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目的建立基于随机扩增多态性DNA分析(Randomly amplified polymorphic DNA,RAPD)的霍乱弧菌简便快速分子分型方法。方法选择16株霍乱弧菌代表性菌株,包括O_1群、O_(139)群和非O_1/非O_(139)群等血清群,通过40条随机引物的RAPD-PCR实验,筛选出理想的随机引物,要求其产生的DNA指纹图谱多态性高,并有足够的分辨力进行分子分型。结果通过一系列RAPD-PCR实验和统计学聚类分析,从40条随机引物中筛选出2条最符合要求的引物。16株霍乱弧菌的分析结果显示,2条引物对于O_1群和O_(139)群产毒株、O_1群非产毒株和非O_1/非O_(139)群均有很好的聚类分辨率。结论本研究筛选获得了2条随机引物并建立了基于RAPD的分子分型方法,该方法简便、省时、省力,适用于地方疾控部门和口岸检疫部门的基层实验室开展霍乱监测和流行病学溯源工作。
Objective To establish a simple and rapid molecular typing method for Vibrio cholerae based on Randomly amplified polymorphic DNA (RAPD). Methods 16 strains of Vibrio cholerae were selected, including serogroup O_1, O_ (139) and non-O_1 / non-O_ (139). Through randomized RAPD-PCR experiments with 40 random primers, Primers require that their DNA fingerprinting patterns be highly polymorphic and have adequate resolution for molecular typing. Results A series of random amplified polymorphic DNA (RAPD) -PCR and statistical clustering analysis were used to screen out the best primers from 40 random primers. The analysis of 16 strains of Vibrio cholera showed that the two primers had good clustering discrimination for O_1 and O_ (139) group producing strains, O_1 non-producing strains and non-O_1 / non-O_ (139) rate. Conclusion This study screened and obtained two random primers and established a molecular typing method based on RAPD. This method is simple, time-saving and labor-saving, and suitable for the local laboratories in the CDC and the port quarantine departments to carry out cholera surveillance and epidemiology Learn from the source of work.