武汉市市售淡水小龙虾中副溶血性弧菌的分离鉴定及多位点序列分型

摘要

采集武汉市各大农贸市场52份小龙虾(即克氏原螯虾,Procambarus clarkii)样品,利用1%氯化钠碱性蛋白胨水(APW)和TCBS对小龙虾样品中副溶血性弧菌(Vibrio parahaemolyticus)进行分离和初步鉴定,用PCR扩增副溶血性弧菌具有种特异性的tl基因来验证疑似菌株. PCR检测方法共检出14株副溶血性弧菌,检出率为26.92%. 在小龙虾中分离得到的14株副溶血性弧菌,对其进行基于dnaE-gyrB-recA-dtdS-pntA-pyrC-tnaA 的多位点序列分型. 其中dtdS的多态性位点比例均高于其他6个基因,为5.2%.7个基因串联后将14株副溶血性弧菌分为12个序列型,其中7株序列型未知,分辨力达0.967.可知武汉市市售淡水小龙虾中副溶血性弧菌呈现出较大的多样性. Neighbor-joining系统进化树将分离得到的14株副溶血性弧菌分为2个群,VP2﹑VP13﹑VP7和VP14同属一个群, 其他10株则属于另外一个群. 其中VP13和已知的临床分离株ST3在进化树上表现出较高的亲缘性(69%).%The Vibrio parahaemolyticus from 52 aquatic samples of Cambarus Clarkii collected from fish markets in Wuhan were isolated and identified by use of APW and Thiosulfate Citrate Bile Salts Agar (TCBS).And then the species specific gene(tl) of Vibrio parahaemolyticus was selected as target gene for PCR amplification and nucleic acid sequencing to examine the suspicious strains.14 strains were Vp positive with the detection rate being 26.92%.These Vp isolated from Cambarus Clarkii were selected for multilocus sequence typing (MLST) based on housekeeping genes(dnaE、gyrB、recA、dtdS、pntA、pyrC、tnaA).The ratio of polymorphic sites of dtdS (5.2%) was higher than the others.Concatenation of these seven genes was able to recognize 12 sequence types with discrimination index of 0.967.The results revealed a diversity of V ibrio para-haemolyticus isolated from Cambarus Clarkii sold in Wuhan.The Neighbor-joining phylogenetic tree divided these 14 strains of V ibrio parahaemolyticus into 2 oups.VP2,VP13,VP7 and VP14 belong to the same group, the others belong to another group.The phylogenetic tree showed a high affinity between one of these strains and the clinical isolate(ST3)(69%).