Molecular detection of rpob gene in bacillus licheniformis via polymerase chain reaction assay / Ahmad Nor Aiman Mohamad Murad

Bacillus species is a rod shaped, Gram-positive bacteria which are abundantly found in various soil environments such as landfill, mangrove and agricultural soils. The bacteria are able to thrive in landfill site due to the high availability of organic and inorganic waste compared to other soils. The aims of this study were to isolate and detect Bacillus licheniformis which possess high potential in bioactivities such as plastic degradation and bioremediation of waste. The common method such as culture media and biochemical tests were used for identification of bacteria isolated from the soils. The landfill soil sample have the highest concentration of bacteria (42.046 x 1010 CFU/ml) compared to mangrove and agricultural soils. The Bacillus detected from these soil samples were Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Paenibacillus macerans and unidentified Bacillus. Among all the Bacillus spp. obtained, B. licheniformis known to have potential in environmental related bioactivities. The molecular approach was used to specifically identify Bacillus licheniformis with the use of bioinformatics tools and PCR. The target organism of Bacillus licheniformis was detected using rpoB gene. rpoB gene is a specific gene of Bacillus licheniformis which responsible for spore production mechanism against heat and encoding beta (β) subunit for most RNA polymerase. The bioinformatics tools used in this study was BLAST, ClustalW, OligoNucleotide and In-silico PCR amplification. The primers obtained was forward primer (5’-GCGTCGGTGATGAGGTTG-3’) and reverse primer (5’-CGTCTTTTACAAGGCGTTCG-3’). The PCR had carried out optimization of parameters for annealing temperature (Ta) and concentration of MgCl2. The results obtained the amplicons at 162 bp with optimal annealing temperature of 55.6 ºC and MgCl2 concentration of 4 mM. In conclusion, the identification of Bacillus licheniformis using PCR assay method via rpoB gene was a rapid, specific and practical method to be used for the species detection.
Keywords: Bacillus licheniformis, Polymerase chain reaction (PCR), PCR optimization, rpoB gene, Bioinformatics tools, landfill soil, mangrove soil