克罗诺杆菌种间鉴定recN基因方法建立

目的 建立一种基于重组和修复蛋白基因(recN)的克罗诺杆菌种间鉴定方法,将不同来源的克罗诺杆菌分离株鉴定到种。方法 扩增recN基因全长序列,选取不同长度的recN基因片段,用MEGA 4.0软件对克罗诺杆菌8个参考菌株进行聚类分析,确定克罗诺杆菌种间鉴定可信度高、序列较短的片段;设计并合成该片段的引物,以8株参考菌株作为参照,构建44株克罗诺杆菌实验菌株的聚类分析图,并用生化反应鉴定对聚类结果进行佐证。结果 基于recN基因上一段640 bp的片段可对克罗诺杆菌不同种的菌株进行区分;以8株参考菌株在聚类图上的位置作为参考,根据遗传距离的远近,44株分离菌中有37株阪崎克罗诺杆菌,3株丙二酸盐阳性克罗诺杆菌,3株苏黎世克罗诺杆菌,1株尤尼沃斯克罗诺杆菌,与生化反应鉴定结果一致。结论 该方法与生化鉴定和基于recN全基因方法相比简便快速,PCR扩增后,一次测序反应就可以将克罗诺杆菌鉴定到种。

Species identification of Cronobacter spp.based on recN gene

Objective To establish a method for species identification of Cronobacter spp.based on recombination and repair protein gene(recN),and to identify Cronobacter strains isolated from different sources at species level by the method established.Methods The whole recN genes of 8 Cronobacter reference strains were amplified and sequenced.Different fragments of recN gene were selected according to their length and location.After comparison and analysis of the phylogenetic tree with MEGA 4.0,a credible and shorter renN gene sequence was chosen for species identification,and then 44 Cronobacter isolates and 8 reference strains were identified at species level and the results were proved by bioochemical reactions.Results Cronobacter strains can be identified at species level based on 640 bp gene sequence of recN.The location of 8 reference strains in the dendrogram was used as a reference according to the genetic distance.All the 44 Cronobacter isolates were identified at species level,including 37 strains of Cronobacter sakazakii,3 strains of Cronobacter malonaticus,3 strains of Cronobacter turicensis,and 1 strain of Cronobacter universalis.The results were coincident with biochemical reaction results.Conclusion The established method could be adopted to identify Cronobacter spp. species by one sequencing after PCR amplification,and it is simple and rapid compared with biochemical identification and the method based on the whole recN gene.