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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。
Objective To detect Vibrio parahaemolyticus (Vibrio parahaemolyticus) in seafood by real-time fluorescence quantitative PCR (RT-PCR) and to improve the detection rate by conventional culture. The molecular typing of Vibrio parahaemolyticus was analyzed by PFGE. Methods On the basis of routine culture, 96 samples were screened by RT-PCR and positive by routine culture method. The coincidence rate and Kappa value of positive likelihood ratio and negative likelihood ratio were used as indexes , The authenticity and reliability of RT-PCR screening were evaluated. 32 strains were subjected to PFGE by restriction enzyme SfiⅠ, and the images were analyzed by Bio Numerics Version 5.01 software. Results The detection rate of 96 samples was 24.00% by routine culture method and 33.33% by RT-PCR / routine culture method. The positive likelihood ratio was 3.88 and the negative likelihood ratio was 0.06. The coincidence rate was 80.21%, which was consistent between them (Kappa value = 0.57). There were 31 PFGE patterns in 32 strains with similarity between 62.1% and 100.0%, which could be divided into 5 clusters. Conclusion RT-PCR can detect Vibrio parahaemolyticus in seafood very well. RT-PCR / routine culture method has high value. Vibrio parahaemolyticus in marine products is distantly related and shows a high degree of polymorphism Sex.