Development of a Taq Man-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses

Virulence. 2020 Jan 1;11(1):707-718. doi: 10.1080/21505594.2020.1771980.

Abstract

With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order Nidovirales. Here, we developed a multiplex TaqMan-probe-based real-time PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and TaqMan fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 102 copies/μL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.

Keywords: Real-time PCR; coronavirus; detection; swine diarrhea virus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Coinfection / diagnosis
  • Coinfection / veterinary
  • Communicable Diseases, Emerging / diagnosis
  • Communicable Diseases, Emerging / veterinary*
  • Coronaviridae / genetics
  • Coronaviridae / isolation & purification*
  • Coronaviridae Infections / diagnosis
  • Coronaviridae Infections / veterinary*
  • Diarrhea / diagnosis
  • Diarrhea / veterinary
  • Open Reading Frames / genetics
  • Polymerase Chain Reaction* / standards
  • RNA, Viral / genetics
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Swine
  • Swine Diseases / diagnosis*

Substances

  • RNA, Viral

Grants and funding

This work was financially supported by the National Natural Science Foundation of Outstanding Youth Fund in China (NSFC Grant no. 31922081), the National Key Research and Development Program of China (Grant no. 2016YFD0500402), National Ten Thousand Talent Program, the Fundamental Research Funds for the Central Universities (Grant no. Y0201900459), Six Talent Peaks Project of Jiangsu Province of China (Grant no. NY-045) and the Bioinformatics Center of Nanjing Agricultural University.