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      Discrimination of Single-Nucleotide Variants Based on an Allele-Specific Hybridization Chain Reaction and Smartphone Detection

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          Abstract

          Massive DNA testing requires novel technologies to support a sustainable health system. In recent years, DNA superstructures have emerged as alternative probes and transducers. We, herein, report a multiplexed and highly sensitive approach based on an allele-specific hybridization chain reaction (AS-HCR) in the array format to detect single-nucleotide variants. Fast isothermal amplification was developed before activating the HCR process on a chip to work with genomic DNA. The assay principle was demonstrated, and the variables for integrating the AS-HCR process and smartphone-based detection were also studied. The results were compared to a conventional polymerase reaction chain (PCR)-based test. The developed multiplex method enabled higher selectivity against single-base mismatch sequences at concentrations as low as 10 3 copies with a limit of detection of 0.7% of the mutant DNA percentage and good reproducibility (relative error: 5% for intra-assay and 17% for interassay). As proof of concept, the AS-HCR method was applied to clinical samples, including human cell cultures and biopsied tissues of cancer patients. Accurate identification of single-nucleotide mutations in KRAS and NRAS genes was validated, considering those obtained from the reference sequencing method. To conclude, AS-HCR is a rapid, simple, accurate, and cost-effective isothermal method that detects clinically relevant genetic variants and has a high potential for point-of-care demands.

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          Most cited references35

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          Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

          Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) that combines isothermal pre-amplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCKv2: 1) 4-channel single reaction multiplexing using orthogonal CRISPR enzymes; 2) quantitative measurement of input down to 2 aM; 3) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxilary CRISPR-associated enzyme; and 4) lateral flow read-out. SHERLOCKv2 can detect Dengue or Zika virus ssRNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.
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            Isothermal Amplification of Nucleic Acids.

            Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.
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              Recombinase polymerase amplification: Basics, applications and recent advances

              Recombinase polymerase amplification (RPA) is a highly sensitive and selective isothermal amplification technique, operating at 37–42°C, with minimal sample preparation and capable of amplifying as low as 1–10 DNA target copies in less than 20 min. It has been used to amplify diverse targets, including RNA, miRNA, ssDNA and dsDNA from a wide variety of organisms and samples. An ever increasing number of publications detailing the use of RPA are appearing and amplification has been carried out in solution phase, solid phase as well as in a bridge amplification format. Furthermore, RPA has been successfully integrated with different detection strategies, from end-point lateral flow strips to real-time fluorescent detection amongst others. This review focuses on the different methodologies and advances related to RPA technology, as well as highlighting some of the advantages and drawbacks of the technique.
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                Author and article information

                Journal
                ACS Sens
                ACS Sens
                se
                ascefj
                ACS Sensors
                American Chemical Society
                2379-3694
                21 February 2022
                25 March 2022
                : 7
                : 3
                : 758-765
                Affiliations
                []Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València , Camino de Vera s/n, 46022 Valencia, Spain
                []Unidad Mixta UPV-La Fe, Nanomedicine and Sensors , Av. Fernando Abril Martorell, 46026 Valencia, Spain
                Author notes
                Author information
                https://orcid.org/0000-0003-4641-4957
                https://orcid.org/0000-0003-4021-5607
                Article
                10.1021/acssensors.1c02220
                8961872
                35188365
                59bcfe66-e727-432a-afc2-b246d101fcca
                © 2022 The Authors. Published by American Chemical Society

                Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 19 October 2021
                : 07 February 2022
                Funding
                Funded by: Ministerio de Economía y Competitividad, doi 10.13039/501100003329;
                Award ID: PID2019-110713RB-I00
                Funded by: European Regional Development Fund, doi 10.13039/501100008530;
                Award ID: NA
                Funded by: Generalitat Valenciana, doi 10.13039/501100003359;
                Award ID: PROMETEO/2020/094
                Funded by: Generalitat Valenciana, doi 10.13039/501100003359;
                Award ID: GVA-FPI-2017
                Categories
                Article
                Custom metadata
                se1c02220
                se1c02220

                dna biosensing,single-nucleotide mutation,allele-specific probe,hybridization chain reaction,cancer biomarker genes

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