动物造模 z-bio 2022-03-23 497

　　Objective To optimize the detection method of porcine endogenous retrovirus copy number in pig genome by using droplet digital PCR technology, and to establish a detection method for PERV copy number.

　　Methods The TaqMan probe double ddPCR method was used, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) were used as internal reference genes, respectively, to determine the specificity of PERV copies. The number detection reaction is best suited for the annealing temperature and the number of reaction cycles. In addition, the optimal template concentration was determined by testing the standard plasmid and porcine cell genome diluted by concentration gradients. Two methods, ddPCR and qPCR, were used to determine the copy number of PERV in porcine-derived cell lines and Bama minipig tissues, and the stability of the two detection methods was compared.

　　Results The optimal annealing temperature for detecting the copy number of PERV based on ddPCR technology was 59.9℃, and the optimal number of reaction cycles was 50; the optimal concentration of the template genome was 1.25-7.5 ng; the coefficient of variation for detecting the copy number of PERV in the same sample-derived genome Between 0.44% and 8.29%.

　　Conclusion In this study, a detection method of PERV copy number based on ddPCR technology was established and the optimal experimental conditions were systematically explored. The determination of PERV copy number in donor animal genome provides a sensitive method and reliable basis.