A low-cost novel lateral flow nucleic acid assay (LFNAA) was developed for rapid and sensitive authentication of yak milk on-site. In this method, a special abasic spacer C3 and tail nucleotide sequence modified reverse primers were innovatively designed for rapid isothermal recombinant polymerase amplification (RPA) to obtain a double-stranded amplicon with a single-stranded DNA tail, which could directly bind to gold nanoparticle (AuNP)-labeled detection probes, and produce a sandwich complex to show a red color on the test line of the strip. Compared to conventional RPA-lateral flow assay based on expensive antibodies, the proposed method in this work demonstrated massive cost reduction, avoided time-consuming and laborious AuNP-antibody conjugation process. The LFNAA revealed high specificity and sensitivity, which allowed detecting as low as 5% of yak milk within 40 min. This LFNAA facilitated non-specialized operators to field determine the authenticity of yak milk by naked eyes, and could be readily applied to other nucleic acid detection such as food safety inspection and point-of-care medical tests.

开发了一种低成本新颖的侧向流核酸测定法（LFNAA），用于现场快速，灵敏地鉴定authentication牛乳。在该方法中，创新设计了特殊的无碱基间隔子C3和尾部核苷酸序列修饰的反向引物，用于快速等温重组聚合酶扩增（RPA），以获得具有单链DNA尾巴的双链扩增子，其可以直接与金纳米粒子结合（AuNP）标记的检测探针，并产生三明治复合物，在检测条的检测线上显示红色。与基于昂贵抗体的常规RPA侧向血流分析相比，本工作中提出的方法证明了成本降低，避免了费时费力的AuNP抗体偶联过程。LFNAA显示出高度的特异性和敏感性，可以在40分钟内检测到5％的牛乳。该LFNAA便利了非专业操作人员通过肉眼现场确定of牛乳的真伪，并且可以很容易地应用于其他核酸检测，例如食品安全检查和即时医疗。