Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2

Virol J. 2021 Jun 2;18(1):110. doi: 10.1186/s12985-021-01559-3.


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.

Keywords: COVID-19; Diagnostics; Internal control; Real-time PCR; SARS-CoV-2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19 / diagnosis*
  • COVID-19 Nucleic Acid Testing / methods*
  • Coronavirus Envelope Proteins / genetics
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Limit of Detection
  • Polyproteins / genetics
  • RNA, Viral / analysis*
  • RNA, Viral / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • SARS-CoV-2 / genetics*
  • SARS-CoV-2 / isolation & purification
  • Sensitivity and Specificity
  • Viral Proteins / genetics


  • Coronavirus Envelope Proteins
  • ORF1ab polyprotein, SARS-CoV-2
  • Polyproteins
  • RNA, Viral
  • Viral Proteins
  • envelope protein, SARS-CoV-2