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Virol J ; 18(1): 110, 2021 06 02.
Article in English | MEDLINE | ID: covidwho-1255943

ABSTRACT

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.


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