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The purpose of this study was to apply temperature-mediated heteroduplex analysis

The purpose of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in isolates and simultaneously differentiate between and gene targets from wild-type and wild-type strains (13 were PZA-resistant strains) and 21 strains (8 were BCG strains). 28). Although cases of PZA-resistant isolates with no mutations have been reported, mutations of and its putative promoter remain the major mechanism of PZA resistance (15, 20). Over 40 different mutations in either the structural gene or its putative promoter associated with PZA resistance in have been described. The changes are either mutations that involve substitution of nucleotides or mutations in the form of nucleotide insertions or deletions (15, 20, 27). In contrast, the natural resistance to PZA demonstrated by strains is uniformly due to a unique single-point mutation (C169G) in (9), the increasing frequency of tuberculosis infections following intravesical instillation of the naturally PZA-resistant BCG strain for the treatment of superficial bladder cancer (1, 17, 19), and the increasing incidence of zoonotic tuberculosis in developing countries due to naturally PZA-resistant (6, 16, 24). Conventional mycobacterial susceptibility testing for PZA is 51-21-8 manufacture dependent on growth of the organism in the presence of the drug. This technique is both time-consuming and potentially unreliable due to the poor growth of in the highly acidic medium required for PZA activity (7, 12). Automated testing systems, such as the BACTEC 460TB and BACTEC MGIT 960 systems, are more sensitive than conventional testing but require from 8 to 12 days to determine antibacterial susceptibility and have the potential for cross-contamination (12, 14, 31). Genotypic assays for the detection of drug resistance have been applied to both cultured isolates and direct patient specimens. Included in these are amplification methods, DNA sequence evaluation, PCR-single-strand conformation polymorphism electrophoresis, and structure-specific cleavage and DNA probe recognition assays, which can handle detecting mutations connected with medication level of resistance (8, 22, 30). Temperature-mediated heteroduplex evaluation (TMHA) using denaturing high-performance liquid chromatography (DHPLC) was originally put on the recognition of particular gene polymorphisms (21). The technology was lately put on the recognition of mutations connected with antituberculosis medication level of resistance (5). The technique used differential retention of homoduplex and heteroduplex DNAs under incomplete denaturing conditions for the identification of mutations in that are responsible for rifampin, isoniazid, streptomycin, ethambutol, and PZA resistance, respectively. Additionally, a separate genetic element (and was found to be problematic. The difficulty of detecting mutations was attributed to the diverse natures of the mutations and their distribution throughout the gene and its putative promoter. It was proposed that the potential for highly stable DNA helices due to increased GC content within specific regions of the gene represented a major technical challenge for TMHA methodology (5). To overcome these difficulties, the analysis conditions of the TMHA assay were reengineered, and a second probe was added. In 51-21-8 manufacture combination, ZNF914 these changes allowed the rapid identification of mutations associated with PZA resistance and the ability to distinguish between the two closely related species of the complex, and complex were studied: 48 strains, 13 of which were PZA resistant, and 21 strains, 8 of which were BCG strains. The PZA-resistant isolates were obtained from either the Tuberculosis Diagnostic Laboratory of the Centers for Disease Control and Prevention (CDC) (20) or the Tuberculosis Diagnostic Section of the Michigan Public Health Laboratory. The genes from 51-21-8 manufacture the 13 PZA-resistant strains had been sequenced and found to contain different mutations distributed throughout the open reading frame, as well as the promoter region (Fig. ?(Fig.1).1). The study isolates included six reference BCG strains (ATCC 35743, ATCC 35744, ATCC 35739, ATCC 35731, ATCC 35738, and ATCC 35748) from the CDC collection. Fifty clinical isolates were obtained from either Creighton University Medical Center (5 and 5 isolates), CDC (4 isolates), or the University of Nebraska Medical Center (UNMC) (4 BCG, and 30 isolates). Clinical isolates were identified as either or as previously described (6, 32) using the standard biochemical reactions, including nitrate reduction, niacin accumulation, and Pzase activity. PZA susceptibility was previously determined for all isolates, with resistance defined by an MIC of >25 g/ml using the proportion method with Middlebrook 7H10 medium (4). Two reference strains were used as probes in the TMHA study, H37Rv,.