A RT-PCR assay for the detection of coronaviruses from four genera

J Clin Virol. 2020 Jul:128:104391. doi: 10.1016/j.jcv.2020.104391. Epub 2020 Apr 30.


Background: During the past two decades, three novel coronaviruses (CoVs) have emerged to cause international human epidemics with severe morbidity. CoVs have also emerged to cause severe epidemics in animals. A better understanding of the natural hosts and genetic diversity of CoVs are needed to help mitigate these threats.

Objective: To design and evaluate a molecular diagnostic tool for detection and identification of all currently recognized and potentially future emergent CoVs from the Orthocoronavirinae subfamily.

Study design and results: We designed a semi-nested, reverse transcription RT-PCR assay based upon 38 published genome sequences of human and animal CoVs. We evaluated this assay with 14 human and animal CoVs and 11 other non-CoV respiratory viruses. Through sequencing the assay's target amplicon, the assay correctly identified each of the CoVs; no cross-reactivity with 11 common respiratory viruses was observed. The limits of detection ranged from 4 to 4 × 102 copies/reaction, depending on the CoV species tested. To assess the assay's clinical performance, we tested a large panel of previously studied specimens: 192 human respiratory specimens from pneumonia patients, 5 clinical specimens from COVID-19 patients, 81 poultry oral secretion specimens, 109 pig slurry specimens, and 31 aerosol samples from a live bird market. The amplicons of all RT-PCR-positive samples were confirmed by Sanger sequencing. Our assay performed well with all tested specimens across all sample types.

Conclusions: This assay can be used for detection and identification of all previously recognized CoVs, including SARS-CoV-2, and potentially any emergent CoVs in the Orthocoronavirinae subfamily.

Keywords: COVID-19; Coronavirus; Emerging; Infectious diseases; SARS-CoV-2.

Publication types

  • Evaluation Study

MeSH terms

  • Animals
  • Betacoronavirus / genetics
  • Betacoronavirus / isolation & purification
  • Bird Diseases / diagnosis*
  • Bird Diseases / virology
  • Birds
  • COVID-19
  • Coronavirus / genetics
  • Coronavirus / isolation & purification*
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / virology
  • Genetic Variation
  • Humans
  • Molecular Diagnostic Techniques / methods*
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • Pneumonia, Viral / virology
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • SARS-CoV-2
  • Severe acute respiratory syndrome-related coronavirus / genetics
  • Severe acute respiratory syndrome-related coronavirus / isolation & purification
  • Swine
  • Swine Diseases / diagnosis*
  • Swine Diseases / virology