三种凝胶电泳分析分枝杆菌热休克蛋白65基因酶切片段的灵敏度比较

A comparison of the sensitivity of three gel electrophoresis methods for the RFLP analysis of mycobacterial heat shock protein 65 gene

目的比较2％（w，v）琼脂糖凝胶、2％（w，v）Metaphor琼脂糖凝胶和10％（w／v）非变性聚丙烯酰胺凝胶分析分枝杆菌热休克蛋白65（hsp65）基因酶切片段的灵敏度。方法分枝杆菌8株，分别制成10^6-10^1/ml菌悬液。采用通用引物对分枝杆菌hsp65基因序列进行PcR扩增，限制性内切酶BstEⅡ和HaeⅢ对扩增产物进行消化，分别采用2％琼脂糖凝胶、2％Metaphor琼脂糖凝胶和10％非变性聚丙烯酰胺凝胶电泳分析分枝杆菌酶切片段。结果经方差分析，3种检测方法的灵敏度差异有统计学意义（F=36．379，P〈O．01）。最小显著差异法（LsD）多重比较结果显示，2％琼脂糖凝胶方法的灵敏度显著低于2％Metaphor琼脂糖凝胶（P〈O．01），亦显著低于10％非变性聚丙烯酰胺凝胶方法（P〈O．01），而2％Metaphor琼脂糖凝胶与10％非变性聚丙烯酰胺凝胶方法间差异无统计学意义（P〉O．05）。结论2％Metaphor琼脂糖凝胶和10％非变性聚丙烯酰胺凝胶分析分枝杆菌酶切图谱的灵敏度均高于2％琼脂糖凝胶。

Objective To compare the performance of 2% （w/v） agarose gel, 2% （w/v） Metaphor agarose gel and 10%（w/v） nondenaturating polyaerylamide gel in the PCR-restriction fragment length polymorphism （RFLP） analysis of mycobacterial heat shock protein 65 （hsp65） gene. Methods This study included 8 Mycobacteria strains, including clinical isolates and standard strains of Mycobacteria tubercu]osis and Mycobacterium intracelluIare. Bacterial suspension of these strains was prepared with the concentration of bacterial cells varying from 10 to 106 per milliliter. PCR was performed to amplify the hsp65 gene with a pair of universal primers followed by the digestion of amplicons with two restriction endonucleases, BstE II and Hae HI. Then, the restriction enzyme-digested fragments were subjected to electrophoresis in 2% agarose gel, 2% Metaphor agarose gel and 10% nondenaturating polyaerylamide gel respectively. Results As analysis of variance showed, the three gel electrophoresis methods were statistically different in sensitivity for the RFLP analysis of mycobacterial hsp65 gene （F = 36.379, P 〈 0.01 ）. Least significance difference （LSD） procedure revealed that the 2% agarose gel-based eleetrophoresis was less sensitive than the 2% Metaphor agarose geland 10% non-denaturing polyacrylamide gel-based electrophoresis （both P 〈 0.01 ）, and no significant differences were observed between the 2% Metaphor agarose geland 10% non-denaturing polyacrylamide gel-based electrophoresis （P 〉 0.05）. Conclusion The 2% Metaphor agarose gel- and 10% nondenaturating polyacrylamide gel-based electrophoresis methods appear to be more sensitive than the 2% agarose gel-based electrophoresis method for the PCR-RFLP analysis of mycobacterial hsp65 gene.