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  

Application of RT-PCR and PFGE methods for detection of vibrio parahaemolyticus in seafood

作　　者：严寒秋[1] 李姗姗[2] 黄瑛[1] 曲梅[1] 张新[1] 吕冰[1] 陈丽娟[1] 王全意[1] 

机构地区：[1]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,北京100013 [2]北京市东城区疾病预防控制中心,北京100009

出　　处：《现代预防医学》2016年第17期3199-3202,共4页Modern Preventive Medicine

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)

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；用限制性内切酶靳I对32株菌进行PFGE分子分型，用BioNumericsVersion5．01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 improve the detection rate of vibrio parahaemolyticus by using the method of real Time PCR （ RT - PCR） , and to conduct the molecular classification analysis on the 32 strains of vibrio parahaemolytieus by using PFGE. Methods On the basis of conventional cuhure method, RT - PCR was applied in a sereening test for 96 samples,then the positive resuhs were verified by culture method. Positive likelihood ratio and negative likelihood ratio,agreement rate and Kappa value were used to evaluate the authenticity and reliability of RT - PCR. 32 strains of vibrio parahaemolyticus were molecularly subtyped by PFGE with the restriction endonuclease Sfi I , and obtained patterns were analyzed by the cluster analysis with BioNumeries Version 5.01 software. Results The positive rate of conventional culture method was 24.0% （23/96）, and in RT- PCR/culture method was 33.33% （32/96）. The positive likelihood rate was 3.88, and the negative likelihood rate was 0.06. Meanwhile,the agreement rate was 80.21% （77/96） ,and the Kappa was 0.57. 31 subtypes existed among 32 strains, and could be grouped into 5 clusters. The similarity was between 62.1% - 100.0%. Conclusion RT - PCR for detection of vibrio parahaemolyticus in seafood plays a very good role in early screening,and RT- PCR/cuhure method has a high value on use. The relationship of vibrio parahaemolytieus in seafood is relative farther,which shows high diversity.

关 键 词：实时荧光定量PCR PFGE 海产品 副溶血性弧菌 

分 类 号：R115[医药卫生—公共卫生与预防医学]