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

目的：建立快速、操作简单、价格相对低廉的驴肉中马源性成分快速可视化检测技术。方法：采用双拖尾重组聚合酶恒温扩增技术(RPA)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RPA引物,引物包含3个功能区域,特异性绑定区域用于RPA扩增反应,spacer 9用于终止聚合酶的扩增,单链拖尾区域用于与试纸条上的探针杂交。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。整个扩增(15 min)和检测(5 min)过程在20 min即可完成,无需复杂的设备,仅需要一台数字化水浴锅。为进一步验证该方法的实际应用性能,对市面上的20份驴肉产品进行测定,并采用PCR(Polymerase Chain Reaction,PCR)结合琼脂糖凝胶电泳的方法进行验证。结果：该方法对马肉的检测限达到0.01%,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份驴肉样品中有2份存在马源性成分。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。

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

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.