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目的对大肠埃希菌ERIC-PCR反应体系进行优化。方法提取大肠埃希菌基因组DNA作为ERIC-PCR反应的模板,通过设计一个L9(34)正交试验对Mg2+浓度、dNTPs浓度、引物浓度和Taq DNA聚合酶4种因素进行优化。结果通过L9(34)正交试验优化的大肠埃希菌ERIC-PCR最佳反应体系(25μl)为:2.5μl 10×Loading buffer,Mg2+浓度2.0mmol/L,dNTPs浓度200μmol/L,引物浓度0.2μmol/L,Taq DNA聚合酶1.0U,模板DNA 40ng。应用该体系进行ERIC-PCR得到的DNA指纹图谱条带丰富、清晰、重复性好。结论采用正交试验优化的ERIC-PCR反应体系结果稳定可靠,对于大肠埃希菌在分子水平上的分型鉴定以及分子流行病学调查具有重要意义。
Objective To optimize the Escherichia coli ERIC-PCR reaction system. Methods The genomic DNA of Escherichia coli was extracted as a template for ERIC-PCR reaction. Four factors, Mg2 + concentration, dNTPs concentration, primer concentration and Taq DNA polymerase, were optimized by designing a L9 (34) orthogonal test. Results The optimum reaction system of Escherichia coli ERIC-PCR (25μl) optimized by L9 (34) orthogonal test was 2.5μl 10 × Loading buffer, the concentration of Mg2 + was 2.0mmol / L, the concentration of dNTPs was 200μmol / μmol / L, Taq DNA polymerase 1.0U, template DNA 40ng. The DNA fingerprinting bands obtained by ERIC-PCR were rich, clear and reproducible. Conclusion The results of ERIC-PCR reaction system optimized by orthogonal experiment are stable and reliable. It is of great significance for the identification of Escherichia coli at the molecular level and molecular epidemiological investigation.