Quantitative Viral Load Determination

qPCR was performed using a previously described duplex assay^{25 (link)} with the primers and probes given in Supplementary Table S3. VLPs were lysed by heating to 95°C for 5 min in a thermocycler before being added to qPCRs. qPCRs (20 μL) used TaqPath™ qPCR Master Mix, CG (ThermoFisher Scientific) with final primer and probe concentrations of 400 and 200 nM, and a sample volume of 1 μL. The reaction mixture was then thermocycled (50°C for 2:00, 95°C for 5:00 and 45 cycles of 95°C for 0:10, and 60°C for 1:00) on a BioRad CFX96 qPCR machine.

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Crone M.A, & Freemont P.S. (2022). Simple Low-Cost Production of DNA MS2 Virus-Like Particles As Molecular Diagnostic Controls. Gen Biotechnology, 1(6), 496-503.

Publication 2022

CFX96 qPCR machine

Primer

Corresponding Organization : Imperial College London

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Variable analysis

independent variables

Heating VLPs to 95°C for 5 min in a thermocycler before adding to qPCRs

dependent variables

QPCR measurements

control variables

Final primer and probe concentrations (400 and 200 nM, respectively)
Sample volume (1 μL)
Reaction mixture thermocycling conditions (50°C for 2:00, 95°C for 5:00 and 45 cycles of 95°C for 0:10, and 60°C for 1:00)
QPCR machine (BioRad CFX96)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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