Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM

Detection of monkeypox virus based on a convenient and sensitive single-step RPA-CRISPR/Cas12a strategy

“…A significant improvement of this study is the use of commercial enzymes that do not require optimization of the LbCas12a protein sequence and the design of no-PAM crRNAs could also be considered when performing a single-step RPA-CRISPR assay to detect dsDNA viruses. 28 …”

“…A signicant improvement of this study is the use of commercial enzymes that do not require optimization of the LbCas12a protein sequence and the design of no-PAM crRNAs could also be considered when performing a single-step RPA-CRISPR assay to detect dsDNA viruses. 28 According to recent reports, most MPXV assays using isothermal amplication and CRISPR detection require a twostep reaction due to the competition between isothermal amplication and the conventional crRNA design principles for CRISPR detection in a single tube. In the two-step process, the product is rst amplied in a separate tube and then transferred to the CRISPR/Cas system for uorescence detection.…”

Loop-Mediated Isothermal Amplification-Integrated CRISPR Methods for Infectious Disease Diagnosis at Point of Care

“…95 A lab-on-paper platform was fabricated to detect SARS-CoV-2 N, S, and RNase P genes simultaneously, 96 and an instrument-free microfluidic system that contained all RPA, CRISPR, and LFA components was clinically validated, demonstrating 94.1% sensitivity and 100% specificity for SARS-CoV-2. 97 Beyond PoC viral disease diagnosis, RPA-integrated CRISPR-based platforms have also been used to diagnose Bordetella pertussis at PoC settings using clinical swab samples through a single-step system, 98 to monitor the presence of pathogens such as S. aureus 99 or Toxoplasma gondii 100 in the environment, and to detect Brucella in blood and milk samples. 101 The integration of isothermal nucleic acid amplification (INAA) strategies with CRISPR/Cas enzymes remains promising for PoC infectious disease diagnosis as well as other microorganism monitoring purposes.…”

Research Progress of Nucleic Acid Detection Technology for Genetically Modified Maize

“…RPA appears to be particularly well suited for multiplexing, where different targets can be verified with different efficiencies; however, it currently requires laborious optimization steps. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPER-associated protein [123], lateral flow assays [124,125] and microfluidics [126] have provided additional options for the on-site point-of-care detection of transgenes (Figure 2). CPA technology is an isothermal DNA amplification system developed by Us-Tar Biotechnology Co., Ltd [106].…”