Abstract: Objective To explore a new method for supplementary evaluation on the efficiency of mycobacteria laboratory's terminal disinfection.Methods First,Mycobacterium complex specific insert sequence IS6110 was used as a target sequence to design mycobacteria specific primer IS6110P independently,and the culture of mycobacteria standard strain H37Rv was used to test the primer's specificity and sensitivity. Second,H37Rv cultivation material pumping filtration membranes were used to evaluate the common nucleotide extraction and genome extraction after formaldehyde terminal disinfection for primer chain amplification. Third,both swab and air suction filter samples were collected to extract nucleotide for primer chain amplification by traditional method and genome extraction method separately. At the same time,mycobacteria were cultured for all of the samples to evaluate the efficiency of different methods.Results Mycobacteria nucleotide could be amplified sensitively and specifically with IS6110P and the limit of the detection was as low as 3.5 colony forming unit (cfu)/R. After terminal disinfection,the PCR results of mycobacteria laboratory's smear samples were still positive with traditional method but negative with genome extraction method;meanwhile the mycobacteria culture tests were all negative. Conclusion Using IS6110P as primers to amplify nucleotide extracted from samples of mycobactria laboratory with genome extraction is a sensitive,convenient and reliable evaluation method for the evaluation on the efficiency of mycobacteria laboratory's terminal disinfection.