Rapid Detection of Genotype II African Swine Fever Virus Using CRISPR Cas13a-Based Lateral Flow Strip

Abstract

The African swine fever virus (ASFV) is a dsDNA virus that can cause serious, highly infectious, and fatal diseases in wild boars and domestic pigs. The ASFV has brought enormous economic loss to many countries, and no effective vaccine or treatment for the ASFV is currently available. Therefore, the on-site rapid and accurate detection of the ASFV is key to the timely implementation of control. The RNA-guided, RNA-targeting CRISPR effector CRISPR-associated 13 (Cas13a; previously known as C2c2) exhibits a "collateral effect" of promiscuous RNase activity upon the target recognition. The collateral cleavage activity of LwCas13a is activated to degrade the non-targeted RNA, when the crRNA of LwCas13a binds to the target RNA. In this study, we developed a rapid and sensitive ASFV detection method based on the collateral cleavage activity of LwCas13a, which combines recombinase-aided amplification (RAA) and a lateral flow strip (named CRISPR/Cas13a-LFD). The method was an isothermal detection at 37 °C, and the detection can be used for visual readout. The detection limit of the CRISPR/Cas13a-LFD was 10¹ copies/µL of p72 gene per reaction, and the detection process can be completed within an hour. The assay showed no cross-reactivity to eight other swine viruses, including classical swine fever virus (CSFV), and has a 100% coincidence rate with real-time PCR detection of the ASFV in 83 clinical samples. Overall, this method is sensitive, specific, and practicable onsite for the ASFV detection, showing a great application potential for monitoring the ASFV in the field.

Keywords: African swine fever; CRISPR/Cas13a; RAA; detection; lateral flow strip.

Figure 1

Capillary electrophoresis of RAA-amplified products, corresponding to five pairs of primers from (A–E). Five pairs of primers were used for RAA amplification; the product was analyzed by capillary electrophoresis.

Figure 2

Establishment of the CRISPR/Cas13a-LFD. (A) Schematic diagram in the whole process of the CRISPR/CRISPR-associated 13 (Cas13a)-LFD detection. The P72 gene in the ASFV genome is amplified by RAA and then transcribed into RNA, which activates the Cas13a nuclease after being recognized by crRNA. The activated Cas13a nuclease then cuts off the reporter molecule, and the reporter molecule can appear as a band on the test strip. (B) Analysis of different crRNAs by CRISPR/Cas13a-LFD. ZL, pUC57-p72 standard plasmid. NTC, negative control.

Figure 3

Sensitivity test of the CRISPR/Cas13a-LFD. A ten-fold serial dilution of pUC57-p72 was used as the detection template. (A) Sensitivity test of different primer and probe combinations; (B) sensitivity of the primer3 + crRNA3 combination after reaction system optimization. Detection reaction was performed independently total twice, with the detection limits of the following: 1:1 × 10⁸ copies/µL, 2:1 × 10⁷ copies/µL, 3:1 × 10⁶ copies/µL, 4:1 × 10⁵ copies/µL, 5:1 × 10⁴ copies/µL, 6:1 × 10³ copies/µL, 7:1 × 10² copies/µL, 8:1 × 10¹ copies/µL.

Figure 4

Specificity test of the CRISPR/Cas13a-LFD. Four DNA viruses (African swine fever virus (ASFV), pseudorabies virus (PRV), porcine circovirus 2 (PCV2), and PCV3) and five RNA viruses (classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome (PRRSV), porcine epidemic diarrhea virus (PEDV), Japanese encephalitis virus (JEV), and encephalomyocarditis virus (EMCV)) were tested.

Figure 5

Clinical sample detection by the CRISPR/Cas13a-LFD. The samples marked with black numbers were ASFV-positive samples, and the samples marked with red numbers were negative samples. Eighty-three samples were used for testing.

Figure 6

Repeated detection of clinical samples by the CRISPR/Cas13a-LFD. The samples marked with black numbers were ASFV-positive samples, and the samples marked with red numbers were negative. Eighty-three samples were used for testing.