Fluorescence resonance energy transfer combined with asymmetric PCR for broad and sensitive detection of porcine reproductive and respiratory syndrome virus 2.

Abstract

With its ever-increasing viral genetic diversity, accurate diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) infection is indispensable for PRRSV control. Here, a sensitive graphene oxide (GO)-based FRET method was developed to detect PRRSV-2 based on the ability of GO to quench fluorophore by fluorescence resonance energy transfer (FRET). Using primers and a fluorophore-labeled ssDNA probe targeting a conserved region between the PRRSV M gene and 3 UTR, asymmetric PCR specifically amplified viral ssDNA that could anneal with probe to generate dsDNA only in the presence of virus. Upon exonuclease III treatment to release the probe fluorophore, which degrades dsDNA with blunt ends or recessed 3´-termini, the ssDNA annealed with other probe to generate enhanced fluorescence. This GO-based FRET assay specifically detected both classical and highly pathogenic PRRSV, with analytical sensitivity approaching 10 copies/μL, similar to that of real-time PCR but greater than that of conventional reverse transcription PCR (RT-PCR). Consistent with real-time RT-PCR detection, the assay developed here exhibited high diagnostic sensitivity for virus detection of sera from experimentally and naturally infected pigs. Thus, this novel GO-based FRET assay combined with asymmetric PCR detection is sensitive and specific and will be valuable for future PRRSV diagnosis.