肉制品中马源性成分重组酶介导链置换等温扩增实时检测方法的建立及应用

Establishment and Application of Recombinase-Mediated Strand Displacement Isothermal Amplification Assay for Rapid Detection of Horse-Derived Components in Meat Products

目的:建立一种快速鉴定肉及肉制品中马源性成分的重组酶介导链置换等温扩增实时检测方法。方法:以马源性ATpase 6基因为靶基因设计多组特异性引物和Exo探针,通过引物筛选及反应参数优化,建立一种采用重组酶介导链置换等温扩增技术检测马源性成分的方法,并对该方法进行特异性、灵敏度和稳定性验证,同时通过对不同掺入比例混合样品、不同加工工艺模拟样品和市售样品进行检测,分析方法的检出限、适用性和准确性。结果:该方法反应迅速、特异性强、灵敏度高。可在39℃恒温条件下16min内完成反应;对23种非目标源性具有良好特异性;目标DNA的检测灵敏度可达到1.8copies/μL水平;对生肉的检出限为0.01%(质量分数,下同),对熟肉制品的检出限为0.1%;对90份市售样品进行检测,结果与标准方法一致。结论:建立的重组酶介导链置换等温扩增方法可用于肉及肉制品中马源性成分的掺假鉴别检测。

Objective:A recombinase-mediated strand displacement isothermal amplification assay for rapid detection of horse-derived components in meat and meat products was established.Methods:A series of specific primers and Exo probes were designed using the horse-derived ATpase 6 gene as the target gene.The primers were screened and the reaction parameters were optimized.The specificity,sensitivity and stability of the assay were evaluated,and the detection limit,applicability and accuracy were analyzed by detecting simulated samples with different mixing ratios and different processing technologies,and commercial samples.Results:The assay was characterized by rapid response,high sensitivity and specificity.The reaction was completed within 16 minutes under a constant temperature of 39℃.The system had good specificity to 23 non-target sources.The detection sensitivity of the target DNA was 1.8 copies/μL.The detection limit was 0.01%for raw meat(mass fraction),and 0.1%(mass fraction)for processed meat products.For 90 commercial samples,the results of this method were consistent with those of the standard method.Conclusion:The recombinase-mediated isothermal amplification assay can be used for the detection of horse-derived components in meat and meat products.