Development Of Real-time PCR Method For Detection Of Duck Enteritis Virus And Differential Analysis Of UL2 Gene Between Virulent Strain And Attenuated Strain

Duck Plague(DP), Also known as duck virus enteritis(DVE), common in ducks, geese, and other en echelon poultry, caused by duck enteritis virus(DEV), is an acute, septic and highly contacting infectious disease. The disease is a kind of infectious diseases for infection of pantropic infection. After infection with high morbidity and mortality, which have generated huge financial burden to waterfowls production industry and restricted the development of poultry industry. Duck plague virus genome is huge.Although the complete genome sequences of several strains have been published in GenBank, but still very little relevant research and analysis for each gene.The test have isolated a strain of virus from a infected duck in a duck farm in Shangdong Gaoqing.The PCR and animal regression methods was identified as duck plague virus.According to the genomic sequence of DEV published in GenBank,a pair of specific primers were designed and synthesized to amplify the UL2 gene,After this DEV and the other virulent DEV isolated in Taian which were preservation in laboratory were picked up DNA,The UL2 gene was amplification,cloned and sequenced and homologous sequence comparison of duck plague virus GenBank gene.The result of sequence analysis showed that,two strains UL2 gene have the same structure with the published DEV virus sequences,but there is a big difference with the Chinese vaccine strain VAC and Clone-03 strain and attenuated strain,the UL2 gene of the attenuated strain are missing a large segments of 528bp.It is showed that the DEV高�'isolates with high virulent strain,suggesting that the UL2gene deleted attenuated the large fragment may be directly related with virulence.Then we chose the relatively sequence of UL44 gene of duck plague virus, using Premier5.0 software to design and synthesis a pair of fluorescent quantitative PCR primers also, the target gene fragment was amplification, cloned then constructed thepositive standard plasmid of duck plague virus. We successfully established a SYBR Green I real-timefluorescent quantitative PCR method to detection DEV. The sensitivity of this method can reach2.7×101copies, compared with the conventional PCR sensitivity increased by 100 times,The specificity test showed that the method only have a positive signal to the DEV template.The Repeatability test showed that the difference is within 0.6 Ct between the Ct values of 5replicates, prove that the method has good repeatability. The results that the method for detection of clinical samples show that this method is better than ordinary PCR in the Sensitivity. It can be an effective means in clinical for rapid diagnosis of duck plague patients.