SREENIVASULU, D (Major)SRILATHA, ChESWARA PRASAD, PSUBHADRA, SUBHRA2016-12-302016-12-302010-12http://krishikosh.egranth.ac.in/handle/1/93700THESESABSTRACT : The present study was taken up to standardize real time reverse transcription polymerase chain reaction (rRT-PCR) for rapid detection and relative quantification of bluetongue virus. Bluetongue virus serotypes 2, 9, 10 and 23 were propagated in BHK21 cell lines, double stranded RNA was extracted and studied the migration pattern of nucleic acid in agarose gel. BTV nucleic acid resolved into nine bands with similar migration pattern in ethidium bromide stained 1% agarose gel. Primers targeting NS3 gene of BTV were designed for standardization of real-time PCR. Conventional RT-PCR was used to check the specificity of the primers before use in real-time PCR. 35 amplification cycles polymerase chain reaction with annealing temperature of 60 0C for 30 seconds at 1.5 mM concentration of MgCl2 and primer extension at 72 0C for 30 seconds were found to be optimum for amplification of NS3 gene sequences of BTV. RT-PCR yielded amplified product of 247 bp which was the expected size of amplification from NS3 gene as template. RT-PCR was applied to various ten-fold dilutions of TCID50/ ml of BTV-2 and detected 3.16×102 TCID50 of virus / ml of tissue culture fluid. RT-PCR was also employed to screen blood samples collected from suspected outbreaks of bluetongue. Out of 32 blood samples screened, 17 samples were found to be positive for the presence of BTV by NS3 specific RT-PCR. Primers targeting NS3 gene of BTV found to be specific for detecting BTV were used for standardization of real-time PCR using SYBR green. A 25 μl real-time PCR reaction mixture containing 2 μl of cDNA, 20 p.moles of primers and annealing temperature of 60 0C was found to be optimum and yielded desired specific amplification of NS3 gene of BTV without any non-specific amplification. Different dilutions of TCID50/ ml of BTV-2 were used as standards. The CT values of the standard samples ranged from 22.1 to 39.8. Lowest CT value of 22.1 was obtained for the standard sample with highest virus titer i.e. 3.16×104 TCID50/ml whereas a CT value of 39.8 was obtained for the standard sample having lowest titer value of 3.16×10-4 TCID50/ml. No template control (NTC) yielded no CT value and remained as undetermined by the test. A standard curve generated by measuring the crossing point of each standard and plotting it against the logarithmic value of titer of the virus was linear and ranged from 3.16×104 to 3.16×10-4 with a linear regression value of - 0.99. The melting curve analysis of the standards indicated the absence of non-specific amplification. Real-time PCR was employed to screen the blood samples collected from sheep during suspected outbreaks of bluetongue, already subjected to conventional PCR. Out of 32 samples screened, the test detected 24 samples to be positive for the presence of BTV. Seven samples detected as negative by conventional PCR were detected as positive by real-time PCR. Real time PCR was also used to quantify the BTV present in the blood samples. The titer of the virus in the clinical samples was calculated using the standard curve and ranged from 1.1×10-2 to 7.7×104 TCID50/ml. In conclusion real-time PCR was found to be specific and more sensitive in detecting BTV than the conventional PCR. Further the test could also be used for relative quantification of BTV in the clinical samples which is not possible by the conventional PCR.enBLUETONGUE VIRUS; DETECTION; REAL-TIME RT-PCR; rRT-PCR;Thesis