Novel diagnostic algorithm using tuf gene amplification and restriction fragment length polymorphism is promising tool for identification of nontuberculous mycobacteria.
Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infections in immunocompromised patients, making the reliable and rapid identification of NTM to the species level very important for the treatment of such patients. Therefore, this study evaluated the usefulness of the novel target genes tuf and tmRNA for the identification of NTM to the species level, using a PCRrestriction fragment length polymorphism analysis (PRA). A total of 44 reference strains and 17 clinical isolates of the genus Mycobacterium were used. The 741 bp or 744 bp tuf genes were amplified, restricted with two restriction enzymes (HaeIII/MboI), and sequenced. The tuf gene-PRA patterns were compared with those for the tmRNA (AvaII), hsp65 (HaeIII/HphI), rpoB (MspI/HaeIII), and 16S rRNA (HaeIII) genes. For the reference strains, the tuf gene-PRA yielded 43 HaeIII patterns, of which 35 (81.4%) showed unique patterns on the species level, whereas the tmRNA, hsp65, rpoB, and 16S rRNA-PRAs only showed 10 (23.3%), 32 (74.4%), 19 (44.2%), and 3 (7%) unique patterns after single digestion, respectively. The tuf gene-PRA produced a clear distinction between closely related NTM species, such as M. abscessus (557-84- 58) and M. chelonae (477-84-80-58), and M. kansasii (141- 136-80-63-58-54-51) and M. gastri (141-136-117-80-58-51). No difference was observed between the tuf-PRA patterns for the reference strains and clinical isolates. Thus, a diagnostic algorithm using a tuf gene-targeting PRA is a promising tool with more advantages than the previously used hsp65, rpoB, and 16S rRNA genes for the identification of NTM to the species level.
Division of Bacterial Respiratory Infection, Center for Infectious Diseases, National Institute of Health, Centers for Disease Control and Prevention, Seoul 122-701, Korea.
Ji-Hyun
JH
Shin
Eun-Jin
EJ
Cho
Jung-Yeon
JY
Lee
Jae-Yon
JY
Yu
Yeon-Ho
YH
Kang
D000465:Algorithms; D054458:Amplified Fragment Length Polymorphism Analysis; D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques / Q000379:methods; D001483:Base Sequence; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics; D004269:DNA, Bacterial / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D009164:Mycobacterium Infections / Q000175:diagnosis* / Q000382:microbiology; D010444:Peptide Elongation Factor Tu / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C074119:heat-shock protein 65, Mycobacterium; C096222:tmRNA; D018833:Chaperonins; D010444:Peptide Elongation Factor Tu