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目的优化金黄色葡萄球菌细菌基因组重复序列PCR(REP-PCR)指纹图谱分型方法,并在临床分离株分型研究中应用,为构建金黄色葡萄球菌同源性追踪体系提供依据。方法试剂盒法提取金黄色葡萄球菌标准菌株ATCC 25923基因组DNA作为模板,根据参考文献设计REP引物,PCR扩增其基因组中靶序列。优化反应体系Mg2+浓度、模板量、引物浓度和退火温度,以得到最佳指纹图谱。以优化好的REP-PCR方法对10株金黄色葡萄球菌临床分离株进行分型。结果PCR反应体系Mg2+浓度2.5 mmol/L,DNA模板量125 ng/25μL,引物REP1R、REP2浓度各0.6μmol/L,退火温度40℃时,指纹图谱条带最清晰、明亮;10株临床分离株指纹图谱均在0.5~1.0 kb之间出现1条相同条带,其中1~3,4,5,8,9号菌株图谱相同;7和10号菌株除在0.5~1.0 kb出现1条条带外,还在1.0~1.5 kb出现1条条带,在1.5~2.0 kb出现1条条带;6号菌株共产生5条条带,分别出现在0.5~1.0 kb(2条),1.0~1.5 kb(2条)和1.5~2.0 kb(1条)。结论成功建立优化的金黄色葡萄球菌REP-PCR指纹图谱分型方法;10株临床分离株用该法分为3型。
Objective To optimize the method of REP-PCR fingerprinting of Staphylococcus aureus and to apply it in the typing analysis of clinical isolates, so as to provide a basis for constructing a homologous tracking system of Staphylococcus aureus. Methods Genomic DNA of Staphylococcus aureus standard strain ATCC 25923 was extracted as a template by the kit method. The REP primers were designed according to the references and the target sequences in the genome were amplified by PCR. Optimum reaction system Mg2 + concentration, template amount, primer concentration and annealing temperature, in order to get the best fingerprint. Ten strains of Staphylococcus aureus clinical isolates were screened by optimized REP-PCR. Results The concentration of Mg2 + in the PCR reaction system was 2.5 mmol / L, the amount of DNA template was 125 ng / 25 μL, the concentrations of REP1R and REP2 were 0.6 μmol / L, and the bands of fingerprint were the clearest and bright at the annealing temperature of 40 ℃. There was one identical band between 0.5 and 1.0 kb in the fingerprints, of which 1, 3, 4, 5, 8 and 9 were the same. The bands of 7 and 10 showed a band of 0.5-1.0 kb 1 band appeared at 1.0-1.5 kb and 1 band appeared at 1.5-2.0 kb. The 6 strains produced 5 bands at 0.5-1.0 kb (2 bands) and 1.0-1.5 kb (2) and 1.5 ~ 2.0 kb (1). Conclusion The optimized method for REP-PCR fingerprinting of Staphylococcus aureus was successfully established. Ten clinical isolates were divided into three types by this method.