Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR‐Cas13a

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) has varied constantly and circulated in the pig industry worldwide. The prevention and control of porcine reproductive and respiratory syndrome (PRRS) is complicated. A visual, sensitive and specific diagnostic method is advantageous to the control of PRRS. The collateral cleavage activity of LwCas13a is activated to degrade non‐targeted RNA, when crRNA of LwCas13a bond to target RNA. The enhanced Cas13a detection is the combination of collateral cleavage activity of LwCas13a and recombinase polymerase amplification (RPA). In this study, the enhanced Cas13a detection for PRRSV was established. The novel method was an isothermal detection at 37°C, and the detection can be used for real‐time analysis or visual readout. The detection limit of the enhanced Cas13a detection was 172 copies/μl, and there were no cross‐reactions with porcine circovirus 2, porcine parvovirus, classical swine fever virus and pseudorabies virus. The enhanced Cas13a detection can work well in clinical samples. In summary, a visual, sensitive and specific nucleic acid detection method based on CRISPR‐Cas13a was developed for PRRSV.

Early diagnosis of rabies virus infection by RPA-CRISPR techniques in a rat model

“…Before invading the brain, RABV replicates slowly, making it difficult to detect by traditional methods. Due to its high sensitivity, RPA-CRISPR has been increasingly used for detecting nucleic acids of viruses, parasites, and cancer cells [24,[30][31][32][33][34][35]. Most recently, RPA-CRISPR was applied to the diagnosis of infection with the betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [36][37][38].…”

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

“…In most of these platforms, a commercially available universal test strip, the HybriDetect-Universal Lateral Flow Assay Kit, was used. This dipstick was originally designed for qualitative or even quantitative rapid testing of proteins, antibodies or gene amplicons, but has been adapted to function in several LFA based CRISPR sensing methods ( Bai et al, 2019 ; Chang et al, 2019 ; Gootenberg et al, 2018 ; Kaminski et al, 2020 ; Mukama et al, 2020b ; Sullivan et al, 2019 ; Tsou et al, 2019 ). This platform offers a Streptavidine line as well as an antibody line that can capture anti-FITC coated AuNPs ( Fig.…”

“… ~120 min 20–37 °C Wang et al, 2020 RPA Recombinase polymerase amplification is a low temperature DNA and RNA amplification technique. 5–60 min 37–42 °C ( Y. Chang et al, 2019 ; English et al, 2019 ; Kellner et al, 2019 ; Khan et al, 2019 ; Sullivan et al, 2019 ; X. Wang et al, 2020 ; Williams et al, 2019 ) SDA Strand Displacement Amplification (SDA) employs a restriction endonuclease, which is capable of nicking of its recognition site, and a DNA exonuclease deficient polymerase, which is capable of initiating synthesis at a nick. 90 min 37–55 °C Zhou et al, 2018 …”

CRISPR-based detection of SARS-CoV-2: A review from sample to result

“…Lateral flow assay (LFA) is the most common colorimetric readout system ( Shao et al, 2019 ; Yuan et al, 2020a ). Many studies adapted the LFAs platforms as their reading system for CRISPR sensing methods ( Bai et al, 2019 ; Chang et al, 2020 ). In addition, the electronic readout is a highly promising approach due to its integration potential ( Bruch et al, 2019 ; Dai et al, 2019 ).…”