CRISPR/Cas12a technology combined with immunochromatographic strips for portable detection of African swine fever virus

Abstract

African swine fever virus (ASFV), the aetiological agent of African swine fever (ASF), causes lethal haemorrhagic fever in domestic pigs with high mortality and morbidity and has devastating consequences on the global swine industry. On-site rapid and sensitive detection of ASFV is key to the timely implementation of control. In this study, we developed a rapid, sensitive and instrument-free ASFV detection method based on CRISPR/Cas12a technology and lateral flow detection (named CRISPR/Cas12a-LFD). The limit of detection of CRISPR/ Cas12a-LFD is 20 copies of ASFV genomic DNA per reaction, and the detection process can be completed in an hour. The assay showed no cross-reactivity with other swine DNA viruses, and has 100% agreement with real-time PCR detection of ASFV in 149 clinical samples. Overall, the CRISPR/Cas12a-LFD method provides a novel alternative for the portable, simple, sensitive, and specific detection of ASFV and may contribute to the prevention and control of ASF outbreaks.

MeCas12a, a Highly Sensitive and Specific System for COVID‐19 Detection

“…Expression and Purification of Recombinant Cas12a Proteins: LbCas12a was expressed and purified as previously described. [11] In brief, the coding sequences of LbCas12a were codon-optimized and synthesized by Genscript (Nanjing, China) and then cloned into pET28a (Novagen) with a C-terminal 10× His tag. The coding sequence of AsCas12 was synthesized by Genscript (Nanjing, China) as previously described.…”

“…Cas12a-Mediated Nucleic Acid Detection: The detection assays were performed as previously reported with minor modifications. [11] In a 20 µL detection assay, with 200 ng LbCas12a protein, 25 × 10 −12 m ssDNA FQ probe sensor, 1 × 10 −6 m crRNA, and 2 µL of the sample in a reaction buffer (100 × 10 −3 m NaCl, 50 × 10 −3 m Tris-HCl, 100 µg mL −1 BSA(bovine serum albumin), pH 7.9) supplied with 10 × 10 −3 m MgCl 2 or MnSO 4 , were incubated at 37°C until detection. A PerkinElmer EnSpire reader with the excitation at 485 nm and emission at 520 nm was used for fluorescence detection.…”

Development and clinical application of a novel CRISPR-Cas12a based assay for the detection of African swine fever virus

“…So the clinical application of this method is limited. Previous studies also showed that CRISPR system can detect ASFV [ 26 , 27 ]. However, they did not compare the sensitivity of CRISPR with qPCR, the most sensitive method in the detection of ASFV until now, indicating that further research is needed in the sensitivity analysis of the CRISPR in ASFV detection.…”

Next-generation pathogen diagnosis with CRISPR/Cas-based detection methods

“…Importantly, the simplicity of the CRISPR/Cas-based detection system allows the rapid development of diagnostic methods for an emergency outbreak of infectious diseases. For example, integrating later flow assay (CORDS, CRISPR/Cas12a-LFD) and naked-eye detection (CRISPR/Cas-based colorimetric platform, fluorescence-based POC system), four groups quickly developed Cas12a-based on-site diagnostic assays for African swine virus (ASFV), an emergence virus for the global swine industry (Table 2) [44][45][46][47]. For the current COVID-19 pandemic [48], several research groups are developing CRISPR/Cas-based diagnostic assay [49][50][51][52][53].…”

“…Isothermal amplification that does not require instruments has been widely integrated into CRISPR-based detection [ 7 ]. For signal readout, more equipment-free methods, such as later flow assay or naked-eye view under light, should be introduced [ 41 , 45 , 46 , 55 ]. Currently, the store and delivery of the CRISPR-based detection system still need a cold chain.…”