In order to identify selected bacterial isolate using 16S rRNA gene amplification, the chromosomal DNA was isolated using
a bacterial DNA extraction kit (Roche Applied Science, Germany). Then amplification by PCR was performed using universal
primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-TACGGTTACCTTGTTACGACT-3’) (27 ) under standard conditions in
a 50 μL volume containing 1X PCR buffer, 1.5 mM MgCl2, 2 mM dNTP mixture, 1 μM primers, 1 μL of Pfu DNA polymerase
(Fermentas, St. Leon-Rot, Germany) and 2 ng of template DNA. Thermocycling conditions were followed by initial denaturation
at 94 °C for 4 min and 30 amplification cycles of denaturation at 94 °C for 1 min, annealing at 59°C for 1 min, primer
extension at 72 °C for 1 min and a final extension at 72 °C for 5 min. After separation of PCR products on 1% agarose gel,
desired DNA fragment was purified from the gel using agarose gel extraction kit (Roche Applied Science, Germany) and sequenced.
For identifying the genus of bacterium, the obtained 16S rRNA gene sequence was compared to the NCBI (National Center for
Biotechnology Information) GenBank database using nucleotide BLAST software ( 28 (link)
).
a bacterial DNA extraction kit (Roche Applied Science, Germany). Then amplification by PCR was performed using universal
primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-TACGGTTACCTTGTTACGACT-3’) (27 ) under standard conditions in
a 50 μL volume containing 1X PCR buffer, 1.5 mM MgCl2, 2 mM dNTP mixture, 1 μM primers, 1 μL of Pfu DNA polymerase
(Fermentas, St. Leon-Rot, Germany) and 2 ng of template DNA. Thermocycling conditions were followed by initial denaturation
at 94 °C for 4 min and 30 amplification cycles of denaturation at 94 °C for 1 min, annealing at 59°C for 1 min, primer
extension at 72 °C for 1 min and a final extension at 72 °C for 5 min. After separation of PCR products on 1% agarose gel,
desired DNA fragment was purified from the gel using agarose gel extraction kit (Roche Applied Science, Germany) and sequenced.
For identifying the genus of bacterium, the obtained 16S rRNA gene sequence was compared to the NCBI (National Center for
Biotechnology Information) GenBank database using nucleotide BLAST software ( 28 (link)
).