Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a Mediated Multiplexable and Portable Detection Platform for GII Genotype Porcine Epidemic Diarrhoea Virus Rapid Diagnosis

Abstract

Porcine epidemic diarrhoea virus (PEDV) is a member of the genus Alphacoronavirus in the family Coronaviridae. It causes acute watery diarrhoea and vomiting in piglets with high a mortality rate. Currently, the GII genotype, PEDV, possesses a high separation rate in wild strains and is usually reported in immunity failure cases, which indicates a need for a portable and sensitive detection method. Here, reverse transcription-recombinase aided amplification (RT-RAA) was combined with the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas12a system to establish a multiplexable, rapid and portable detection platform for PEDV. The CRISPR RNA (crRNA) against Spike (S) gene of GII PEDV specifically were added into the protocol. This system is suitable for different experimental conditions, including ultra-sensitive fluorescence, visual, UV light, or flow strip detection. Moreover, it exhibits high sensitivity and specificity and can detect at least 100 copies of the target gene in each reaction. The CRISPR/Cas12a detection platform requires less time and represents a rapid, reliable and practical tool for the rapid diagnosis of GII genotype PEDV.

Keywords: CRISPR/Cas12a; GII genotype; lateral flow strip; porcine epidemic diarrhoea virus; rapid diagnosis.

Figure 1

Establishing the CRISPR/Cas12a-LFS. (A) Schematic diagram of PEDV visual detection, the method combines RAA assay, the CRISPR/Cas12a-FDS, and lateral flow strips (LFSs). The ssDNA reporter was labeled with FAM and digoxin (ssDNA-FD-reporter) at the 5′ and 3′ termini, respectively. The immunochromatographic strip using Au-NPs anti-FAM antibody to show the readout. The sample band was only shown when the ssDNA-FD-reporter was cleaved by CRISPR/Cas12a, which is activated by PEDV cDNA. Both the control line and the test line showed when the ssDNA-FD-reporter was partially cleaved, and only the test line showed when the ssDNA-FD-reporter was completely cleaved. (B) The specificity of different porcine viruses was detected by CRISPR/Cas12a-LFS. (C) The LOD of PEDV S gene was detected by CRISPR/Cas12a-LFS. Serially diluted synthetic pUC57-PEDV-S was used as the template.

Figure 2

Design of clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA) of porcine epidemic diarrhoea virus (PEDV) Spike gene and schematic diagram of CRISPR/Cas12a-fluorescent detection system (FDS). (A) Schematic diagram of the CRISPR/Cas12a-FDS assay. Specific crRNAs targeting the PEDV Spike gene were designed for PEDV genome detection. When CRISPR/Cas12a proteins were bound to double-stranded DNA (dsDNA) guided by specific crRNA, it nonspecifically activated its trans-cleavage activity, the quenched fluorescent ssDNA was cleaved and thus stimulating fluorescence. F, fluorophore; Q, quencher. (B) Five crRNAs targeting Spike gene selected for PEDV detection and the relative positions of these crRNAs in S gene. (C) Sequence alignment of Spike genes from 19 strains of PEDV targeted by crRNAs. The nucleotide variants from the consensus sequence were highlighted with red color.

Figure 3

The optimization of the CRISPR/Cas12a-FDS assay. (A) The time course of GII PEDV/JSX2014 was detected by five crRNAs (crRNA1–crRNA5) respectively in CRISPR/Cas12a-FDS. (B) The time course of GI PEDV/CV777 was detected by five crRNAs (crRNA1–crRNA5) in CRISPR/Cas12a-FDS. (C) The time course of the GII PEDV/JSX2014 genome was detected by CRISPR/Cas12a-FDS when the molar range of ssDNA-FQ reporter was 0–2 pmol. (D) The time course of the GII PEDV/JSX2014 genome was detected by CRISPR/Cas12a-FDS when the molar range of Cas12a protein was 0–10 pmol. (E) The time course of the GII PEDV/JSX2014 genome was detected by CRISPR/Cas12a-FDS when the ratio of Cas12a concentration to crRNA concentration ranged from 1:0 to 1:10. (F) The time course of the GII PEDV/JSX2014 genome was detected by CRISPR/Cas12a-FDS when the temperature of reaction ranged from 15 to 42°C. Error bars in panels (A–F) represent the mean ± SD, where n = 3 replicates. (G) The cDNA of PEDV detection with CRISPR/Cas12a-FDS at 37°C in 45 min. No fluorescent amplification was detected for the nucleic acids of other tested porcine viruses, GI PEDV, TGEV, PDCoV, FMDV, SVA, and ASFV. (H) Sensitivity of the CRISPR/Cas12a-FDS. The serially diluted pUC57-PEDV-S plasmid was used as a template.

Figure 4

Establishing the Recombinase Aided Amplification (RAA)-CRISPR-FDS. (A) Schematic diagram of PEDV visual detection, the method combines RAA assay, and the CRISPR/Cas12a-FDS. (B) Sensitivity of the CRISPR/Cas12a-FDS combined with RAA. The serially diluted pUC57-PEDV-S plasmid was used as a template. (C) After samples were detected by CRISPR/Cas12a-FDS at 37°C, the fluorescence of different porcine viruses was observed under UV light by the gel imaging system at the 30th min. Fluorescence could only be observed in PEDV/JSX2014 tubes. (D) Sensitivity of the CRISPR/Cas12a-FDS visual observations. The serially diluted pUC57-PEDV-S plasmid was used as a template. (E) Sensitivity of the CRISPR/Cas12a-FDS combined with RAA visual observations. The serially diluted pUC57-PEDV-S plasmid was used as a template.

Figure 5

Detection results of clinical samples. (A) Detection of PEDV cDNA in 72 rectal swab samples using CRISPR/Cas12a-LFS. The test line and control line on the lateral strip were marked with arrows. (B) Detection of PEDV cDNA in 72 rectal swab samples using quantitative real-time PCR (qRT-PCR). (C) The Venn diagram shows the consistency between the CRISPR/Cas12a-LFS and qRT-PCR assays.