Detection of non-tuberculous mycobacteria DNA through droplet digital PCR: a preliminary study

Introduction: Traditional methods for nontuberculous mycobacteria diagnosis are time-consuming or characterized by low sensitivity. Here, we attempted a droplet digital PCR (ddPCR) method to rapidly identify and quantify Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MAB) DNA. Materials and Methods: Analytical sensitivity of MAB and MAC ddPCR assays (MAB: 16S and 23S; MAC: 23S and rpoB) was evaluated using 7 serial dilutions (one to 100,000 copies) of NTM DNA in two independent experiments. Probit analysis calculated the limit of detection (LOD) for a 95% positive result. Results: The intra-run coefficient of variation (CV) was 0.1% and 2.3% for MAC 23S and rpoB, and 2.8% and 3.1% for MAB 16S and 23S. The inter-run CV was 2.3% and 5.8% for the MAC targets, and 4.0% and 3.4% for the MAB targets. MAC and MAB assays showed a good linear correlation between the expected and observed results. The mean R2 were 0.998 and 0.993 for the MAC and MAB intra-runs, respectively, and 0.988 and 0.992 for the MAC and MAB inter-runs, respectively. The assay detected and quantified NTM DNA until at concentrations as low as 102 copies/80µl of eluate for both assays. In particular, LOD for MAC was 169 copies/80µl for rpoB and 148 copies/80µl for 23s, while for MAB was 50 copies/80µl for 16s and 83 copies/80µl for 23s. Conclusion: Preliminary results show good reproducibility for both tests. Thus, ddPCR has the potential to provide a highly sensitive, accurate, and rapid method for the diagnosis of NTM infection.