摘要
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 an enterobacterial repetitive intergenic consensus（ERIC）sequence-based polymerase chain reaction（PCR）system to identify and track Escherichia coli. Methods With extracted E.coli genomic DNA as a template,an L9（3^4）orthogonal experiment was designed to examine the effects that four factors,i.e.,Taq DNA polymerase,the Mg^2＋ concentration,the primer concentration,and the dNTP concentration,had on the results of ERIC-PCR amplification. Results The orthogonal experiment resulted in an optimized ERIC-PCR system as follows：a 25-μl ERICPCR system with 2.5μl of 10× PCR buffer,2.0mmol/L of Mg^2＋,200lmol/L of dNTP,0.2μlmol/L of primer,1.0U of Taq DNA polymerase,and 40 ng of template DNA.Taq DNA polymerase was the factor that most significantly influenced the experimental results,followed by the Mg^2＋concentration.In contrast,the primer concentration and dNTP concentration had little influence on the results.The DNA fingerprint obtained through use of this ERIC-PCR system displayed an abundance of distinct bands,and the technique was highly repeatable. Conclusion The optimized ERIC-PCR system was found to be consistent and reliable,which is important in molecular typing and molecular epidemiological study of E.coli.
出处
《中国病原生物学杂志》
CSCD
北大核心
2014年第9期788-790,共3页
Journal of Pathogen Biology
基金
山东省自然科学基金项目(No.ZR2012HQ005)
青岛大学引进人才资助项目(No.063-06300510)
