The Quadruplex Real‐Time PCR Assay for Simultaneous Detection of GETV, PRRSV, JEV, and PCV2 in Swine

This study aimed to establish a quadruplex real‐time PCR assay for the simultaneous detection of Getah virus (GETV), Porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), and Porcine circovirus type 2 (PCV2). Specific primers and probes were designed based on comparative analysis of conserved gene sequences from each pathogen. The optimal reaction system and conditions were determined through systematic optimization, standard curves were established, and the assay’s specificity, sensitivity, repeatability, and clinical application performance were evaluated. The standard curves all exhibited good linear relationships, with R 2 values of 0.9993, 0.9972, 0.9976, and 0.9995, respectively. The amplification efficiencies were 101.286%, 109.863%, 98.626%, and 122.211%, respectively. The limits of detection (at 95% confidence) for GETV, PRRSV, JEV, and PCV2 were 7.454, 7.111, 24.876, and 7.7 copies, respectively. The results demonstrated no cross‐reactivity with 12 other common swine pathogens, including pseudorabies virus (PRV) and classical swine fever virus (CSFV). Both intra‐assay and inter‐assay coefficients of variation (CV) were below 1.4%. Testing of 151 clinical samples using this assay revealed positive rates of 18.54%, 14.57%, 3.31%, and 10.60% for GETV, PRRSV, JEV, and PCV2, respectively, with 100% concordance compared to established standard methods. The developed quadruplex real‐time PCR assay offers advantages of high throughput, sensitivity, specificity, and stability, providing a technical foundation for rapid differential diagnosis and epidemiological surveillance of GETV, PRRSV, JEV, and PCV2 in swine populations.