ACS sensorsJan 28, 2026

Fast One-Step Test for Identifying African Swine Fever Virus Variants Without Sample Extraction

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Feb 1, 2026

Abstract

The assay achieved 97.9% accuracy with 96 field samples for detecting African swine fever virus variants.

Detection sensitivity reached 6 copies/μL, indicating ultrasensitive performance.
The assay utilizes a one-pot method combining multiplex RPA and CRISPR/Cas12a.
An extraction-free protocol was developed for testing serum and saliva samples.
The entire sample-to-answer process is completed within 1 hour using freeze-dried reagent microspheres.
This system is designed for rapid on-site surveillance of ASFV variants and could be adapted for other pathogens.

Simplified

African swine fever virus (ASFV) causes devastating outbreaks in swine populations worldwide. The co-existence of wild-type and emerging gene-deleted variants (e.g., ASFV-ΔI177L) necessitates rapid on-site diagnostic tools for strain identification, which is critical for timely disease control and tailored farm management. Here, we developed a field-deployable, extraction-free one-pot assay (CORDSv2) that combines multiplex RPA and CRISPR/Cas12a to target conserved ASFV sequences and specific fluorescent markers (eGFP/mCherry) of gene-deleted variants. The assay achieved ultrasensitive detection (LOD: 6 copies/μL) and demonstrated 97.9% accuracy with 96 field samples. To streamline field operations, we integrated an extraction-free protocol (for serum/saliva) with freeze-dried reagent microspheres, enabling stable storage and direct use with minimal manual handling. When paired with a portable fluorometer (hippoCORDS), the system completes the entire sample-to-answer workflow within 1 h with a single step: addition of lysate to the microspheres. This robust, portable system addresses the urgent need for simple, on-site ASFV variant surveillance and is adaptable for other high-threat pathogens.

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Fast One-Step Test for Identifying African Swine Fever Virus Variants Without Sample Extraction

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