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

目的? 建立快速、简便、经济的双拖尾重组聚合酶恒温扩增结合核酸杂交试纸条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? 采用多重双拖尾重组聚合酶恒温扩增技术(multiplex recombinase polymerase amplification,mRPA)与核酸杂交试纸条相结合。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、蓝三种颜色的金纳米探针和试纸条上的检测探针杂交,形成肉眼可见的红、黄、蓝三色条带,整个RPA扩增(15 min)和试纸条检测(5 min)过程在20 min内即可完成。结果? 该方法对牛肉中鸡、鸭、猪肉的检测限达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PCR结合凝胶电泳的检测结果具有很好的一致性。结论? 双拖尾重组聚合酶恒温扩增技术结合核酸杂交试纸条具有特异性强、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。

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

Objective: To establish a fast, simple, and economical visual detection technology for the rapid detection of chicken, duck, and pork derived components in beef by double tailed recombinase polymerase amplification (RPA) combined with nucleic acid hybridization lateral flow strip (NAH-LFS). Methods: The assay was established by combining triplex double tailed RPA with NAH-LFS. The double tailed RPA primers were designed from mitochondrial D-loop region of chicken,duck and pork, which assisted RPA product containing two ssDNA. The trailing sequences of RPA products from three species were hybridized with gold nanoprobes of red, yellow, and blue colors and three detection probes on test strips, forming visible red, yellow, and blue bands. The entire amplification (15 minutes) and detection (5 minutes) process can be completed within 20 minutes. The detection limit of this method for chicken, duck, and pork was 0.01%, and only sepcific amplifications with chickens, ducks, and pigs nucleic acid was found and there was no cross reaction with other 10 animals species. 50 commercially available beef samples were collectand and the authenticity was tested with this RAP-NAH-LFS : one of 10 beef fillings was detected with pork adulteration, and one of 10 beef slices was detected with chicken adulteration. And the detection results of this method were consistant with PCR combined with agarose gel electrophoresis. Conclusion: This method has the advantages of strong specificity, high sensitivity, and simple operation. It can be used for visual and rapid detection of chicken, duck, and pig adulteration in beef.