Quantitative Detection Method for Ovine-derived Materials in Meat Products using Single Copy Nucleus Genes by Droplet Digital PCR
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摘要: GeneBank搜索牛、绵羊、山羊、猪、马、驴、鸡、鸭、鹅、火鸡、狗、猫、鼠、兔、貂等15种动物的单拷贝核基因组序列信息，应用生物信息学分析筛选15种动物共有的种间保守区域，设计一对可同时扩增15种动物源性DNA的内参基因引物和探针；同时分析筛选绵羊和山羊共有且和其余动物种内特异性区域，设计一对只能扩增羊源性DNA的特异性基因引物和探针。基于微滴数字PCR技术，引入内参基因校正羊种属特异性基因测定方法，建立科学准确的肉制品中羊源性成分量化判定方法。结果表明，所建立方法具有良好的特异性和通用性，内参基因和羊种属特异性基因的最低检出限分别为32和26 copies/μL，模拟添加样品的正确度偏差均值为8.66%，符合数字PCR方法制定指南要求不得大于25%，说明量化判定方法结果具有较高的准确性。Abstract: 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.
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