Use of novel specific primers targeted to pheS and tuf gene for species and subspecies identification and differentiation of the Bacillus subtilis subsp. subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis

To clearly delimit the members of the Bacillus subtilis group (BSG), is difficult using common phenotypic and genotypic methods. This study described the use of pheS and tuf gene as targets for interspecies discrimination within the BSG, and also to develop specific PCR and SNP primers for species and subspecies identification and differentiation. The average sequence similarity values of the pheS and tuf gene among type strains were 85.1 and 94.7%, respectively, and all members of the BSG could be clearly distinguished based on phylogenetic analyses of pheS gene sequence. In addition, the specific primers were designed according to pheS and tuf gene sequence. The primers were shown to specifically identify B. subtilis subsp. subtilis, B. amyloliquefaciens and Bacillus licheniformis, and clearly differentiate the subspecies of B. amyloliquefaciens using specific-PCR, combined with two-plex minisequencing method. In conclusion, we have successfully established a comparative sequence analysis and rapid molecular diagnosis techniques for determination of interspecies within the BSG. Key words: Bacillus subtilis group (BSG), species and subspecies discrimination, comparative sequence analysis, specific Pcr, two-plex minisequencing.