钩端螺旋体脉冲场凝胶电泳标准化技术的建立及谱型特征初步分析

Establishment of standardized method for Pulsed-field gel electrophoresis on Leptospira interrogans and analysis on their patterns

目的建立中国钩端螺旋体(钩体)脉冲场凝胶电泳(PFGE)的标准化操作程序及中国15群15型代表菌株的图谱数据库。方法参照目前美国疾病控制中心及亚太地区PulseNet提供的其他病原体PFGE标准化操作程序,结合钩体菌株的特性,对菌体基因组染色体DNA纯化技术、限制性内切酶消解方案及脉冲场电泳参数进行优化。结果利用标准化PFGE操作程序,建立了中国15群15型钩体代表菌株基因组DNA的NotⅠ酶切图谱,并对历年从四川、安徽省钩体病监测工作现场分离的部分黄疸出血群菌株进行PFGE分析,结果发现中国15群15型钩体代表菌株各自具有独特的谱型特征,24株黄疸出血群分离株存在3种谱型特征,91.67%(22/24)的分离株与黄疸出血群赖型(56601)的谱型匹配。结论建立的PFGE标准化操作程序制作钩体菌株的酶切图谱,具有图像清晰、分辨率高、各种大小的片段分布均匀等特点,能较好地反映出中国钩体菌株的分子遗传学特征,与传统的血清学分类存在较好的相关性。

Objective To establish a standardized operation procedure for pulsed-field gel eleetrophoresis （PFGE） on Leptospira interrogans as well as a figure digital database to develop the Chinese representative reference strains. Methods Under the characteristics of strains and referring to the other SOPs of PFGE on pathogens provided by CDC and PulseNet Asia Pacific, genomie chromosome DNA purification, restriction endonuelease digestion and the parameters for running PFGE were optimized. Results Not Ⅰ digestion patterns of leptospiral genome for the Chinese representative strains were established and partial isolates of serogroup ieterohaemorrhagiae from the leptospirosis surveillance in Siehuan and Anhui provinces were analyzed by PFGE. Results showed that each of all the 15 Chinese representative strains had a unique pattern. 91.67% （22/24） of the 24 isolates identified as serogroup ieterohaemorrhagiae matched to the map of the reference strain 56601 （serogroup ieterohaemorrhagiae serovar lai）. Conclusion The PFGE figures were clear with high resolution and the fragments were equally distributed by this standardized operating procedure so as to reveal the molecular-genetic characteristics of Leptospira interrogans. The patterns had high relativity with the serological identification and seemed to be very important for genetic analysis of strains in studying the outbreak of leptospirosis.