建立多重液相基因芯片方法快速检测狐狸、水貂成分

Development of a Multiplex Liquidchip Assay for Rapid Identification of Fox and Mink Ingredients

基于xMAP液相芯片新型生物技术平台,分别以狐狸线粒体DNA D-loop区序列、水貂线粒体DNA细胞色素b基因序列为模板设计特异扩增引物和探针,并对探针进行锁核酸修饰,建立了二重xMAP液相基因芯片方法,用于快速检测狐狸和水貂源性成分。该法能准确鉴定鉴别狐狸和水貂DNA,对其他18种动物物种DNA均呈检测阴性,对狐狸、水貂DNA的检测低限分别为2.8pg/μl、0.9pg/μl,对肉类混样检出限为0.05%(m/m)。对目标源性DNA含量为1%(V/V)的32份饲料与食品核酸添加样本均呈对应目标检测阳性。结果表明,该方法特异性强、灵敏度高,适用于食品与饲料领域相关原料和产品的质量与安全检验。

A novel nucleic acid detection method,based on the xMAP(flexible multi-analyte profiling)technology platform,was developed for high-throughput and simultaneous identification of fox and mink ingredients in products of animal origin.A duplex xMAP assay was established,the primers and probes were designed targeting a fox mitochondrial D-loop gene or a mink mitochondrial cytochrome b gene.The probes were incorporated with locked nucleic acids to improve detection efficiency.The assay accurately identified fox and mink DNA,without cross reactions with DNA samples of eighteen nontarget animal species.The LOD on purified fox and mink DNA was evaluated as 2.8pg/μl and 0.9pg/μl,respectively.The detection sensitivity on samples of experimental meat mixtures was demonstrated to be 0.05%(m/m).The assay successfully detected 32 mocked positive DNA sample of food and feeds,which each was made by adding fox DNA or mink DNA at 1%(V/V)proportion of the same concentration.In conclusion,the duplex xMAP assay provided rapid identification of fox and mink ingredients with high specificity and high sensitivity.The technique is suitable to be applied in food and feeds quality assurance systems and safety inspections.