Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory.
OBJECTIVE
To facilitate efficient identification of commonly encountered mycobacteria species (Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium fortuitum complex, Mycobacterium chelonae/abscessus, Mycobacterium kansasii, Mycobacterium gordonae) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated.


RESULTS
Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40 M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88.9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1.1 with an average turn around time of less than 3 days.


CONCLUSIONS
A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting.


CONCLUSIONS
With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.
Tuberculosis Reference Laboratory, Public Health Laboratory Service Branch, Centre for Health Protection, Department of Health, Hong Kong Special Administrative Region, Kowloon, Hong Kong.
K L
KL
Leung
C W
CW
Yip
W F
WF
Cheung
A C T
AC
Lo
W M
WM
Ko
K M
KM
Kam
D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes; D004269:DNA, Bacterial / Q000302:isolation & purification; D007753:Laboratories; D009161:Mycobacterium / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D010865:Pilot Projects; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013045:Species Specificity
D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S