动物实验,巨细胞,荧光定量PCR z-bo 2020-10-12 316

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

　　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 determine the specificity, sensitivity, reproducibility and stability of the method . Verify gender and apply this method to detect adult MCMV mouse blood samples and 409 mouse blood samples submitted for testing in 2018.

　　The standard curve gradient 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. When rat cytomegalovirus, salivary cytomegalovirus, human herpes simplex virus, pseudorabies virus, and frost herpes virus type I are used as templates, there is no amplification curve, and they have excellent specificity. The coefficient of variation within the method group and between the method group is 0.39%-0.68% and 0.48%-1.01%, respectively, which has good reproducibility and stability. The maximum dilution of MCMV virus in human blood samples of spiked mice was 1:1000 (100.75TCID50/0.1 mL), and all 409 mouse blood samples were negative.

　　Conclusion: The established mouse cytomegalovirus fluorescence quantitative PCR method has excellent sensitivity, specificity and stability. It can effectively detect mouse MCMV, monitor experimental mouse MCMV, and supplement and improve related standards.