A Quadruplex RT-qPCR for the Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, and Porcine Pseudorabies Virus.

Simple Summary: African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine pseudorabies virus (PRV) are important pathogens circulating in pig herds in many countries. Pigs infected with these viruses show similar signs, such as increased body temperature and loss of appetite, and are prone to respiratory symptoms, digestive symptoms, neurological symptoms, and/or abortion in pregnant sows. To accurately detect these viruses and differentially diagnose these diseases, four pairs of specific primers and TaqMan probes were designed aiming at the B646L (p72) gene of ASFV, the 5′ untranslated region (5′UTR) of CSFV, the ORF6 gene of PRRSV, and the gB gene of PRV. After optimizing the reaction conditions, a multiplex real-time quantitative RT-PCR (RT-qPCR) assay was developed for the differential detection of ASFV, CSFV, PRRSV, and PRV. The assay was further validated to test 3116 clinical samples, and the positivity rates of ASFV, CSFV, PRRSV, and PRV were 10.84% (338/3116), 0.80% (25/3116), 14.92% (465/3116), and 1.38% (43/3116), respectively. The assay provides a highly specific, sensitive, and reproducible method for the detection of ASFV, CSFV, PRRSV, and PRV. African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine pseudorabies virus (PRV) induce similar clinical signs in infected pigs, including hyperthermia, anorexia, hemorrhage, respiratory distress, neurological symptoms, and/or abortions in pregnant sows. The differential diagnosis of these diseases relies on laboratory examinations. In this study, a quadruplex RT-qPCR was established using four pairs of specific primers and probes aimed at the B646L (p72) gene of ASFV, the 5′ untranslated region (5′UTR) of CSFV, the ORF6 gene of PRRSV, and the gB gene of PRV for the detection and differentiation of ASFV, CSFV, PRRSV, and PRV. The assay exhibited great sensitivity with limits of detection (LODs) of 134.585, 139.831, 147.076, and 142.331 copies/reaction for ASFV, CSFV, PRRSV, and PRV, respectively. The assay exclusively identified ASFV, CSFV, PRRSV, and PRV, yielding negative results for the other control swine viruses used in this study. The intra-assay and inter-assay coefficients of variation (CVs) were not higher than 1.12%, indicating good reproducibility of the assay. The quadruplex RT-qPCR assay was used to analyze 3116 clinical tissue samples from pigs in Guangxi province, China, from April 2023 to September 2024. ASFV, CSFV, PRRSV, and PRV had positivity rates of 10.84% (338/3116), 0.80% (25/3116), 14.92% (465/3116), and 1.38% (43/3116), respectively, demonstrating a coincidence rate of ≥99.45% with the previously described RT-qPCR assays, which were also used to test these same samples. The established assay was rapid, sensitive, and accurate in detecting and differentiating ASFV, CSFV, PRRSV, and PRV. [ABSTRACT FROM AUTHOR]