SEQUENCING APPLICATION FOR MOLECULAR BACTERIAL IDENTIFICATION IN MCBAD JEPARA

DNA sequencing is a technique of determining the nucleotide sequences in a DNA molecule. The sequence is known as a DNA sequence, which is the most basic information of a gene or genome because it contains the instructions needed for the formation of living organisms. DNA sequencing can be used to determine the bacteria identity by comparing it's sequence with other DNA sequences which are already known in GenBank. Conventional bacterial identification (based on morphology, staining and biochemical tests) require a long time and the result is determined by the accuracy of the analyst, while the molecular identification of bacteria require a much faster and more accurate results than conventional bacterial identification.

In this study, we used 2 set of primers, namely V16S-F: 5'-ACA TGA TCC TGG GTT CTC AG-3 'and V16S-R: 5'-CGG TAC TTA TGT CCT GAC TT-3' to amplify Vibrio 16SrDNA gene and 24F: 5'-AGA TGG GTT TCA TGA CT-3 'and 1540R: 5'-AAG GTG GAG CCG CAA ATC CA-3' to amplify non Vibrio 16SrDNA gene. Pure bacterial isolates on TCBSA media or nutrient agar (NA) or nutrient broth (NB) extracted using Wizard genomic DNA purification kit following the manufacture protocol. The quantity and quality of amplification results (PCR product) analyzed using nanodrop or electrophoresis, prior to sequencing using a 3500 Genetic Analyzer.

Thirty five samples consisting of 5 Vibrio bacteria samples and 30 non Vibrio bacteria samples successfully sequenced using the two primer sets mentioned above. Purity of bacteria as well as the quantity and quality of PCR products is the key to success in the sequencing process. Bacteria isolates that are not purely giving wrong results in identification because in this study, we used a universal primer.