Abstract: Objective To develop a PCR-microplate reverse hybridization("simulant chip") method for the detection and identification of several species of mycobacteria in cutaneous infection.Methods DNA was extracted from reference strains of 5 species(M.tuberculosis,M.avium,M.intracellulare,M. kansasii,M.fortuitum)of mycobacteria,and amplified by PCR targeting 16s rRNA.The coating concentra-tion and time of probes in wells,time and temperature of hybridization,and dilution fold of avidin-horseradish peroxidase were optimized.Five standard and 9 clinical strains of mycobacterium were dectected by"simulant chip"according to the optimized condition to test the sensitivity and specificity of this method.Results The followings were the optimum values:of concentration of probe,100 nmol/ml,for coated time,15 minutes, time and temperature of hybridization,45 minutes and 55℃,respectively,and of dilution fold of avidin-horseradish peroxidase,1:800.The detection limit of"simulant chip"was 1×10¹～1×10² cells/mL for these mycobacteria.No cross reaction was observed for the probes and other species of mycobacterium.Conclusion The"simulant chip"is a rapid,sensitive,specific and convenient method for the diagnosis of cutaneous mycobacterium infection.

Key words: Mycobacterium, Two-hybrid system techniques, Oligonucleotide array sequence analysis