金黄色葡萄球菌REP-PCR指纹图谱分型方法优化

Optimization of REP-PCR fingerprint map classification for Staphylococcus aureus

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 reaction conditions of repetitive extragenic palindromic PCR （REP-PCR） fingerprint map classification for Staphylococcus aureus and to apply the method in the classification of S. aureus clinic isolates and supply data for S. aureus homology tracing. Methods Genomic DNA of S. aureus reference strain ATCC 25923 was abstracted with bacteria DNA abstract kit and used as the template for PCR. Target sequences in S. aureus genomic DNA were amplified with the primers designed according to the reference. To obtain optimum fingerprint maps, template, Mg2^＋ , and primer concentrations of the reaction system and annealing temperature of PCR were optimized. Then 10 clinic strains of S. aureus were classified by optimum PCR. Results The electrophoresis bands of amplified products were most clear when 25μL of the reaction system contained 125 ng template,2.5 mmol/L Mg2^＋ ,0.6 p.mol/L each primer,and primers were annealed at 40 ℃. Each fingerprint maps of ten strains of clinic isolates showed a band between 0.5 kb to 1.0 kb. The fingerprint maps of number 1,2,3,4,5,8 ,and 9 strain were same,and the maps of number 7 and 10 strain were also same. The fingerprint map of number 7 and 10 strain showed 3 bands, one in 5.0 kb - 1.0 kb, one in 1.0 - 1.5 kb and one in 1.5 - 2. 0 kb. The fmgerprint map of number 6 strain showed 4 bands, of which two laid between 0. 5 kb and 1.0 kb, one laid between 1.0 kb and 1.5 kb and one between 1.5 kb and 2. 0 kb. Conclusion The optimum REP-PCR fingerprint map classification method of S. aureus was obtained and ten clinic strains of S. aureus were classified into 3 types by the classification method.