Study On The Detection Method Of Mouse Norovirus

Whether animal experiments can proceed(progress)smoothly is closely related to the quality of the laboratory animals. It is required by our country that the SPF level laboratory mice must be excluded from11kinds of virus, nevertheless, the microorganism carried by the themselves can still interfere the result of the experiments.Murine Norovirus was isolated from the accidental died mice, which can induce inflammation and even death in the immunodeficient mice. The reports on murine norovirus virus are still relatively infrequent in domestic, understanding of it’s pathogenicity and the influence on the experiment is still not entirely clear.To make the detection method for MNV fast, accurate and high-efficiency implementation, four methods is established in this research:One step RT-PCR, Real-time qPCR, IFA and ELISA, and then investigate the status of Beijing laboratory mice infect MNV, specific work is as follows:1. The establishment of a standardized detection method for MNV(1)One step RT-PCR:Through analyzing the whole genome sequence of MNV, design the optimal amplification primer, optimize the reaction system and reaction conditions, make the sensitivity of system optimized, ultimately, determine the sensitivity of the method can achieve102copies when the primer concentration of the reaction system was0.3μmol/L, Mg2+concentration was1.5mmol/L;(2)Real-time qRT-PCR:Optimized and evaluated the reaction condition, and finally determined the concentration of primer and probe in the reaction system are respectively0.30.3μmol/and0.3μmol/L, with Ct value of35as the detection limit, sensitivity of the method can achieve a few copy number;(3)indirect immunofluorescence assay(IFA):Use virus particles as antigen. Strictly control the amount of positive cell on the antigen slides, use this method to test80serums, results showed that positive27, negative53, coincidence rate was96.0%.(4)Enzyme linked immunosorbent assay(ELISA):Use VP1as antigen. Board method is used to get the optimal sensitivity, determine the antigen concentration and the serum dilutability. ELISA method for determination of positive32, negative48, coincidence rate was97.5%. 2. the status of Beijing laboratory mice infect MNV is investigatedIn the current study, a totally600mouse serum samples, which had been submitted to our center by different facilities from Beijing area in the last6years, were tested by ELISA and IFA. Based on the random selected600serum sample, the prevalence of MNV by year ranged from9.1%to47.5%in the last6years, and there was not any variation pattern of the MNV prevalence based on the current data. In addition, most MNV positive samples were derived from experimental facilities (69/223). Only one mouse serum sample from animal vendors was detected MNV positive (1/377). Based on the current analysis, the prevalence of MNV is higher in the laboratory animal facilities, especially in the experimental facilities in Beijing area of China.