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Abstract
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°C and M. avium complex (MAC; M. avium and M. intracelluare) below 48°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°C, 66.06°C, and 57.58°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.
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Table 1.
Primers and probes used in real-time polymerase chain reaction and melting curve analysis
Table 2.
Melting point temperatures of the 16S rRNA gene fragment in the 705 nm channel
Table 3.
Melting point temperatures of the hsp65 gene fragment in the 640 nm channel
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