Study Of Isolation And Nested-PCR Identification On Mycoplasma Gallisepticum

Mycoplasma gallisepticum(MG) is a primary pathogen causing chronic respiratory disease in chicken,which is characterized by coughing,nasal discharge,rattling and inflammation of the saccus pneumaticus.MG infection is easily contagious and often lead to the disease of chicken flock,but the mortality is not very high,for it is often the subclinical infection chickens that lead toegg production of layer dropped and hatchability reduced,which cause serious losses in the poultry industry.Isolation and identification were carried out for the suspicious infected chi-ckens came from 24 poultry farms in the area of Chongqing.,which was the purpose of this study.After being soaked for 24h in 25%solution of penicillin,200μL culture were taken out and added to improved Hartleys medium with thallium acetate to amplify the bacteria.After 2 or 3 days,the colony was o-bserved with the appearance of fried egg.There were small globular,short rh-abditiform and irregular blue bacteria from the test under microscope by Wrig-ht staining.Therefore the isolates were initially diagnosed as mycoplasma.Bioc-hemical reactions showed that the isolates had the ability to break down glucose,which changed the slightly alkaline solution into acid solution,at the same time,the colour of the liquid medium with phenolsulfonphthalein turned from red to yellow.The isolates were able to absorb the red blood cell of chicken and were also identified with antiserum of anti MG-S6 strain.On the basis of the criterion that metabolism inhibition titer between 1:80 andl:1280 was positive,9 strains of mycoplasma gallisepticum were initially screened from 17 isolates in this study.Based on the conserved sequence of Mycoplasma gallisepticum of 16S/23S rRNA intergenic spacer region,two pairs of primers were designed by primer premier 5.0.The exterior sequence of 577bp and the interior sequence of 283bp were respectively amplified by Nest-PCR.The conditions of PCR were explored and changed until the optimal result of amplification was reached.The bands were little dark from the first amplification.The products of the first amplification were then diluted by 500 times and used as the template for the secondary amplification.The bright bands were clearly seen in the result,whereas the control group didn't amplify any band.Therefore the suspicious Mycoplasma gallisepticum strains isolated from Chongqing in the experiment were identified as Mycoplasma gallisepticum isolates.The products of the secondary amplification were then analyzed by the restriction fragment length polymorphism(RFLP) method,in which the restriction endonuclease RasI was used. Mycoplasma gallisepticum isolates showed two different RFLP patterns by electrophoretic analysis.All the mvcoplasma gallisepticun strains were 255/28 type,that was to say,there was only one RsaI site in the product which was digested into two fragments of 255bp and 28bp.The results suggest that there may be no difference in genotype of mvcoplasma gallisepticun strains.The present domestic and foreign methods of detecting and extracting bacterial plasmid were used to study Mycoplasma gallisepticum.The alkaline lysis method was first used to extract mvcoplasma gallisepticun culture.The extraction kit of extracting bacterial plasmid,which was ripe at home,was then used to test the extraction.Both the methods had the same result that there were no any bands by electrophoretic analysis,whereas the reference group had several different bands.Results initially suggest that there is no plasmid in Mycoplasma gallisepticum or the specificity of the method isn't so good that the plasmid of Mycoplasma gallisepticum can't be extracted.