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Graphene oxide and self-avoiding molecular recognition systems-assisted recombinase polymerase amplification coupled with lateral flow bioassay for nucleic acid detection

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Abstract

A new nucleic acid detection technique, termed Nano-SAMRS-RPA, is reported which employed carbon nanomaterial (graphene oxide, GO) and self-avoiding molecular recognition systems (SAMRS) to improve the specificity of recombinase polymerase amplification (RPA). In the presence of GO and SAMRS primers, the assay artifacts, including primer-dimers, nonspecific products, off-target hybrids, and non-canonical folds, are completely suppressed and eliminated, which makes the creation of RPA-based methods faster by simplifying the primer design and eliminating the need for primer optimization and complex probe. Moreover, a lateral flow bioassay (LFB) was also devised for simply and rapidly indicating the Nano-SAMRS-RPA results. Particularly, the new detection system only requires a single-labeled primer, eliminating the false-positive result from hybridization (the labeled probe and reverse primer) and the use of real-time instrument, more complex enzymatic solutions, and probes. As a result, GO, SAMRS primers, and LFB convert RPA from a technique suited only for the research laboratory into one that has a practical value in clinical settings, field environments, and at points-of-care testing. Human papillomaviruses (HPV) genotypes 16 and 18 were applied as model analytes to test the assay’s availability. The initial data indicated that Nano-SAMRS-RPA could detect down to 10 copies per reaction, and the sensitivity (14/14 samples collected from HPV16 and HPV 18 patients) and specificity (75/75 samples collected from non-HPV patients) for clinical sample detection were 100%. The proof-of-concept technique can be reconfigured to detect various nucleic acid sequences by redesigning the specific RPA primers.

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Funding

This work was supported by the Beijing Talent Fund (No. 2016000021223ZK38), by the Beijing Health System High-level Health Technical Personnel Foundation (no. 2015-3-081), by Beijing Nova Program (Z161100004916079), and by Cultivation Fund Project of the National Natural Science Foundation in Beijing Children’s Hospital, Capital Medical University (GPMS201904).

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Authors and Affiliations

  1. Key Laboratory of Major Diseases in Children, Ministry of Education, National Key Discipline of Pediatrics (Capital Medical University), National Clinical Research Center for Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Disease, Beijing Pediatric Research Institute, Beijing Children’s Hospital, National Center for Children’s Health, Capital Medical University, Beijing, 10045, People’s Republic of China

    Yi Wang, Wei-wei Jiao, Ya-cui Wang, Chen Shen, Hui Qi & A-Dong Shen

  2. Department of Clinical Laboratory, The First People’s Hospital of Guiyang, Guiyang, 550003, Guizhou, People’s Republic of China

    Yu Wang

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  1. Yi Wang
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  2. Wei-wei Jiao
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  3. Yu Wang
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  4. Ya-cui Wang
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  5. Chen Shen
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  6. Hui Qi
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  7. A-Dong Shen
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Correspondence to A-Dong Shen.

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Wang, Y., Jiao, Ww., Wang, Y. et al. Graphene oxide and self-avoiding molecular recognition systems-assisted recombinase polymerase amplification coupled with lateral flow bioassay for nucleic acid detection. Microchim Acta 187, 667 (2020). https://doi.org/10.1007/s00604-020-04637-5

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