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      Establishment of RPA-Cas12a-Based Fluorescence Assay for Rapid Detection of Feline Parvovirus

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      Author(s): 1 , 1 , 1 , 1 , 3 , 2 , , 3 ,
      Publication date (Electronic, pub):
      Publication date (Electronic, collection):
      Journal: Polish Journal of Microbiology
      Publisher: Sciendo
      Keywords: CRISPR-Cas12a, detection, feline parvovirus, RPA, real-time fluorescence

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          Abstract

          Feline parvovirus (FPV) is highly infectious for cats and other Felidae and often causes severe damage to young kittens. In this study, we incorporated recombinase polymerase amplification (RPA) and Cas12a-mediated detection and developed an RPA-Cas12a-based real-time or end-point fluorescence detection method to identify the NS1 gene of FPV. The total time of RPA-Cas12a-based fluorescence assay is approximately 25 min. The assay presented a limit of detection (LOD) of 1 copies/μl (25 copies/per reaction), with no cross-reactivity with several feline pathogens. The clinical performance of the assay was examined using total genomic DNA purified from 60 clinical specimens and then compared to results obtained with qPCR detection of FPV with 93.3% positive predictive agreement and 100% negative predictive agreement. Together, the rapid reaction, cost-effectiveness, and high sensitivity make the RPA-Cas12a-based fluorescence assay a fascinating diagnostic tool that will help minimize infection spread through instant detection of FPV.

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          Most cited references24

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          Nucleic acid detection with CRISPR-Cas13a/C2c2

          Jonathan S. Gootenberg, Omar O Abudayyeh, Jeong Wook Lee … (2017)
          Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting CRISPR effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous RNAse activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed SHERLOCK ( S pecific H igh Sensitivity E nzymatic R eporter Un LOCK ing), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage, and readily reconstituted on paper for field applications.
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            Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

            Omar Abudayyeh, Max J. Kellner, Julia Joung … (2018)
            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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              Recombinase polymerase amplification: Basics, applications and recent advances

              Ivan Lobato, Ciara O'Sullivan (2017)
              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

                Contributors
                Jian Liu
                Rong Xu
                Journal
                Journal ID (nlm-ta): Pol J Microbiol
                Journal ID (iso-abbrev): Pol J Microbiol
                Journal ID (publisher-id): pjm
                Journal ID (doi): pjm
                Title: Polish Journal of Microbiology
                Publisher: Sciendo
                ISSN (Print): 1733-1331
                ISSN (Electronic): 2544-4646
                Publication date (Electronic, pub): 4 March 2024
                Publication date (Electronic, collection): March 2024
                Volume: 73
                Issue: 1
                Pages: 39-48
                Affiliations
                [1 ]School of Biological and Pharmaceutical Engineering, Shaanxi University of Science and Technology , Xi’an, China
                [2 ]Shanghai Animal Disease Prevention and Control Center , Shanghai, China
                [3 ]Ningbo Municipal Center for Disease Control and Prevention , Ningbo, China
                Author notes
                Corresponding authors: J. Liu, Shanghai Animal Disease Prevention and Control Center, Shanghai, China; R. Xu, Ningbo Municipal Center for Disease Control and Prevention, Ningbo, China
                [#]

                Ting Wang and Hao Zeng contributed equally to this study.

                Author information
                https://orcid.org/0000-0001-8402-6838
                Article
                Publisher ID: pjm-2024-005
                DOI: 10.33073/pjm-2024-005
                PMC ID: 10911697
                PubMed ID: 38437470
                SO-VID: 5ac5bb7f-893c-41c3-b6f5-4cffb38a5288
                Copyright © © 2024 Ting Wang et al., published by Sciendo
                License:

                This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

                History
                Date received : 27 September 2023
                Date accepted : 29 December 2023
                Page count
                Pages: 10
                Funding
                This work was supported by Xi’an Science and Technology Plan Project (23LLRHZDZX0012, 22GXFW0007),, and Key Research and Development Plan Project in Shaanxi Province (2023-YBNY-170).
                Categories
                Subject: Original Paper

                Keywords: crispr-cas12a,detection,feline parvovirus,rpa,real-time fluorescence
                Data availability:
                Keywords: crispr-cas12a, detection, feline parvovirus, rpa, real-time fluorescence

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