非脱羧勒克菌脉冲场凝胶电泳分子分型方法建立

Establishment of molecular typing by pulsed-field gel electrophoresis for Leclercia adcarboxglata

目的利用不同的内切酶和电泳条件评价非脱羧勒克菌的脉冲场凝胶电泳(PFGE)实验方法,建立一系列非脱羧勒克菌的PFGE方法。方法 2013—2015年从甘肃省某婴儿奶粉加工企业车间的环境和原料中分离非脱羧勒克菌,利用溶菌酶和蛋白酶K对其裂解后,选用不同的内切酶对胶块内的核酸进行酶切,脉冲场电泳后评价分析电泳图谱,得到最佳实验方案。结果经过几种内切酶的对比试验后,发现SpeⅠ酶在本试验方案中反应稳定,酶切后条带大约为17~21条,且分布均匀,更适合用于图谱分析。结论本研究确定了非脱羧勒克菌的PFGE方法,可以为非脱羧勒克菌在食品、药品等加工过程的污染以及医院感染监测,提供溯源技术手段。

Objective To evaluate experimental methods of pulsed-field gel electrophoresis（PFGE） for Leclercia adcarboxglata strains by using different restriction enzymes and electrophoreses conditions, to establish series PFGE methods.Methods Leclercia adcarboxglata strains were isolated from the environment and raw materials in a factory processing baby milk powder in Gansu province and then lysed by lysozyme and protease K. Electrophoresis was taken after digestion of nucleic acid with different restriction enzymes. Optimized experimental scheme was gotten after analyzing and evaluating the electrophoretogram of PFGE. Results Enzyme SpeⅠ displayed stable reaction in the experimental scheme after contrast test of several restriction enzymes. Aouht 17 to 21 bands distributed evenly after the digestion with different restriction enzymes and were more suitable for analysis of electrophoretogram. Conclusions PFGE test of Leclercia adcarboxglata established in this study provides traceability technology for surveillance of food and medicine pollution and nosocomial infection.