LAMP技术快速检测海产品副溶血弧菌的条件优化

Optimize of Loop-mediated isothermal amplification technology in detection of Vibrio parahaemolyticus

摘要: 本研究以副溶血弧菌不耐热溶血素tlh为目标基因设计特异性引物,优化反应条件,并对特异性、灵敏度进行了验证。LAMP最佳反应条件为,外内引物浓度比为1∶8,Mg2+反应浓度为4 mmol/L,d NTPs浓度为3.5 mmol/L,温度为65℃,反应时长为1 h,只对副溶血弧菌产生扩增反应,与其余细菌不产生扩增反应,细菌基因组DNA和纯培养物的灵敏度约为5.45×101 fg/μL和140 cfu/m L,对人工污染食品样品进行检测,检出限为2.8×102 cfu/m L。该方法反应时间短、特异性强、灵敏度高。

Abstract: In this study,two pairs of primers were designed according to the heat-sensitive hemolysin gene tlh of V. parahaemolyticus and the LAMP amplification reaction conditions were optimized to develop the rapid detection method of V. parahaemolyticus,and verified the specificity and sensitivity. These results suggested that the LAMP mixture containing concentration ratio for outer primer and inner primer was 1 to 8,4 mmol/L Mg2+,3.5 mmol/L d NTPs,performed at 65 ℃ for 1 h. No crossreaction was found with other common bacteria which showed a good specificity. The detection limits of LAMP assay and pure cultures were 5.45 ×101fg/μL LAMP mixture of genomic DNA and 140 cfu/m L respectively. The detection limit of V. parahaemolyticus in food samples were 2.8 ×102cfu/m L. The result indicated that LAMP had the advantage of rapidity,specificity,and sensitivity.