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目的:建立多重PCR(Multiplex PCR,MPCR)结合变性高效液相色谱(Denaturing High-Performance Liquid Chromatography,DHPLC)检测副溶血性弧菌的方法,并了解该菌携带毒力基因的情况。方法:根据副溶血性弧菌的毒力基因tdh、trh和种特异性基因toxR进行引物设计,以其单一PCR扩增产物在DHPLC出现的色谱峰作为标准色谱峰,建立MPCR-DHPLC的检测方法。结果:该方法能有效扩增副溶血性弧菌的tdh、trh和toxR基因,25株副溶血性弧菌的MPCR扩增产物经DHPLC分析所得的色谱峰在位置和峰型与标准色谱峰有很好的符合性,其他菌属的菌株均无特异性扩增,对模拟污染样品可正确检测,灵敏度达到1*103CFU/ml,特异性达到100%。结论:本文建立的MPCR-DHPLC方法可准确检测副溶血性弧菌,并了解该菌携带毒力基因的情况。
OBJECTIVE: To establish a multiplex PCR (MPCR) coupled with Denaturing High-Performance Liquid Chromatography (DHPLC) for the detection of Vibrio parahaemolyticus and to understand the virulence genes of this strain. Methods: According to the virulence genes tdh, trh of Vibrio parahaemolyticus and toxR of species-specific gene, the single peak of DHPLC was used as a standard chromatographic peak to establish the detection method of MPCR-DHPLC . Results: This method can effectively amplify the tdh, trh and toxR genes of Vibrio parahaemolyticus. The MPCR amplification products of 25 strains of Vibrio parahaemolyticus were analyzed by DHPLC at the position and peak type and the standard chromatographic peaks Very good compliance, other strains of bacteria have no specific amplification, the correct detection of simulated contaminated samples, the sensitivity of 1 * 103CFU / ml, the specificity of 100%. Conclusion: The MPCR-DHPLC method established in this paper can accurately detect Vibrio parahaemolyticus and understand the virulence genes of this strain.