Validasi Desain Primer Deteksi Gen ndmA Enzim Demetilase pada Isolat Bakteri Pendegradasi Kafein secara in Vitro

Abstract

Caffeine-degrading bacteria possess the N-demethylase enzyme, which plays a role in catalyzing the conversion of caffeine into simpler compounds. This enzyme is involved in the N-demethylation process, a mechanism of caffeine degradation that removes the methyl group from caffeine. The N-demethylase enzyme is encoded by the ndmA gene. The ndmA gene is one of the target genes that can be used to detect a bacterium’s ability to degrade caffeine. The presence of this gene in the bacteria can be analyzed using PCR. This study aims to validate the designed PCR primers used to detect the ndmA gene in caffeine-degrading bacteria in vitro. The methods employed included bacterial genomic DNA extraction, amplification of the target gene using PCR with the designed primers, and analysis of the amplification products via electrophoresis. Primer validation was performed to observe the presence of a DNA band of the target size. The results showed that the primers used were capable of producing a clear, single DNA band of approximately 200 bp in isolate KAFS 34. The optimal PCR conditions used for the run were as follows: pre-denaturation at 95 °C for 3 minutes; denaturation at 95 °C for 3 minutes; annealing at 56 °C for 30 seconds; extension at 72 °C for 1 minute; and final extension at 72 °C for 5 minutes. The PCR run was performed over 30 cycles with a final storage temperature of 4 °C. The successful amplification of the Pseudomonas plecoglossicida KAFS 34 isolate in this study indicates that the PCR reaction conditions used were optimal, allowing the primers to bind specifically to the target gene.