TITLE: Development of a multiplexed RT-qPCR for the surveillance of SARS-CoV-2 variants of world-wide concern
AUTHORS: Ellinor Marving, Alonzo Alfaro-Núñez, Sofie Holdflod Nielsen, Michelle Jørgensen, Anna S. Fomsgaard, Shila Mortensen, Morten Rasmussen, Ria Lassaunière, Charlotte Polacek Strandh, Claus Nielsen, Maiken Worsøe Rosenstierne, Arieh Cohen, Anders Fomsgaard, Katja Spiess

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Multiple SARS-CoV-2 variants have evolved that increase the transmissibility of the virus and/or cause its escape from immune response. Whole genome sequencing remains the gold standard and is implemented into the national surveillance programs worldwide to detect these novel variants of concern. However, this technique cannot immediately be scaled up when a fast action is needed to limit the spread of SARS-CoV-2 variants every time a new outbreak occurs, as this can be laborious and time-consuming.</div><div class = "text-block">Two key mutations with multiple reassurance in the SARS-CoV-2 spike glycoprotein have been identified to have a distinct signature enabling to detect five SARS-CoV-2 variants (B1.351, B1.1.7, P1 and N439K/Y453F variants). Three of them are SARS-CoV-2 variants of major concern worldwide. We developed a specific multiplexed RT-qPCR to detect these key mutations, the H69/V70deletion and the N501Y mutation and increased the sensitivity of the PCR by using LNA probes targeting the N501Y mutation carrying a single amino acid exchange. Additionally, we can exclude target failure and secure SARS-CoV-2 detection by a primer/probe pair detecting the Wt sequence (SARS-CoV-2; Wuhan lineage) at aa position 501. Our multiplexed RT-qPCR assay offers an alternative strategy for SARS-CoV-2 variant surveillance detecting five SARS-CoV-2 variants, which can build up on to detect new emerging SARS-CoV-2 variants.</div></div>

[STEPS]
?. ProtocolWork on ice Vortex and centrifuge reagents before use, except the Luna WarmStart® RT Enzyme Mix here only mix carefully by pipetting or short vortexingDilute the primer and probe stock (100µM) 1:5 with nuclease free water to a working concentration of 20µMPrepare mastermix on ice containing the following:     Luna® Universal Probe One-Step RT-qPCR Kit      per reaction        Luna® Universal Probe One-Step RT-qPCR Kit 2x      12.5   µl        Luna WarmStart® RT Enzyme Mix (20X)      1.25   µl        SNP_501_F_2   (20µM)      0,5   µl        SNP_501_R_2   (20µM)      0,5   µl        Probe4_Wt_n_501_LNA (20µM)       0,25   µl        Probe4_B117_y_LNA (20µM)       0,25   µl        SARS-CoV-2_deletion_F (20µM)      0.5   µl        SARS-CoV-2_deletion_R (20µM)      0.5   µl        SARSCoV2_deletion_P (20µM)      0,25 µl        H2O      3.5 µl        Total      20 µl        Sample      5µl        Total      25µl   Add 20µL of mastermix to each well/ PCR tube Add 5µl of the 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 and nuclease free water) to the corresponding wells containing the mastermix and mix by pipettingAdd 5µl of extracted RNA (unknown sample) to the designated wells and mix by pipettingSeal the plates/tubes Centrifuge to remove air bubblesRun the PCR with the following cycler conditions and set the thermocycler to read the HEX, FAM and Cy5 channels:Cycler protocol     Cycle      Time      Temperature        1 cycle      10min      550C        1 cycle      3min      950C        45 cycles      15s      950C               30s      580C   Data evaluationH69/70 deletionHEX channel: no Ct value = no detection of the H69/70 deletion, Ct ≤ 38 H69/70 deletion detectedN501Y mutationMark negative control (NTC) as NTC in plate set-up, otherwise the wild type and the N501Y mutant will not be grouped correctly.Correct undershooting curves (often for weak samples) by adjusting ”Cycles to analyze”. This also helps the software to group wildtype and the N501Y mutant correctly. In the tab Allelic discrimination: Set Selected Fluorophores (right low corner) to X: FAM, Y: Cy5Allele 1 –FAM probe detects the N501Y mutant. Allele 2 – Cy5 detects the SARS-CoV-2 (Wuhan lineage). Sometimes the Wt (Cy5) is marked as Heterozygote and not Allele 2.   Luna® Universal Probe One-Step RT-qPCR Kit    per reaction    Luna® Universal Probe One-Step RT-qPCR Kit 2x    12.5
  µl    Luna WarmStart® RT Enzyme Mix (20X)    1.25
  µl    SNP_501_F_2
  (20µM)    0,5
  µl    SNP_501_R_2
  (20µM)    0,5
  µl    Probe4_ 501_Y_mutant (20µM)     0,25
  µl    Probe4_ N_501 (20µM)     0,25
  µl    SARS-CoV-2_deletion_F (20µM)    0.5
  µl    SARS-CoV-2_deletion_R (20µM)    0.5
  µl    SARSCoV2_deletion_P (20µM)    0,25 µl    H2O    3.5 µl    Total    20 µl    Sample    5µl    Total    25µl  Add 20µL of mastermix to each well/ PCR tube Add 5µl of the 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 and nuclease free water) to the corresponding wells containing the mastermix and mix by pipettingAdd 5µl of extracted RNA (unknown sample) to the designated wells and mix by pipettingSeal the plates/tubes Centrifuge to remove air bubblesRun the PCR with the following cycler conditions and set the thermocycler to read the HEX, FAM and Cy5 channels:Cycler protocol     Cycle      Time      Temperature        1 cycle      10min      550C        1 cycle      3min      950C        45 cycles      15s      950C               30s      580C   Data evaluationH69/70 deletionHEX channel: no Ct value = no detection of the H69/70 deletion, Ct ≤ 38 H69/70 deletion detectedN501Y mutationMark negative control (NTC) as NTC in plate set-up, otherwise the wild type and the N501Y mutant will not be grouped correctly.Correct undershooting curves (often for weak samples) by adjusting ”Cycles to analyze”. This also helps the software to group wildtype and the N501Y mutant correctly. In the tab Allelic discrimination: Set Selected Fluorophores (right low corner) to X: FAM, Y: Cy5Allele 1 –FAM probe detects the N501Y mutant. Allele 2 – Cy5 detects the SARS-CoV-2 (Wuhan lineage). Sometimes the Wt (Cy5) is marked as Heterozygote and not Allele 2. Cycler protocol  Cycle    Time    Temperature    1 cycle    10min    55°C    1 cycle    3min    95°C    45 cycles    15s    95°C         30s    58°C  Data evaluationH69/70 deletionHEX channel: no Ct value = no detection of the H69/70 deletion, Ct ≤ 38 H69/70 deletion detectedN501Y mutationMark negative control (NTC) as NTC in plate set-up, otherwise the wild type and the N501Y mutant will not be grouped correctly.Correct undershooting curves (often for weak samples) by adjusting ”Cycles to analyze”. This also helps the software to group wildtype and the N501Y mutant correctly. In the tab Allelic discrimination: Set Selected Fluorophores (right low corner) to X: FAM, Y: Cy5Allele 1 –FAM probe detects the N501Y mutant. Allele 2 – Cy5 detects the SARS-CoV-2 (Wuhan lineage). Sometimes the Wt (Cy5) is marked as Heterozygote and not Allele 2. N501Y mutationMark negative control (NTC) as NTC in plate set-up, otherwise the wild type and the N501Y mutant will not be grouped correctly.Correct undershooting curves (often for weak samples) by adjusting ”Cycles to analyze”. This also helps the software to group wildtype and the N501Y mutant correctly. In the tab Allelic discrimination: Set Selected Fluorophores (right low corner) to X: FAM, Y: Cy5Allele 1 –FAM probe detects the N501Y mutant. Allele 2 – Cy5 detects the SARS-CoV-2 (Wuhan lineage). Sometimes the Wt (Cy5) is marked as Heterozygote and not Allele 2. Interpreting results  Result    69/70del (HEX)    501 mutant (FAM)    501 wildtype
  (Cy5)    Potentially&nbsp; B.1.1.7 (UK)    Ct
  &lt;45    Grouped as allele 1 (RFU FAM &gt; RFU Cy5 at Ct 45)         Potentially B.1.351 (South Africa) or P.1 (Brazil)    Ct
  Undetected    Grouped as allele 1 (RFU FAM &gt; RFU Cy5 at Ct 45)         Potentially B.1.258 (N439K mutation), other lineages    Ct
  &lt;45         Grouped as allele 2 or heterozygote (RFU Cy5 &gt;
  RFU FAM at Ct 45)    Wildtype, other lineages    Ct
  Undetected         Grouped as allele 2 or heterozygote (RFU Cy5 &gt;
  RFU FAM at Ct 45)
Luna® Universal Probe One-Step RT-qPCR Kit    per reaction    Luna® Universal Probe One-Step RT-qPCR Kit 2x    12.5
  µl    Luna WarmStart® RT Enzyme Mix (20X)    1.25
  µl    SNP_501_F_2
  (20µM)    0,5
  µl    SNP_501_R_2
  (20µM)    0,5
  µl    Probe4_ 501_Y_mutant (20µM)     0,25
  µl    Probe4_ N_501 (20µM)     0,25
  µl    SARS-CoV-2_deletion_F (20µM)    0.5
  µl    SARS-CoV-2_deletion_R (20µM)    0.5
  µl    SARSCoV2_deletion_P (20µM)    0,25 µl    H2O    3.5 µl    Total    20 µl    Sample    5µl    Total    25µl
Cycle    Time    Temperature    1 cycle    10min    55°C    1 cycle    3min    95°C    45 cycles    15s    95°C         30s    58°C
Result    69/70del (HEX)    501 mutant (FAM)    501 wildtype
  (Cy5)    Potentially&nbsp; B.1.1.7 (UK)    Ct
  &lt;45    Grouped as allele 1 (RFU FAM &gt; RFU Cy5 at Ct 45)         Potentially B.1.351 (South Africa) or P.1 (Brazil)    Ct
  Undetected    Grouped as allele 1 (RFU FAM &gt; RFU Cy5 at Ct 45)         Potentially B.1.258 (N439K mutation), other lineages    Ct
  &lt;45         Grouped as allele 2 or heterozygote (RFU Cy5 &gt;
  RFU FAM at Ct 45)    Wildtype, other lineages    Ct
  Undetected         Grouped as allele 2 or heterozygote (RFU Cy5 &gt;
  RFU FAM at Ct 45)