Preparation of positive control

Cultures of M. tuberculosis H37Ra [there are 18 copies of IS6110 in the genome of M. tuberculosis H37Ra (NCBI Reference Sequence: {"type":"entrez-nucleotide","attrs":{"text":"NZ_CP016972.1","term_id":"1130465480","term_text":"NZ_CP016972.1"}}NZ_CP016972.1)] were maintained on modified Roche Medium Base (Qiao Yu Biotechnology Co., Ltd., Shanghai, China). DNA was insolated based on the CTAB-phenol-chloroform method described by Mir AW, et al. (2008) with some modifications [21]. Briefly, the M. tuberculosis cells were harvested and re-suspended in 200 μL of TE buffer (Tris–EDTA), followed by incubation at 100°C for 10 min, mixing with 30 μL of 10% SDS and 10 μL Proteinase k (20 mg/ml), and finally incubation at 37°C for 1 h. Then, 100 μL of 5 M NaCl and 80 μL of 1.8% CTAB (cetyl trimethyl ammonium bromide) were added and incubated at 65°C for 10 min. After incubation, an equal volume of chloroform: isoamyl alcohol (24:1) was added, mixed thoroughly and centrifuged at 12,000 rpm for 5 min. The supernatant was carefully separated, an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added and then centrifuged at 12,000 rpm for another 5 min. The supernatant was transferred into a new tube and mixed with 500 μL of isopropanol, mixed and spun at 12,000 rpm for 5 min, the supernatant was discarded, and the pellets were washed with 500 μL of 75% ethanol, dried and re-suspended in 25 μL of TE buffer. Other extracted DNA samples from five non-tuberculous mycobacteria strains, M. phlei (ATCC 607), M. smegmatis (ATCC 19420), M. gastri (ATCC 15754), Escherichia coli O157 (ATCC 35150) and Staphylococcus aureus (ATCC 29213) were used in this study.