Graphene oxide and self-avoiding molecular recognition systems-assisted recombinase polymerase amplification coupled wit
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ORIGINAL PAPER
Graphene oxide and self-avoiding molecular recognition systems-assisted recombinase polymerase amplification coupled with lateral flow bioassay for nucleic acid detection Yi Wang 1 & Wei-wei Jiao 1 & Yu Wang 2 & Ya-cui Wang 1 & Chen Shen 1 & Hui Qi 1 & A-Dong Shen 1 Received: 18 March 2020 / Accepted: 6 November 2020 # Springer-Verlag GmbH Austria, part of Springer Nature 2020
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-SAMRSRPA 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 NanoSAMRS-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 proofof-concept technique can be reconfigured to detect various nucleic acid sequences by redesigning the specific RPA primers. Keywords Graphene oxide . Carbon nanomaterial . Recombinase polymerase amplification . Lateral flow bioassay . Nucleic acid detection
Introduction Nucleic acid amplification in vitro has become a useful tool in basic research, biotechnology, and human health [1]. The PCRbased methods are the most popular amplification technologies, which require expensive laboratory apparatuses, and are time* A-Dong Shen [email protected] 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
2
Department of Clinical Laboratory, The First People’s Hospital of Gu
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