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This study aimed to develop a rapid,simple,and cost-effective method for the differentiation of mycobacterium species.A total of 40 clinical mycobacterial isolates belonging to 12 different species and 16 reference strains of 16 different species were differentiated by the simplex real-time PCR coupled with melting temperature calling analysis.By comparing their melting profiles with those of reference strains,all clinical mycobacterial isolates were differentiated as Mycobacterium tuberculosis complex or nontuberculous mycobacteria,and the latter were further divided into 5 groups.In comparison with 16S-23S internal transcribed spacer sequencing method as the gold standard method,sensitivity,specificity,positive predictive value and negative predictive value of the assay were all 100% when it was used for the differentiation between Mycobacterium tuberculosis complex and nontuberculous mycobacteria.The simplex real-time PCR coupled with melting temperature calling analysis could be an alternative method for the differentiation between Mycobacterium tuberculosis complex and nontuberculous mycobacteria.