实时荧光定量PCR和PFGE在检测海产品中副溶血性弧菌的应用

发布时间：2017-12-27 02:08

本文关键词：实时荧光定量PCR和PFGE在检测海产品中副溶血性弧菌的应用　出处：《现代预防医学》2016年17期 　论文类型：期刊论文

【摘要】：目的应用实时荧光定量PCR(RT-PCR)筛检海产品中副溶血性弧菌,结合常规培养以提高检出率;应用PFGE对32株副溶血性弧菌进行分子分型分析。方法在常规培养的基础上,应用RT-PCR对96件样品增菌液进行初筛,再用常规培养法确证阳性;用阳性似然比与阴性似然比、符合率与Kappa值做为指标,评价RT-PCR筛检的真实性和可靠性;用限制性内切酶SfiⅠ对32株菌进行PFGE分子分型,用Bio Numerics Version 5.01软件对图像进行聚类分析。结果 96件样品常规培养法检出率24.00%,RT-PCR/常规培养法检出率33.33%。阳性似然比为3.88,阴性似然比为0.06。符合率为80.21%,中、高度一致(Kappa值=0.57);32株菌得到31个PFGE图谱类别,相似性在62.1%~100.0%之间,可分5个聚类群。结论 RT-PCR在检测海产品中副溶血性弧菌有很好的初筛作用,RT-PCR/常规培养法使用价值高;海产品中副溶血性弧菌亲缘关系较远,显示高度多态性。
[Abstract]:Objective to detect Vibrio parahaemolyticus in seafood by real-time fluorescence quantitative PCR (RT-PCR), and to improve the detection rate by routine culture. 32 strains of Vibrio parahaemolyticus were analyzed by PFGE. Methods on the basis of routine training on the application of RT-PCR in 96 samples of broth were screened, and the conventional culture method confirmed positive; positive likelihood ratio and negative likelihood ratio, the coincidence rate as the index and Kappa value, authenticity and reliability evaluation of RT-PCR screening; 32 strains were PFGE molecular typing with the restriction endonuclease Sfi, cluster analysis was used to detect Bio Numerics Version 5.01 software. Results the detection rate of the conventional culture of 96 samples was 24%, and the detection rate of the conventional culture method of RT-PCR/ was 33.33%. The positive likelihood ratio was 3.88, and the negative likelihood ratio was 0.06. The coincidence rate was 80.21%, middle and high (Kappa value =0.57); 32 strains obtained 31 PFGE map categories, similar to 62.1%~100.0%, can be divided into 5 cluster groups. Conclusion RT-PCR has a good initial screening effect in detecting Vibrio parahaemolyticus in seafood. RT-PCR/ has a high value in routine culture. The Vibrio parahaemolyticus in seafood is far away from each other, showing high polymorphism.
【作者单位】：北京市疾病预防控制中心食物中毒诊断溯源技术北京市重点实验室;北京市东城区疾病预防控制中心;
【基金】：国家科技重大专项(2012ZX10004215-003-001) 北京市高层次卫生技术人才培养计划(2013-3-099)
【分类号】：R440;R155.5
【正文快照】： 副溶血性弧菌(Vibrio parahaemolyticus,VP)属于弧菌科弧菌属,广泛存在于海产品中,是引起感染性腹泻和食源性疾患的重要病菌[1-2]。VP的首次检出就是1953年日本学者从海鱼中培养到的[3],截止到目前VP的检测主要依赖传统的常规培养法,也是公认的“金标准”。该方法检测能力有限

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