Rapid and sensitive detection of Ebola RNA in an unamplified sample based on CRISPR-Cas13a and DNA roller machine

CRISPR-based nucleic acid diagnostics for pathogens

“…Nowadays, the outbreak caused by viruses — e.g. , Zika virus (ZIKV), African swine fever virus (ASFV), Ebola virus and SARS-CoV-2 — is a persistent threat to public health and global security [ [78] , [79] , [80] ]. The mostly recently viral epidemic outbreak is the COVID-19 caused by pathogenic SARS-CoV-2.…”

“…In addition to person-to-person transmission, SARS-CoV-2 enables person-to-material/material-to-person transmissions [ 81 ], highlighting the demand for rapid and low-cost diagnostic methods. Therefore, CRISPR/Cas-based nucleic acid diagnostics have frequently reported for SARS-CoV-2, and other reported targeting viruses include ASFV [ 64 , 82 ], ZIKV [ 9 , 79 , 83 ], Dengue virus (DENV) [ 9 ], Ebola virus [ 80 ] and hepatitis B virus (HBV) [ 84 ].…”

“…A DNA walker principle was built as an alternative strategy to amplify Cas13 cleavage reaction. By combining with a DNA walker machine for amplifying the output signal, a preamplification-free Cas13a assay allowed to detect Ebola RNAs with a LOD of 291 aM (∼175 copies RNA/μL) [ 80 ]. Nevertheless, the design of DNA walker could complicate the nucleic acid assay.…”

CRISPR-Cas13a system: A novel tool for molecular diagnostics

“…They also detected EBOV in the whole blood, urine, and saliva samples using this approach ( Barnes et al, 2020 ). Recently, a novel method called the Cas-Roller was reported, which combines the Cas13a reaction with a DNA roller reaction to detect Ebola RNA and achieved an LOD of 291 aM within 40 min ( Hang et al, 2022 ). In addition, the RPA-SHERLOCK method was employed to detect the Crimean-Congo hemorrhagic fever virus (CCHFV), another severe hemorrhagic fever virus ( Bente et al, 2013 ), realizing an LOD of 1 copy/μl within 30–40 min and discriminating CCHFV from a series of CCHFV-related viruses ( Li et al, 2022a ).…”

Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA

“…Besides, upon binding of targets, some Cas proteins, such as Cas12 and Cas13, unleash trans-cleavage (also called collateral cleavage) to degrade non-specific single-stranded nucleic acids, endowing Cas-based methods with improved sensitivity ( Chen et al, 2018 ; Cox et al, 2017 ; Gootenberg et al, 2018 ; Li et al, 2018 ). In addition, such CRISPR-based tools could provide an easy-to-use readout using minimal instrumentation ( Chen et al, 2021a ; de Puig et al, 2021 ; Hang et al, 2022 ; Liang et al, 2019 ). Both RT-LAMP and RPA have been combined with CRISPR-Cas for clinical detection of SARS-CoV-2 (i.e.…”