检测创伤弧菌两种毒力基因的环介导等温扩增检测方法的建立

摘要

为建立快速检测致病性创伤弧菌两种毒素的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,分别以创伤弧菌溶血素A基因(hemolysin gene A,HA)和多重毒素(repeats in toxin,RTX)毒力基因的保守序列作为靶序列设计内、外引物.通过肉眼观察白色沉淀初步判断检测结果,通过琼脂糖电泳最终确定检测结果.LAMP检测创伤弧菌的两种基因的灵敏度都达到10 CFU/mL.特异性结果表明致病性创伤弧菌的结果为阳性,而其他几种常见弧菌和食源菌检测结果为阴性.在人工模拟样品检测中,LAMP结果显示,采用水煮法提取DNA,从样品处理到报告结果耗时1.5 h,鱼肉中创伤弧菌的检出限为1×102CFU/g(RTX)和1×104CFU/ g(HA);采用同样方法提取 DNA进行普通PCR,其检出限为1×103CFU/g(RTX)和1×104CFU/ g(HA),耗时4 h.结果表明,本研究建立的LAMP方法检测创伤弧菌两种毒力基因时具有灵敏度高、特异性强、方法简便等优点,适用于食品和养殖业中的现场快速检测.%In the present study, we developed and validated a loop-mediated isothermal amplification (LAMP) assay for detection of two toxins genes of Vibrio vulnificus.The inner and outer primers of LAMP were designed based on the conserved sequences of V.vulnificus hemolysin gene A (HA) and repeats-in-toxin (RTX) virulent gene. The detection method was preliminary evaluated by formation of the white deposition and confirmed through agarose gel electrophoresis. The sensitivity of this detection method was 10 CFU/mL for two LAMP genes of V.vulnificus.This method showed good specificity to V.vulnificus as it did not react with any other bacterial species.The bacterial DNA was extracted by water-boiling method and it took 1.5 h from sample processing to obtaining results. The detection limits were 102CFU/g for RTX and 104CFU/g for HA. On the contrast, the detection limit of common PCR method was 103CFU/g for RTX and 104CFU/g for HA and it took 4 h to complete test.This LAMP method may be used for on-site diagnostics of V.vulnificus in the food and agricultural industries.