Development of a Novel PCR Primer to Differentiate and Identify Bacillus subtilis and Closely Related Species Isolated from Thai Fermented Foods

A novel PCR assay based on 16S-23S internal transcribed spacers (ITS) length polymorphism was developed for rapid differentiation and identification of the Bacillus subtilis group, especially B. subtilis, B. licheniformis and B. amyloliquefaciens, the most frequently isolated bacilli from fermented foods. A new group-specific conserved primer pair, CITS-F and CITS-R, was designed for specific amplification of the ITS region in B. subtilis and closely related species. The fingerprints of seven reference species, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus, B. atrophaeus, B. vallismortis and B. mojavensis using CITS-F and CITS-R primers showed the same four signature bands of 227, 400, 542 and 650 bp. They are different from those of other genera and species tested. Therefore, these four signature bands could be used to differentiate and identify the B. subtilis group. Moreover, the sequence of the 227 bp signature band could also be used to distinguish closely related species of the B. subtilis group used in this study. A novel PCR assay based on ITS length polymorphism pattern using CITS-F and CITS-R could be considered as a rapid and easy method for the primary differentiation of the B. subtilis group.