Development Of The Real Time PCR Application To Detect Duck Plague Targetted TK Gene

Duck plauge caused acute, febricity and septico-infectious disease. They were characterized as generally prevalence, rapid transmission, high mortality and morbidity, which have generated huge financial burden to waterfowls production industry and restricted the development of poultry industry. Firstly, we downloaded several TK gene fragment of DPV, then aligned them by using Oligo6.0version. After that, we designed primers and probes according to the result of multiple alignment, targetted the TK gene of DPV. The target gene fragment was amplified by PCR, purified and cloned into PMD-18T for preparing standard plasmid. Then we optimised the reaction time, reannealing and elongation temperature by using standard plasmids as template. In order to explore the sensitivity of the methods we developed, we serially diluted PMD-TK to4.8×10-1copies/ul. In this section, the detection limits of real-tim PCR could be down to5copies/ul of PMD-TK per reaction. For evaluation the specificity of the protocol we developed, we extracted the total DNA of NDV, Salmonella (No.50338),Ecoli.078, and DHV. And there was just one postive result, templated by DPV, could be observed. In the section of repeat experimental analysis, the Ct value of each different reaction was less than5%, which demonstrated that this method was characterized by stability and repeatability.