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克罗诺杆菌是一种发现较晚的食源性致病菌。快速的分型方法对于食源性疾病暴发中致病菌的溯源和种内遗传变异具有重要的作用。本研究中,在充分考虑基因片段的长度、同源性、酶切位点及PCR扩增效率等因素的基础上,从65个候选基因(53个鞭毛相关基因和12个菌毛相关基因)中,筛选出2个菌毛相关基因(命名为P3和P4)作为限制性片段长度多态性(RFLP)分型的靶标基因。选择4种限制性内切酶,分别对2种基因进行限制性酶切,经非变性丙烯酰胺凝胶电泳和软件分析,对阪崎克罗诺杆菌进行8种PCR-RFLP分型,并分别构建系统树。将分型结果与BOX-PCR分型作比较,P3基因经限制内切酶HaeIII的分型结果与BOX-PCR分型结果具有较高的一致性。利用限制性内切酶HaeIII对P3基因进行酶切所建立的PCR-RFLP分型方法,可用于阪崎克罗诺杆菌的初步分型。
Cronobacter is a food-borne pathogen found later. The rapid typing method plays an important role in traceability and intraspecific genetic variation of pathogenic bacteria in outbreaks of foodborne diseases. In this study, 65 candidate genes (53 flagella-related genes and 12 pili-related genes) were selected based on the full consideration of the length, homology, restriction sites and PCR amplification efficiency of the gene fragments. Two pili-related genes (named P3 and P4) were screened as target genes for restriction fragment length polymorphism (RFLP) typing. Four kinds of restriction enzymes were selected and restriction endonuclease digestion was performed on the two genes respectively. Eight types of PCR-RFLP were detected by non-denaturing acrylamide gel electrophoresis and software analysis, respectively Build a system tree. Comparing the typing results with BOX-PCR typing, the genotyping result of P3 gene by restriction endonuclease HaeIII was highly consistent with BOX-PCR typing results. The PCR-RFLP typing method for restriction analysis of P3 gene by restriction endonuclease HaeIII can be used for the preliminary typing of Corynebacterium konjacs.