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。
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.fluvicdis, V. dam- sela, V. cholerae, K harveyi, and V. ichthyoenteri, and the negative position in orange were observed in the reaction products added with fluores- cent 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.
出处
《黑龙江畜牧兽医》
CAS
北大核心
2014年第3期39-42,209,共5页
Heilongjiang Animal Science And veterinary Medicine
基金
中央财政支持地方高校发展专项(CXTD16)
江苏省水产三项工程项目(DY2012-3-7
D2013-5-4)
连云港市科技攻关项目(CG1134)
2011江苏高校优势学科建设工程项目(无编号)
