Development of Visual Detection of African Swine Fever Virus Using CRISPR/AapCas12b Lateral Flow Strip Based on Viral Major Capsid Protein Gene B646L

TL;DR

A new rapid and visual test using CRISPR technology was developed to detect African swine fever virus in clinical samples with high sensitivity and specificity.

Contribution

A CRISPR/AapCas12b-based lateral flow strip method for on-site detection of African swine fever virus targeting the B646L gene.

Findings

The method detected the B646L gene with a sensitivity of 6 copies/µL and completed analysis within an hour.

The assay showed high specificity against common porcine pathogens and matched real-time PCR results in 34 clinical samples.

The test is suitable for on-site use and has potential for early monitoring and control of African swine fever.

Abstract

Numerous diagnostic techniques for African swine fever virus identification have been established, including virus isolation, PCR, and serological assays including enzyme-linked immunosorbent assay. However, these methods require expensive instruments and professional operators, making them less suitable for on-site applications. In this study, we established an RPA-CRISPR/AapCas12b-LFS method for the detection of African swine fever virus by selecting the B646L gene as the target. This method achieved a sensitivity threshold of 6 copies/µL for B646L gene detection, completing analysis within an hour. It can be used for visual detection of African swine fever virus in clinical samples and has potential significance for early monitoring, prevention, and control of African swine fever.

African swine fever (ASF), induced by the African swine fever virus (ASFV), is an acute hemorrhagic disease characterized by high fever, systemic hemorrhages, and elevated mortality. Current diagnostic techniques including PCR and ELISA present limitations in field applications due to requirements for specialized equipment and prolonged processing duration. Therefore, rapid and accurate detection of ASFV has become a key link in ASF prevention and control. This study established a rapid and precise visual diagnostic approach by integrating the CRISPR/AapCas12b system with lateral flow strip (LFS) technology, specifically targeting the B646L gene encoding the major capsid protein p72. The CRISPR/AapCas12b-LFS platform achieved a sensitivity threshold of 6 copies/µL for B646L gene detection, completing analysis within an hour. Validation study confirmed exceptional specificity against common porcine pathogens including PRRSV, CSFV, PRV, PPV4, and PCV3. The developed assay demonstrated complete concordance with real-time PCR results when analyzing 34 clinical specimens including three heart samples, three liver samples, three spleen samples, three lung samples, three kidney samples, three lymph node samples, five serum samples, five blood samples, and five oral swab samples for ASFV detection. Overall, this method is sensitive, specific, and practicable onsite for ASFV detection, showing a great application potential for monitoring ASFV in the field.

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