PoRV is a significant etiological agent of neonatal diarrhea in piglets, resulting in substantial economic losses within the global swine industry due to elevated mortality rates and reduced productivity. To address the urgent need for accessible and rapid diagnostics in resource-limited settings, we have developed a CRISPR/Cas12a-based assay integrated with recombinase polymerase amplification (RPA) for the visual detection of PoRV. This platform specifically targets the conserved VP6 gene using optimized RPA primers and crRNA, harnessing Cas12a’s collateral cleavage activity to enable dual-readout via fluorescence or lateral flow dipsticks (LFDs). The assay demonstrates a detection limit of 10² copies/μL within 1 h, exhibiting no cross-reactivity with phylogenetically related pathogens such as Transmissible Gastroenteritis Virus (TGEV). By eliminating reliance on thermal cyclers or specialized equipment, this method is fully deployable in swine farms, veterinary clinics, or field environments. The lateral flow format provides immediate colorimetric results that require minimal technical expertise, while the fluorescence mode allows for semi-quantitative analysis. This study presents a robust and cost-effective platform for decentralized PoRV surveillance in swine populations, addressing the critical need for portable diagnostics in resource-limited settings and enhancing veterinary health management.

Global climate change has increased the frequency and intensity of heat waves, posing a significant threat to livestock production. During heat exposure, the disruption of intestinal barrier integrity is a pivotal event in the pathogenesis of heat stress-induced intestinal injury. Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are key consequences of heat stress at the cellular level. However, direct causal evidence linking ER stress to mitochondrial dysfunction in heat-stressed enterocytes remains limited. To investigate this, we used an integrated transcriptomic, metabolomic, and functional validation strategy to assess mitochondrial bioenergetics and cellular ultrastructure in porcine intestinal epithelial (IPEC-J2) cells under acute heat stress. Transcriptomic analysis revealed extensive reprogramming, highlighting the significant enrichment of pathways related to protein processing in the endoplasmic reticulum, apoptosis, and MAPK signaling. Untargeted metabolomics identified significant perturbations in amino acid and energy metabolism, as well as altered bile acid profiles. Functional assessments confirmed that heat stress severely impaired mitochondrial bioenergetics, as evidenced by reduced maximal respiration and ATP production, and induced ultrastructural damage to mitochondria. The pharmacological inhibition of ER stress by 4-phenylbutyric acid (4-PBA) significantly attenuated the mitochondrial bioenergetic impairment and ultrastructural damage, whereas ER stress induction recapitulated these defects. We demonstrate that heat stress induces profound transcriptional and metabolic remodeling characterized by ER stress activation, which critically mediates subsequent mitochondrial bioenergetic dysfunction and ultrastructural damage. Our findings suggest that targeting ER stress may represent a promising therapeutic strategy to ameliorate enterocyte mitochondrial dysfunction and mitigate heat stress-induced intestinal injury in livestock.