Author: Chantal B.F. Vogels; Anderson F. Brito; Anne Louise Wyllie; Joseph R Fauver; Isabel M. Ott; Chaney C. Kalinich; Mary E. Petrone; Marie-Louise Landry; Ellen F. Foxman; Nathan D. Grubaugh
Title: Analytical sensitivity and efficiency comparisons of SARS-COV-2 qRT-PCR assays Document date: 2020_4_1
ID: 6mdimxnk_34
Snippet: To make a fair comparison between nine primer-probe sets ( Table 1) , we used the same qRT-PCR reagents and conditions for all comparisons. We used the Luna Universal One-step RT-qPCR kit (New England Biolabs, Ipswich, MA, USA) with standardized primer and probe concentrations of 500 nM of forward and reverse primer, and 250 nM of probe for all comparisons. PCR cycler conditions were reverse transcription for 10 minutes at 55°C, initial denatura.....
Document: To make a fair comparison between nine primer-probe sets ( Table 1) , we used the same qRT-PCR reagents and conditions for all comparisons. We used the Luna Universal One-step RT-qPCR kit (New England Biolabs, Ipswich, MA, USA) with standardized primer and probe concentrations of 500 nM of forward and reverse primer, and 250 nM of probe for all comparisons. PCR cycler conditions were reverse transcription for 10 minutes at 55°C, initial denaturation for 1 min at 95°C, followed by 40 cycles of 10 seconds at 95°C and 20 seconds at 55°C on the Biorad CFX96 qPCR machine (Biorad, Hercules, CA, USA). We calculated analytical efficiency of qRT-PCR assays tested with corresponding RNA transcript standards using the following formula 17, 18 : 100 × (10 ) E = −1/slope − 1
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