What if African swine fever could be screened in thirty minutes, without specialized equipment, using either visible fluorescence or a simple lateral-flow strip? This paper combines RPA with CRISPR/Cas12a to target the KP177R gene. Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus transmission.

The p22 protein encoded by the ASFV KP177R gene has no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. African swine fever is an acute, febrile, and virulent infectious disease in pigs, clinically characterized by high fever and hemorrhage, with a lethality rate of up to one hundred percent.

The virus can be transmitted through soft ticks of Ornithodoros species or through direct contact with infected pigs, making prevention and control relevant worldwide. To date, no credible commercial vaccine has been developed worldwide, and there are no effective treatments for infected pigs.

Early detection of the virus in the environment, equipment, and infected pigs is critical for its elimination. Antibody testing is delayed: immunoglobulin M can be detected four days after infection, while immunoglobulin G can be detected six to eight days after infection.

By the time antibodies can be detected, the virus has been infecting, replicating, and proliferating for at least one week and has been discharged into the environment. PCR, real-time quantitative PCR, hemadsorption testing, and viral antigen detection are used, but the methods rely on laboratory instruments, complicated operation steps, and trained professional technicians.

The need is a simple ASFV detection method requiring few reagents, no specialized instrumentation, high sensitivity, and suitability for on-site use in farms. Targeting the KP177R gene, a visual ASFV detection assay based on CRISPR/Cas12a combined with RPA is intended for screening viruses in the environment or infected pigs as early as possible.

It may also distinguish future candidate KP177R gene-deleted ASFV vaccines from field virus strains. The KP177R RPA-CRISPR/Cas12a assay system consisted of the RPA amplification product of the target double-stranded DNA KP177R gene, Cas12a protein, NEBuffer 2.1, single-stranded DNA reporter, guide RNA, and water.

The complete fifty-microliter assay included KP177R RPA amplification product, Cas12a protein, guide RNA, fluorescein-labeled single-stranded DNA reporter, buffer, and water, while four reaction systems lacking individual components served as controls. For a visual assay, the reaction was performed in a metal bath at thirty-seven degrees Celsius for thirty minutes, and fluorescence was observed with the naked eye under blue light.

For the lateral-flow format, the fluorescein-labeled reporter was replaced with a biotin-labeled reporter, and the reaction product was applied to a lateral-flow dipstick to determine the target gene. The biotin reporter reaction system was performed in a metal bath at thirty-seven degrees Celsius for zero, ten, twenty, thirty, forty, fifty, or sixty minutes.

Fifty microliters of reaction product were added dropwise to the lateral-flow dipstick and incubated for ten minutes, after which the test and control line densities were measured and their ratios calculated. To determine the optimal reaction temperature and time, the fluorescein-labeled reporter was used in a fifty-microliter assay in a ninety-six-well microplate.

The tested temperatures were twenty, twenty-five, thirty, thirty-five, thirty-seven, forty, and forty-two degrees Celsius, with fluorescence measured every two minutes for one hour. A twenty-microliter reaction was also tested in PCR tube strips for thirty minutes at the same temperatures, with the optimal temperature determined from fluorescence under blue light.

Figure one documents the construction and initial validation of the Cas12a-based assay. Panels A and B show amplification of the Cas12a gene and restriction-digestion identification of the recombinant plasmid, including the labeled three-thousand-six-hundred-eighty-four-base-pair fragment.

Panel C compares four RPA primer pairs for the KP177R target, with the F1/R1 product corresponding to the expected one-hundred-twenty-base-pair fragment. Finally, SDS-PAGE and anti-His Western blotting in panels D and E identify Cas12a protein at the labeled one-hundred-forty-three-kilodalton position.

Figure two maps the assay’s operating conditions. Panels A through C compare temperature and reaction time using complete reactions alongside controls lacking gRNA, KP177R, Cas12a, or single-stranded DNA, with fluorescence measured instrumentally or by blue-light inspection.

Panel D examines Cas12a and gRNA concentration combinations, while panel E shows the reaction reformulated in a twenty-milliliter volume for room-temperature visualization after incubation at thirty-seven degrees Celsius for thirty minutes. Together, these tests support the authors’ selection of practical parameters for KP177R detection.

The gRNA-CRISPR/Cas12a assay system can work stably at thirty-seven degrees Celsius and has the strongest fluorescence response, while targeted cleavage of the ASFV KP177R gene shows specific green fluorescence. To assess specificity, nucleic acids of PRRSV, PCV2, PEDV, PDCoV, and PRV were used as controls.

ASFV and the five other virus types were simultaneously amplified by RPA, then measured with fluorescein- and biotin-labeled reporter systems to determine assay specificity. Figure four tests whether the KP177R RPA–CRISPR/Cas12a assay reacts specifically to ASFV rather than other swine viruses or the no-template control.

In the visual fluorescence image and microplate readout, only the ASFV group, sample one, produces a clear signal, while the other groups remain near baseline. The lateral-flow strips likewise show a T-line only for ASFV, and the densitometry summarizes the T- and C-line signals, supporting the assay’s reported specificity.

The positive plasmid pMD18-T-KP177R was tenfold diluted from 6.83 times ten to the tenth copies per milliliter through 6.83 times ten to the zero copies per milliliter to obtain different sample DNA concentrations. These concentrations were used as templates for RPA amplification, and the RPA products were used as target double-stranded DNA for CRISPR/Cas12a detection.

Sensitivity was determined by observing fluorescence color development and the lateral-flow dipstick test-to-control ratios. Figure five tests the KP177R RPA-CRISPR/Cas12a assay across tenfold serial dilutions of ASFV DNA. Green fluorescence is visible by eye on dipsticks one through nine, corresponding to concentrations from 6.83 times 10 to the power of eight down to 6.83 times 10 to the power of zero copies per milliliter, while the no-template control remains negative.

The microplate fluorescence curves and lateral-flow strip measurements provide instrumental confirmation, showing why the assay is relevant for sensitive ASFV detection. Under blue light, a visual green fluorescent response was observed at a minimum template concentration of 6.83 times ten to the zero copies per microliter.

The same minimum concentration produced a fluorescence signal in the microplate reader and a test-line red blot with the lateral-flow dipstick. These results indicate a detection limit of 6.83 times ten to the zero copies per microliter of viral nucleic acid for the fluorescein- and biotin-labeled reaction systems.

Figure six tests reproducibility by examining the KP177R RPA-CRISPR/Cas12a assay across three months. Panel A shows dipsticks for strongly, moderately, and weakly positive samples, with the caption reporting that the T-line signal did not change over time at each concentration.

Panel B quantifies the T- and C-line intensities and their relative ratios, supporting the authors’ conclusion that the assay produces consistent results without relying on specialized instrumentation. Ten KP177R gene fragments from clinical samples were detected using fluorescein-labeled and biotin-labeled KP177R RPA-CRISPR/Cas12a assays and RT-qPCR.

The fluorescein-labeled assay found six positive and four negative samples, and the biotin-labeled assay showed identical positive and negative results. The fluorescein- and biotin-labeled assays were compatible for testing each KP177R gene fragment, while the sentence reports that these ten samples were also detected by RT-qPCR.

Figure seven compares the KP177R RPA–CRISPR/Cas12a assay with RT-qPCR using clinical samples. In the fluorescence and dipstick formats, samples one through six show positive signals, while the designated negative samples and no-template controls do not; the RT-qPCR panel likewise shows positive curves for samples one through six and negative results for seven through ten, with sample eleven as the control.

This agreement supports the assay’s potential for instrument-light ASFV detection. The diagnostic coincidence rate for both assays was one hundred percent, with a kappa value of one point zero zero zero and a p-value less than zero point zero zero one.

The six positive samples had viral nucleic acid contents of 10 to the 3.97, 10 to the 3.81, 10 to the 3.36, 10 to the 2.93, 10 to the 2.59, and 10 to the 2.49 copies per microliter, respectively. Detection assays suitable for little instrumentation and equipment and not requiring specialized technicians are crucial for detecting viruses in the environment and infected pigs in the first instance.

To reduce dependence on thermal cycling instruments and skilled operators, the study adopted isothermal amplification of nucleic acids, which amplifies gene fragments at lower temperatures with simple heating equipment. Representative isothermal amplification techniques include loop-mediated isothermal amplification and recombinase-mediated isothermal amplification, including RPA.

The assay targets KP177R by integrating RPA amplification and CRISPR/Cas12a precision gene cutting. The assay is rapid and requires neither specialized equipment nor highly qualified personnel. Detection results can be determined by visual fluorescence or lateral-flow dipstick coloration.

The KP177R RPA-CRISPR/Cas12a assay is especially suitable for clinical sample testing sites with poor experimental conditions and can provide technical support for rapid ASFV screening. The KP177R RPA-CRISPR/Cas12a assay provided visual fluorescence and lateral-flow detection, detected as few as 6.83 times ten to the zero copies per microliter, and matched RT-qPCR across ten clinical samples.