SARS-CoV-2 Viral Load Quantification via RT-qPCR

Stool samples, reagent-only negative controls, and mock community positive controls (Zymo Research) were extracted using either the AllPrep PowerFecal DNA/RNA 96 Kit (Qiagen) or the Maxwell HT 96 gDNA Blood Isolation System (Promega) [27 (link)]. SARS-CoV-2 viral load was quantified as per CDC guidelines [28 ] using the 2019-nCoV N1 primer and probe set [28 ], as well as human RNaseP as an internal control. Each RT-qPCR reaction contained TaqPath™ 1-Step RT-qPCR Master Mix (Thermo Fisher), RNA template, the CDC N1 or RNaseP forward and reverse primers (IDT), probe, and RNase-free water to a total reaction volume of 10 μl. Viral copy numbers were quantified using N1 quantitative PCR (qPCR) standards (IDT) in tenfold dilutions to generate a standard curve. The assay was run in triplicate for each sample with three no-template control wells per 384 well plate.

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Nguyen L.H., Okin D., Drew D.A., Battista V.M., Jesudasen S.J., Kuntz T.M., Bhosle A., Thompson K.N., Reinicke T., Lo C.H., Woo J.E., Caraballo A., Berra L., Vieira J., Huang C.Y., Das Adhikari U., Kim M., Sui H.Y., Magicheva-Gupta M., McIver L., Goldberg M.B., Kwon D.S., Huttenhower C., Chan A.T, & Lai P.S. (2023). Metagenomic assessment of gut microbial communities and risk of severe COVID-19. Genome Medicine, 15, 49.

Publication 2023

mock community positive controls AllPrep PowerFecal DNA/RNA 96 Kit Maxwell HT 96 gDNA Blood Isolation System TaqPath™ 1-Step RT-qPCR Master Mix

2019 ncov Assay Blood system Dilutions Human Isolation Primers Promega Rnase Rt pcr Stool

Corresponding Organization :

Other organizations : Harvard University, Massachusetts General Hospital, Ragon Institute of MGH, MIT and Harvard

Top 5 similar protocols

Variable analysis

independent variables

Extraction method (AllPrep PowerFecal DNA/RNA 96 Kit or Maxwell HT 96 gDNA Blood Isolation System)

dependent variables

SARS-CoV-2 viral load

control variables

Stool samples
Reagent-only negative controls
Mock community positive controls (Zymo Research)
CDC 2019-nCoV N1 primer and probe set
Human RNaseP as an internal control
TaqPath™ 1-Step RT-qPCR Master Mix
N1 quantitative PCR (qPCR) standards (IDT) in tenfold dilutions to generate a standard curve
No-template control wells

positive controls

Mock community positive controls (Zymo Research)

negative controls

Reagent-only negative controls
No-template control wells

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