Author: Jennifer A. Doudna
Title: Blueprint for a Pop-up SARS-CoV-2 Testing Lab Document date: 2020_4_12
ID: modtthxx_61
Snippet: To experimentally determine our limit of detection, we performed serial dilutions of SARS-CoV-2 positive control RNA (see Methods) in triplicate, from 5 x 10 4 to 1 x 10 2 copies/ml in 500 copies/ml steps. At each dilution, we determined the Ct (cycle threshold) of three SARS-CoV-2 genes (N-gene, S-gene and ORF1ab) as well as spiked-in MS2 bacteriophage nucleic acid, an internal control for nucleic acid extraction efficiency and qPCR amplificatio.....
Document: To experimentally determine our limit of detection, we performed serial dilutions of SARS-CoV-2 positive control RNA (see Methods) in triplicate, from 5 x 10 4 to 1 x 10 2 copies/ml in 500 copies/ml steps. At each dilution, we determined the Ct (cycle threshold) of three SARS-CoV-2 genes (N-gene, S-gene and ORF1ab) as well as spiked-in MS2 bacteriophage nucleic acid, an internal control for nucleic acid extraction efficiency and qPCR amplification. The Ct value is inversely proportional to the amount of starting target RNA, and represents the PCR cycle number at which the fluorescent signal of the reaction crosses a set threshold. This threshold was set manually based on background fluorescence of the reaction mixture.
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