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RESEARCH ARTICLEas the matrix. The gel was visualized under UV transilluminator(Hoefer, USA) and the results weredocumented on gel documentation system.RESULTS AND DISCUSSIONProcessing and detection of avianMycoplasma in clinical samplesThe samples were thoroughly agitated in thebroth and kept at 37°C under 5-10 percent CO 2tension in screw capped tubes. The agar platescontaining the same samples were also kept undersimilar conditions in a humid atmosphere. One ml ofbroth culture from each sample was taken after aperiod of four hours and processed for the extractionof genomic DNA. Remaining broth cultures wereincubated as mentioned above. The agar plates wereobserved daily under microscope (10x) for thepresence of any colonies. The genomic DNA of E.coli, S. aureus and P. multocida were also extractedfor checking the specificity of the selected primers.After extraction of the genetic material, PCR wascarried out using Mycoplasma genus specificprimers.Mycoplasma genus specific polymerasechain reactionThe primers GPO3 and MGSO were used inthis study for the amplification of DNA fromMycoplasma.The amplified products were visualized bysub-marine agarose gel electrophoresis. A positivePCR result for Mycoplasma was indicated by thepresence of a 270 bp fragment in electrophoresed gelunder UV transillumination, whereas, noamplification indicated a negative result. In negativecontrol, amplification was not detected and inpositive control (reference strain 6/85) 270 bp bandwas observed. The DNA prepared from E. coli, S.aureus and P. multocida did not evidence anyamplification with these primers, indicating thespecificity of the selected primers (Fig. 1).Out of the total 50 samples (collected inbroth and agar) subjected to genus-specific PCR, 12were found positive. These included eight samplescollected both in PPLO broth and streaked on to agar.Four more samples from agar plates also gave apositive result. Thus, a total of 12 samples werepositive for Mycoplasma genus specific PCR.Species differentiation of avianMycoplasma from clinical samplesThe positive samples in genus specific PCRwere subsequently subjected to a species specificPCR using another set of selected primers.Mycoplasma species specific PCR samplescollected in PPLO brothThe primers MG IGSR F and MG IGSR Rcould not amplify the DNA isolated from E. coli, S.aureus and P. multocida.Agar gel electrophoresis of the amplifiedPCR products were carried out along with a positivecontrol and negative control in 0.5 × TBE buffer. Apositive PCR was indicated by the presence of a bandin the 660 bp region in the test and positive controlsamples. Out of the eight genus-specific PCRpositive samples subjected to species specific PCR,only one sample was fount positive.Mycoplasma species specific PCR samplescultured on PPLO agarThe individual colonies observed after 3days on the 25 agar plates streaked with sampleswere picked up and put in PPLO broth and kept at37°C under 5-10 per cent CO2tension. The brothtubes were daily examined for colour change frompink to orange or yellow. Samples from those tubeswith colour change were used for extraction of DNA.The species specific PCR was carried out in abovementioned conditions.Among the twenty five samples swabbedonto agar, 12 were positive in genus specific PCRand these samples were further subjected to speciesspecific PCR. Out of these samples, three werepositive in species specific PCR. Specificity of theprimers was indicated by the absence ofamplification of DNA from other bacterial speciestested. (Fig. 2).JIVA Vol. 10 Issue 1 April 20127