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Background: Tuberculosis (TB) remains an important cause of morbidity and mortality throughout the world. The surge of TB has been accompanied by an increase in multi-drug-resistant tuberculosis (MDR-TB). In this study, we developed a denaturing HPLC (DHPLC) method for detecting rpoB gene mutation as a rifampin resistance based on sequence., Methods: In this study, we used 99 mycobacterial isolates grown in Ogawa media. At first, we used a PCR method that can amplify the 235 bp and 136 bp rpoB DNAs of Mycobacterium tuberculosis complex (MTB) and Non-tuberculous mycobacteria (NTM). And then, PCR-restriction fragment length polymorphism (RFLP) of rpoB DNA (342 bp), which comprises the Rif(T) region, was used for the differential identification of Mycobacteria. Finally, we detected these amplicons by DHPLC, compared to PCR-RFLP results, and performed sequencing., Results: Among 99 mycobacterial isolates, 80 (81%) were MTB and 19 (19%) were NTM. NTM were identified to 7 different species by DHPLC and PCR-RFLP. rpoB mutation was detected in 9 (11%) of the MTB specimens. These results were confirmed by using sequencing., Conclusions: DHPLC provided a rapid, simple, and automatable performance for detection of rifampin resistant Mycobacterium tuberculosis complex and would be helpful as a supplemental method in high-throughput clinical laboratories.

Background: The aim of our study was to optimize and establish erythropoietin (EPO) enzyme linked immunosorbent assay (ELISA) system., Methods: We prepared several monoclonal and polyclonal antibodies specific to human-EPO. The best combinations of antibodies for coating and detecting antibodies were selected for the establishment of ELISA. We tested several methods such as a competitive EIA and a sandwich ELISA., Results: The best sandwich ELISA was optimized compared to competitive EIA when purified polyclonal antibody (PoAb) was used as a coating antibody and biotinylated PoAb as a detecting antibody. This sandwich ELISA easily detected EPO when PoAb pairs were used compared to the ELISA using monoclonal antibody and PoAb. There were no significant differences between the effects of various blocking solutions on the performance of sandwich ELISA using biotinylated antibody. The ELISA system using PBST containing 3% BSA as a blocking solution can sensitively detect EPO (10 mU/mL) in a broad range of EPO concentrations (10-2,000 mU/mL) and there were cross-reactions with other cytokines)., Conclusions: EPO can be easily determined by using biotinylated PoAb as a detecting antibody and another PoAb as a coating antibody.