Abstract: ZHANG Cai-ping, WANG Hong-sheng*, FENG Yu-miao, LIN Lin, CUI Pan-gen, CHEN Min, WU Qin-xue. *Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding authors: LIN Lin, Email: dllon3@yahoo.com.cn；WANG Hong-sheng, Email:whs33@vip.sina.com 【Abstract】 Objective To compare the performance of 2% （w/v） agarose gel, 2% （w/v） Metaphor agarose gel and 10%（w/v） nondenaturating polyacrylamide 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 tuberculosis and Mycobacterium intracellulare. 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, BstEⅡand Hae Ⅲ. Then, the restriction enzyme-digested fragments were subjected to electrophoresis in 2% agarose gel, 2% Metaphor agarose gel and 10% nondenaturating polyacrylamide 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 electrophoresis was less sensitive than the 2% Metaphor agarose gel- and 10% non-denaturing polyacrylamide gel-based electrophoresis （both P < 0.01）, and no significant differences were observed between the 2% Metaphor agarose gel- and 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.