AimsThe purpose of this study was to evaluate the usefulness of REBA Myco-ID (R) assay based on the principle of reverse blot hybridization assay (REBA) for the rapid detection of Mycobacterium tuberculosis (MTB), the differentiation of MTB from nontuberculous mycobacteria (NTM) and the identification of NTM species in liquid cultures. Methods and ResultsA total of 274 mycobacterial liquid cultures that cultured from respiratory specimens including 94 acid-fast bacilli (AFB) smear positives and 180 AFB smear negatives were used in this study. The results of PCR-REBA were compared with those of real-time PCR and PCR-Restriction fragment length polymorphism (RFLP) assays. Sensitivity of 195 MTB and 79 NTM strains determined using DNA isolated from liquid cultures was 100% and 975%, respectively. However, two of the liquid culture NTM specimens were not detected by PCR-REBA and were identified as Rhodococcus jostii and R.erythropolis by PRA Myco-ID and rpoB gene sequence analysis. Frequently identified NTM species in the liquid cultures were Mycobacterium avium complex (n=50, 633%) and Mycobacterium abscessus complex (n=11, 139%). The PCR-REBA is able to identify mycobacterial strains more rapidly than conventional tests and does not require specialized instruments and is possible to differentiate between Myc.abscessus and Mycobacterium massiliense strains. ConclusionsPCR-REBA assay showed rapid, highly sensitive and specific results for the identification of MTB and NTM and discriminated between NTM species from mycobacterial liquid cultures. Significance and Impact of the StudyEven though the use of molecular diagnostic technologies is more expensive than the use of conventional methods, the clinical and economic benefit of saving time in regard to expenses remains to be elucidated. Therefore, the PCR-REBA may provide the essential information to accelerate therapeutic decisions for appropriate antibiotic treatments in the acute phase of mycobacterial diseases.