Development of a gold nanoparticle-based oligonucleotide microarray for simultaneous detection of seven swine viruses.

Abstract

A gold nanoparticle (GNP) based oligonucleotide microarray assay (GNMA) that combined GNP-based multiplex asymmetric PCR with silver enhancement detection, was developed for simultaneous detection of seven important swine viruses in intensive swine production. Multiplex PCR was first performed to enable the target fragments of seven viruses containing a universal sequence, which were labeled simultaneously with GNPs by multiplex asymmetric PCR in the presence of excess GNP-conjugated primer. Target labeled products were captured by virus-specific oligonucleotide probes immobilized on microarrays, followed by silver staining for signal enhancement. Black image of microarray spots was easily detected by the naked eye or a simple flatbed scanner and quantified. The results for purified plasmid constructs indicated that the assay was highly specific for detecting the seven viruses in single or multiple infections, and as few as 6-80 copies/μl of specific viral target fragments were detected successfully. Fifty-seven archived samples were tested by this assay, and the results were 100% consistent with previous results based on real-time PCR and those obtained by conventional PCR/RT-PCR and sequencing. The assay is appropriate for the routine diagnosis of viral infections in pigs due to its simplicity, low-cost, high specificity and sensitivity.