Establishment of a One–Pot RAA–CRISPR/Cas13a Assay-Based TGEV S Gene Detection

Abstract

Porcine transmissible gastroenteritis virus (TGEV) is a highly contagious pathogen causing severe diarrhea in pigs, particularly piglets, leading to significant economic losses. Distinguishing TGEV from the genetically similar porcine respiratory coronavirus (PRCV) remains challenging due to their high genomic homology. In this study, we developed a one–pot assay combining recombinase-aided amplification (RAA) and CRISPR/Cas13a technology, targeting the TGEV S gene. This method was optimized for sensitivity and specificity, with orthogonal tests determining the optimal reagent concentrations. The assay achieved a detection limit of 4.13 copies/µL within 40 min at 37 °C, demonstrating no cross-reactivity with other porcine viruses. Clinical validation on 140 samples showed 100% concordance with RT–qPCR and RT–PCR results. Since the established method is completed in a single reaction tube, it eliminates the need for step-by-step operations, simplifying the process and reducing the risk of cross–contamination and false positives in subsequent tests. Overall, this assay shows promising potential for TGEV detection.

Keywords

• TGEV
• RAA
• CRISPR/Cas13a
• detection method