Rapid differentiation of mycobacteria by simplex real-time PCR with melting temperature calling analysis

AbstractAims: This study aimed to develop a rapid, simple and cost-effective methodfor the differentiation of Mycobacterium species.Methods and Results: A total of 80 clinical mycobacterial isolates belonging to12 different species and 16 reference strains of 16 different species weredifferentiated by the simplex real-time PCR coupled with melting temperaturecalling analysis. By comparing their melting profiles with those of the referencestrains, all clinical mycobacterial isolates were differentiated as Mycobacteriumtuberculosis complex or nontuberculous mycobacteria, and the latter werefurther divided into five groups. In comparison with 16S–23S internaltranscribed spacer sequencing method as the gold standard method, bothsensitivity and specificity of the assay were 100% when it was used for thedifferentiation between Myco. tuberculosis complex and nontuberculousmycobacteria.Conclusions: The simplex real-time PCR coupled with melting temperaturecalling analysis could be an alternative method for the differentiation betweenMyco. tuberculosis complex and nontuberculous mycobacteria.Significance and Impact of the Study: Rapid differentiation of mycobacteriacould shorten the diagnostic time of mycobacterial diseases. It is also helpfulfor achieving optimal therapy and appropriate patient management.IntroductionMycobacterium tuberculosis (MTB) is the causative agentof tuberculosis (TB) which is one of the leading infec-tious causes of death in developing countries (Parsonset al. 2011). Although tuberculosis is not endemic indeveloped countries, nontuberculous mycobacteria(NTM) are responsible for the majority of mycobacterialinfections in immunocompromised and immunocompe-tent individuals (Richardson et al. 2009). The differentia-tion and identification of Mycobacterium species isrequired for achieving optimal therapy and appropriatepatient management because of the differences in antimi-crobial susceptibilities among various Mycobacterium spe-cies. However, it is historically done using traditionalbiochemical method which is slow, cumbersome andtime-consuming. Several in-house and commercial assaysfor rapid identification of Mycobacterium species has beendeveloped, such as molecular biology tests, chromatogra-phy methods and matrix-assisted laser desorption/ioniza-tion time-of-flight mass spectrometry (MALDI-TOF-MS)(Hettick et al. 2006; Bille et al. 2012). At present, themajority of molecular biology tests for identification ofMycobacterium species are based on the utility of specificoligonucleotide probes (Barken et al. 2007). Thus, thesetests are laborious and costly like chromatography meth-ods although they are technically excellent.Recently, a duplex real-time PCR assay which targetsrpoB gene of mycobacteria has been developed for differ-entiating MTC from NTM (Williams et al. 2007). Theassay only uses SYBR green dye for melt curve analysisand does not require additional probes for hybridization.Primer sets are used to amplify a 235 bp region of theMTC rpoB gene and a 136 bp region of the NTM rpoBgene respectively. The mycobacterial strain could be dif-ferentiated as MTC or NTM according to the melting