TITLE: Large scale screening of SARS-CoV-2 variants of worldwide concern by RT-qPCR
AUTHORS: Katja Spiess , Ellinor  Marving, Alonzo Alfaro-Núñez, Vithiagaran Gunalan, Sofie Holdflod Nielsen, Michelle G. P. Jørgensen, Anna S. Fomsgaard, Søren M. Karst, Shila Mortensen, Ria Lassaunière, Jannik Fonager, Morten Rasmussen, Maiken Worsøe Rosenstierne, Charlotta Polacek Strandh, Anne-Marie Vangsted, Claus Nielsen, Arieh S. Cohen, Anders Fomsgaard

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Multiple SARS-CoV-2 variants have evolved increasing the transmissibility of the virus and/or cause its escape from immune response. Whole genome sequencing remains the gold standard and is implemented into national surveillance programs worldwide to detect these novel variants of concern. However, this technique can have a high turnaround time and it might not be possible to immediately be scaled up when fast action is required to limit the spread of SARS-CoV-2 variants every time a new local outbreak occurs. </div><div class = "text-block">Four key mutations H69/V70 deletion, L452R, E484K and N501Y with multiple reassurance in the SARS-CoV-2 spike (S) glycoprotein were identified to have a distinct signature enabling to detect SARS-CoV-2 variants of worldwide concern (Alpha [B.1.1.7], Alpha [B.1.17 + E484K], Beta [B.1.351], Gamma [P1], Delta [B.1.617,2]) as well as variants of interest/notes. We developed RT-qPCRs to detect these key mutations and increased the sensitivity of the PCRs by using modified probes targeting the 452R, 484K and 501Y mutations, with a single nucleotide exchange present, leading to an amino acid substitution. Large scale screening of the SARS-CoV2 variants of concern was included into the national surveillance program in Denmark, where the RT-qPCRs run as multiplexed (H69/V70 and N501Y mutation) - or as a single reactions detecting all four key mutations in parallel in a 384-well format. </div><div class = "text-block">Our RT-qPCR systems offer a fast and flexible testing strategy to detect SARS-CoV-2 variants of concern and of interest/note,  especially needed in countries where whole genome sequencing in large scale is too cost- and labour intensive.</div></div>

[STEPS]
?. Protocol for 96 and 384 well PCR formatPrepare three different mastermixes. Make sure to vortex all reagents before use.Mastermix 1, for detection of H69/V70 and N501Y, contains the following: Mastermix 2, for detection of E484K, contains the following: Mastermix 3, for detection of L452R, contains the following: Add 20µL (96 well) or 10µL (386 well) of mastermix to each well.Add 5µl of extracted RNA (unknown sample) to each of the three mastermixes.Add 5µl of control (positive controls such as virus cultures, sequenced patient samples with known information about the SARS-CoV-2 variant or TWIST synthetic RNA controls and negative controls such as negative tested patient samples or nuclease free water) to the corresponding mastermixes.Seal the plates/tubes Centrifuge to remove air bubblesCycler protocolRun the PCR with the following cycler conditions and set the thermocycler to read the FAM, HEX, ROX and Cy5 channels: Data evaluationMark negative control (NTC) as NTC in plate set-up, otherwise the wild type and the mutant will not be grouped correctly.Correct undershooting curves (often for weak samples). This also helps the software to group wildtype and mutant correctly. Set correct fluorophores (depending on which mastermix is being analyzed) in allelic discrimination chart.Use a threshold RFU>200 for all fluorophores in case one of the probes in the allelic discrimination pair fails.Perform allelic discrimination at Ct = 45 for all SNPs.Mastermix 1, for detection of H69/V70 and N501Y, contains the following: Mastermix 2, for detection of E484K, contains the following: Mastermix 3, for detection of L452R, contains the following: Add 20µL (96 well) or 10µL (386 well) of mastermix to each well.Add 5µl of extracted RNA (unknown sample) to each of the three mastermixes.Add 5µl of control (positive controls such as virus cultures, sequenced patient samples with known information about the SARS-CoV-2 variant or TWIST synthetic RNA controls and negative controls such as negative tested patient samples or nuclease free water) to the corresponding mastermixes.Seal the plates/tubes Centrifuge to remove air bubblesCycler protocolRun the PCR with the following cycler conditions and set the thermocycler to read the FAM, HEX, ROX and Cy5 channels: Data evaluationMark negative control (NTC) as NTC in plate set-up, otherwise the wild type and the mutant will not be grouped correctly.Correct undershooting curves (often for weak samples). This also helps the software to group wildtype and mutant correctly. Set correct fluorophores (depending on which mastermix is being analyzed) in allelic discrimination chart.Use a threshold RFU>200 for all fluorophores in case one of the probes in the allelic discrimination pair fails.Perform allelic discrimination at Ct = 45 for all SNPs.  Luna® Universal Probe One-Step RT-qPCR Kit    Volume per reaction (µL) 96 well format    Volume per reaction (µL) 384 well format  Luna® Universal Probe One-Step RT-qPCR Kit 2x    12.5    7.5  Luna WarmStart® RT Enzyme Mix (20X)    1.25  0.75SARS-CoV-2 N501Y F  (100µM)    0.1  0.1SARS-CoV-2 N501Y R (100µM)    0.1  0.1SARS-CoV-2 501Y_mutant MGB P (100µM)     0.05  0.05SARS-CoV-2 N501 WT MGB P (100µM)     0.05  0.05SARS-CoV-2_H69/V70 F (100µM)    0.1  0.1SARS-CoV-2_H69/V70  R (100µM)    0.1  0.1SARS-CoV-2_H69/V70 P (100µM)    0.05  0.05H2O  5.7  1.2Total    2010Sample    5  5Total    2515Mastermix 2, for detection of E484K, contains the following: Mastermix 3, for detection of L452R, contains the following: Add 20µL (96 well) or 10µL (386 well) of mastermix to each well.Add 5µl of extracted RNA (unknown sample) to each of the three mastermixes.Add 5µl of control (positive controls such as virus cultures, sequenced patient samples with known information about the SARS-CoV-2 variant or TWIST synthetic RNA controls and negative controls such as negative tested patient samples or nuclease free water) to the corresponding mastermixes.Seal the plates/tubes Centrifuge to remove air bubblesCycler protocolRun the PCR with the following cycler conditions and set the thermocycler to read the FAM, HEX, ROX and Cy5 channels: Data evaluationMark negative control (NTC) as NTC in plate set-up, otherwise the wild type and the mutant will not be grouped correctly.Correct undershooting curves (often for weak samples). This also helps the software to group wildtype and mutant correctly. Set correct fluorophores (depending on which mastermix is being analyzed) in allelic discrimination chart.Use a threshold RFU>200 for all fluorophores in case one of the probes in the allelic discrimination pair fails.Perform allelic discrimination at Ct = 45 for all SNPs.  Luna® Universal Probe One-Step RT-qPCR Kit    Volume per reaction (µL) 96 well format    Volume per reaction (µL) 384 well format  Luna® Universal Probe One-Step RT-qPCR Kit 2x    12.5    7.5  Luna WarmStart® RT Enzyme Mix (20X)    1.25  0.75SARS-CoV-2_E484K F  (100µM)    0.1  0.1SARS-CoV-2_E484K R (100µM)    0.1  0.1SARS-CoV-2_484K_mutant MGB FAM P (100µM)     0.15  0.15SARS-CoV-2_E484K_WT MGB P (100µM)     0.05  0.05H2O    5.85  1.35Total    20  10Sample    5  5Total    25 15Mastermix 3, for detection of L452R, contains the following: Add 20µL (96 well) or 10µL (386 well) of mastermix to each well.Add 5µl of extracted RNA (unknown sample) to each of the three mastermixes.Add 5µl of control (positive controls such as virus cultures, sequenced patient samples with known information about the SARS-CoV-2 variant or TWIST synthetic RNA controls and negative controls such as negative tested patient samples or nuclease free water) to the corresponding mastermixes.Seal the plates/tubes Centrifuge to remove air bubblesCycler protocolRun the PCR with the following cycler conditions and set the thermocycler to read the FAM, HEX, ROX and Cy5 channels: Data evaluationMark negative control (NTC) as NTC in plate set-up, otherwise the wild type and the mutant will not be grouped correctly.Correct undershooting curves (often for weak samples). This also helps the software to group wildtype and mutant correctly. Set correct fluorophores (depending on which mastermix is being analyzed) in allelic discrimination chart.Use a threshold RFU>200 for all fluorophores in case one of the probes in the allelic discrimination pair fails.Perform allelic discrimination at Ct = 45 for all SNPs.  Luna® Universal Probe One-Step RT-qPCR Kit    Volume per reaction (µL) 96 well format    Volume per reaction (µL) 384 well format  Luna® Universal Probe One-Step RT-qPCR Kit 2x    12.5    7.5  Luna WarmStart® RT Enzyme Mix (20X)    1.25  0.75SARS-CoV-2_L452R F  (100µM)    0.1  0.1SARS-CoV-2_L452R R (100µM)    0.1  0.1SARS-CoV-2_452R_mutant BHQ+ P  (100µM)     0.05  0.05SARS-CoV-2_L452_WT BHQ+ P (100µM)     0.05  0.05H2O   5.95  1.45Total    20  10Sample    5  5Total    25  15Add 20µL (96 well) or 10µL (386 well) of mastermix to each well.Add 5µl of extracted RNA (unknown sample) to each of the three mastermixes.Add 5µl of control (positive controls such as virus cultures, sequenced patient samples with known information about the SARS-CoV-2 variant or TWIST synthetic RNA controls and negative controls such as negative tested patient samples or nuclease free water) to the corresponding mastermixes.Seal the plates/tubes Centrifuge to remove air bubblesCycler protocolRun the PCR with the following cycler conditions and set the thermocycler to read the FAM, HEX, ROX and Cy5 channels: Data evaluationMark negative control (NTC) as NTC in plate set-up, otherwise the wild type and the mutant will not be grouped correctly.Correct undershooting curves (often for weak samples). This also helps the software to group wildtype and mutant correctly. Set correct fluorophores (depending on which mastermix is being analyzed) in allelic discrimination chart.Use a threshold RFU>200 for all fluorophores in case one of the probes in the allelic discrimination pair fails.Perform allelic discrimination at Ct = 45 for all SNPs.Cycler protocolRun the PCR with the following cycler conditions and set the thermocycler to read the FAM, HEX, ROX and Cy5 channels:  Cycle    Time    Temperature    1 cycle    10min    55°C    1 cycle    3min    95°C    45 cycles    15s    95°C    30s    58°C  Data evaluationMark negative control (NTC) as NTC in plate set-up, otherwise the wild type and the mutant will not be grouped correctly.Correct undershooting curves (often for weak samples). This also helps the software to group wildtype and mutant correctly. Set correct fluorophores (depending on which mastermix is being analyzed) in allelic discrimination chart.Use a threshold RFU>200 for all fluorophores in case one of the probes in the allelic discrimination pair fails.Perform allelic discrimination at Ct = 45 for all SNPs.SARS-CoV-2 variant H69/V70 HEX501Y FAM vs. N501 Cy5484K FAM vs. E484 ROX452R HEX vs. L452 ROXWILDTYPECt ≥ 38RFU Cy5 > RFU FAMRFU ROX > RFU FAMRFU ROX > RFU HEXAlphaCt RFU FAM > RFU Cy5 RFU ROX > RFU FAMRFU ROX > RFU HEXBetaCt ≥ 38RFU FAM > RFU Cy5 RFU FAM > RFU ROXRFU ROX > RFU HEXGammaCt ≥ 38RFU FAM > RFU Cy5 RFU FAM > RFU ROXRFU ROX > RFU HEXDeltaCt ≥ 38RFU Cy5 > RFU FAMRFU ROX > RFU FAMRFU HEX > RFU ROX
Luna® Universal Probe One-Step RT-qPCR Kit    Volume per reaction (µL) 96 well format    Volume per reaction (µL) 384 well format  Luna® Universal Probe One-Step RT-qPCR Kit 2x    12.5    7.5  Luna WarmStart® RT Enzyme Mix (20X)    1.25  0.75SARS-CoV-2 N501Y F  (100µM)    0.1  0.1SARS-CoV-2 N501Y R (100µM)    0.1  0.1SARS-CoV-2 501Y_mutant MGB P (100µM)     0.05  0.05SARS-CoV-2 N501 WT MGB P (100µM)     0.05  0.05SARS-CoV-2_H69/V70 F (100µM)    0.1  0.1SARS-CoV-2_H69/V70  R (100µM)    0.1  0.1SARS-CoV-2_H69/V70 P (100µM)    0.05  0.05H2O  5.7  1.2Total    2010Sample    5  5Total    2515
Luna® Universal Probe One-Step RT-qPCR Kit    Volume per reaction (µL) 96 well format    Volume per reaction (µL) 384 well format  Luna® Universal Probe One-Step RT-qPCR Kit 2x    12.5    7.5  Luna WarmStart® RT Enzyme Mix (20X)    1.25  0.75SARS-CoV-2_E484K F  (100µM)    0.1  0.1SARS-CoV-2_E484K R (100µM)    0.1  0.1SARS-CoV-2_484K_mutant MGB FAM P (100µM)     0.15  0.15SARS-CoV-2_E484K_WT MGB P (100µM)     0.05  0.05H2O    5.85  1.35Total    20  10Sample    5  5Total    25 15
Luna® Universal Probe One-Step RT-qPCR Kit    Volume per reaction (µL) 96 well format    Volume per reaction (µL) 384 well format  Luna® Universal Probe One-Step RT-qPCR Kit 2x    12.5    7.5  Luna WarmStart® RT Enzyme Mix (20X)    1.25  0.75SARS-CoV-2_L452R F  (100µM)    0.1  0.1SARS-CoV-2_L452R R (100µM)    0.1  0.1SARS-CoV-2_452R_mutant BHQ+ P  (100µM)     0.05  0.05SARS-CoV-2_L452_WT BHQ+ P (100µM)     0.05  0.05H2O   5.95  1.45Total    20  10Sample    5  5Total    25  15
Cycle    Time    Temperature    1 cycle    10min    55°C    1 cycle    3min    95°C    45 cycles    15s    95°C    30s    58°C
SARS-CoV-2 variant H69/V70 HEX501Y FAM vs. N501 Cy5484K FAM vs. E484 ROX452R HEX vs. L452 ROXWILDTYPECt ≥ 38RFU Cy5 > RFU FAMRFU ROX > RFU FAMRFU ROX > RFU HEXAlphaCt RFU FAM > RFU Cy5 RFU ROX > RFU FAMRFU ROX > RFU HEXBetaCt ≥ 38RFU FAM > RFU Cy5 RFU FAM > RFU ROXRFU ROX > RFU HEXGammaCt ≥ 38RFU FAM > RFU Cy5 RFU FAM > RFU ROXRFU ROX > RFU HEXDeltaCt ≥ 38RFU Cy5 > RFU FAMRFU ROX > RFU FAMRFU HEX > RFU ROX