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The present study was taken up with a view to undertake plaque purification of BTV-4 isolate available in the department, to confirm its purity. BTV-4 isolate was adapted to Vero cell line and had a titer of 106.46/ml after 8 passages in the process of plaque purification which was determined as 104.833/ml prior to adaption. Plaque purification was carried for three times using agarose overlaying method in 6 well plate. Each time plaques were observed from 4th day of infection of approximately not more than 1mm diameter. | Int.J.Curr.Microbiol.App.Sci 2018 7 4 2837-2844 EXCELLENT PUBLISHERS International Journal of Current Microbiology and Applied Sciences ISSN 2319-7706 Volume 7 Number 04 2018 Journal homepage http www.ijcmas.com Original Research Article https doi.org 10.20546 ijcmas.2018.704.323 Plaque Purification of Bluetongue Virus Serotype-4 BTV-4 M. Srikanth Reddy1 Kalyani Putty1 Y. Narasimha Reddy1 P.P. Rao2 M. Ramakoti Reddy3 Uma2 Sunil R. Patil1 Susmitha Birru1 Abdul Muzeer Shaik1 and Dhanalakshmi Kancharlapally1 1College of Veterinary Science Rajendranagar Hyderabad-500030 Telangana India 2Ella Foundation Genome Valley Hyderabad-500 078 Telangana India directorate of Poultry Research Rajendranagar Hyderabad-500030 Telangana India Corresponding author ABSTRACT Keywords BTV-4 Vero cells TCID50 Plaque purification Sea plaque agarose RT-PCR Article Info Accepted 23 March 2018 Available Online 10 April 2018 The present study was taken up with a view to undertake plaque purification of BTV-4 isolate available in the department to confirm its purity. BTV-4 isolate was adapted to Vero cell line and had a titer of 106 46 ml after 8 passages in the process of plaque purification which was determined as 104.833 ml prior to adaption. Plaque purification was carried for three times using agarose overlaying method in 6 well plate. Each time plaques were observed from 4th day of infection of approximately not more than 1mm diameter. One of the plaques from third round of purification was grown in T25 flask and the resultant cell culture fluid was used for RNA isolation and RT-PCR through which the respective plaque was confirmed as BTV-4. Therefore it was confirmed that there was no other serotype other than BTV-4 and the plaque purified virus can be used either to study pathogenesis or to raise hyperimmune serum. Introduction Bluetongue BT an infectious non-contagious viral disease of sheep and other domestic and wild ruminants caused by bluetongue virus BTV is a segmented .
