溶藻弧菌相关分离株的分子及VITEK鉴定  

Molecular and VITEK identification of Vibrio alginolyticus-related isolates

作　　者：郭静[1] Liswaniso Gadafii 郭安南[1] 阎永伟[1] 张德民[1] 裘琼芬[1] 黄素文[2] 戴海平 

机构地区：[1]宁波大学应用海洋生物技术教育部重点实验室,浙江宁波315211 [2]宁波出入境检验检疫局,浙江宁波315012 [3]浙江省宁波市鄞州区渔业技术管理服务站,浙江宁波315012

出　　处：《水产学报》2012年第3期383-390,共8页Journal of Fisheries of China

基　　金：国家自然科学基金项目(30970104);浙江省自然科学基金人才培养项目(R305333);宁波市科技计划项目(2010C10013);宁波大学人才工程项目(2010859);宁波大学科研基金(xkl116)

摘　　要：哈维群弧菌是弧菌属的核心菌群,包括溶藻弧菌在内的6个种在表型和遗传型上均十分相似,要准确鉴定各种有一定难度。看家基因的研究及生化鉴定系统的出现为弧菌鉴定提供了多种方法,本文比较了16S rRNA基因、toxR基因和pyrH基因以及VITEK 2 COMPACT GN鉴定卡对溶藻弧菌相关分离株的分辨力。哈维群弧菌基因组内16S rRNA基因是多拷贝的,且拷贝间序列差异大于种间差异,不适用于种的鉴定。单拷贝基因toxR和pyrH序列种内差异均小于种间差异。基于toxR基因相似性比较可清楚地将18个疑似溶藻弧菌分离株和5个参比株归并到4个种;toxR基因系统发育学分析也显示,哈维群弧菌各种独立地聚类分支,且溶藻弧菌种内存在两个明显的聚类分支,说明两个分支的溶藻弧菌具有独立的进化方向。pyrH基因的相似性比较和发育学分析也得到类似的结果,但pyrH基因具有较强的保守性,因而分辨力稍低于toxR基因。VITEK 2 COMPACT GN鉴定卡在鉴定溶藻弧菌时也有一定的错误率,且鉴定谱较窄。因此,建议在实际应用中采用toxR基因比对作为溶藻弧菌快速鉴定的主要手段,可再选用pyrH基因对鉴定结果进行验证。Vibrio alginolyticus,a member of the core group of the genus Vibrio,also called V.harveyi group,shares high phenotypic and genotypic resemblance with the other species of this group.It is very difficult to identify strains of V.alginolyticus from others.The housekeeping genes and various automated systems based on biochemical characteristics of bacteria provide more alternatives for identification.In this study,we compared the resolution of 16S rRNA gene,toxR gene,pyrH gene and VITEK 2 COMPACT GN card on V.alginolyticus-related isolates.There are multiple copies of 16S rRNA gene in the genome of Vibrio and the intragenomic heterogeneity among different copies might be higher than intergenomic heterogeneity in some strains.These facts make the 16S rRNA gene not suitable for the discrimination of V.harveyi group species.Both of single-copy genes toxR and pyrH show a considerable gap between the maximal interspecies similarity and the minimal intraspecies similarity.The toxR gene similarity analyses of eighteen isolates and five reference strains can clearly group them into four species.The phylogenetic tree based on toxR gene shows that strains of individual species clustered together independently,and there are two different clusters within V.alginolyticus,indicating that they have different evolving paths.The similarity and phylogenetic analyses based on pyrH gene come to the same result of toxR gene.The discriminatory power of pyrH gene is slightly lower than that of toxR gene.VITEK 2 COMPACT GN card presents one misidentification among 9 isolates of V.alginolyticus and its species database does not include V.campbellii,V.rotiferianus and V.natriegens.We suggested that toxR gene could be the first choice for fast identification of V.alginolyticus isolates,and pyrH gene may be used for verification.

关 键 词：溶藻弧菌 16S RDNA toxR pyrH VITEK2COMPACT 

分 类 号：S917[农业科学—水产科学] Q93-331[生物学—微生物学]