Abstract
Infections caused by non-tuberculous mycobacteria (NTM) is increasing wordwide. Due to the difference in treatment of NTM infections and tuberculosis, rapid species identification of mycobacterial clinical isolates is necessary for the effective management of mycobacterial diseases treatment and their control strategy. In this study, a cost-effective technique, real-time PCR coupled with high-resolution melting (HRM) analysis, was developed for the differentiation of Mycobacterial species using a novel rpoBC sequence. A total of 107 mycobacterial isolates (nine references and 98 clinical isolates) were subjected to differentiation using rpoBC locus sequence in a real-time PCR-HRM assay scheme. From 98 Mycobacterium clinical isolates, 88 species (89.7%), were identified at the species level by rpoBC locus sequence analysis as a gold standard method. M. simiae was the most frequently encountered species (41 isolates), followed by M. fortuitum (20 isolates), M. tuberculosis (15 isolates), M. kansassi (10 isolates), M. abscessus group (5 isolates), M. avium (5 isolates), and M. chelonae and M. intracellulare one isolate each. The HRM analysis generated six unique specific groups representing M. tuberculosis complex, M. kansasii, M. simiae, M. fortuitum, M. abscessus–M. chelonae group, and M. avium complex. In conclusion, this study showed that the rpoBC-based real-time PCR followed by HRM analysis could differentiate the majority of mycobacterial species that are commonly encountered in clinical specimens.
Highlights
The genus Mycobacterium encompass several acid-fast bacilli (AFB), including Mycobacterium tuberculosis complex, Mycobacterium leprae, and non-tuberculous mycobacteria (NTM) (Bottai et al, 2014)
Multiplex real time PCR technique has been used for identification of mycobacterial species (Mokaddas and Ahmad, 2007; Richardson et al, 2009; Ngan et al, 2011; Nasr Esfahani et al, 2012), which a three or four species could be identified in a reaction at maximum
We developed an in-house PCR-High-resolution melting curve (HRM) assay targeting rpoBC locus, which could successfully differentiate the predominant Iranian clinical mycobacterial species, including M. tuberculosis, M. avium complex, M. fortuitum, M. kansasii, M. simiae, and
Summary
The genus Mycobacterium encompass several acid-fast bacilli (AFB), including Mycobacterium tuberculosis complex, Mycobacterium leprae, and non-tuberculous mycobacteria (NTM) (Bottai et al, 2014). HRM assay has been used as a simple, low cost and rapid method in mycobacterial research works such as investigation of drug resistance among M. tuberculosis (Pietzka et al, 2009; Ramirez et al, 2010), or mycobacterial species identification (Perng et al, 2012; Issa et al, 2014; Chen et al, 2017). Most of the latter analyses could only discriminate NTM into group or complex level. This target is used for the first time for species identification of mycobacteria
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