High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system

Abstract

Here we report the development of a high throughput, all-solution phase, and isothermal detection system to detect African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with fluorescence-based point-of-care (POC) system we developed for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 hrs. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 hrs of incubation, resulting in an improvement of the detection limit to 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact, multiplexing, and simple ASFV detection, intended for low resource settings.

CRISPR‐Cas system for biomedical diagnostic platforms

“…He et al presented a sophisticated technology for efficient detection of African swine fever virus (ASFV), a highly severe pathogen that results in almost a hundred percent death rate in domestic pigs 55 . Since ASFV possesses a double‐stranded DNA genome, CRISPR‐Cas12a together with a crRNA is employed to detect the target DNA by cutting ssDNA probes after hybridizing the ASFV target.…”

“…(B) Diagram of fluorescent sensor specific units. Reprinted with permission from Elsevier 55 . (C) Schematic illustration of diagnosis mechanism based on CRISPR‐Cas12a.…”

RAA-Cas12a-Tg: A Nucleic Acid Detection System for Toxoplasma gondii Based on CRISPR-Cas12a Combined with Recombinase-Aided Amplification (RAA)

“…For Cas orthologues, Cas12a and Cas13 are the main nucleases for the development of CRISPR-Cas-based nucleic acid detection method with higher sensitivity and specificity for the capability of recognizing a nucleic acid target, and they also have been developed to combine with isothermal amplification technologies, such as Recombinase-aided Amplification (RAA), to enhance the diagnostic accuracy [ 29 , 30 , 31 ]. Depending on the type of nucleic acid substrate (DNA or RNA), CRISPR-Cas12a/Cas13-based systems have been alternatively applied to the detection of different pathogens, including viruses [ 26 , 29 , 32 , 33 , 34 ], bacteria [ 31 , 35 , 36 , 37 ], parasitic protozoa Plasmodium spp. [ 38 ] and Cryptosporidium spp.…”