Development and application of a novel Bio–Plex suspension array system for high–throughput multiplexed nucleic acid detection of seven respiratory and reproductive pathogens in swine

Abstract

The aim of this study was to develop a multiple PCR assay based on the suspension array system for the simultaneous detection of respiratory and reproductive pathogens in swine. Pseudorabies virus (PRV), Japanese encephalitis virus (JEV), classic swine fever virus (CFSV), African swine fever virus (ASFV), porcine circovirus type 2 (PCV–2), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV) are the major respiratory and reproductive viral pathogens in pig farms. Seven pairs of specific primers and probes were designed, and the multiple PCR was performed, with the PCR products hybridized to beads coupled to probes, which were then detected by Bio–Plex suspension array system. The limit of detection, specificity and repeatability of this method was determined. The assay was further tested using 137 clinical samples, and the results were compared with conventional PCR to evaluate the ability of the method to diagnose porcine viruses. The results showed that the assay had a high degree of specificity and repeatability, and the simultaneous detection limit for the seven viruses reached 103 copies/μL. Forty-nine of the clinical samples tested positive for at least one of the viruses, the principal viral infections in the clinical samples were PCV–2 and PRRSV. The suspension method represented a rapid, specific and high–throughput tool for single or mixed detection of the seven porcine viruses simultaneously, and has great significance for the development of liquid chip techniques for the diagnosis of diseases in animals.