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Related Experiment Video

Updated: Nov 12, 2025

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A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for RT-qPCR.

Lelde Kalnina1, Àngels Mateu-Regué1, Stephanie Oerum2

  • 1Center for Genomic Medicine, Rigshospitalet, Copenhagen, Denmark.

APMIS : Acta Pathologica, Microbiologica, Et Immunologica Scandinavica
|March 17, 2021
PubMed
Summary

A simplified, safe, and rapid RNA extraction method using guanidinium isothiocyanate, phenol, and chloroform allows for sensitive SARS-CoV-2 detection via RT-qPCR, expanding diagnostic capacity.

Keywords:
RNA extractionRT-qPCRSARS-CoV-2SARS-CoV-2 RT-qPCRclinical microbiologymolecular microbiologyrapid diagnostic methodsvirologyvirus inactivation

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Area of Science:

  • Molecular Biology
  • Virology
  • Biochemistry

Background:

  • The COVID-19 pandemic necessitates accessible and reliable SARS-CoV-2 RNA detection methods.
  • Commercial RNA extraction kits present challenges including cost, limited availability, and incomplete viral inactivation.
  • There is a critical need for safer, in-house SARS-CoV-2 RNA extraction protocols using standard laboratory resources.

Purpose of the Study:

  • To optimize and simplify an RNA extraction protocol for SARS-CoV-2 detection.
  • To establish a safe and efficient method for preparing viral RNA for RT-qPCR analysis.
  • To enhance diagnostic capabilities in laboratories without specialized viral containment facilities.

Main Methods:

  • Optimized a simplified RNA extraction protocol using acidic guanidinium isothiocyanate (GITC), phenol, and chloroform.
  • Determined GITC/RNA dilution thresholds for efficient two-step RT-qPCR of B2M mRNA in nasopharyngeal (NP) and oropharyngeal (OP) swabs.
  • Developed and validated a one-step RT-qPCR assay for SARS-CoV-2 using NP and OP samples, including testing a viral dilution series.

Main Results:

  • Achieved high sensitivity for SARS-CoV-2 detection, with a limit of approximately 4 viral RNA copies per RT-qPCR.
  • The optimized protocol yields PCR-ready viral RNA in approximately 30 minutes from swab.
  • Demonstrated successful SARS-CoV-2 detection in RT-qPCR from inactivated samples, enabling use in non-viral classified labs.

Conclusions:

  • The developed RNA extraction method is simple, safe, and cost-effective.
  • This protocol significantly expands the capacity for SARS-CoV-2 testing by utilizing readily available reagents and equipment.
  • The method provides a sensitive and rapid alternative to commercial kits for viral RNA extraction and detection.