Development of a method for molecular subtyping Bacillus anthracis using HRM PCR

Abstract

Introduction. Bacillus anthracis is the causative agent of anthrax, a pathogen characterized by high genetic monomorphism that complicates differentiation of strains. Thus, molecular methods for pathogen typing require the improvement. The aim of the study. To select marker SNPs for new genetic groups of B. anthracis and to develop a method for their laboratory identification using HRM PCR. Materials and methods. The core genome of 222 strains of B. anthracis from the GenBank database and 66 strains from the collection of pathogenic microorganisms of the Stavropol Anti-Plague Institute was aligned using the parsnp software. A dendrogram based on 7242 core genome SNPs was built in MEGA X software. The strains for validation of the HRM method included representatives of various genetic groups. The HRM PCR reaction was performed using the "Type-it HRM PCR Kit" and "KAPA HRM FAST qPCR Kit" and a Rotor Gene DNA ther-mocycler with HRM function. Data analysis and visualization were performed using custom scripts in the Python and R development environments. Results and discussion. Marker SNPs for 6 genetic groups have been identified, which make it possible to determine whether strains belong to one of 7 new subclusters. Pairs of primers were selected for the loci containing them, HRM PCR parameters were optimized for discrimination of different alleles of SNP loci, and an analysis scheme was developed. Conclusion. Thus, marker SNPs were selected to determine the genetic subclusters A.Br.CEA, A.Br.STI, A.Br. Tsiankovskii, B.Br.Europe, B.Br.Siberia, B.Br.Asia, B.Br.018, and a new laboratory method was developed for molecular subtyping of B. anthracis using HRM PCR.