Development of multiplex PCR assays based on the 16S-23S rRNA internal transcribed spacer for the detection of clinically relevant nontuberculous mycobacteria

作者：Ngan G J Y; Ng L M; Jureen R; Lin R T P; Teo J W P^*

来源：Letters in Applied Microbiology, 2011, 52(5): 546-554.

DOI：10.1111/j.1472-765X.2011.03045.x

• diagnosis
• mycobacteria
• polymerase chain reaction

Aims:
To accelerate the identification and differentiation of clinically relevant nontuberculous mycobacteria (NTM) with two sets of multiplex PCR (mPCR) targeting the 16S-23S rRNA internal transcribed spacer (ITS) region for timely patient management.
Methods and Results:
Two mPCR assays were developed: Slow-Growers (SG) mPCR was used for the detection of slow-growing mycobacteria, which included Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium gordonae and Mycobacterium xenopi whilst the other mPCR assay labelled as Fast-Growers (FG) mPCR was used for the detection of Mycobacterium fortuitum complex, Mycobacterium abscessus and Mycobacterium chelonae. In these assays, a common forward primer based on a conserved section of the 16S rRNA region was used in conjunction with species-specific reverse primers. The mPCRs were tested against 247 clinical mycobacterial isolates and demonstrated 100% specificity and sensitivity. Identification of the mycobacterial species was also validated by DNA sequencing of the 16S-23S ITS region and when further confirmation was needed, hsp65 sequencing was performed.
Conclusions:
The mPCR assays could be a potentially useful diagnostic tool for the rapid and accurate identification of clinically relevant NTM.
Significance and Impact of the Study:
In this study, we looked at the frequency of hospital isolated NTM over the last 5 years (2005-2010), and an mPCR targeting the ITS region was developed for NTM species that appeared to be more prevalent in the context of Singapore.

• 出版日期2011-5