Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy

Abstract

It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.

KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus

“…The length of the RPA primers should preferably be 30−35 bp; otherwise, recombinase activity will be affected. Consecutive polyguanine should be avoided at the 5’ end so as not to interfere with the recombination of the amplified fragments; cytosine and guanine are preferred at the 3’ end to improve the amplification performance of the primers ( 41 , 42 ). At the same time, screening elimination was needed to determine the most suitable primers for RPA; the more primers screened, the greater the chance of finding the most suitable primers for RPA ( 43 ).…”

A Novel CRISPR/Cas12a-Mediated Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive and Reliable Detection of Circulating Tumor Deoxyribonucleic Acid

“…When activated by matching target DNA, it can cut any nontarget ssDNA indiscriminately, showcasing remarkable self-signal amplification capability. , To further boost target DNA detection sensitivity, a common approach is to combine the CRISPR/Cas12a system with various nucleic acid amplification strategies (e.g. ; LAMP, EXPAR, RPA), which has become a mainstream trend. − Chen et al developed a strategy called DETECTR by combining CRISPR/Cas12a and isothermal amplification to detect DNA . Compared with the strategy of only CRISPR/Cas12a, it could realize the sensitivity of attomolar .…”

Development of a reverse transcription loop-mediated isothermal amplification based clustered regularly interspaced short palindromic repeats Cas12a assay for duck Tembusu virus

“…Subsequently, the Cas12a performs the collateral cleavage on nonspecific single-stranded DNA (ssDNA) (e.g., the fluorescent ssDNA reporter HEX-N12-BHQ1) when the Cas12a, crRNA, and target DNA form a ternary complex ( Gootenberg et al, 2018 ; Li et al, 2018b ). Based on this collateral cleavage activity, a number of Cas12a-based molecular diagnostic methods coupled with isothermal amplification techniques containing RPA, RCA, RAA and LAMP have been developed to detect pathogens, including RNA viruses, DNA viruses and bacteria ( Li et al, 2018b ; Xu et al, 2020 ; Mayuramart et al, 2021 ; Chen et al, 2022 ; Qin et al, 2022 ; de Sousa et al, 2023 ; Fan et al, 2023 ; Mao et al, 2023 ; Wu et al, 2023 ; Zhao et al, 2023 ). For instance, an RT-RAA-based CRISPR-Cas12a assay was established to detect SARS-CoV-2 and influenza viruses, and the assay was effective for screening of these viruses ( Mayuramart et al, 2021 ).…”