副溶血性弧菌PMA-LAMP方法的建立

Development of Loop-mediated Isothermal Amplification (LAMP) Assay Based on Propidium Monoazide (PMA) for Detecting Vibrio parahaemolyticus

摘要:

副溶血性弧菌（Vibrio parahemolyticus）是一种常见的食源性致病菌，在海鲜等食物中检出率很高，食用没有煮熟的带菌食物或者腌制品，极易引起食物中毒。目前用以检测副溶血性弧菌的快速检测方法都不能区别死活菌，容易出现假阳性结果。本研究基于DNA结合染料叠氮溴化丙锭（propidium monoazide, PMA），利用荧光染料钙黄绿素（Calcein）的特性，建立了一种实时的可视化环介导等温扩增方法（Loop-mediated isothermal amplification, LAMP），并与qPCR方法进行比较。实时可视化LAMP方法的反应结果，既可以肉眼直接观测，也可以借助荧光分析设备实时监测，并对半定量分析进行初探，为之后向定量分析深入研究打下基础。实验结果表明，实时可视化PMA-LAMP方法的灵敏度可达5.0×102 CFU/mL，模拟食样灵敏度为1.9×102 CFU/mL，与PMA-qPCR方法结果一致。除去前增菌时间，整个检测过程只需2 h，且准确率高，为副溶血性弧菌快速检测的开展提供了有效的技术支持。

Abstract:

Vibrio parahaemolyticus is a common foodborne pathogen frequently detected in seafood, and consumption of raw, pickled, or uncooked food contaminated with V. parahaemolyticus can cause food poisoning. Nearly all current methods for rapidly detecting V. parahaemolyticus can not distinguish live cells from dead cells, resulting in false-positive results. In this study, a real-time visual loop-mediated isothermal amplification (LAMP) method was developed based on the characteristics of the DNA binding dye propidium monoazide (PMA) and fluorescent dye calcein to detect viable V. parahaemolyticus strains. This method was compared to quantitative polymerase chain reaction (qPCR). The results of real-time visual PMA-LAMP assay could be observed by the naked eye and monitored in real-time using fluorescence analysis instruments. A preliminary semi-quantitative analysis was conducted, providing a basis for in-depth quantitative analysis. The results indicated that the sensitivities of the real-time visual PMA-LAMP assay in the sensitivity experiment and artificial contamination experiment were 5.0 × 102 CFU/mL and 1.9 × 102 CFU/mL, respectively, which were consistent with the values from PMA-qPCR. The entire detection process required only 2 h, excluding the pre-enrichment step. Furthermore, the method showed high accuracy and may be an effective technical method for rapid detection of V. parahaemolyticus.