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

Michel, Janine; Neumann, Markus; Krause, Eva; Rinner, Thomas; Muzeniek, Therese; Grossegesse, Marica; Hille, Georg; Schwarz, Franziska; Puyskens, Andreas; Förster, Sophie; Biere, Barbara; Bourquain, Daniel; Domingo, Cristina; Brinkmann, Annika; Schaade, Lars; Schrick, Livia; Nitsche, Andreas

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12985-021-01559-3.