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No. 85 (PMB 11)
Detection of Mycobacterium kansasii using amplicons generated by the COBAS
TaqMan 48 Analyzer
Arizumi Kikuchi 1 , Yuko Wakayama 1 , Takahiro Sawamura 1 , Satsuki Nakamura 2 , Shu Taga 2 , Yuji
Itoh 2 , Takeshi Seki 2 , Hiroya Sakuma 1,2 , Osami Daimaru 1,2 , Takeo Nakakita 1,2 , Shinichi Itoh 3
1 Daiyukai Second Medical and Science Research Laboratories, 2 Daiyukai General Hospital, 3 Daiyukai
First Hospital, Ichinomiya, Aichi, Japan
Mycobacterium detection using genetic techniques are rapid tests that are mainly based on nucleic acid
amplification. Many laboratories in Japan use the COBAS TaqMan 48 Analyzer (Roche Diagnostics), a
real-time PCR-based system that uses TaqMan hydrolysis probes. This system is efficient and easy to
use and has good sensitivity and specificity. However, this system can only detect the M. tuberculosis
and M. avium complex among mycobacteria. We examined methods to detect M. kansasii using the
amplicon generated by the COBAS TaqMan 48 Analyzer by real-time PCR using a hydrolysis probe.
We used the type strains of 7 mycobacterial species: M. avium, M. fortuitum, M. gastri, M. intracellular,
M. kansasii, M. marinum, and M. tuberculosis. We extracted the total nucleic acids from these type
strains and performed the COBAS TaqMan MTB Test (Roche Diagnostics) or the COBAS TaqMan
MAI Test (Roche Diagnostics) on a COBAS TaqMan 48 Analyzer. After the reaction, the amplicons
were cleaned using the High Pure PCR Product Purification Kit (Roche Diagnostics). The M. kansasii
detection primer pair was designed from the inner sequence of 16S rRNA, which was used in the
COBAS TaqMan 48 Analyzer. The detection probe used was highly specific for M. kansasii, and the
locked nucleic acids were incorporated into the probe to increase specificity. Real-time PCR was
performed using Light Cycler 1.5 (Roche Diagnostics).The primer and probe for M. kansasii detection
accepted the amplification of M. kansasii and M. gastri. M. kansasii lung disease shows the highest
response to chemotherapy, and the method for its treatment is better established than that for other
non-tuberculous mycobacterial infectious diseases. Since early and rapid detection of M. kansasii is
very important, we suggested that our method can be employed to detect M. kansasii using COBAS
TaqMan 48 Analyzer in routine laboratory work.
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