TITLE:  SARS-CoV-2 Omicron detection RT-qPCR assay with BA.1 and BA.2/BA.3 differentitation
AUTHORS: Nikita Yolshin  , Kirill  Varchenko, Andrey  Komissarov

[DESCRIPTION]
Omicron lineage (B.1.1.529) – SARS-CoV-2 variant of concern designated on 26 November 2021 by WHO. This variant has a large number of mutations, some of which are concerning. There is already enough evidence that Omicron lineage connected with increased risk of reinfection with this variant and reduced neutralization by convalescent and vaccinated sera, as compared to other VOCs. Omicron subvariants includes several subvariants with strikingly different genetic characteristics: BA.1, BA.2, BA.3.
We developed RT-qPCR specific to omicron lineage with detemination of BA.1 and BA.2/BA.3 for epidemiological reasons. Omicron lineage B.1.1.529 and its sublineages BA.1 and BA.2/BA.3 have some indels that turned out to be a good target for its detection. Using annotations of amino acid substitutions and indels for more than 5 million samples in 1513 Pango lineages from the GISAID database available on 2021-12-03, relative frequencies of substitutions and indels by each lineage were calculated with Python v3.9.2 programming language, Pandas v1.3.5 and Numpy v1.3.5 tools. Using this data we found indels highly specific for BA.1 and BA.2/BA.3: ERS31del for Omicron (B.1.1.529) lineage, including BA.1, BA.2 and BA.3; and Ins214EPE specific only to BA.1. For these targets have been developed assays, that were then multiplexed.
Assay for detection BA.1 have been already published at protocols.io. Here we publish multiplex RT-qPCR detecting all Omicron subvariants with the opportunity to differentiate BA.1 and BA.2/BA.3.

    
Characteristics and analysis of specificity of Ins214EPE were already published in previous protocol. We only can add that limit of detection of the test is 1000 copies/ml

Here we describe features of newly introduced ERS31del assay. 

10-fold serial dilutions of SARS-CoV-2 RNA of Omicron lineage were used to assess ERS31del assay amplification efficiency. The amplification efficiency was 105% (R2 = 0,99). 

  
Developed RT-qPCR assay demonstrates high specificity. It was tested on 27 clinical samples (RNA extracted from oropharyngeal swabs) with previously characterized viruses belonging to 10 different SARS-CoV-2 lineages.
 
                      RP  2-SARS-ORF  NdeI  Ins214    1  c-                2  P.1  35,35  15,45          3  B.1.351  39,91             4  B.1     15,69          5  B.1  33,84  26,69          6  B.1  24,26  26,98          7  B.1.1.7  25,27  20,64          8  B.1.1.7  30,54  28,05          9  B.1.1.7  26,99  31,25          10  AT.1  30,30  33,07          11  AT.1  30,02  31,33          12  AT.1  28,00  33,00          13  B.1.1.523  29,12  27,09          14  B.1.1.523  27,39  23,46          15  B.1.1.523  30,02  27,09          16  B.1.1.317  25,22  25,31          17  B.1.1.317  27,58  22,71          18  B.1.1.317  28,70  23,91          19  B.1.617.2  26,66  34,03          20  B.1.617.2  25,72  28,43          21  B.1.617.2  26,78  25,42          22  AY.122  23,96  28,95          23  AY.122  31,16  35,05          24  AY.122  24,19  34,93          25  BA.1  27,98  22,20  23,57  22,08    26  BA.1  27,08  24,93  27,16  24,65    27  BA.1  25,19  27,48  29,83  25,57    28  BA.2  26,50  19,64  21,07       29  BA.2  24,70  20,81  22,70     

Specific signal was detected only in samples with SARS-CoV-2 Omicron lineage RNA: BA.1 and BA.2 (confirmed by whole-genome sequencing). Specificity was additionally tested on clinical samples positive for other respiratory viruses from the collection of Smorodintsev Research Institute of Influenza - influenza A and B, human seasonal coronaviruses, hRSV, rhinoviruses - with no false-positive results.

Analytical sensitivity
Limit of detection of the test is 1000 copies/ml

[STEPS]
1.  Order oligonucleotides with following sequences 5'->3':
            target  name    sequence    ORF1  2-SARS-CoV-2-ORF-1-F  AGAGCTATGAATTGCAGAC    ORF1  2-SARS-CoV-2-ORF-1-R  GGGAAATACAAAATTTGGACA    ORF1  2-SARS-CoV-2-ORF-1-P  FAM-AATTGGCAAAGAAATTTGACACCTTCA-BHQ1    ESR31del  N31-33del F    GTTTGGTGGACCCTCAGATT    ESR31del  N31-33del R    CAAGACGCAGTATTATTGGGTAAAC    ESR31del  N31-33del P    HEX-AGTAACCAGAATGGTGGGGCGCG-BHQ1    Ins214EPE  Ins214EPE F    ATATTCTAAGCACACGCCTATT    Ins214EPE  Ins214EPE R    GGCAAATCTACCAATGGTTCTA    Ins214EPE  Ins214EPE P    ROX-TGCGTGAGCCAGAAGATCTCCCT-BHQ2    Human RP  RP-CB-F    AGATTTGGACCTGCGAGCG    Human RP  RP-CB-R    GAGCGGCTGTCTCCACAAGT    Human RP  RP-CB-P    Cy5-TTCTGACCTGAAGGCTCTGCGCG-BHQ2  
 
2. Prepare oligonucleotides mix for 100 reactions of multiplex RT-qPCR

 
          100 rxn      2-SARS-CoV-2 F  5  100 pmol/mcl    2-SARS-CoV-2 R  5  100 pmol/mcl    2-SARS-CoV-2 P  2,5  100 pmol/mcl    N31-33del F  2,5  100 pmol/mcl    N31-33del R  2,5  100 pmol/mcl    N31-33del P  1,25  100 pmol/mcl    Ins214 F  10  100 pmol/mcl    Ins214 R  10  100 pmol/mcl    Ins214 P  5  100 pmol/mcl    RP F  5  100 pmol/mcl    RP R  5  100 pmol/mcl    RP P  5  100 pmol/mcl    water  61,25     
 

3. Briefly vortex and centrifuge reagents before use.
Use oligonucleotides mix from previous step.
Prepare the PCR reaction mixture following the specifications below:

 
        2X   RT-qPCR Buffer  12,5    oligonucleotides   mix  1,2    25 x RT-qPCR   enzyme mix  1    RNA template  5    nuclease-free   water  5,3  
 
4. Perform the amplification in a qPCR thermocycler with appropriate temperature profile:

  

5. Interpretation of the results: 
detection of fluorescence of FAM probe (2-SARS-CoV-2-ORF-1 probe) means the presence of SARS-CoV-2 RNA in the sample,
detection of fluorescence in HEX channel (ERS31del probe) means the presence of B.1.1.529/omicron (BA.1/BA.2/BA.3) RNA, 
detection of fluorescence in ROX channel (Ins214EPE probe) means the presence of BA.1 RNA.
detection of fluorescence in Cy5 channel (RP probe)

  
  
              2-SARS-CoV-2  N31-33del  Ins214EPE  RP      FAM  HEX  ROX  Cy5      +  -  -  +  non-omicron lineage    +  +  -  +  BA.2    +  +  +  +  BA.1    -  -  -  +  SARS-COV-2 negative   sample    -  -  -  -  bad swab