动物造模,荧光定量PCR z-bio 2021-10-28 346

　　Objective: To establish a real-time fluorescent quantitative PCR method for detecting mouse hepatitis virus (MHV), and use it to detect experimental nude mice and other rodents first.

　　Method: Select the conserved region of the MHVE gene, design and synthesize specific primers and probes, establish a fluorescent quantitative PCR method, and determine the specificity, sensitivity, linearity, repeatability and method of the method, and verify the stability. At the same time, using established methods, MHV tests were performed on 79 clean mice, 35 SPF mice, 63 nude mice, 10 gerbils, 20 golden hamsters and 4 squirrels.

　　Result: We successfully established the MHV real-time fluorescent quantitative PCR method. The linear range of this method is (1x101? 1x109) replication/μL, and it is compatible with Sendai (SV) virus and reovir. Type 3 (Reo3), mouse hepatitis virus (PVM) and bovine coronavirus (BCV) are non-cross-reactive, and the detection sensitivity of this method is 10 copies/L. Reproducibility and stability tests show that the coefficient of variation between experiments is less than 5%. The test results showed that 63 nude mole mice, 35 SPF mice, 10 gerbils, 20 golden hamsters and 4 ground squirrels were all negative. The MHV positive rate of 79 clean mice was 22.78%. Compared with RT-PCR, the reliability of this method is 97.47%.

　　 Conclusion: The established MHV fluorescence quantitative PCR method is specific and sensitive, and can detect MHV carried by various rodents.