动物造模,荧光定量PCR z-bio 2022-05-07 364

　　Objective To establish a real-time quantitative PCR detection method for mouse hepatitis virus (MHV), and apply it to the detection of rodents such as nude-waisted mice.

　　Methods Select the conserved region of MHVE gene to design and synthesize specific primers and probes, establish a real-time quantitative PCR method, and verify the specificity, sensitivity, linearity, repeatability and stability of the method. At the same time, 79 clean mice, 35 SPF mice, 63 naked mole rats, 10 gerbils, 20 golden hamsters and 4 chinchillas were tested for MHV by the established method.

　　Results The MHV real-time fluorescence quantitative PCR method was successfully established; the linear range of the method was (1×101~1×109) copies/μL, which was comparable to Sendai (SV) virus, reovirus type 3 (Reo3), mouse pneumonia virus (PVM) and bovine coronavirus (BCV) have no cross-reaction, and the detection sensitivity of the method is 10 copies/L. Repeatability and stability tests showed that the coefficient of variation between experiments was less than 5%. The test results showed that 63 naked mole rats, 35 SPF mice, 10 gerbils, 20 golden hamsters and 4 chinchillas were all negative. The positive rate of MHV in 79 clean mice was 22.78%. Compared with RT-PCR, the established method has a reliability of 97.47%.

　　Conclusion The established MHV real-time PCR method is specific and sensitive, and can detect MHV carried in a variety of rodents.