Comparative evaluation of primers and DNA extraction techniques for PCR amplification of 16s rDNA of bacterial isolates from petrochemical industrial effluent

Abstract

The investigation delved into a comparative assessment of primers employed in the polymerase chain reaction (PCR) for amplifying bacterial DNA sourced from diverse sampling locations within the effluent of Eleme Petrochemical Industry. Two extraction methodologies, namely ethanol precipitation and guanidium thiocyanate ethylenediaminetetraacetate (EDTA) sarcosyl buffer (GES), were scrutinized. Comparative analysis of DNA yield and quality indicated a superior recovery with the GES method in contrast to ethanol extraction. The prominent bacterial isolates, Pseudomonas and Bacillus, exhibited yields of 611.6 and 615.5 ng/μl and DNA quality of 25.35 and 24.55 ng/μl (260/280), 28.99 and 31.43 (260/230) using GES. Conversely, ethanol precipitation yielded 90.2 and 0.2 ng/μl with DNA quality of 1.65 and 0.21 (260/280), 1.57 and 0.00 (260/230) for Pseudomonas and Bacillus, respectively. Successful amplification, validated through ethidium bromide on 1% agarose gel electrophoresis, was achieved. Genus-level identification of isolated bacterial organisms encompassed Pseudomonas, Bacillus, Serratia, Micrococcus, Escherichia, Klebsiella, Vibrio, Proteus, Achromobacter, Citrobacter, and Flavobacterium. The comparative assessment of PCR primers unveiled V6V8F and V6V8R as the most suitable for amplifying bacterial DNA. These primers facilitated the amplification of 16S rDNA fragments from all bacterial isolates with an anticipated size of 500 base pairs.