Quantitative Detection Method for Ovine-derived Materials in Meat Products using Single Copy Nucleus Genes by Droplet Digital PCR

GeneBank was used to search single-copy nuclear genome sequences of 15 animals including cow, sheep, goat, pig, horse, donkey, chicken, duck, goose, turkey, dog, cat, rat, rabbit, mink, etc. A pair of internal reference gene primers and probes were designed based on screening for interspecific conserved areas common to 15 animals applied bioinformatics. A pair of species-specific gene primers and probes were designed based on screening for sheep and goats are common, but separate from other animal species. Based on microdroplet droplet digital PCR technology, internal reference gene was used to correct specific genes of sheep species, a scientific and accurate quantitative determination method for sheep-derived components in meat products was established. The results showed that the method has good specificity and universality. The minimum detection limits of internal reference genes and sheep species specific genes were 32 and 26 copies/μL, respectively. The mean deviation of accuracy of the simulated added samples was 8.66%, and the deviation of accuracy meeting the requirements of the digital PCR method should not be more than 25%, indicating that the results of the quantitative determination method had high accuracy.