Use of PCR-restriction fragment length polymorphism analysis of the hsp65 gene for rapid identification of mycobacteria in Brazil.
Polymerase chain reaction amplification of part of the gene coding for the heat shock protein hsp65 followed by restriction enzyme analysis (PRA) is a recently described tool for rapid identification of mycobacteria. In this study, the speed and simplicity of PRA for identification of isolates of mycobacteria from patients with clinical symptoms of tuberculosis was evaluated and compared with identification results obtained by commercially available methods. Established PRA patterns were observed for nineteen isolates of Mycobacterium tuberculosis, eleven belonging to the complex M. avium-intracellulare, four of M. kansasii, one of M. fortuitum, one of M. abscessus, three of M. gordonae and one of the recently described species M. lentiflavum, as identified by commercially available methods. Two isolates of M. fortuitum and one of M. gordonae had unique and so far undescribed PRA patterns, suggesting geographically-related intra-species variation within the hsp65 sequence. We propose the inclusion of these new patterns in the PRA identification algorithm and have defined more accurately the molecular weight values of the restriction fragments. This is the first report on the isolation of M. lentiflavum in Brazil suggesting that identification by means of PRA could be useful for detection of mycobacterial species that are usually unnoticed. Where the use of several commercial techniques in combination was necessary for correct identification, PRA demonstrated to be a simple technique with good cost-benefit for characterization of all mycobacterial isolates in this study.
da Silva Rocha
A
A
Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
da Costa Leite
C
C
Torres
H M
HM
de Miranda
A B
AB
Pires Lopes
M Q
MQ
Degrave
W M
WM
Suffys
P N
PN
D001426:Bacterial Proteins; D001938:Brazil; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics; D009164:Mycobacterium Infections / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D014376:Tuberculosis / Q000382:microbiology
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; C074119:heat-shock protein 65, Mycobacterium; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases; C032823:GGTNACC-specific type II deoxyribonucleases; D018833:Chaperonins