Detection Of Novel Duck Reovirus And Adaptability In The Cells

Novel Duck Reovirus is emerging in China in recent years, with irregular necrosis of the liver and spleen, bleeding and cardiac muscle, bursa bleeding as the main feature of the new disease, commonly known as "the new duck liver disease", "duck white spot disease " " duck necrotizing hepatitis disease ", etc. The disease has been identified by the NDRV cause. In order to improve the accuracy, sensitivity and effectiveness of the diagnosis of NDRV infection, this study established a NDRV fluorescence quantitative RT-PCR, RT-LAMP method and detection methods, as well as in different cells the adaptation of the virus were analyzed, and the virus in chicken embryo fibroblast(CEF) was purified and passaged clones,which laid the foundation for the development of attenuated vaccine.1. The detection of fluorescence quantitative RT-PCRassay of NDRVA pair of specific primers targeted to gene S1 of novel duck reovirus was designed and a real-time RT-PCR assay based on SYBR GreenⅠfluorescent was developed for the quantization of novel duck reovirus. The standard curve was plotted based on the linear relationship between the amount of plasmid DNA and the cycle threshold(CT). The detect limit of the RT-LAMP assay with high specificity and reproducibility was 100 times higher than RT-PCR.Different organs of artificially infected ducklings were detected by this method,and we found higher virus amount in the spleen and liver.2. The detection of RT-LAMP assay of NDRVAn one-step reverse transcription loop-mediated isothermal amplification(RT-LAMP)assay for detecting novel duck reovirus(NDRV) was established with four primers based on six conserved positions of the S3 gene. The process of assay was completed by using Bst DNA within 30 min at 63℃. The detect limit of the RT-LAMP assay was 100 times higher than RT-PCR, and no amplification was found with the samples of 6 other common duck diseases. The clinical application results showed the coincidence rate between the assay and the virus isolation and identification is 98%. The assay was a potential useful technique for NDRV detection in field condition.3. The different adaptability of NDRV in CellsPassaged to adapt after stabilization in CEF, DEF, Vero, BHK-21 and DF1 cells, the virus was harvested at different time period and measured the TCID50, which was depended on to draw the growth curve of NDRV, and Screening out its most comfortable cell——CEF cells. The NDRV in CEF cells were cloned, purified and passaged, increasing the virus titer from 103.7/0.1m L to 106.4/0.1m L.