摘要
目的优化沙门菌基因间共有重复序列-PCR(ERIC-PCR)分子分型方法,分析沙门菌标准菌株及流行分离株基因组DNA ERIC-PCR指纹图谱。方法提取鼠伤寒沙门菌基因组DNA作为模板,根据ERIC核心片段设计引物,运用ERIC-PCR技术扩增沙门菌基因组中的靶序列,PCR产物经琼脂糖凝胶电泳后用凝胶成像分析仪对图谱进行观察分析。反应体系中模板量、Mg2+浓度、引物浓度和退火温度的优化实验则采用对优化项目设置梯度、其它条件不变的方法进行。以优化好的ERIC-PCR方法对16株沙门菌及1株大肠杆菌进行分析。结果在总体积为25μl的反应体系中,选择DNA模板量为100ng/25μl,Mg2+浓度为2.0mmol/L,引物ERIC1R、ERIC2浓度分别为0.4μmol/L,退火温度为52℃时,扩增产物的电泳图谱条带最清晰完整。不同血清群、型和来源的沙门菌株间基因组DNA ERIC-PCR指纹图谱带型差异较大,可在250～5000bp范围内出现3～13条条带,并在250bp处出现一条特征条带。结论优化了沙门菌ERIC-PCR的重要反应条件。ERIC-PCR法能区分不同来源的沙门菌,可用于沙门菌病的流行病学调查和同源性追踪,弥补传统分型方法的不足。
Objective To optimize the reaction conditions of enterobacteia repetitive intergenic consensus sequences-based PCR（ERIC-PCR）,a molecular typing method of Salmonella,study the genomic DNA ERIC-PCR fingerprint maps of Salmonella standard strains and epidemic isolates,and supply data for a system of Salmonella epidemiology investigation and homology tracing.Methods Genomic DNA of S.typhimurium was abstracted and used as the template for PCR.Enterobacteia repetitive intergenic consensus sequences were used as primers to amplify the target sequences in S.typhimurium genomic DNA.Amplification products were separated by agarose gel electrophoresis,and electrophoresis maps were analyzed by gel image analysis system.To optimize template concentration,Mg 2＋ concentration,primers concentration and annealing temperature of PCR,the factor to be optimized was designed in different concentration grads and other factors were fixed.Analyzed 16 Salmonella strains and one E.coli strain by PCR conditions optimized.Results The electrophoresis bands of amplified products were entire and most clear when template concentration was 100ng/25μl,Mg 2＋ concentration was 2.0mmol/L,primers concentrations were 0.4μmol/L respectively in the total volume of 25μl of the reaction system,and annealing temperature was 52℃.The ERIC-PCR fingerprint maps of different Salmonella strains with different sources were different.From 250 to 5000bp,there were 3 to 13 bands,and in those there was a specific 250bp band.Conclusion Important reaction conditions of ERIC-PCR had been optimized in this study.ERIC-PCR technique could discriminate Salmonella strains isolated from different regions.It could be used in Salmonella epidemiology investigation and homology tracing,so as to make up for the flaws of the traditional classification methods for salmonella.
出处
《卫生研究》
CAS
CSCD
北大核心
2008年第5期609-612,共4页
Journal of Hygiene Research
基金
四川大学华西公共卫生学院青年科学研究基金资助
关键词
沙门菌
基因间共有重复序列-PCR
流行病学
Salmonella,enterobacteia repetitive intergenic consensus sequences-based PCR
