title: Comparative evaluation of polymerase chain reaction and restriction enzyme analysis: two amplified targets, hsp65 and rpoB, for identification of cultured mycobacteria.
abstract: The increasing incidence of tuberculosis and other mycobacterial infections due to AIDS epidemic resulted in the need of rapid and accurate identification of isolated mycobacteria. The correct identification result leads to the selection of an appropriate therapeutic regimen. Polymerase chain reaction and restriction enzyme analysis (PCR-REA) has been developed since 1992 and used as the rapid method for identifying mycobacteria. Several genes or sequences have been used as an amplified target for PCR-REA. The present study aims to evaluate the potential use of PCR-REA of gene-encoding heat shock protein 65 kDa (hsp65) and beta-subunit RNA polymerase (rpoB) for the identification of mycobacteria compared with conventional biochemical identification. Two hundreds clinical isolates, consisting of 50 isolates of Mycobacterium tuberculosis and 150 isolates of nontuberculous mycobacteria (NTM), were submitted for identification using PCR-REA and biochemical method. The results demonstrated that PCR-REA identified 188 isolates of both M. tuberculosis and NTM concordantly with biochemical identification. Discordant identification results obtained from 12 isolates, comprised of 8 M. scrofulaceum, 1 M. avium complex, 1 M. malmoense, 1 M. terrae complex, and 1 M. chelonae/abscessus. Overall, the concordant percentage of results obtained from PCR-REA compared with biochemical method was 100%, 98.8%, and 83.3% for M. tuberculosis complex, rapidly growing, and slowly growing mycobacteria, respectively, and the results of hsp65 PCR-REA was in agreement with those obtained from rpoB PCR-REA. From this study, PCR-REA appears to be a simple, rapid, and reliable method for identifying mycobacteria in a routine microbiology laboratory.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Wattana
authors.initials: W
authors.lastname: Cheunoy
authors.forename: Therdsak
authors.initials: T
authors.lastname: Prammananan
authors.forename: Angkana
authors.initials: A
authors.lastname: Chaiprasert
authors.forename: Suporn
authors.initials: S
authors.lastname: Foongladda
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques / Q000379:methods*; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D015870:Gene Expression; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping / Q000379:methods*; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; C074119:heat-shock protein 65, Mycobacterium; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D018833:Chaperonins