Single tube multiplexed molecular detection of endemic porcine viruses in combination with a background screening for transboundary diseases

Detection of several pathogens with multiplexed real-time polymerase chain reaction (qPCR) assays in a one step set-up, allows the simultaneous detection of two endemic porcine and four different selected transboundary viruses. Reverse transcription qPCR (RT-qPCR) systems for the detection of Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine circovirus virus type 2 (PCV2), two of the most economically important diseases 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. The 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 as well clinical specimens were used for a complete evalution. 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 like fever or hemorrhages. The new system could significantly improve the early detection of the most important notifiable diseases of swine and could lead to a new approach in syndromic surveillance.