Multi-probe real-time PCR identification of common Mycobacterium species in blood culture broth.
Mycobacterium tuberculosis complex, M. avium, and M. intracellulare are the most common causes of systemic bacterial infection in AIDS patients. To identify these mycobacterial isolates in primary blood culture broths, we developed a multiple hybridization probe-based real-time PCR assay using the LightCycler system. The primers were designed to amplify a 320-bp fragment of Mycobacterium 16S rRNA genes. Reaction specificity was evaluated using PCR amplification curves along with specific melting temperatures of probes on DNA extracted from 13 Mycobacterium species. In this study, results showed 100% accuracy for the selected bacterial panel. Detection limits were 350, 600, and 650 colony-forming unit (CFU)/ml blood culture broths for M. tuberculosis complex, M. avium, and M. intracellulare, respectively (1 to 2 CFU/reaction). To evaluate clinical applicability, 341 acid-fast bacilli in blood culture broths were analyzed. In total, 327 (96%) were positively identified: 54.5% M. tuberculosis complex, 37.5% M. avium, and 3.8% M. intracellulare. Results can be available within 3 hours of receiving a broth sample, which makes this rapid and simple assay an attractive diagnostic tool for clinical use.
Foongladda
Suporn
S
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address: sisfm@mahidol.ac.th.
Pholwat
Suporn
S
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Eampokalap
Boonchuay
B
Microbiology Laboratory, Bamrasnaradura Institute, Ministry of Public Health, Nonthaburi, Thailand.
Kiratisin
Pattarachai
P
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Sutthent
Ruengpung
R
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
D015373:Bacterial Typing Techniques; D001483:Base Sequence; D001769:Blood; D003470:Culture Media; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D009164:Mycobacterium Infections / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013997:Time Factors
D003470:Culture Media; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S