Molecular Identification of Rice Bran Isolate Bacteria Based on 16S rRNA Gene: A Comparative Study of DNA Extraction Methods

Abstract

DNA extraction serves as a critical preliminary step for molecular identification, and the application of such molecular techniques has become indispensable for analyzing bacterial diversity in rice bran isolates. This study aimed to confirm bacterial species based on 16S rRNA gene analysis. Two DNA isolation methods were compared: a chemical method utilizing Cetyl Trimethyl Ammonium Bromide (CTAB/NaCl) and an enzymatic method employing a commercial kit. The CTAB/NaCl method yielded DNA with a purity of 1.83 for isolate BR, while the enzymatic method produced higher purity values (2.04 for isolate TR). Amplification of the 16S rRNA gene was performed using universal primers 27F and 1492R. Sequence data were analyzed using BLAST-N and phylogenetic tree reconstruction software. BLAST-N analysis revealed that both BR and TR isolates belong to the genus Bacillus, with 99% identity values. Phylogenetic analysis further demonstrated that both isolates are closely related to Bacillus methylotrophicus strain CBMB205. The novelty of this research lies in the comparative evaluation of DNA extraction methods specifically optimized for rice bran isolates, a substrate with complex composition that poses unique challenges for DNA isolation, as well as the first molecular confirmation of Bacillus methylotrophicus from Indonesian rice bran.