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目的对5省份屠宰场鸡胴体中分离的空肠弯曲菌进行鉴定和分子分型,为建立空肠弯曲菌食物中毒的追踪溯源数据库提供科学数据。方法采用生化和分子生物学方法筛检和鉴定鸡专项监测网报送的鸡胴体中分离株。利用属特异16S rRNA基因和种特异的MapA、CeuE基因引物,建立多重PCR反应,鉴定空肠弯曲菌;参照国际认可的PFGE方法,选用内切酶SmaⅠ和KpnⅠ分别进行单酶切,并利用BioNumerics软件对分离株的电泳指纹图谱进行聚类分析,所得结果收入溯源数据库。结果对81株屠宰场鸡胴体分离株进行生化和PCR鉴定,确证72株为空肠弯曲菌,9株为结肠弯曲菌;脉冲场凝胶电泳分子分型结果表明,这72株空肠弯曲菌共产生48种带型。通过聚类分析,按照63.9%相似度将这些菌株分为A~M共13个群。每群包含1~11个带型,72株菌的带型分布具有完全地域同源性,即同一带型的菌株均来自同一省份。结论采用SmaⅠ和KpnⅠ单酶切进行PFGE分型,结合聚类分析,可提高PFGE分型的分辨率,增加菌株分型的准确性和溯源的可信性。
Objective To identify and molecularly classify Campylobacter jejuni isolated from carcass of slaughterhouse in five provinces and provide scientific data for establishing tracing and tracing database of Campylobacter jejuni food poisoning. Methods The biochemical and molecular biological methods were used to screen and identify the isolates from chicken carcasses submitted by the chicken special monitoring network. Multiplex PCR was performed to identify Campylobacter jejuni using 16S rRNA gene and species-specific MapA and CeuE gene primers. According to the internationally accepted PFGE method, single enzyme digestion was performed with restriction endonucleases SmaI and KpnⅠ, respectively. BioNumerics software The electrophoresis fingerprinting of the isolates was clustered and the results were collected into a traceability database. Results Eight hundred and seventy slaughterhouse chicken carcasses were identified by biochemical and PCR analysis, of which 72 strains were Campylobacter jejuni and 9 were Campylobacter coli. The molecular typing of pulsed-field gel electrophoresis showed that 72 strains of Campylobacter jejuni were co-produced 48 kinds of belt type. By cluster analysis, these strains were divided into A ~ M groups according to 63.9% similarity. Each group contains 1 ~ 11 bands, 72 strains of the band distribution has complete regional homology, that is the same band type strains are from the same province. Conclusion PFGE typing using SmaⅠ and Kpn Ⅰ digestion can improve the resolution of PFGE typing and increase the accuracy of typing and the credibility of traceability.