摘要
为获得肉制品中牛、羊、猪、鸡、鸭等动物源性成分的快速高效多重PCR检测方法,利用5对牛、羊、猪、鸭和鸡等动物源性成分特异性PCR引物(靶基因序列来源于线粒体基因组),对多重PCR反应条件引物浓度和退火温度进行优化,并确定该检测方法的检出限。结果表明,多重PCR方法最佳反应条件为鸭源特异性引物浓度0.12μmol·L^(-1),鸡源特异性引物浓度0.16μmol·L^(-1),猪源特异性引物浓度0.16μmol·L^(-1),羊源特异性引物浓度0.32μmol·L^(-1),牛源特异性引物浓度0.24μmol·L^(-1),退火温度58℃,d NTPs浓度0.20 mmol·L^(-1),Taq DNA聚合酶添加量5 U,循环次数40。在最佳反应条件下,所建立的牛、羊、猪、鸡和鸭等5种动物源性成分多重PCR的g DNA检出限达到20 pg。应用优化后的多重PCR方法对21份市售肉制品样本进行盲样检测,验证鉴别方法的准确性,结果与现行标准的检测结果一致,掺假率达42.86%。本试验结果为肉制品中该5种动物源性成分的快速检测提供了技术支撑。
In order to obtain an efficient multiplex PCR method for the detection of adulterated meat species,specific multiplex PCR amplification primers,based on the homologous and specific sites of mitochondrial genes,were designed and optimized in five animal samples, including bovine, ovine, pork, duck and chicken. The optimal primer concentrations were 0. 12,0. 16,0. 16,0. 32,0. 24 μmol·L-1 for duck,chicken,pork,ovine and bovine samples,respectively. The other optimal conditions were 0. 200 mmol·L-15 U of Taq DNA polymerase,annealing temperature was 58℃,and the PCR cycle times 40. Under the optimal conditions,the determined g DNA detection limit for these five-meat species was 20 picograms,and the established method was verified by 21 kinds of meat samples（ as blind sample） in local super market. The verification results showed that the method was able to distinguish accurately the fake meat（ the adulteration ratio was 42. 86%） from other samples,which was consistent with issued current standards.The results in this research could provide technology support for rapid identification and detection of meat samples.
出处
《核农学报》
CAS
CSCD
北大核心
2018年第3期506-514,共9页
Journal of Nuclear Agricultural Sciences
基金
闵行区产学研合作计划项目(2016MH256)
关键词
动物源性成分
五重PCR
优化
检出限
animal species, five-plex PCR, optimization, detection limit
