多重聚合酶链式反应技术在热加工肉制品真实性检测中的应用

Application of multiplex polymerase chain reaction technology in the authenticity detection of heat-processed meat products

中文关键词:  多重PCR  物种鉴定  灵敏度  掺假检测  肉制品

英文关键词:multiplex polymerase chain reaction  species identification  sensitivity  adulteration detection  meat products

中文摘要:

      目的 建立多重聚合酶链式反应(polymerase chain reaction, PCR)技术在单一反应体系中同时鉴别多种肉类的高效检测方法。方法 基于动物线粒体基因序列的物种特异性差异, 采用多软件协同设计与筛选引物, 构建八重PCR体系, 实现对羊肉、鸡肉、兔肉、鸽肉、鹅肉、火鸡肉、鸵鸟肉和狗肉8种肉类来源的同步检测。通过条件优化和DNA测序验证扩增结果的特异性, 并在多物种DNA混合模板及掺假模型肉样中对方法的灵敏度与适用性进行评价。结果 多重PCR可在单一反应管内稳定扩增8种目标物种DNA, 未发现非特异性扩增; 测序结果与对应物种一致。灵敏度结果表明, 在生肉及热加工肉样品中, 各目标物种DNA的最低检出限为0.01~0.05 ng, 在掺假样品中目标肉类含量低至0.10%仍可检出。结论 所建立的多重PCR方法可成功应用于市售肉制品样品的真实性检测, 能够实现多种肉类来源的快速、准确鉴别, 具有良好的实际应用价值, 可为食品安全监测与市场监管提供技术支撑。

英文摘要:

      Objective To establish n method for simultaneously identifying multiple meat species within a single reaction system by efficient multiplex polymerase chain reaction (PCR) technology. Methods Based on species-specific variations in animal mitochondrial gene sequences, primers were designed and screened using multiple bioinformatics tools, and an octuple PCR assay was developed for the simultaneous detection of 8 kinds of meat species, namely mutton, chicken, rabbit, pigeon, goose, turkey, ostrich and dog. The specificity of the amplification products was confirmed through reaction condition optimization and DNA sequencing. The sensitivity and applicability of the method were further evaluated by multi-species DNA mixtures and adulterated meat model samples. Results The octuple PCR assay enabled stable and simultaneous amplification of DNA from all 8 kinds of target species in a single reaction tube, with no nonspecific amplification observed. Sequencing results were consistent with the corresponding target species. Sensitivity analysis showed that, in both raw and heat-processed meat samples, the limits of detection for the target species ranged from 0.01 to 0.05 ng of genomic DNA. In adulterated samples, target meat components were detectable at levels as low as 0.10%. Conclusion The established octuple PCR method can be successfully applied to the authenticity assessment of commercially available meat products, allowing rapid and accurate identification of multiple meat species. This method demonstrates good practical applicability and provides effective technical support for food safety surveillance and market regulation.