RAO, V.D.P (Major)SREENIVASULU, DSRILATHA, ChSURYANARAYANA, V.V.SLAKSHMI KAVITHA, K2016-12-202016-12-202003-08http://krishikosh.egranth.ac.in/handle/1/91638THESESABSTRACT : In the present investigation, an attempt was made to develop a radiolabelled Egg Drop Syndrome-76 (EDS-76) viral DNA probe by using local isolate which enables accurate diagnosis of EDS virus infection in poultry especially in breeder stocks because of its vertical transmission. In this study, VN 1 isolate of EDS-76 virus was cultivated in duck embryos and purified by using 10-40 percent sucrose density gradients. The isolate was serologically similar to EDS-76 strain 127 by Haemagglutination Inhibition (HI), Agar gel immunodiffusion test (AGID), Counter immuno electrophoresis (CIE) with standard EDS-127 strain virus antiserum. From the purified virus, DNA was extracted by proteinase K-phenol: chloroform method and the results provided evidence to the fact that the isolate consisted of DNA of molecular weight of 34.2kb. Restriction endonuclease pattern using Bam HI resulted into four fragments viz., 16kb,11kb, 4kb and 2kb. Of these the 2kb fragment was found to be specific for EDSV by nucleotide sequence analysis. The 2kb fragment was cloned into pBluescript II KS+ vector and the transformation efficiency was 2x104 /μg of plasmid. The nucleotide sequence of 2kb insert revealed that the 2kb fragment codes for two genes, the fibre protein and pVIII protein genes. The homology with the published sequence was found to be 96 per cent. The fragment was labeled with -33P ATP and the total incorporation was 2.4x107dpm/20ng DNA. The labeled DNA when used as a probe, detected EDSV specific genome with a sensitivity level of 1.25 pg genomic DNA. Proteinase K treated, phenol:chloroform extracted DNA when denatured by alkali enabled quick diagnosis of faecal samples. However the hybridization efficiency was found to be less in case of alkali denatured proteinase K treated samples, which showed positive signals after long time of exposure. No signals could be obtained from direct denatured samples. On comparison with HI, AGID and CIE, the dot blot hybridization using labeled probe, proved to be more sensitive, specific and able to detect virus in faecal samples of infected birds as early as 24hr post inoculation .enEgg Drop Syndrome-76; EDS-76; DNA probe; poultryThesis