Related Experiment Video
Updated: Nov 3, 2025
Author Spotlight: Advancing Pathogen Diagnostics with Standardized LAMP
Published on: September 8, 2023
Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2
Janine Michel1, Markus Neumann1, Eva Krause1
1Centre for Biological Threats and Special Pathogens, Highly Pathogenic Viruses (ZBS 1), Robert Koch Institute, Seestraße 10, 13353, Berlin, Germany.
Background:
The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply.
Aim:
Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol).
Methods:
Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen.
Results:
Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits.
Conclusion:
The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.
