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

BackgroundThe 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.AimHere 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).MethodsTwo 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.ResultsLimit 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.ConclusionThe 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.

Automated summary

The presented RKI/ZBS1 SARS-CoV-2 protocol offers a cost-effective alternative for detecting SARS-CoV-2 RNA, especially when facing shortages of commercially available ready-to-use kits. By running two duplex real-time RT-PCR assays simultaneously, the protocol enables testing specimens in duplicate and increases testing capacity while saving reagents.