TITLE: Multiplexed RT-qPCR to screen for SARS-COV-2 B.1.1.7, B.1.351, and P.1 variants of concern
AUTHORS: Chantal Vogels, Joseph Fauver, Nathan Grubaugh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">With the emergence of SARS-CoV-2 variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for targeted molecular surveillance methods. While sequencing is the gold standard, it cannot always be immediately scaled or implemented in some settings to detect variants when their frequencies are low. The Applied Biosystems TaqPath COVID-19 assay (ThermoFisher), a PCR test, was discovered to have a distinct signature (spike gene target failure, [SGTF]) when testing viruses containing the Δ69/70 HV deletion, like the B.1.1.7 variant first detected in the UK. However, a sample with a SGTF is not definitive for B.1.1.7, and cannot detect other variants of concern that lack the Δ69/70 HV deletion, such as B.1.351 detected in South Africa and P.1 recently detected in Brazil. We developed a multiplexed RT-qPCR assay that can detect all three variants by targeting the Δ3675-3677 SGF deletion in the ORF1a gene, which has not yet been widely detected in other SARS-CoV-2 lineages. Furthermore, by also targeting the Δ69/70 HV deletion in the spike gene, our assay can differentiate B.1.1.7 from B.1.351 and P.1. Finally, we include the CDC N1 primer and probe set in our multiplexed assay as a control to ensure that target failures are likely due to the presence of the ORF1a and/or spike deletions and that there is sufficient virus RNA for sequencing confirmation. Our multiplexed RT-qPCR assay can be rapidly scaled to support SARS-CoV-2 variant surveillance.</div><style>
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[STEPS]
?. [RT-qPCR Protocol]
Briefly vortex and centrifuge reagents before use.
?. [RT-qPCR Protocol]
Prepare 20 µM working stocks of the primers and probes, by adding 20 µL of 100 µM stock to 80 µL nuclease-free water.
?. [RT-qPCR Protocol]
Use the 20 µM working stocks to prepare primer-probe-water mix containing the following:ComponentVolume (1 reaction) Volume (100 reactions)CDC_N1_F (200 nM/reaction)0.2 µL20 µLCDC_N1_R (200 nM/reaction)0.2 µL20 µLCDC_N1_P (100 nM/reaction)0.1 µL10 µLYale_69/70del_F (400 nM/reaction)0.4 µL40 µLYale_69/70del_R (400 nM/reaction)0.4 µL40 µLYale_69/70del_P (200 nM/reaction)0.2 µL20 µLYale_ORF1a-del_F (400 nM/reaction)0.4 µL40 µLYale-ORF1a-del_R (400 nM/reaction)0.4 µL40 µLYale-ORF1a-del_P (200 nM/reaction)0.2 µL20 µLNuclease-free water1.5 µL150 µL
ComponentVolume (1 reaction) Volume (100 reactions)CDC_N1_F (200 nM/reaction)0.2 µL20 µLCDC_N1_R (200 nM/reaction)0.2 µL20 µLCDC_N1_P (100 nM/reaction)0.1 µL10 µLYale_69/70del_F (400 nM/reaction)0.4 µL40 µLYale_69/70del_R (400 nM/reaction)0.4 µL40 µLYale_69/70del_P (200 nM/reaction)0.2 µL20 µLYale_ORF1a-del_F (400 nM/reaction)0.4 µL40 µLYale-ORF1a-del_R (400 nM/reaction)0.4 µL40 µLYale-ORF1a-del_P (200 nM/reaction)0.2 µL20 µLNuclease-free water1.5 µL150 µL
?. [RT-qPCR Protocol]
Diagram sample, standard, and control positions on a 96-well plate map.
?. [RT-qPCR Protocol]
ComponentVolume in 20 µL reactionTube label = MM10 µLTube label = RT1 µLTube label = primer-probe-water mix4 µLViral RNA, positive control, or negative control5 µL (do not add to master mix)
ComponentVolume in 20 µL reactionTube label = MM10 µLTube label = RT1 µLTube label = primer-probe-water mix4 µLViral RNA, positive control, or negative control5 µL (do not add to master mix)
?. [RT-qPCR Protocol]
Add 15 µL of mastermix to each well (on ice).
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Add 5 µL of positive control (Twist synthetic RNA control at 100 copies/µL) and no-template control (NTC - water) to the designated wells (on ice). Mix by pipetting (avoid bubbles).
?. [RT-qPCR Protocol]
Add 5 µL of viral RNA to the designated wells (on ice). Mix by pipetting (avoid bubbles).
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Cover with plate sealer. Centrifuge to remove bubbles, if present.
?. [RT-qPCR Protocol]
Set the thermocycler to read FAM, HEX, and Cy5 fluorophores.
?. [RT-qPCR Protocol]
Run the following thermocycler conditions:StepTemperatureTime155°C 10 min295°C 1 min395°C 10 sec455°C 30 sec5Read plateRepeat steps 3-5 for 39 cycles.
StepTemperatureTime155°C 10 min295°C 1 min395°C 10 sec455°C 30 sec5Read plateRepeat steps 3-5 for 39 cycles.
?. [RT-qPCR Protocol]
Interpreting results: ResultCDC_N1 (FAM)Yale_69/70del (HEX)Yale_Orf1a-del (Cy5)Potentially B.1.1.7CT ≤ 35UndetectedUndetectedPotentially B.1.351 or P.1CT ≤ 35CT ≤ 35UndetectedPotentially B.1.375, B.1.258, or other lineagesCT ≤ 35UndetectedCT ≤ 35Other lineagesCT ≤ 35CT ≤ 35CT ≤ 35InconclusiveCT > 35 or undetectedAny valueAny value
ResultCDC_N1 (FAM)Yale_69/70del (HEX)Yale_Orf1a-del (Cy5)Potentially B.1.1.7CT ≤ 35UndetectedUndetectedPotentially B.1.351 or P.1CT ≤ 35CT ≤ 35UndetectedPotentially B.1.375, B.1.258, or other lineagesCT ≤ 35UndetectedCT ≤ 35Other lineagesCT ≤ 35CT ≤ 35CT ≤ 35InconclusiveCT > 35 or undetectedAny valueAny value