Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay

Figure 1. Screening of highly active sgRNAs for detection of PEDV, TGEV, PDCoV, and SADS-CoV. Identification of the highly active sgRNA targeting PEDV ORF3 gene (A), TGEV N gene (B), PDCoV N gene (C), and SADS-CoV N gene (D) by CRISPR/Cas12a cleavage assay; DNA template represents the amplification fragment of ORF3 or N gene as the template; ssDNA activator represents the complementary single-stranded DNA of the sgRNA. No excitation light stands for white light. PEDV, porcine epidemic diarrhea virus; TGEV, transmissible gastroenteritis virus; PDCoV, porcine deltacoronavirus; SADS-CoV, swine acute diarrhea syndrome coronavirus.

Figure 2. Sensitivity of CRISPR/Cas12a combined with LAMP assay for portable detection of PEDV, PDCoV, TGEV, and SADS-CoV. (A,C,E,G) Colorimetric/fluorescence signals from a series of 10-fold dilutions of plasmid DNA by CRISPR/Cas12a coupled with LAMP assay. (B,D,F,H) Sensitivity of the CRISPR/Cas12a coupled with LAMP assay quantified by a multi-functional microplate reader (n = 3). No excitation light stands for white light; PEDV, porcine epidemic diarrhea virus; TGEV, transmissible gastroenteritis virus; PDCoV, porcine deltacoronavirus; SADS-CoV, swine acute diarrhea syndrome coronavirus; NC, negative control. Data are represented as mean ± SEM.

Figure 3. Specificity of LAMP-Cas12a assay for detection of four porcine coronaviruses. (A) The agarose gel electrophoresis for detection of specific LAMP amplicons; Lane M: 2000 DNA Ladder; bp: base pairs; Lane 1: PEDV-ORF3 plasmid as DNA template; Lane 2: TGEV-N plasmid as DNA template; Lane 3: PDCoV-N plasmid as DNA template; Lane 4: SADS-CoV-N plasmid as DNA template; Lane 5: non-template control (NTC). (B) The colorimetric/fluorescence signal detection under the blue (470 nM) and UV light; LAMP-amplified products of PEDV-ORF3 plasmid (1), TGEV-N plasmid (2), PDCoV-N plasmid (3), SADS-CoV-N plasmid (4), non-template control (5). No excitation light stands for white light; PEDV, porcine epidemic diarrhea virus; TGEV, transmissible gastroenteritis virus; PDCoV, porcine deltacoronavirus; SADS-CoV, swine acute diarrhea syndrome coronavirus.

Figure 4. One-tube detection of four porcine coronaviruses in clinical samples based on CRISPR/Cas12a combined with RT-LAMP assay. (A) Diagram of principle of the contamination-free one-tube CRISPR/Cas12a combined with RT-LAMP assay detection of clinical samples. (B) Colorimetric/fluorescence readout of four porcine CoVs by one-tube multiplex RT-LAMP-Cas12a assay. CRISPR, clustered regularly interspaced short palindromic repeats; RT-LAMP, reverse transcription-loop-mediated isothermal amplification; No excitation light stands for white light; PEDV, porcine epidemic diarrhea virus; TGEV, transmissible gastroenteritis virus; PDCoV, porcine deltacoronavirus; SADS-CoV, swine acute diarrhea syndrome coronavirus; NC, negative control.

Figure 5. Establishment of one-tube multiplex LAMP-Cas12a assay for detection of four porcine diarrhea coronaviruses. (A) Schematic diagram experimental flow and principle of contamination-free one-tube multiplex LAMP-Cas12a assay. (B) Colorimetric/fluorescence readout of four porcine coronaviruses by one-tube multiplex LAMP-Cas12a assay. 1: plasmid DNA mixture of PEDV, TGEV, PDCoV, and SADS-CoV (1:1:1:1) as templates; 2: non-template control (NTC). sgRNA, small guide RNA; LAMP, loop-mediated isothermal amplification; ROX-BHQ2, ROX-labeled ssDNA-FQ reporter; No excitation light stands for white light; PEDV, porcine epidemic diarrhea virus; TGEV, transmissible gastroenteritis virus; PDCoV, porcine deltacoronavirus; SADS-CoV, swine acute diarrhea syndrome coronavirus.