动物造模,荧光定量PCR检测,巨细胞病毒 z-bio 2021-10-19 432

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

　　Method: Select the conservative sequence of the MCMV Smith strain DNA polymerase gene published by NCBI, design primers and probes, and establish a fluorescence quantitative PCR method for MCMV. The specificity, sensitivity, reproducibility and stability of the method are. Established. .. This method is used to detect adult MCMV mouse blood samples and 409 mouse blood samples submitted for testing in 2018.

　　Result: The slope of the established standard curve of the MCMV real-time PCR method was -3.418, the R2 value was 0.999, and the amplification efficiency was 96.137. The minimum content of MCMV that can be quantitatively detected is 47 copies/μl. .. When using rat cytomegalovirus, simian cytomegalovirus, human herpes simplex virus, pseudorabies virus, and type I herpes virus as templates, there is no amplification curve and good specificity. The coefficient of variation within the method group and between the method group was 0.39? 0.68? 0.48? 1.01? Good reproducibility and stability. The maximum dilution factor of MCMV virus that can be detected in human and mouse blood samples is 1:1000 (100.75TCID50/0.1 mL), and all 409 mouse blood samples are negative.

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