amplification temperature was 63℃ and the reaction only took 35 min. The amplification products could not only be detected by agarose gel electrophoresis with ladder-like pattern bands, but also could be visualized using calcein with naked eye directly. Forty-five strains were tested for the specificity of LAMP assay, and all the V. vulnificus strains were identified correctly while other strains were negative results. The sensitive of the new LAMP assay was 100-fold more sensitive than the conventional PCR. Meanwhile, all the V. vulnificus strains were detected correctly in spiked, clinical and environmental samples by the new LAMP assay. Compared with other well-known techniques, the new LAMP assay targeted to vvhA gene was extremely rapid, simple, sensitive and specific for V. vulnificus identification.
- Vibrio vulnificus /
- loop-mediated isothermal amplification /
- vvhA gene

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参考文献(24)

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利用扩增vvhA基因的环介导等温扩增技术快速，特异而敏感地检测创伤弧菌

doi: 10.1007/s13131-018-1182-8

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Rapid, specific and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification targeted to vvhA gene

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