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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

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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 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.

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Acknowledgements

We acknowledge the financial supports of the grants (Mega Project of Research on the Prevention and Control of HIV/AIDS, Viral Hepatitis Infectious Diseases 2013ZX10004-101 to Changyun Ye) from the Ministry of Science and Technology, People’s Republic of China, and grant (No. 2015SKLID507 to Changyun Ye) from State Key Laboratory of Infectious Disease Prevention and Control, China CDC.

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Authors and Affiliations

  1. State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention, Beijing, 102206, China

    Yi Wang, Yan Wang, Jianguo Xu & Changyun Ye

  2. Department of Microbiology, Guizhou Medical University, Guiyang, 550004, China

    Hui Li

  3. The Sixth People’s Hospital of Zhengzhou, Zhengzhou, 450000, China

    Huaqing Xu

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  1. Yi Wang
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  2. Hui Li
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  3. Yan Wang
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  5. Jianguo Xu
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  6. Changyun Ye
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Correspondence to Changyun Ye.

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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

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Wang, Y., Li, H., Wang, Y. et al. 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. Nano Res. 11, 2632–2647 (2018). https://doi.org/10.1007/s12274-017-1893-z

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  • DOI: https://doi.org/10.1007/s12274-017-1893-z

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