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.

中文翻译：

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开发用于鉴定梅花鹿和马鹿及其杂交种的四引物 ARMS-PCR。

准确鉴定鹿源成分对于食品和药品的真实性具有重要意义。多年来，已经开发了多种方法来验证这些产品；然而，确定雌鹿产品是否是杂交品种具有挑战性。本研究对梅花鹿（Cervus nippon）、马鹿（Cervus elaphus）及其杂交后代的锌指蛋白X连锁（ZFX）基因序列进行了扩增和测序，获得了X221和X428物种特异性单核苷酸多态性（验证了SNP）位点，并开发了四引物扩增难治性突变系统（T-ARMS-PCR）测定法来鉴定雌性梅花鹿、马鹿及其杂交鹿的亲本。基于X221位点开发的T-ARMS-PCR可以根据PCR产物大小分别为486 bp、352 bp和179 bp的大小来识别梅花鹿、马鹿及其杂交后代，与X428一样该基因座可以根据 PCR 产物大小分别为 549 bp、213 bp 和 383 bp 的情况来识别梅花鹿、马鹿及其杂交后代。利用该方法对随机购买的40种标记有鹿源性成分的产品进行检测，结果表明，基于两个SNP位点的鉴定结果与实际来源完全一致。该方法准确、简便、专属性强，为鹿产品品种鉴定提供了重要的技术参考。也是对现有鹿产品原始成分鉴别方法的重要补充。