Establishment Of Rabbit Splenic Fibroblasts And RHDV Strain Establishment Of QRT-PCR Method For Genome SYBR Green Fluorescence Detection

Rabbit viral hemorrhagic disease(RHD)is also known as rabbit sputum.It is an acute,septic,highly contagious,infectious and lethal and extremely high,with the main clinical features of rabbits with substantial organ bleeding.RHDV is a single-stranded positive-strand RNA virus with a genome length of approximately 7.5 Kb.At present,there is no systematic genomic monitoring method and stable cultured cell system for this RHDV,which seriously affects the research on the mechanism of cell infection and prevention and control of RHDV.Therefore,this paper establishes the detection method of SYBR Green fluorescent quantitative RT-PCR of RHDV genome and establishes the spleen source cells of rabbits,and conducts preliminary infection test of RHDV of established cells.The specific contents are as follows:1.According to the gene structure of RHDV,in this paper,seven pairs of primers were designed to amplify the genome of RHDV SCH07 strain preserved in this laboratory.A set of RHDV genome SYBR Green fluorescence quantitative RT-PCR assays was established by optimizing the optimal reaction conditions.The sensitivity,specificity,and repeatability tests were tested.The method has no cross-reaction with Pasteurella rabbit,Salmonella rabbit,Rabbit Escherichia coli and RHDV b.The minimum detection value is 104 copies/?L,and the intra-and inter-assay coefficients of variation are less than 3%.The results show that the method has the advantages of high sensitivity,strong specificity,good stability,high precision and fast detection.2.In the process of culturing the primary cells of the lactating rabbit,a fibroblast-like cell is isolated from the spleen tissue,and a cell with a different morphology is formed on the surface of the primary cell during the cultivation process,and the growth rate is faster than that.Primary cells were isolated from primary cells by cell separation and designated as RS17-2.The cell growth characteristics and biological characteristics were studied by measuring the subculture conditions of RS17-2 cells,detecting cell marker proteins,cell growth curves,and cell karyotypes.The results showed that the cell line had serum lazyness,positive fibroblast marker protein detection,and normal karyotype,and the cells can still grow stably after passing through 150 generations.This provides cellular material for studies related to RHDV infection mechanisms.3.The RS17-2 cell line was infected with RHDV SCH07 strain and the SCH07 strain RNA was transfected into the cell line.After infection,the CPE of the cells was observed and the fluorescence quantitative method established by the cells was used for detection.The cells showed CPE in the first infection,but with the continued blind transmission,the cell CPE decreased,and the quantitative detection of viral nucleic acid decreased with the increase of the number of passages.The cell virus nucleic acid was negative after the sixth pass of blind transmission;RNA transfection After the cell line,the cells shrink more and more with time,but the CPE difference between the time gradients is not obvious,and the fluorescence nucleic acid content of the cells is positive.It provides a scientific reference for further research on the mechanism of RHDV infection in cells.