Fishery
GENOMIC CHARACTRIZATION OF AEROMONAS HYDROPHILA ISOLATES USING RAPD-PCR TECHNIQUE by Mitali Dhiman and S. S. Mishra Central Inland Fisheries Resear
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′.
Selected A.hydrophila isolates were used in RAPD-PCR analysis. First, a set of 20 oligo primers (Operon A & B set) were screened to see the suitability of primers in producing polymorphic bands. The amplification was carried out using Thermal cycler using the following programme : one cycle of initial denaturation at 94oC x 5 min. followed by 45 cycles at 94oC for 1min., 36oC x1min. and 72oC x 2min. and final extension at 72oCx10 min. PCR amplified products were analyzed on 1.5% agarose gel (Sambrook et al., 1989). Accordingly 2 primers (OPA5 and OPA 9), which produced good polymorphic bands were selected and used in RAPD-PCR analysis of all A.hydrophila. The banding pattern and RAPD profiles were analyzed using the Gel documentation system (Geldoc 2000, BioRad) and TFPGA software.
Results and Discussions
Out of 32 isolates on RS medium screened, 20 were identified as Aeromonas species. However, when these bacteria were screened using PCR for aerolysin gene, only 12 isolates and the standard MTCC strain produced 209 bp specific DNA band indicating these to be A.hydrophila . No amplification was seen in other isolates and in negative controls. This indicated that PCR to be more specific and accurate for detection of A.hydrophila. Phylogenetic analysis of 12 A.hydrophila isolates was carried out using RAPD-PCR. Out of 20 primers screened to test their suitability to produce polymorphic bands, 2 primers (OPA5 and OPA9) were selected and used for RAPD-PCR. The banding pattern was estimated using the gel documentation system and the genetic similarity index was calculated as per the formula of Nei and Li using TFPGA software. Dendogram analysis of all isolates revealed high genetic variability among the isolates regardless of their source of origin. RAPD assays has been proved useful for identification and classification of a variety of microbial pathogens of humans and animals (Berg et al., 1994). However, RAPD profiles of A.hydrophila strains were scattered, demonstrating the genomic variety of motile aeromonads. There was no species specific fragments among profiles of A.hydrophila strains.
About the Author
Mitali Dhiman, Research Scholar. Dr.S.S.Mishra, Principal Scientist, Biotechnology Laboratory, Regional Centre, Central Inland Fisheries Research Institute, 24, Panna Lal Road, Allahabad – 211 002 (Uttar Pradesh) INDIA
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