随机扩增多态性DNA分子标记技术在肠球菌分型中的应用

Application of Random Amplified Polymorphic DNA Molecular Marker Technique in Typing of Enterococcus

该研究旨在探索随机扩增多态性DNA(Random Amplified Polymorphic DNA,RAPD)分子标记技术在肠球菌种间快速分型的效果。试验通过筛选模板、单引物、双引物及RAPD反应体系,构建肠球菌种间分型体系,对32株已知肠球菌分型确定分型相似度标准,并通过对46株未知肠球菌进行种间分型以及管家基因rpoA测定验证分型效果。结果显示微波法提取基因组DNA满足实验需要,筛选出7条单引物用于双引物配对组合,最适的双引物反应体系为:2×PCR Taq Mix 12.5μL,模板DNA 1μL,引物各1μL,ddH_(2)O 9.5μL;分型效果最好的双引物组合为Primer1+Primer5,该引物组合可以在80%的相似度下区分肠球菌种间差异,Primer1+Primer3组合在500 bp位置扩增出海氏肠球菌的特征条带,可以作为海氏肠球菌的鉴定指标,用以快速鉴定海氏肠球菌。

The effect of random amplified polymorphic DNA(RAPD)molecular marker technique on rapid typing of Enterococcus species was investigated.An interspecific typing system of Enterococcus was constructed through screening the template,single primer,double primer and RAPD reaction system.The standard of strain similarity typing was established by typing 32 known strains of Enterococcus.Interspecific typing of 46 unknown strains of Enterococcus and testing of the house-keeping gene rpoA were performed to verify the effect of typing.The results showed that microwave-assisted extraction of the genomic DNA satisfied the experimental requirements,and seven single primers were selected for the paired combination of two primers.The optimal two-primer reaction system was:2×PCR Taq Mix 12.5μL;Template DNA and primer 1μL;ddH_(2)O 9.5μL.The optimal two-primer pair was Primer 1 and Primer 5,which allowed the distinguishment of Enterococcus species with 80%similarity.The primer pair of Primer1 and Primer3 can amplify the characteristic DNA segment of Enterococcus hirae at 500 bp,which can be used as the identification index for the rapid identification of Enterococcus hirae.