嗜水气单胞菌双重荧光定量PCR检测方法的建立与初步应用

发布时间：2019-04-29 18:41

【摘要】：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。
[Abstract]:Objective to establish a double fluorescence quantitative PCR method for rapid and accurate detection of pathogenic Aeromonas hydrophila. Methods specific primers and TaqMan probes were designed for 16s rDNA and aerA sequences of Aeromonas hydrophila to optimize the dual fluorescence quantitative PCR reaction conditions. The clinical samples were detected and compared with routine PCR method and isolation culture identification. Results the sensitivity of the fluorescent quantitative PCR reaction system to the plasmid standard was 10 copies / reaction, which was 100 times higher than that of the conventional PCR method, and there was no false positive in the detection of 12 other species of bacteria by this method. The sensitivity of the actual sample is also higher than that of the ordinary PCR,. The copy number of the target gene is proportional to the isolation rate of the target bacteria in the sample. Conclusion the method has high sensitivity and specificity, and can be used for the diagnosis, disease control and epidemiological study of pathogenic Aeromonas hydrophila infection.
【作者单位】：军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室;吉林农业大学食品科学与工程学院;长春中医药大学附属医院;
【基金】：国家科技部传染病重大专项(No.2013ZX10004-217-002) 吉林省科技计划项目(No.20140101032JC,20150101110JC)联合资助~~
【分类号】：R440

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