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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

I have a thermal cycler question for laboratory automation it is too long to put in here so read the details p?

A PCR thermal cycler uses a computer chip and input output circuits to control the thermal cycling sequence. the inputs and outputs are voltage signals. the computer system can read a voltage input and modify a voltage output. what type of device would be connected to the imput and what would be connected to the output? and why?

The input can be a thermocouple. The thermocouple outputs a millivolt proportional to temperature. Now you can quantify the temperature. You can then use a Proportional Integral Derivative (PID) routine to control an output device based upon a set point temperature. The output can be typically a 0-5VAC scaled output to control a device that allows heating or cooling, depending what you require. The output device can be fan which can speed up or slow down based on the setpoint. Or it can be a valve which can be actuated to open and shut allowing cooling air in or not to allow for cooling.

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