Development Of C-ELISA And Fluorescence NASBA Detection System For Classical Swine Fever Virus

Classical Swine Fever(CSF)is a viral,epidemic,contact infectious disease caused by Classical Swine Fever Virus(CSFV).CSFV is listed in the list of animal diseases that must be declared by the OIE.In Chinese“National Medium-and Long-Term Animal Disease Control Plan”,it is listed as one of the five priority types of animal disease prevention and control.The outbreak of CSFV always cause significant economic losses,so it is important to establish a rapid,timely and effective detection method for the virus.Competitive ELISA(C-ELISA)can sensitively detect CSFV antibodies in pigs,and is usually used to evaluate the effect of CSFV vaccine vaccination.The sensitivity and specificity of the method can be greatly improved by optimizing the raw antigens,antibodies and detection procedures in the ELISA kit.NASBA is an isothermal amplification detection method.Compared with RT-PCR,it only requires conventional equipment such as water bath and microplate reader.It is a fast and low-cost diagnostic system.G-quadruplex(G4)as a specific nucleic acid structure combined with Thioflavin T(ThT)can not only promote the folding of the G4 structure,but also significantly enhance the fluorescence properties of ThT.Based on these principles,a more sensitive and specific competitive ELISA antibody detection method and a G4-ThT-NASBA fluorescence detection system for CSFV RNA were constructed in this study.In this study,we expressed and purified about 4 mg E2 recombinant protein by infecting SF9 cells.We also obtained 4 mg E2 monoclonal antibody by E2 monoclonal antibody hybridoma cells.Next,the optimal antigen coating mass was determined by the checkerboard method to be 0.05μg/well,and the optimal antibody dilution factor was 1:5000.After repeated verification,it was confirmed that 3%BSA PBST was used as the optimal blocking solution,PBST was the optimal serum dilution solution,and37°C for 10 min was selected as the reaction conditions after adding TMB.Accelerated experiments demonstrated that the assembled kit could be stored at 4℃for 8 months to1 year.The clinical serum samples collected from pig farms were tested by ELISA,and the results showed that the negative and positive detection rates of the self-made kits were 97.83%and 90%.the detection sensitivity and specificity are higher than those of the commercially available kits in ROC curve.Next,the study constructed a G4-ThT-NASBA fluorescence detection system to detect CSFV RNA.The fluorescence spectra of different G4 nucleic acids and non-G4nucleic acids reacted with ThT were tested,and it was found that when some G-quadruplexes were combined with the fluorescent complex ThT,they would emit light at around 490 nm under the excitation light of 425 nm wavelength.Significant fluorescence enhancements can be detected by light,and the components of the NASBA reaction do not contribute to the fluorescence.After optimization,it was determined that the best forward primer was F-GG29,the best final concentration of ThT was 4μM,and no additional K~+was needed.Through the G4-ThT-NASBA system combining the G4-ThT fluorescent biosensor with NASBA,we can detect the target RNA at a minimum of 2 copies,and the detection process will not be interfered by other swine-derived viral RNAs.The detection of cell samples and serum samples with CSFV proves the application value of this method in the detection of CSFV infection in clinical practice.Further studies confirmed the possibility of this system for real-time fluorescence detection.It has been verified that adding ThT to the NASBA amplification system in advance will not affect the amplification reaction,and an exponential growth curve can be drawn according to the fluorescence value of the amplification product at different reaction times.The optimization experiment showed that the optimal reaction time of the real-time G4-ThT-NASBA system was 120 min,and the optimal final concentration of ThT was 6μM.The optimized real-time fluorescent G4-ThT-NASBA system can also detect a minimum of 2 copies of target RNA,and can stably detect infected cell samples and clinical serum samples.The two types of methods constructed in this study include serological diagnostic methods and nucleic acid detection methods currently occupying the mainstream in the field of virus detection.Among them,the competitive ELISA kit can rapidly detect the antibody level in CSFV vaccine immunized pigs,and the G4-ThT-NASBA system can economically,sensitively and rapidly detect the RNA of CSFV wild strains.Through the simultaneous detection and flexible application of these two methods,we can achieve more effective prevention and control of CSFV,and provide effective detection tools for the purification of CSFV in China.