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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2.4×10~1 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。
In this study, 22 virulence-associated genes of Vibrio anguillarum isolated from turbot, halibut and carp were detected and the molecular biological method was established. The distribution of 8 virulence-related genes was detected by PCR. The results showed that all the 22 strains of pathogenic Vibrio anguillarum could amplify the target bands of 6 genes (empA, vah1, vah4, flaA, rtxA and tonB) without amplification VirA and angM genes were amplified. Two primers were designed for vah4 and rtxA. The two PCR products were amplified by the same PCR reaction, with sensitivity of 2.4 × 103 CFU / ml and no amplified bands of control. The primers of vah4 were designed to amplify LAMP. The pathogenic Vibrio anguillarum could amplify the ladder-like bands, showing a positive reaction. The six control strains had no ladder-like amplification bands and showed a negative reaction. The LAMP amplification sensitivity was 2.4 × 10 ~ 1 CFU / ml. LAMP detection sensitivity is 100 times that of double PCR. Compared with PCR, LAMP technology is simple and fast in operation, high in sensitivity and does not require expensive instruments. The LAMP method for detecting Vibrio anguillarum is more suitable for practical application in breeding production.