[ODP-021] NGSを併用したNested Multiplex PCRによるらい菌の薬剤耐性および型別の迅速同定法
Mycobacterium leprae is the main causative agent of leprosy worldwide, and its genotype can be classified into four single nucleotide polymorphism (SNP) types and 16 subtypes. Determining drug resistance and genotype of M. leprae is typically conducted by PCR and Sanger DNA sequencing, however, it requires a huge amount of effort. In this study, we developed a rapid method for identification of M. leprae drug resistance and SNP genotype directly from clinical specimens by combining nested multiplex PCR with next generation sequence analysis. We used this method to analyze clinical samples from two paucibacillary, nine multibacillary, and six type-undetermined leprosy patients. From these, we amplified drug resistance determining regions (DRDR) of folP1, rpoB, gyrA and gyrB, and regions of 84 SNP-InDels in the M. leprae genome and their sequences were determined. The results showed that seven samples were subtype 1A, three were 1D, and seven were 3K. Three samples of the subtype 3K had folp1 mutation. Of the three, two had an A to G substitution at nucleotide 157 (Thr53Ala) in folp1 and one had a C to T substitution at nucleotide 164 (Pro55Leu) in folp1. No mutations were detected in the DRDRs of rpoB, gyrA, and gyrB in all the samples. This method can be implemented as more rapid genetic analyses of M. leprae in clinical samples.