GENOMIC CHARACTRIZATION OF AEROMONAS HYDROPHILA ISOLATES USING RAPD-PCR TECHNIQUE
Aeromonas hydrophila are involved in various disease problems in humans and aquatic animals and are known to be phenotypically, serologically and genetically quite diverse. Development and use of a sensitive and specific diagnostic test is warranted for detection and characterization of this pathogen.In the present study, Fish and water samples from river Hooghly were streaked onto Aeromonas selective growth medium (Rimler-shotts Medium, HiMedia, Mumbai). After incubation at 30oC for 24 hrs plates were observed for characteristic colony development. The bacteria were then identified using a battery of standard cultural and biochemical tests (Buchanan and Gibbons., 1988). Bacterial DNA was extracted following the method of Sambrook et al., (1989). Primers specific for ‘aerolysin' gene (209 bp product, Ozbas et al., 2000) were used as the target gene for PCR amplification. PCR mix of 50 µl containing 1.0U Taq polymerase, 5 μl 10X assay buffer, 0.1μm each of forward and reverse primers, 200 μm dNTPs and 1μl bacterial DNA sample was amplified using Thermal cycler (Gene Amp2400 PCR system, Applied Biosystem). After 35 cycles of amplification (at 94oC 2 min, 56oC 2 min and 72o C 2 min), the products were electrophoresed on 2% agarose gel and visualized under Gel documentation system. The sequence of primers used was : Forward primer - 5'- CCA-AGG-GGT-CTG-TGG-CGA-CA-3', Reverse primer -5'- TTT-CAC-CGG-TAA-CAG-GAT-TG-3'.
Aeromonas hydrophila are involved in various disease problems in humans and aquatic animals and are known to be phenotypically, serologically and genetically quite diverse. Development and use of a sensitive and specific diagnostic test is warranted for detection and characterization of this pathogen.In the present study, Fish and water samples from river Hooghly were streaked onto Aeromonas selective growth medium (Rimler-shotts Medium, HiMedia, Mumbai). After incubation at 30oC for 24 hrs plates were observed for characteristic colony development. The bacteria were then identified using a battery of standard cultural and biochemical tests (Buchanan and Gibbons., 1988). Bacterial DNA was extracted following the method of Sambrook et al., (1989). Primers specific for ‘aerolysin' gene (209 bp product, Ozbas et al., 2000) were used as the target gene for PCR amplification. PCR mix of 50 µl containing 1.0U Taq polymerase, 5 μl 10X assay buffer, 0.1μm each of forward and reverse primers, 200 μm dNTPs and 1μl bacterial DNA sample was amplified using Thermal cycler (Gene Amp2400 PCR system, Applied Biosystem). After 35 cycles of amplification (at 94oC 2 min, 56oC 2 min and 72o C 2 min), the products were electrophoresed on 2% agarose gel and visualized under Gel documentation system. The sequence of primers used was : Forward primer - 5'- CCA-AGG-GGT-CTG-TGG-CGA-CA-3', Reverse primer -5'- TTT-CAC-CGG-TAA-CAG-GAT-TG-3'.
