基于PCR-RFLP技术鉴定常见食源性致病菌

Identification of common food-borne pathogens based on PCR-RFLP techniques

根据细菌核糖体基因16S rRNA保守端400 bp大小区段特征设计引物,应用聚合酶链式反应连接的限制性片段长度多态性(PCR-RFLP)方法建立一种新型灵敏的食源性致病菌鉴定技术。首先用特异性引物对9属19种细菌基因进行PCR扩增,扩增产物应用7种限制性内切酶Eco52Ⅰ、HindⅢ、MboⅠ、MluⅠ、PstⅠ、SalⅠ和XbaⅠ进行消化酶切,再利用琼脂糖凝胶电泳分辨酶切DNA片段大小数目,分析不同种属细菌酶切产物态型,从而进行基因型鉴别,针对16S rRNA基因片段利用RFLP进行分析,结果表明:19种致病菌表现出10种特异性的RFLP图谱,可准确鉴定区分9属细菌,最低检测限可以达到1 pg/μL。这证明PCR-RFLP技术可用于常见食源性致病菌的快速鉴定。

This study adopted a polymerase chain reaction-based restriction fragment length polymorphism （PCR-RFLP） method to analyze food-borne pathogens mitochondrial.16S rRNA from 19 kinds food-borne pathogens were amplified. With ribosomal gene 16S rRNA as the special attractant, a trail to digest the target gene by restrictive enzymes Eco52 Ⅰ, Hind Ⅲ, Mbo Ⅰ, Mlu Ⅰ, Pst Ⅰ, Sal Ⅰ and Xba Ⅰ was carried out after PCR amplification of a specific 400 bp region of this gene,and the restriction fragments were analyzed by agarose gel electrophoresis. The digested products of the 16S rRNA of different food-borne pathogens manifested in specific patterns. The lowest detection limit is 1 pg/μL. These results showed that the genotype verification of 16S rRNAs by PCR-RFLP method proved to be a good tool for detecting foodborne pathogens.