Single-Tube Multiplexed Molecular Detection of Endemic Porcine Viruses in Combination with Background Screening for Transboundary Diseases

Abstract

Detection of several pathogens with multiplexed real-time quantitative PCR (qPCR) assays in a one-step setup allows the simultaneous detection of two endemic porcine and four different selected transboundary viruses. Reverse transcription (RT)-qPCR systems for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), two of the most economically important pathogens of swine worldwide, were combined with a screening system for diseases notifiable to the World Organization of Animal Health, namely, classical and African swine fever, foot-and-mouth disease, and Aujeszky's disease. Background screening was implemented using the identical fluorophore for all four different RT-qPCR assays. The novel multiplex RT-qPCR system was validated with a large panel of different body fluids and tissues from pigs and other animal species. Both reference samples and clinical specimens were used for a complete evaluation. It could be demonstrated that a highly sensitive and specific parallel detection of the different viruses was possible. The assays for the notifiable diseases were even not affected by the simultaneous amplification of very high loads of PRRSV-and PCV2-specific sequences. The novel broad-spectrum multiplex assay allows in a unique form the routine investigation for endemic porcine pathogens with exclusion diagnostics of the most important transboundary diseases in samples from pigs with unspecific clinical signs, such as fever or hemorrhages. The new system could significantly improve early detection of the most important notifiable diseases of swine and could lead to a new approach in syndromic surveillance.

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction

“…A previous report has described a mRT-qPCR for the simultaneous detection of ASFV, CSFV, FMDV, Suid herpesvirus 1 (pseudorabies; SuHV-1), PRRSV, and PCV-2. 25 Unlike the 4-plex RT-qPCR described herein, the mRT-qPCR from the previous study was not evaluated using OF samples and utilizes the same reporter dye for ASFV, CSFV, FMDV, and SuHV-1 detection, such that positive amplification reported by the dye requires follow-up with 4 single-target RT-qPCR assays for confirmatory diagnosis. 25 The goal of this proof-of-concept study was to assess the feasibility of OF sampling from pigs infected with ASFV, CSFV, or FMDV and to develop a single test for their detection.…”

“…25 Unlike the 4-plex RT-qPCR described herein, the mRT-qPCR from the previous study was not evaluated using OF samples and utilizes the same reporter dye for ASFV, CSFV, FMDV, and SuHV-1 detection, such that positive amplification reported by the dye requires follow-up with 4 single-target RT-qPCR assays for confirmatory diagnosis. 25 The goal of this proof-of-concept study was to assess the feasibility of OF sampling from pigs infected with ASFV, CSFV, or FMDV and to develop a single test for their detection. In keeping with the 3 Rs (replacement, reduction, and refinement) of institutional animal care and use guidelines, this study sought to reduce the number of animals required by leveraging demonstration studies for foreign animal disease infections used to educate veterinary pathologists and diagnosticians.…”

Development and Validation of a Multiplex, Real-Time RT PCR Assay for the Simultaneous Detection of Classical and African Swine Fever Viruses

“…Multiplex, gel-based PCR assays for detection of CSFV and ASFV [10], [11] and RT-PCR assays for singleplex CSFV [11]–[18] or ASFV detection [19]–[21] have been described. However, to date, a multiplex real-time RT-PCR for simultaneous detection of CSFV and/or ASFV has not been reported.…”

A multiplex PCR for simultaneous detection of classical swine fever virus, African swine fever virus, highly pathogenic porcine reproductive and respiratory syndrome virus, porcine reproductive and respiratory syndrome virus and pseudorabies in swines

“…One or more viruses can infect swine and cause the same syndrome such as reproductive and/or respiratory failure and high fever. As far as the authors knows, mPCR has been used to detect ASFV, CSFV, PRRSV and PRV infections of swine in a single reaction before (Wernike et al 2013), but the authors could not distinguish HP-PRRSV and PRRSV. In this study, we developed and evaluated a multiplex PCR for not only simultaneous detection of nucleic acid from four viruses: CSFV, ASFV, PRRSV, PRV, but also differentiated HP-PRRSV and PRRSV.…”