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目的建立快速准确检测致病性嗜水气单胞菌的双重荧光定量PCR方法。方法针对嗜水气单胞菌的16S rDNA和气溶素基因aerA的序列设计特异性引物及TaqMan探针,优化双重荧光定量PCR反应条件,并结合常规PCR方法及分离培养鉴定对临床样品进行检测和对比验证。结果荧光定量PCR反应体系对质粒标准品的敏感性为10拷贝/反应,是常规PCR检测方法的100倍;该方法检测12种其他种属细菌时未出现假阳性;对实际样品的检测结果其敏感性同样高于普通PCR,定量检测目的基因的拷贝数与样品中目的菌的分离率成正比。结论本方法敏感性高,特异性好,可用于致病性嗜水气单胞菌感染的诊断、疾病防控及流行病学研究。
Objective To establish a rapid and accurate double-fluorescence quantitative PCR method for the detection of pathogenic Aeromonas hydrophila. Methods Specific primers and TaqMan probes were designed according to the sequence of 16S rDNA and aerA of Aeromonas hydrophila, and the conditions of double fluorescence quantitative PCR were optimized. The clinical samples were detected by conventional PCR and isolation and culture identification Contrast verification. Results The sensitivity of the PCR system to plasmid standard was 10 copies / reaction, which was 100 times higher than that of the conventional PCR method. The detection of 12 kinds of bacteria from other species did not show any false positive. The detection results of the real samples The sensitivity is also higher than that of ordinary PCR, and the copy number of the target gene for quantitative detection is directly proportional to the isolation rate of the target bacteria in the sample. Conclusion The method is sensitive and specific and can be used for the diagnosis, disease prevention and epidemiology of pathogenic Aeromonas hydrophila.