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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 被引量:1

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
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摘要 Here, we report a novel and universal methodology,termed "ntarctic 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 nudeic acid sequences by redesigning the specific MCDA primers. Here, we report a novel and universal methodology,termed "ntarctic 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 nudeic acid sequences by redesigning the specific MCDA primers.
出处 《Nano Research》 SCIE EI CAS CSCD 2018年第5期2632-2647,共16页 纳米研究(英文版)
关键词 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) 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)
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