Rapid Visual Detection of African Swine Fever Virus with a CRISPR/Cas12a Lateral Flow Strip Based on Structural Protein Gene D117L

Figure 1. Schematic diagram of RPA-CRISPR/Cas12a-LFS detection of ASFV. The ssDNA probe labeled by 5′-gold-Ab-FAM and 3′-biotin is used for lateral flow strip (LFS) detection, whereas the ssDNA probe labeled by 5′-FAM and 3′-BHQ is used for blue light, UV detection, and fluorescence detection.

Figure 2. Two crRNAs of the ASFV D117L gene and the crRNA-mediated CRISPR/Cas12a reactions. (A) PCR amplification products of ASFV D117L gene in agarose gel. (B) In vitro transcribed D117L crRNA1 and crRNA2 in agarose gel. (C,D) Two-crRNA-mediated LbCas12a cleavage of D117L PCR products in non-denatured PAGE with silver staining. (E,F) Two-crRNA-mediated LbCas12a cleavages of pCold-I-D117L plasmids in agarose gels. (G) Detection of crRNA-LbCas12a reactions by blue light and ultraviolet light, respectively. (H) Detection of crRNA-LaCas12a reactions by fluorescence signal. NC denotes the reactions without template DNA. The Student’s t test; * p < 0.05 and ** p < 0.01 denote significant and very significant difference, respectively.

Figure 3. ASFV D117L-crRNA1-mediated CRISPR/Cas12a-LFS. The CRISPR/Cas12a reactions were set up in different samples of 1–6, as indicated in panel (B). The Cas12a reactions were subjected to lateral flow strip (LFS) detection (A) and also verified by blue light and UV light detection (B), as well as fluorescence detection (C).

Figure 4. The sensitivity of RPA-CRISPR/Cas12a-LFS detection. (A) RPA products of D117L amplified from pCold-I-D117L with gradient concentrations from 2 × 1010 copies/uL to 2 × 100 copies/uL (sample Nos. 1–11), with NC denoting negative control. The Cas12a-LFS detection of sensitivity was presented in (B) and also verified by blue light detection (C) and fluorescence detection (D).

Figure 5. The specificity of CRISPR/Cas12a-LFS detection. (A) The specific gene PCR products of nine swine viruses as indicated were verified by 2% agarose gel electrophoresis. The NC denotes negative control. The specificity of Cas12a-LFS detection was presented in (B) and also verified by blue light detection (C) as well as fluorescence detection (D).

Figure 6. RPA-Cas12a-LFS detection of clinical samples. (A) Results of RPA-Cas12a-LFS detection of first batch of 30 clinical samples, with 8 positive (Nos. 1–8) and 22 negative (Nos. 9–30). (B) Results of RPA-Cas12a-LFS detection of second batch of 38 clinical samples, with 10 positive (Nos. 1–10), 3 weak positive (Nos. 11–13), and 25 negative (Nos. 14–38). The NC denotes the negative control.