Application and evaluation of a probe melting analysis-based assay for detection of Isoniazid-resistant mutations in Mycobacterium tuberculosis

Objective To validate the performance of a PMA-based assay for detection of INH-resistant mutations in MTB and investigate the mutation characteristic of INH-resistance.Methods The MTB standard strain H37Rv was from National Tuberculosis Reference Laboratory,1 wild-type strain and 1 katG S315T ACC mutant strain were from Xiamen CDC,7 MTB INH-resistant strains with known INH resistance mutations were from Shenzhen Center for Chronic Disease Control,Henan CDC,No.309 Hospital of PIA and Xiamen CDC.707 MTB clinical isolates were from Xiamen CDC,Xiamen No.1 Hospital and Zhangzhou CDC,126 sputum samples were from Xiamen Tongan CDC.The genomic DNA of the MTB standard strain H37Rv,7 MTB INH-resistant strains and 833 clinical samples,were extracted with Xiamen Zeesan Mycobacterium tuberculosis Isoniazid-resistance Mutation Test Kit using the thermal lysis method.The genomic DNA of 1 wild-type strain and 1 katG S315T ACC mutant strain were extracted with AxyPrep Bacterial Genomic DNA Miniprep Kit.The mutations were discriminated by the △Tm between the samples and wild type control in katG315 codon,inhA promoter -17 to -8 region,ahpC promoter region -44 to -30 and - 15 to 3,and inhA94 codon.A 10-fold dilution series of MTB DNA from 3 × 105 to 300 copies/ reaction obtained from a wild-type strain and a katG S315T ACC mutant strain,respectively,were prepared to determine the analytical sensitivity.Seven MTB INH-resistant strains with 9 predetermined mutations were used for the analytical specificity assay,and 5 mutants of which were used for the repeatability assay.The clinical detection performance of PMA assay were confirmed by the sequencing method in 833 samples.Results Results could be obtained within 3 hours from DNA extraction to PMA assay,including 46 samples in a standard 96-well real-time PCR instrument simultaneously.The analytical sensitivities of PMA were 300 copies/reaction for both the wild-type strain and katG S315T ACC mutant strain.Nine INH-resistant point mutations could be discriminated and 5 of which had standard deviations of melting temperature less than 0.5 ℃.Fully concordant results of mutant locus between PMA assay and sequencing were obtained in all 162 mutant samples.INH-resistant mutations in the four loci were found in 19.4％ (162/833) samples by PMA assay in Xiamen and Zhangzhou.Among the 14 lNH-resistant mutant types detected,katG S315T ( AGC→ACC),inhA promoter - 15C→T and katG S315N (AGC→AAC) accounted for 83.3％ (135/162) of the overall mutations. Key words: Mycobacterium tuberculosis ;  Isoniazid;  Drug resistance, bacterial;  Mutation;  Polymerase chain reaction