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目的对单核细胞增生李斯特菌脉冲场凝胶电泳(PFGE)分子分型的相关影响因素进行分析和探讨,并建立该菌的PFGE双酶切技术。方法分别利用限制性内切酶AscⅠ和ApaⅠ对1/2a、1/2b、1/2c、3a和4b型的单核细胞增生李斯特菌进行PFGE分析(包括1种品牌的AscⅠ和5种品牌的ApaⅠ),通过指纹图谱的比较,优化各种内切酶的实验参数。结果AscⅠ酶切产生11条左右清晰的DNA指纹图谱条带,ApaⅠ酶切产生的条带数量与之相似,但其中约10%的菌株(1/2a、1/2b血清型)均产生消化不完全的DNA片段。同时用5种品牌的ApaⅠ酶消化,并改变消化温度、提高酶浓度、延长酶消化时间均无法克服此现象。结论AscⅠ酶适于1/2a、1/2b、1/2c、3a和4b血清型的单核细胞增生李斯特菌的分型,但ApaI酶适于多数1/2a和1/2b血清型的分型。
Objective To analyze and discuss the related factors of molecular typing of Listeria monocytogenes pulsed-field gel electrophoresis (PFGE) and to establish PFGE double-digestion technique. Methods PFGE analysis of 1 / 2a, 1 / 2b, 1 / 2c, 3a and 4b Listeria monocytogenes were carried out by restriction endonucleases AscI and ApaI (including 1 brand of AscI and 5 brands ApaI), through the comparison of fingerprints, to optimize a variety of endonuclease experimental parameters. Results A total of 11 clear DNA fingerprinting bands were generated by AscⅠ digestion. The numbers of bands produced by ApaⅠ digestion were similar, but about 10% of the strains (1 / 2a and 1 / 2b serotypes) Complete DNA fragment. At the same time with five brands of Apa Ⅰ enzyme digestion, and change the digestion temperature, increase enzyme concentration, prolonged enzyme digestion time can not overcome this phenomenon. Conclusion The Asc Ⅰ enzyme is suitable for the typing of 1 / 2a, 1 / 2b, 1 / 2c, 3a and 4b serotypes of Listeria monocytogenes, but the ApaI enzyme is suitable for most 1 / 2a and 1 / 2b serotypes Type.