病原性鳗弧菌(Vibrio anguillarum)双重PCR与LAMP检测方法的建立

本研究检测了分离自发病大菱鲆、半滑舌鳎及鲤鱼的22株病原鳗弧菌(Vibrio anguillarum)毒力相关基因的携带情况，并建立了病原鳗弧菌的分子生物学检测方法。以PCR方法检测8个毒力相关基因的分布，结果显示，22株病原鳗弧菌均可扩增出6个基因(empA、vah1、vah4、flaA、rtxA和tonB)目的条带，未扩增出virA和angM基因；针对vah4和rtxA设计引物进行双重PCR扩增，同一PCR反应体系可扩增出两条目的条带，灵敏度为2.4×103 CFU/ml，对照菌无任何扩增条带；以vah4设计引物进行LAMP扩增，病原鳗弧菌可扩增出阶梯状条带，呈现阳性反应，6株对照菌无阶梯状扩增条带且呈现阴性反应，LAMP扩增灵敏度为2.4×101 CFU/ml。LAMP检测灵敏度是双重PCR的100倍，LAMP技术与PCR比较，操作简便、快速、灵敏度高且不需昂贵仪器，LAMP检测鳗弧菌的方法更适合于养殖生产实际应用。

Detection of Pathogenic Vibrio anguillarum by Using Duplex PCR and LAMP Assays

Vibrio anguillarum widely exists in aquatic environments and has been recognized as one of the prominent waterborne pathogens that undermine the aquaculture industry worldwide. In this study, we investigated the prevalent distribution of eight virulence-associated genes in the V. anguillarum strains isolated from Scophthalmus maximus, Cynoglossus semilaevis and Cyprinus carpio, and improved the detection of V. anguillarum by using duplex PCR and LAMP assays. Six genes (empA, vah1, vah4, flaA, rtxA, and tonB) were detected in all 22 pathogenic strains of V. anguillarum, but virA and angM were not detected. The duplex PCR assay was established with vah4 and rtxA genes as molecular markers. Two gene fragments from the chromosomal DNA of V. anguillarum were detected in one PCR reaction with the detection limit of 2.4×103 CFU/ml. The assay in 6 other control strains generated negative results. The LAMP assay was established with vah4 as the molecular marker. The positive reaction was shown as stair-step amplified bands and the detection limit was 2.4×101 CFU/ml. The assay produced negative reactions in 6 types of control pathogenic bacteria (no amplified bands). The LAMP method was 100 times more sensitive than the PCR method. Therefore we concluded that the LAMP assay could be a sensitive, rapid and simple tool for the detection of V. anguillarum, and recommend to employ this method in the early diagnosis of V. anguillarum infection in aquatic animals.