关键词: 黄牛；水牛；牦牛；三重real-time PCR

Abstract: This study aimed to establish a polymerase chain reaction (PCR)-based method for accurately and quickly identifying yellow cattle-, buffalo- and yak-derived components. The species-specific primers and probes were developed and used to establish simplex, duplex and triplex real-time PCR methods for the simultaneous identification of the three kinds of bovines. The ability of each primer/probe set to detect target species was determined using a DNA dilution panel. In the simplex real-time PCR, the limits of detection (LODs) for the three bovines were all 0.025 ng/μL; in the duplex real-time PCR assay, the sensitivity for detection of yellow cattle was reduced to 0.1 ng/μL, while those of the buffalo and yak were still at 0.025 ng/μL. Under optimal condition with the ratio of three primer/probe sets at 3:3:2= yellow cattle:buffalo:yak, the triplex real-time PCR assay showed LOD of 0.1 ng/μL for both yellow cattle and buffalo, but 0.025 ng/μL for yak. However, these detection abilities in the duplex and triplex real-time PCR were all based on that the tested sample DNA, which was a mixture of the target species DNAs mixed in an equal proportion. But if the proportions of the three bovines were quite different, especially if there was great difference between yellow cattle and yak (9:1), the species with the lowest proportion probably would be undetectable. Therefore, triplex real-time PCR is an effective way to simultaneously detect the three animal-derived components in samples of whole meat, while simplex real-time PCR is recommended for use on ground meat.

Key words: yellow cattle; buffalo; yak; triplex quantitative real-time polymerase chain reaction