[Usefulness of multiplex real-time PCR and melting curve analysis in identification of nontuberculous mycobacteria].
BACKGROUND
Nontuberculous mycobacteria (NTM) should be correctly identified to the species level, because of different treatment plans among NTM species. This study was performed to assess the usefulness of real-time PCR and melting curve analysis in the identification of NTM.


METHODS
One hundred fifty-two clinical NTM isolates were identified to the species level by PCR-restriction fragment length polymorphism analysis (PRA). Those strains were then identified by multiplex real-time PCR and melting curve analysis on the 16S rRNA gene and hsp65 gene.


RESULTS
In the 16S rRNA gene fragment analysis, M. abscessus-M. chelonae group showed melting point at temperatures above 65 degrees C and M. avium complex (MAC; M. avium and M. intracelluare) below 48 degrees C, which differentiated M. abscessus-M. chelonae group and MAC from other NTM. In the hsp65 gene fragment analysis, M. abscessus-M. chelonae group was clearly divided into M. abscessus type I, M. abscessus type II, and M. chelonae according to the melting points at 61.25 degrees C, 66.06 degrees C, and 57.58 degrees C, respectively.


CONCLUSIONS
With the multiplex real-time PCR and melting curve analysis of 16S rRNA and hsp65 genes, M. abscessus and M. chelonae were readily identified and MAC were differentiated from other NTM. Especially, M. abscessus and M. chelonae, which were not differentiated from each other with the 16S rRNA gene fragment analysis, were identified with hsp65 gene fragment analysis.
Kang
Seong-Ho
SH
Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea.
Yoo
Kwang Cheol
KC
Park
Kyoung Un
KU
Song
Junghan
J
Kim
Eui Chong
EC
D001426:Bacterial Proteins / Q000235:genetics; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics; D003199:Computer Systems; D004269:DNA, Bacterial / Q000737:chemistry; D009170:Nontuberculous Mycobacteria / Q000235:genetics / Q000302:isolation & purification*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; C074119:heat-shock protein 65, Mycobacterium; D018833:Chaperonins