Ultrasensitive Detection of Porcine Epidemic Diarrhea Virus from Fecal Samples Using Functionalized Nanoparticles

PLoS One. 2016 Dec 9;11(12):e0167325. doi: 10.1371/journal.pone.0167325. eCollection 2016.

Abstract

Porcine epidemic diarrhea virus (PEDV) is the main causative agent of porcine diarrhea, which has resulted in devastating damage to swine industry and become a perplexed global problem. PEDV infection causes lesions and clinical symptoms, and infected pigs often succumb to severe dehydration. If there is not a timely and effective method to control its infection, PEDV will spread rapidly across the whole swine farm. Therefore, preclinical identification of PEDV is of great significance for preventing the outbreak and spread of this disease. In this study, a functionalized nanoparticles-based PCR method (UNDP-PCR) specific for PEDV was developed through systematic optimization of functionalized magnetic beads and gold nanoparticles which were further used to specifically enrich viral RNA from the lysate of PEDV stool samples, forming a MMPs-RNA-AuNPs complex. Then, oligonucleotides specific for PEDV coated on AuNPs were eluted from the complex and were further amplified and characterized by PCR. The detection limitation of the established UNDP-PCR method for PEDV was 25 copies in per gram PEDV stool samples, which is 400-fold more sensitive than conventional RT-PCR for stool samples. The UNDP-PCR for PEDV exhibited reliable reproducibility and high specificity, no cross-reaction was observed with other porcine viruses. In 153 preclinical fecal samples, the positive detection rate of UNDP-PCR specific for PEDV (30.72%) was much higher than that of conventional RT-PCR (5.88%) and SYBR Green real-time RT-PCR. In a word, this study provided a RNA extraction and transcription free, rapid and economical method for preclinical PEDV infection, which showed higher sensitivity, specificity and reproducibility, and exhibited application potency for evaluating viral loads of preclinical samples.

MeSH terms

  • Animals
  • Coronavirus Infections / diagnosis
  • Coronavirus Infections / veterinary*
  • Coronavirus Infections / virology
  • Feces / virology*
  • Gold / chemistry
  • Metal Nanoparticles / chemistry
  • Polymerase Chain Reaction / methods
  • Porcine epidemic diarrhea virus / genetics
  • Porcine epidemic diarrhea virus / isolation & purification*
  • RNA, Viral / genetics
  • RNA, Viral / isolation & purification*
  • Swine / virology*
  • Swine Diseases / diagnosis*
  • Swine Diseases / virology

Substances

  • RNA, Viral
  • Gold

Grants and funding

This study was funded by grants from the scientific and technological project in Shaanxi province (Grant No. 2015NY155), the innovation project of science and technology plan projects in Shaanxi province (2016KTCL02-13), Key project of Shaanxi province science and technology innovation team (2013KCT-28). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.