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当前位置: 首页 > 文章 > 基于tlh基因病原副溶血弧菌LAMP检测方法的建立 黑龙江畜牧兽医 2014 (5) 39-42+209
Position: Home > Articles > Construction of the Loop- mediated isothermal amplification( LAMP) method for detection of pathogenic Vibrio parahaemolyticus based on the tlh gene Heilongjiang Animal Science and Veterinary Medicine 2014 (5) 39-42+209

基于tlh基因病原副溶血弧菌LAMP检测方法的建立

作  者：
秦国民;白雪松;毕可然;阎斌伦;秦蕾;张晓君
单  位：
淮海工学院海洋学院江苏省海洋生物技术重点实验室
关键词：
副溶血弧菌;tlh基因;环介导恒温扩增(LAMP)技术;荧光染料SYBR GreenⅠ
摘  要：
为了研究副溶血弧菌(V.parahaemolyticus)的快速分子生物学检测方法,试验以副溶血弧菌特异性tlh基因为分子靶标设计引物,利用环介导恒温扩增(Loop-mediated isothermal amplification,LAMP)技术建立病原副溶血弧菌的快速检测方法。结果表明:特异性检测仅副溶血弧菌基因组DNA可扩增出阶梯状条带,且反应产物中加入荧光染料SYBR GreenⅠ后呈现明显的绿色阳性反应;而鳗弧菌、河口弧菌、美人鱼弧菌、霍乱弧菌、哈氏弧菌、鱼肠道弧菌这6种对照病原菌均未出现任何扩增条带,且反应产物中加入荧光染料SYBR GreenⅠ后呈现橙色阴性反应;灵敏度检测的最低检测限为42 cfu/mL;对人工染菌的9种水产品检测均可获得阳性扩增结果;该方法检测时间从核酸抽提到结果分析仅需1 h左右,较传统方法操作简单、耗时短、不需昂贵仪器。说明该方法可用于针对副溶血弧菌的进出口检验检疫、食品安全检测及由该菌引起的水产动物疾病的诊断与分子流行病学调查。
译  名：
Construction of the Loop- mediated isothermal amplification( LAMP) method for detection of pathogenic Vibrio parahaemolyticus based on the tlh gene
作  者：
QIN Guo - min;BAI Xue - song;BI Ke - ran;YAN Bin - lun;QIN Lei;ZHANG Xiao - jun;Jiangsu Key Laboratory of Marine Biotechnology,College of Oceanology Huaihai Institute of Technology;
关键词：
Vibrio parahaemolyticus;;tlh gene;;loop-mediated isothermal amplification(LAMP) technology;;fluorescent dye SYBR Green Ⅰ
摘  要：
To study a rapid molecular detection method for Vibrio parahaemolyticus( V. parahaemolyticus),species- specific tlh genes of V. parahaemolyticus were used as molecular targets to design the primers. A rapid detection method for pathogenic V. parahaemolyticus was established using the loop- mediated isothermal amplification( LAMP) technology. The results showed that the stepped bands were amplified only in the V. parahaemolyticus genome DNA,and an obvious positive reaction in green was observed in the reaction product added with fluorescent dye SYBR Green Ⅰ. There were no stepped bands detected in 6 kinds of control pathogenic bacteria including V. anguillarum,V. fluvialis,V. damsela,V. cholerae,V. harveyi,and V. ichthyoenteri,and the negative position in orange were observed in the reaction products added with fluorescent dye SYBR Green Ⅰ. The limit of sensitivity detection was 42 cfu / mL,while the positive reactions were observed in artificial infected nine aquatic foods 9 using LAMP detection system. The LAMP method took only 1 h or so to finish the detection from nucleic acid extraction to the result analysis. Compared with the traditional method,it had advantages of simple operation,short time- consuming,and without expensive equipment. The results indicate that the method can be used in the import and export inspection and quarantine and food safety testing for V. parahaemolyticus,diagnosis and molecular epidemiology for aquatic animal disease caused by V. parahaemolyticus.

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