Development of a nanoparticle-assisted triplex RT-PCR assay for porcine reproductive and respiratory syndrome virus detection

Background: Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped, single-stranded, positive sense RNA virus, has constantly varied and circulated in the pig herd causing severe economic losses worldwide. In this study, a nanoparticle-assisted RT-PCR (Nano-RT-PCR) assay for PRRSV detection was established by the combination of nano-particles with traditional RT-PCR technology. Results: : A rapid detection method based on PRRSV Nsp2 gene sequences to identify classical, highly pathogenic, and NADC30-like strains was developed, and its specificity and sensitivity were evaluated. The results showed that no cross-reaction was observed for the triplex Nano-RT-PCR when detecting related viruses, so as classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), swine transmissible gastroenteritis virus (TGEV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine parvovirus (PPV). The assay was more sensitive than the conventional RT-PCR assay with detection limits of 3.32×10 2 , 6.18×10 3 , and 3.26×10 3 copies/μL Nsp2 recombinant plasmid of classical, highly pathogenic, and NADC30-like strains which is 10 times, 1 time, and 10 times higher than conventional PCR, respectively. Moreover, the results of 80 clinical samples showed 71.25% were determined PRRSV positive by our Nano RT-PCR assay, compared with 60% for conventional RT-PCR, and that NADC30-like strain had become the main endemic strains of PRRSV in China and there was a mixed infection of two or three strains. Conclusions: : A sensitive and specific Nano-RT-PCR detection method was developed which can be applied in clinical diagnosis and field surveillance of PRRSV infection and determined the strain type.