动物造模,巨细胞病毒,荧光定量PCR检测 z-bo 2020-09-25 344

　　Objective: To establish a fluorescence quantitative PCR method for mouse cytomegalovirus (MCMV) and apply it in advance.

　　Method: Select the conservative sequence of the DNA polymerase gene of MCMV Smith strain published by NCBI to design primer probes, establish a fluorescent quantitative PCR method for MCMV, and the specificity, sensitivity and repeatability of the method. , And applied this method to verify the stability and detect fake MCMV mouse blood samples and 409 mouse blood samples submitted for testing in 2018.

　　The standard curve slope of the established MCMV real-time PCR method is -3.418, the R2 value is 0.999, the amplification efficiency is 96.137%, and the minimum quantitatively detectable MCMV content is 47 copies/μL. Using rat cytomegalovirus, salivary cytomegalovirus, human herpes simplex virus, pseudocanine virus and type I frost herpes virus as templates, there is no amplification curve, and it has good specificity. The coefficient of variation within the method group and between the method group was 0.39%-0.68% and 0.48%-1.01%, respectively, with good reproducibility and stability. The maximum dilution of MCMV virus in human blood samples spiked into mice was 1:1000 (100.75TCID50/0.1 mL), and all 409 mouse blood samples were negative. Conclusion Conclusion The established mouse cytomegalovirus fluorescence quantitative PCR method has high sensitivity, strong specificity, and stability. It can effectively detect MCMV in mice, and can monitor MCMV in experimental mice and supplement relevant standards.