Detection of SARS-CoV-2 from raw patient samples by coupled high temperature reverse transcription and amplification

PLoS One. 2020 Nov 2;15(11):e0241740. doi: 10.1371/journal.pone.0241740. eCollection 2020.

Abstract

SARS-CoV-2 is spreading globally with unprecedented consequences for modern societies. The early detection of infected individuals is a pre-requisite to contain the virus. Currently, purification of RNA from patient samples followed by RT-PCR is the gold standard to assess the presence of this single-strand RNA virus. However, these procedures are time consuming, require continuous supply of specialized reagents, and are prohibitively expensive in resource-poor settings. Here, we report an improved nucleic-acid-based approach to detect SARS-CoV-2 with the ability to detect as little as five viral genome equivalents. The approach delivers results without the need to purify RNA, reduces handling steps, minimizes costs, and allows evaluation by non-specialized equipment. The use of unprocessed swap samples is enabled by employing a heat-stable RNA- and DNA-dependent DNA polymerase, which performs the double task of stringent reverse transcription of RNA at elevated temperatures as well as PCR amplification of a SARS-CoV-2 specific target gene. As results are obtained within 2 hours and can be read-out by a hand-held LED-screen, this novel protocol will be of particular importance for large-scale virus surveillance in economically constrained settings.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Betacoronavirus / genetics*
  • Betacoronavirus / isolation & purification
  • COVID-19
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / virology
  • Humans
  • Nasopharynx / virology
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • Pneumonia, Viral / virology
  • RNA, Viral / genetics
  • RNA, Viral / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • SARS-CoV-2
  • Temperature

Substances

  • RNA, Viral

Grants and funding

TB acknowledges support from the Konstanz Research School Chemical Biology (KoRS-CB) and AM support by the German Research Foundation (DFG) within SPP 1784. RK acknowledges support by MyPOLS Biotech GmbH, which provided support in the form of salary for RK. The funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.