基于颜色判定的环介导恒温扩增法快速检测副溶血性弧菌  

Colorimetric Detection of Vibrio parahaemolyticus by Loop Mediated Isothermal Amplification Method

作　　者：刘威[1] 邹大阳[1] 尹志涛[1] 刘大伟[1] 李雪莲[1] 魏晓[1] 王雪松[1] 姜铮[1] 袁静[1] 黄留玉[1] 

机构地区：[1]军事医学科学院疾病预防控制所,北京100071

出　　处：《中国科学：生命科学》2011年第10期1037-1042,共6页Scientia Sinica(Vitae)

摘　　要：利用DNA环介导恒温核酸扩增法(LAMP)针对副溶血性弧菌特异基因tlh基因设计4条引物,通过引物特异性识别tlh基因上的6个独立区域来快速检测副溶血性弧菌.LAMP反应的过程中会产生白色沉淀焦磷酸镁,故可以通过监测浊度来判定反应结果.实时浊度仪监测反应结果表明,LAMP反应在60～65℃恒温条件下50min内完成;如果在反应前添加羟基萘酚兰(HNB),蓝色的阳性结果很明显区别于紫色阴性结果;LAMP方法的最低检出限为9.74pg/μL,PCR方法最低检出限为97.4pg/μL,LAMP方法检测灵敏度是PCR方法检测灵敏度的10倍,且具有良好的特异性.LAMP方法用于快速检测副溶血性弧菌具有检测过程简单、实验装置简便、反应结果肉眼可辨别、灵敏度高和特异性强的特点,所以LAMP方法检测副溶血性弧菌特别适合用于现场和基层检疫及医疗单位的快速诊断.A loop-mediated isothermal amplification（LAMP） assay was developed and validated for the specific detection of Vibrio parahaemolyticus.One set of primers were designed for recognizing six distinct sequences on Vibrio parahaemolyticus-specific gene tlh.LAMP reaction process will produce a white precipitate of magnesium pyrophosphate,and the reaction results can be judged by monitoring the turbidity.The real-time turbidity monitoring results show that LAMP reaction can be completed in a constant temperature of 60-65°C in 50 min.If adding hydroxy naphthol blue（HNB） before the reaction,blue-positive results are significantly different from the purple-negative results.The sensitivity of LAMP with detection limits of 9.74 g/μL is 10 times than that of PCR,and the primers designed had a good specificity too.The LAMP method reported here demonstrates a potential and valuable means for detection of Vibrio parahaemolyticus,especially for its rapidity,simplicity and low cost.The LAMP assay is suitable for on-site and primary quarantine,medical units for rapid diagnosis.

关 键 词：tlh基因 DNA介导恒温 核酸扩增法 快速检测 副溶血性弧菌 

分 类 号：R155.5[医药卫生—营养与食品卫生学]