Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

PLoS One. 2021 Feb 26;16(2):e0247792. doi: 10.1371/journal.pone.0247792. eCollection 2021.

Abstract

Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COVID-19 / diagnosis*
  • COVID-19 Nucleic Acid Testing / methods*
  • Chlorocebus aethiops
  • Endopeptidase K / chemistry*
  • Hot Temperature
  • Humans
  • Molecular Diagnostic Techniques / methods
  • Nucleic Acid Amplification Techniques / methods
  • SARS-CoV-2 / genetics
  • SARS-CoV-2 / isolation & purification*
  • Sensitivity and Specificity
  • Specimen Handling / methods
  • Vero Cells

Substances

  • Endopeptidase K

Supplementary concepts

  • LAMP assay

Grants and funding

This work was supported by: - Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (ANPCyT). Grant: PICT 2016-0828 to V.L. https://www.argentina.gob.ar/ciencia/agencia - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Grant: PIP 2014-11220130100121CO to V. L. https://www.conicet.gov.ar/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.