Here, we report a novel and universal methodology, termed “Antarctic thermolabile uracil-DNA-glycosylase (AUDG)-supplemented nucleic acid amplification techniques (NAAs) using a labeled-based nanoparticle lateral flow biosensor (LFB)” (AUDG-NAAs-LFB), which merges enzymatic (AUDG) digestion of contaminant amplicons with different nucleic acid amplification techniques (NAAs), and uses a lateral flow biosensor (LFB) for the rapid and visual confirmation of the presence of a target nucleic acid sequence. AUDG-NNAs-LFB is a one-pot, closedvessel assay, that can effectively eliminate false-positive signals arising from either carryover contaminants or the interaction between labeled primers. A new LFB was devised for detecting three targets (two amplicons generated from amplification of target sequences, and a chromatography control), without the need for probe-hybridization or additional incubation steps. As a proof of concept, multiple cross displacement amplification (MCDA), which is a specific, sensitive, and rapid isothermal amplification method, was selected as the model amplification technique to demonstrate the feasibility of AUDG-NAAs-LFB. As a result, we demonstrate the applicability of the AUDG-MCDA-LFB method for simultaneously detecting high-risk human papillomaviruses genotypes 16 and 18, which are the most and second-most prevalent strains of the virus reported in women worldwide. We also confirm the principle behind the AUDG-MCDA-LFB assay and validate its sensitivity, reproducibility, and specificity using serial dilutions of the type-specific plasmids, as well as clinical samples. This proof-of-concept method (AUDG-MCDA-LFB) can be easily reconfigured to detect various nucleic acid sequences by redesigning the specific MCDA primers.

中文翻译：

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南极热不稳定的尿嘧啶-DNA-糖基化酶辅助的多重交叉置换扩增，使用基于标记的纳米颗粒侧向流动生物传感器，可同时检测核酸序列并消除残留污染

在这里，我们报告了一种新颖的通用方法，称为“使用基于标记的纳米颗粒侧向流生物传感器（LFB）的南极不耐热尿嘧啶DNA-糖基化酶（AUDG）-补充的核酸扩增技术（NAA）”（AUDG-NAAs-LFB ），将污染物扩增子的酶促（AUDG）消化与不同的核酸扩增技术（NAA）合并在一起，并使用侧向流生物传感器（LFB）快速和目视确认目标核酸序列的存在。AUDG-NNAs-LFB是一种单罐封闭容器测定法，可以有效消除残留污染物或标记引物之间相互作用产生的假阳性信号。设计了一种新的LFB，用于检测三个靶标（通过扩增靶标序列和色谱控制获得两个扩增子），无需探针杂交或其他孵育步骤。作为概念验证，选择了一种特定，灵敏且快速的等温扩增方法的多重交叉位移扩增（MCDA）作为模型扩增技术，以证明AUDG-NAAs-LFB的可行性。结果，我们证明了AUDG-MCDA-LFB方法可同时检测高风险的人类乳头瘤病毒基因型16和18，这是全世界女性中报告的该病毒的最高和第二高流行株。我们还确认了AUDG-MCDA-LFB分析背后的原理，并使用类型特异性质粒和临床样品的系列稀释液验证了其灵敏度，可重复性和特异性。