Abstract: Objective To develop a rapid method for the detection of Mycobacterium tuberculosis(MTB) durg-resistant genes of isonizai,rifampicin,streptomycin,ethambutol,pyrazinamide,and quinolones.Methods We designed 12 pairs primers and 54 probes by Oligo6.0,constructed a gene membrane chip for MTB drug-resistant gene detection by the combinatiion of multiplex PCR and reverse dot blot hybridization,and detected 52 clinical isolates of MTB.Results The 12 primes were divided into 4 reactons to establish a multiplex PCR reaction system under the same conditions.Then with reverse dot blot hybridization,a gene membrane chip of 54 oligonucleotide probes was developed and the chip included 36 wild-type probes,16 mutant probes,and a positive and a negative probe.The sensitivity of the MTB drug-resistant gene detection with the chip was 95.4% (41/43) and the specificity was 100%.Conclusion The gene membrane chip developed with the combination of multiplex PCR and reverse dot blot hybridization could be used to detect MTB drug-resistant gene effectively,and the method is rapid and convenient,and with good sensitivity and specificity for grassroot application.