双拖尾重组聚合酶恒温扩增技术结合核酸杂交侧流条快速检测牛肉中的鸡、鸭、猪源性成分

Nucleic acid hybridization-lateral flow strip combined with double tailed recombinase polymerase amplification for rapid detection of chicken, duck and pig adulteration in beef

中文关键词:  肉类鉴定  双拖尾重组聚合酶扩增  核酸杂交试纸条  可视化检测

英文关键词:meat authenticity  double tailed recombinase polymerase amplification  nucleic acid hybridization- lateral flow strip  visual detection

中文摘要:

      目的 建立双拖尾重组聚合酶恒温扩增(recombinase polymerase amplification, RPA)结合核酸杂交侧流条(nucleic acid hybridization-lateral flow strip, NAH-LFS)快速检测牛肉中鸡源、鸭源、猪源性成分的可视化检测技术。方法 采用多重双拖尾RPA技术与NAH-LFS相结合, 对鸡、鸭、猪线粒体D环区域设计特异性拖尾RPA引物, 扩增出双拖尾的RPA产物。鸡、鸭、猪3个物种RPA产物的拖尾序列分别与红、黄、蓝3种颜色的金纳米探针和侧流条上的检测探针杂交。结果 鸡、鸭、猪3个物种RPA产物与探针杂交后形成肉眼可见的红、黄、蓝3色条带, 整个RPA扩增(15 min)和侧流条检测(5 min)过程在20 min内即可完成。该方法对牛肉中鸡、鸭、猪肉的检出限达0.01%, 且仅对鸡、鸭、猪肉DNA有特异性, 与其他10个物种的DNA均无交叉反应。采集50份市售牛肉样品, 使用该方法进行真实性检测: 10份牛肉干、10份生牛肉、10份酱牛肉中未检测出其他动物源性成分, 10份牛肉馅料中检测出1份含猪源性成分, 10份牛肉片中检测出1份含鸡源性成分。该方法与聚合酶链式反应结合凝胶电泳的检测结果具有很好的一致性。结论 RPA技术结合NAH-LFS具有特异性强、灵敏度高、操作简单的优点, 可实现牛肉中鸡、鸭、猪源性成分的同时可视化快速检测。

英文摘要:

      Objective To establish a visual detection technology for the rapid detection of chicken, duck, and pig derived components in beef by double tailed recombinase polymerase amplification (RPA) combined with nucleic acid hybridization-lateral flow strip (NAH-LFS). Methods By combining multiple double tailed RPA technology with NAH-LFS, specific trailing RPA primers were designed for D-loop region of chicken, duck and pig mitochondria, and double trailing RPA products were amplified. The trailing sequences of RPA products of chicken, duck and pig hybridized with gold nano-probes of red, yellow and blue and detection probes on lateral flow strips respectively. Results The RPA products of chicken, duck and pig hybridize with the probe to form red, yellow and blue bands visible to the naked eye. The whole process of RPA amplification (15 min) and lateral flow strip detection (5 min) could be completed within 20 min. The limit of detection for chicken, duck and pork in beef was 0.01%, and it was only specific for chicken, duck and pork DNA, and there was no cross reaction with the DNA of other 10 species. The 50 samples of beef sold in the market were collected, and the authenticity was tested by this method. No other animal-derived components were detected in 10 dried beef, 10 raw beef and 10 sauce beef, 1 pork-derived component was detected in 10 beef stuffing, and 1 chicken-derived component was detected in 10 beef slices. The results of this method were in good agreement with those of polymerase chain reaction combined with gel electrophoresis. Conclusion RPA technology combined with NAH-LFS has the advantages of strong specificity, high sensitivity and simple operation, which can realize the simultaneous visual and rapid detection of chicken, duck and pig-derived components in beef.