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目的建立霍乱弧菌多重PCR-变性高效液相色谱(DHPLC)快速分型方法。方法分别合成扩增霍乱弧菌胶原酶基因(vcc基因)、O1群和O139群的毒力基因(ctxA基因和tcpA基因)以及O139群毒力基因(LPSgt基因),并对4对引物的PCR退火温度进行优化,建立多重PCR-DHPLC分型方法。结果4种基因可同时被扩增,应用多重PCR-DHPLC方法对霍乱弧菌进行分型的结果与预期的一致。结论多重PCR-DHPLC分型方法可用于快速、准确地鉴定细菌纯培养物是否为霍乱弧菌以及具体的菌群。
Objective To establish a multiplex PCR-denaturing high performance liquid chromatography (DHPLC) rapid typing method for Vibrio cholerae. Methods The virulence genes (ctxA gene and tcpA gene) and O139 group virulence gene (LPSgt gene) of Vibrio cholerae (VCC) gene, O1 group and O139 group were amplified by polymerase chain reaction (PCR) Annealing temperature to establish multiple PCR-DHPLC typing method. Results Four genes could be amplified at the same time. The results of multiplex PCR-DHPLC typing of V. cholerae were in accordance with expectation. Conclusion The multiplex PCR-DHPLC typing method can be used to quickly and accurately identify whether the pure culture of bacteria is Vibrio cholerae and specific bacterial flora.