三引物PCR检测牛羊猪源性成分的方法

A tri-primer multiplex PCR for simultaneous detection of cattle, sheep and pig

建立了三引物PCR鉴定牛羊猪源性成分的方法,为肉类及产制品掺假鉴定或肉品溯源提供了简单易行的方法。通过对比牛羊猪的线粒体DNA(mt DNA)全序列,在保守区设计通用引物,在特异性区域设计特异引物并结合三引物PCR技术,扩增3个物种的特异性片段,羊、牛和猪产生片段大小分别为570、549、420 bp,依据片段大小可以在一次PCR中鉴定3个物种。建立的方法简单易行,特异性和灵敏度好,可以在肉类及产制品鉴定和溯源中应用,提高了检测效率。

The purpose was to establish a sensitive, reliable and cost-effective detection method for meat adulteration. In the present study, a novel tri-primer PCR method for the simultaneous detection of pig, sheep and or goat and cattle was developed. The method was based on the conserved region and specific sequences of mt DNA from pig, cattle and sheep. Three primers were designed in this study including a piece of downstream primer for three species, one upstream primer for cattle and sheep, the third primer for pig specific mt DNA sequence. After optimization for the PCR reaction system, 570 bp, 549 bp and 420 bp PCR fragments were obtained for sheep, cattle and pig, respectively. The tri-primer PCR system had no nonspecific reaction with chicken, duck, hare, donkey and fish. The sensitivity for this method was 20 pg DNA in a total of 20 μL system. The data indicated that the proposed method could be used as a reliable routine screening assay of various food products for the presence of fraud ingredients.