基于双拖尾重组聚合酶恒温扩增技术的核酸杂交试纸条快速检测驴肉中的马源性成分

Rapid determination of horse-derived components in donkey meat by nucleic acid hybridization strip based on double trailing recombinant polymerase constant temperature amplification technology

中文关键词:  肉类真实性  双拖尾扩增  核酸杂交试纸条  马源性成分  可视化检测

英文关键词:meat authenticity  double trailing  nucleic acid hybridization lateral flow strip  horse-derived components  visual detection

中文摘要:

      目的 建立快速、操作简单、价格相对低廉的重组聚合酶恒温扩增(recombinase polymerase amplification, RPA)技术结合核酸杂交试纸条快速检测驴肉中马源性成分的可视化检测技术。方法 将双拖尾重组聚合酶恒温扩增技术与核酸杂交试纸条相结合。针对马线粒体细胞色素b (cytb)基因设计拖尾RPA引物, 这对特殊引物使RPA扩增子带有两个单链拖尾序列, 这两个单链拖尾序列可以特异性地被金纳米探针和试纸条上的检测探针识别并捕获, 形成一条肉眼可见的红色条带, 从而对马源性成分进行检测。结果 该方法整个扩增(15 min)和检测(5 min)过程在20 min即可完成, 对马肉的检出限达到0.010%, 且仅对马肉核酸有特异性扩增, 与其他8个物种的核酸均无交叉反应。20份市售驴肉样品中有2份存在马源性成分, 且该方法与PCR结合凝胶电泳的检测结果具有很好的一致性。结论 该方法特异性强、灵敏度高, 可实现驴肉中马源性成分的可视化快速检测。

英文摘要:

      Objective To establish a rapid, simple and relatively low-cost visual detection technology for rapid determination of horse-derived components in donkey meat by recombinase polymerase amplification (RPA) combined with nucleic acid hybridization lateral flow strip. Methods Double tailed RPA was combined with nucleic acid hybridization lateral flow strip. The double tailed RPA primers were designed from horse mitochondrial cytochrome b (cytb) gene. This special primers enabled the RPA products carry two single-stranded trailing sequence, which could be specially recognized and captured by the probes on gold nanoparticles and test line forming a red line visible to the naked eyes. Results The whole process of amplification (15 min) and detection (5 min) was completed within 20 min. The detection limit of horsemeat reached 0.010%, and there was only specific amplification of horsemeat nucleic acid, and no cross reaction with nucleic acid of other 8 species. Two of 20 commercial donkey meat samples were detected with horse meat, and this result was in good agreement with that of PCR combined with gel electrophoresis. Conclusion This method has high specificity and sensitivity, and can be used for visual and rapid detection of horse derived components in donkey meat.