Abstract: Objective To explore the application value of fluorescent PCR melting curve method in the identification of non-tuberculous mycobacteria (NTM), and to provide scientific basis for rapid and accurate diagnosis. Methods A total of 71 suspected NTM clinical isolates with positive BACTEC MGIT 960 liquid culture and negative MPB64 rapid antigen test were collected from Xi'an Chest Hospital from April to October 2020. Each clinical isolate was identified using the same specimen by fluorescent PCR melting curve method, gene chip method and 16S rRNA gene sequencing. The 16S rRNA gene sequencing results were used as reference standards to evaluate the detection efficiency of the fluorescent PCR melting curve method for strain identification. Results Detected by the melting curve method, the 67 NTM strains were mainly Mycobacterium tortoise/abscess (37.3%, 25/67) and Mycobacterium intracellular (25.3%, 17/67). The sensitivity, specificity and coincidence rate of Mycobacterium tortoise/abscess by fluorescence PCR melting curve method were 100.0% (24/24), 97.7% (42/43) and 98.5% (66/67), respectively; and those of Mycobacterium intracellular were 94.4% (17/18), 100.0% (49/49) and 98.5% (66/67), respectively. The consistency was 97.0% (65/67) rate between the fluorescence PCR melting curve method and the 16S rRNA gene sequencing method, and was 83.6% (56/67) between the gene chip method and the 16S rRNA gene sequencing method. Conclusion The fluorescence PCR melting curve method has high consistency with the gene sequencing method, it could be used for the rapid detection of mycobacterial species identification with good clinical application value.

Key words: Mycobacteria, Polymerase chain reaction, Microarray analysis, Gene sequencing