Development of a tetra-primer ARMS-PCR for identification of sika and red deer and their hybrids

Accurate identification of deer-derived components is significant in food and drug authenticity. Over the years, several methods have been developed to authenticate these products; however, identifying whether female deer products are hybrids is challenging. In this study, the zinc finger protein X-linked (ZFX) gene sequences of sika deer (Cervus nippon), red deer (Cervus elaphus) and their hybrid offspring were amplified and sequenced, the X221 and X428 species-specific single nucleotide polymorphisms (SNP) loci were verified, and a tetra-primer amplification refractory mutation system (T-ARMS-PCR) assay was developed to identify the parent-of-origin of female sika deer, red deer, and their hybrid deer. The T-ARMS-PCR developed based on the X221 locus could identify sika deer, red deer, and their hybrid offspring according to the presence or absence of PCR product sizes of 486 bp, 352 bp, and 179 bp, respectively, just as X428 locus could identify sika deer, red deer, and their hybrid offspring according to the presence or absence of PCR product sizes of 549 bp, 213 bp, and 383 bp, respectively. Forty products labeled deer-derived ingredients randomly purchased were tested using this assay, and the results showed that the identification results based on the two SNP loci were utterly consistent with the actual sources. In addition, this method was found to be accurate, simple, convenient, and with high specificity, thus providing an essential technical reference for deer product species identification. It is also an important supplement to the identification methods of the original ingredients of existing deer products.