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Nano Research

Article Title

Antarctic thermolabile uracil-DNA-glycosylase-supplemented multiple cross displacement amplification using a label-based nanoparticle lateral flow biosensor for the simultaneous detection of nucleic acid sequences and elimination of carryover contamination

Keywords

Antarctic thermolabile uracil-DNA-glycosylase (AUDG), nucleic acid amplification techniques (NAAs), multiple cross displacement amplification (MCDA), lateral flow biosensor (LFB), limit of detection (LOD), human papillomaviruses (HPV)

Abstract

ABSTRACT 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 probehybridization 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-MCDALFB assay and validate its sensitivity, reproducibility, and specificity using serial dilutions of the type-specific plasmids, as well as clinical samples. This proofof-concept method (AUDG-MCDA-LFB) can be easily reconfigured to detect various nucleic acid sequences by redesigning the specific MCDA primers.

Graphical Abstract

Publisher

Tsinghua University Press

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