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根据副溶血弧菌种特异性基因(tox R)、耐热直接溶血素基因(tdh)和相对耐热直接溶血素基因(trh)为靶基因分别设计引物,进行PCR扩增及反应条件的优化,建立了检测含有溶血素毒力基因的致病性副溶血弧菌的多重PCR方法。3对引物能分别特异性地扩增出368、269、486 bp的目的片段。副溶血弧菌均能扩增出tox R基因,不含溶血素基因的菌株(tdh-/trh-)仅扩增出1条目的片段,而含不同溶血素基因的致病性副溶血弧菌则分别扩增出2条(tdh+/trh-或tdh-/trh+)和3条(tdh+/trh+)特异性条带。检测其他非副溶血弧菌的供试菌,则不出现任何扩增条带。人工模拟样品检测结果显示对致病性副溶血弧菌的最低检测浓度为103CFU/m L。结果表明该多重PCR检测方法具有较好的特异性和灵敏性,对检测致病性副溶血弧菌有重要意义。
According to the target genes of toxR, tdh and trh, we designed the primers for PCR amplification and optimization of the reaction conditions , A multiplex PCR method for detecting pathogenic Vibrio parahaemolyticus containing hemolysin virulence genes was established. Three pairs of primers were able to amplify the specific fragments of 368,269,486 bp respectively. Vibrio parahaemolyticus was able to amplify tox R gene, only 1 fragment was amplified from the strain without hemolysin gene (tdh- / trh-), whereas pathogenic Vibrio parahaemolyticus with different hemolysin genes Two specific bands (tdh + / trh- or tdh- / trh +) and three (tdh + / trh +) bands were amplified respectively. Test other strains of non-Vibrio parahaemolyticus, then no amplification bands. The artificial simulated sample test results showed that the lowest detectable concentration of pathogenic Vibrio parahaemolyticus was 103 CFU / m L. The results showed that the multiplex PCR detection method has a good specificity and sensitivity for the detection of pathogenic Vibrio parahaemolyticus is of great significance.