Development of a PCR based marker system for easy identification and classification of aerobic endospore forming bacilli

Restriction fragment length analysis of 16S rRNA gene of 52 different aerobic endospore forming Bacilli (AEFB) strains with HaeIII enzyme has revealed the presence of a 460 bp long fragment in 50 AEFB strains. BLAST analysis revealed that the fragment was 463 bp long and it was located at 3' end of 16S rRNA gene. Further specificity of this fragment for AEFB strains was checked by PCR and in silico methods. In PCR based method a primer pair (463 F and 463R) specific to this fragment was designed and this primer pair has shown amplification of 463 bp fragment in AEFB strains only. In in silico methods homology of primer pair and presence of restriction enzyme site in 16S rRNA genes were checked in 268 species of AEFB. Almost all species of AEFB have shown positive results for both of the tests. Further multiple alignments of 463 bp sequences of different species of AEFB have shown that it is a good marker for identification and classification of AEFB.