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.
【저자키워드】 COVID-19, SARS-CoV-2, diagnostics, Real-time PCR, Internal control, 【초록키워드】 protocol, Genome, diagnostic, RNA, nucleic acid, PCR, management, Patient, SARS-CoV-2 RNA, crisis management, real-time RT-PCR, PCR assays, information, genomic, SARS-CoV-2 PCR, COVID-19 crisis, supplementary material, reaction, house, reagents, sequence, specimen, market, PCR assay, RT-PCR assays, reagent, regions, Result, approach, assays, addition, indicate,