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Letter to the Editor| Volume 83, ISSUE 1, e19-e21, July 2021

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Development of a multiplex RT-qPCR using the drop out strategy to screen the SARS-CoV-2 South African 501Y.V2 variant

Published:May 03, 2021DOI:https://doi.org/10.1016/j.jinf.2021.04.035
Development of a multiplex RT-qPCR using the drop out strategy to screen the SARS-CoV-2 South African 501Y.V2 variant
Previous ArticleEffectiveness of BNT162b2 vaccine against the B.1.1.7 variant of SARS-CoV-2 among healthcare workers in Brescia, Italy.
Next ArticleGastrointestinal manifestations are associated with severe pediatric COVID-19: A study in tertiary hospital
      Emergence of SARS-CoV-2 variants may have implications for virus transmissibility and immune response, as well as for the vaccination strategy, creating the need to rapidly screen for the presence of variants in COVID-19 samples(
      • Davies N.G.
      • Barnard R.C.
      • Jarvis C.I.
      • Kucharski A.J.
      • Munday J.
      • Pearson C.A.B.
      • Russell T.W.
      • Tully D.C.
      • Abbott S.
      • Gimma A.
      • Waites W.
      • Wong K.L.M.
      • van Zandvoort K.
      • Eggo R.M.
      • Funk S.
      • Jit M.
      • Atkins K.E.
      • Edmunds W.J.
      • Houben R.
      • Meakin S.R.
      • Quilty B.J.
      • Liu Y.
      • Flasche S.
      • Lei J.
      • Sun F.Y.
      • Krauer F.
      • Lowe R.
      • Bosse N.I.
      • Nightingale E.S.
      • Sherratt K.
      • Abbas K.
      • O'Reilly K.
      • Gibbs H.P.
      • Villabona-Arenas C.J.
      • Waterlow N.R.
      • Medley G.
      • Brady O.
      • Williams J.
      • Rosello A.
      • Klepac P.
      • Koltai M.
      • Sandmann F.G.
      • Foss A.M.
      • Jafari Y.
      • Prem K.
      • Chan Y.W.D.
      • Hellewell J.
      • Procter S.R.
      • Jombart T.
      • Knight G.M.
      • Endo A.
      • Quaife M.
      • Showering A.
      • Clifford S.
      Estimated transmissibility and severity of novel SARS-CoV-2 Variant of Concern 202012/01 in England.
      medRxiv. 2020; https://doi.org/10.1101/2020.12.24.20248822
      ,
      • Lauring A.S.
      • Hodcroft E.B.
      Genetic Variants of SARS-CoV-2 - What Do They Mean?.
      JAMA - J. Am. Med. Assoc. 2021; https://doi.org/10.1001/jama.2020.27124
      ). Among them, much attention has been paid to the 501Y.V1 (also named B.1.1.7), 501Y.V2 and 501Y.V3 (also named P1) variants detected in UK, South Africa and Brazil, respectively(
      • Tang J.W.
      • Tambyah P.A.
      • Hui D.S.
      Emergence of a new SARS-CoV-2 variant in the UK.
      J. Infect. 2021; https://doi.org/10.1016/j.jinf.2020.12.024
      ,
      • Tegally H.
      • Wilkinson E.
      • Giovanetti M.
      • Iranzadeh A.
      • Fonseca V.
      • Giandhari J.
      • Doolabh D.
      • Pillay S.
      • San E.J.
      • Msomi N.
      • Mlisana K.
      • von Gottberg A.
      • Walaza S.
      • Allam M.
      • Ismail A.
      • Mohale T.
      • Glass A.J.
      • Engelbrecht S.
      • van Zyl G.
      • Preiser W.
      • Petruccione F.
      • Sigal A.
      • Hardie D.
      • Marais G.
      • Hsiao M.
      • Korsman S.
      • Davies M.A.
      • Tyers L.
      • Mudau I.
      • York D.
      • Maslo C.
      • Goedhals D.
      • Abrahams S.
      • Laguda-Akingba O.
      • Alisoltani-Dehkordi A.
      • Godzik A.
      • Wibmer C.K.
      • Sewell B.T.
      • Lourenço J.
      • Alcantara L.C.J.
      • Kosakovsky Pond S.L.
      • Weaver S.
      • Martin D.
      • Lessells R.J.
      • Bhiman J.N.
      • Williamson C.
      • de Oliveira T.
      Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa.
      medRxiv. 2020; https://doi.org/10.1101/2020.12.21.20248640
      ,
      • Voloch C.M.
      • da Silva Francisco R.
      • de Almeida L.G.P.
      • Cardoso C.C.
      • Brustolini O.J.
      • Gerber A.L.
      • Guimarães A.P.de C.
      • Mariani D.
      • da Costa R.M.
      • Ferreira O.C.
      • Frauches T.S.
      • de Mello C.M.B.
      • Leitão I.de C.
      • Galliez R.M.
      • Faffe D.S.
      • Castiñeiras T.M.P.P.
      • Tanuri A.
      • de Vasconcelos A.T.R.
      Genomic characterization of a novel SARS-CoV-2 lineage from Rio de Janeiro, Brazil.
      J. Virol. 2021; (</bib>)https://doi.org/10.1128/JVI.00119-21
      ). High throughput sequencing (HTS) technologies represent the gold standard for variantsidentification, but they are resource intensive, require expertise and have a long turnaround time(i.e. several days or weeks).
      It is therefore essential to combine the routine genomic surveillance of SARS-CoV-2 variants with targeted methods such as RT-PCR that are simpler, more rapid and less expensive to implement.Recent PCR-based protocols targeted different mutant regions within the SARS-CoV-2 genome, in particular ORF1 and Spike (S) genes. Common mutations (e.g. Δ3675–3677 in ORF1a gene; N501Y in S gene) were used to detect all three variants of interest, without distinction(
      • Perchetti G.A.
      • Zhu H.
      • Mills M.G.
      • Shrestha L.
      • Mohamed Bakhash S.
      • Lin M.
      • Xie H.
      • Huang M.L.
      • Bedford T.
      • Jerome K.R.
      • Greninger A.L.
      2021. Specific allelic discrimination of N501Y and other SARS-CoV-2 mutations by ddPCR 1 detects B.1.1.7 lineage in Washington State 2 3 Running Title: ddPCR assay for SARS-CoV-2 N501Y mutation 32.
      medRxiv. 2021; 03 (10.21253321)https://doi.org/10.1101/2021.03.10.21253321
      ,
      • Vogels C.B.
      • Breban M.I.
      • Alpert T.
      • Petrone M.E.
      • Watkins A.E.
      • Ott I.M.
      • Goes de Jesus J.
      • Morales Claro I.
      • Magalhães Ferreira G.
      • Crispim M.A.
      • CADDE Genomic Network B.-.U.
      • Singh L.
      • Tegally H.
      • Anyaneji U.J.
      • Hodcroft E.B.
      • Mason C.E.
      • Khullar G.
      • Metti J.
      • Dudley J.T.
      • MacKay M.J.
      • Nash M.
      • Wang J.
      • Liu C.
      • Hui P.
      • Murphy S.
      • Neal C.
      • Laszlo E.
      • Landry M.L.
      • Muyombwe A.
      • Downing R.
      • Razeq J.
      • de Oliveira T.
      • Faria N.R.
      • Sabino E.C.
      • Neher R.A.
      • Fauver J.R.
      • Grubaugh N.D.
      2021a. PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern.
      medRxiv. 2021; (01.28.21250486.)https://doi.org/10.1101/2021.01.28.21250486
      ,
      • Vogels C.B.
      • Fauver J.
      • Grubaugh N.
      Multiplexed RT-qPCR to screen for SARS-COV-2 B1.1.7, B.1.351, and P.1 variants of concern V.3 Coronavirus Method Development Community.
      medrxiv. 2021; https://doi.org/10.17504/protocols.io.br9vm966
      ). Specific detection of some SARS-CoV-2 variants were also investigated, using unique mutations or deletions (e.g. Δ69/70 HV for UK variant) (
      • Vogels C.B.
      • Fauver J.
      • Grubaugh N.
      Multiplexed RT-qPCR to screen for SARS-COV-2 B1.1.7, B.1.351, and P.1 variants of concern V.3 Coronavirus Method Development Community.
      medrxiv. 2021; https://doi.org/10.17504/protocols.io.br9vm966
      ), but so far no protocol has been specifically developed for the South African variant.
      To differentiate between501Y.V2and the two other main variants (501Y.V1 and 501Y.V3),literatureexamination and nucleotide alignment of the SARS-CoV-2 reference towardsthe three main variantsgenomes allowedus to design primers flanking the specific Δ242–244 LLA deletion from the 501Y.V2 spike gene, and a probe that covered the deletion.This strategy, also named “drop-out”,allowes for amplification and detects all SARS-CoV-2 viruses that do not contain the deletion, while the South African 501 V.V2 variant is not amplified. A multiplex RT-qPCR was thus developed to detect four targets: i) the N1/N2 genes of SARS-CoV-2; ii) the drop-out approach for 501 V.V2 variant (Δ242–244 LLA deletion); iii) the drop out approach for 501 V.V1 variant (Δ69/70 HV deletion) developed in
      • Vogels C.B.
      • Fauver J.
      • Grubaugh N.
      Multiplexed RT-qPCR to screen for SARS-COV-2 B1.1.7, B.1.351, and P.1 variants of concern V.3 Coronavirus Method Development Community.
      medrxiv. 2021; https://doi.org/10.17504/protocols.io.br9vm966
      b); and iv) RNA quality control using Rnase P gene (Table 1).
      Table 1Primer and probe sequences tested for the proposed SARS-CoV-2 multiplex detection (N1/N2, 501 V.V1 and 501 V.V2 variants, RNase P control).
      Set nameNt positionsTMPrimers/probeSequence
      CDC N128,287–28,30653.6Forward primerGAC CCC AAA ATC AGC GAA AT
      28,335–28,35857.7Reverse primerTCT GGT TAC TGC CAG TTG AAT CTG
      28,309–28,33263.3ProbeFAM-ACC CCG CAT TAC GTT TGG ACC-BHQ1
      CDC N229,164–29,18352.3Forward primerTTACAA ACA TTG GCC GCA AA
      29,213–29,23054.1Reverse primerGCG CGA CAT TCC GAA
      29,188–29,21063.2ProbeFAM-ACA ATT TGC CCC CAG CGC TTC AG-BHQ1
      Liege 242/244del22,239–22,26155.2Forward primerTGG TAG ATT TGC CAA TAG GTA TT
      22,365–22,38755.1Reverse primerAAA AGT CCT AGG TTG AAG ATA AC
      22,274–22,29853.5ProbeROX-TTT CAA ACT TTA CTT GCT TTA CAT A-BHQ2
      Yale 69/70del21,710–21,73359.3Forward primerTCA ACT CAG GAC TTG TTC TTA CCT
      21,795–21,81757.4Reverse primerTGG TAG GAC AGG GTT ATC AAA C
      21,755–21,77961.2ProbeCy5-TTC CATG CTA TAC ATG TCT CTG GGA-BHQ2
      Rnase P56.5Forward primerAGA TTT GGA CCT GCG AGC G
      59.5Reverse primerGAG CGG CTG TCT CCA CAA GT
      63.5ProbeHEX-TTC TGA CCT GAA GGC TCT GCG CG-BHQ1
      The multiplex assay was tested on fifty-eight SARS-CoV-2 samples: twenty samples belonging to a variety of strains without the Δ242–244 LLA andΔ69/70 HV deletions), twenty samples belonging to 501Y.V1 lineage(Δ69/70 HV deletion), eight samples belonging to the 501Y.V3 lineage, and ten samples belonging to 501Y.V2 lineage (Δ242–244 LLA deletion). All samples were nasal or nasopharyngeal swabs, and nucleic acid was extracted from 300 µL viral transport medium and eluted in 50µL using the Maxwell® RSC Viral TNA kit (Promega, USA). SARS-CoV-2 lineage was firstly determined by Whole Genome Sequencing using Minion (Oxford Nanopore Technologies, USA)(
      • Artesi M.
      • Bontems S.
      • Göbbels P.
      • Franckh M.
      • Maes P.
      • Boreux R.
      • Meex C.
      • Melin P.
      • Hayette M.P.
      • Bours V.
      • Durkin K.
      A recurrent mutation at position 26340 of SARS-CoV-2 is associated with failure of the E Gene quantitative reverse transcription-PCR utilized in a commercial dual-target diagnostic assay.
      J. Clin. Microbiol. 2020; 58https://doi.org/10.1128/JCM.01598-20
      ). The multiplex RT-qPCR was performed in duplicates using the TaqMan™ Fast Virus 1-Step Master Mix (Applied Biosystems – Life Technologies, USA) with 400 nM of each primer and 200 nM of each probe per reaction, and 5 µL of RNA in a total reaction volume of 20 µL. PCR cycler conditions were reverse transcription for 10 min at 55 °C, initial denaturation for 1 min at 95 °C, followed by 45 cycles of 10 s at 95 °C and 1min15 at 55 °C, and a final cooling of 30 s at 40 °C on the LightCycler480 (Roche, USA).
      The results showed that our 242/244 del “drop-out” primer/probe set can serve as a screening tool to specifically detect viruses with the Δ242–244 deletion, in multiplex with the detection of the UK 501 V.V1 variant and the standard detection of SARS-CoV-2 N1/N2 genes proposed by the centre for Disease Control and Prevention (CDC) (Table 2). No amplification was observed for any 501 V.V2 samples, while all other SARS-CoV-2 virusesincluding 501 V.V1 and 501 V.V3 samples were amplified. Concerningsensitivity of this multiplex RT-qPCR,fifty-eight infected samples with Cycle threshold (Ct) values ranging from 7.78 to 31.25 for the N1/N2 genes were tested and they were properly detected by the 242/244del primer/probe set with a mean delay of 3.78Ct values compared to the standard N1/N2 genes detection (see Table 2).Sensitivity limit of themethod was also assessed and samples were properly amplified down to 100 viral copies per ml. Testing samples below this threshold could lead to false drop-out results.Moreover, this method could potentially detect other viruses with the Δ242/244 LLA deletion.
      Table 2Validation results of the multiplexed 501 V.V2 variant screening RT-qPCR assay on different SARS-Cov-2 lineages. Cycle threshold values are indicated for the four targets: N1/N2 genes, drop-out 242/244 deletion, drop-out 69/70 deletion and the RNase P control. No detection was indicated by “ND”.
      Samples IDSARS-CoV-2

      Lineages      		Detection (Ct values)
      N1/N2Liege 242/244delYale 67/70delRNase P
      Liege-1B.1.351 501 V.V2



      South African variant      		7.78ND10.8131.45
      Liege-211.20ND14.8728.82
      Liege-312.70ND14.2829.76
      Liege-415.31ND18.1726.42
      Liege-515.36ND19.7725.33
      Liege-616.83ND18.0827.18
      Liege-717.42ND18.7828.34
      Liege-817.78ND19.1628.11
      Liege-922.50ND24.8729.02
      Liege-1022.86ND26.2323.45
      Liege-11P1 501 V.V3

      Brazilian variant

      		9.1714.7712.4532.14
      Liege-129.7315.2212.9932.07
      Liege-1313.4318.7317.2129.1
      Liege-1414.1718.716.8831.58
      Liege-1514.2318.5916.9927.93
      Liege-1614.5520.8918.6327.17
      Liege-1717.2322.3720.4925.93
      Liege-1818.822.0520.3631.93
      Liege-19B.1.1.7 501.V1

      UK variant      		11.6915.95ND34.13
      Liege-2012.1615.99ND30.79
      Liege-2112.4517.35ND30.61
      Liege-2213.5318.88ND22.64
      Liege-2317.2719.76ND32.81
      Liege-2417.4620.80ND31.44
      Liege-2517.6021.38ND26.61
      Liege-2617.8820.76ND32.63
      Liege-2718.1620.51ND33.64
      Liege-2818.9623.15ND25.79
      Liege-2919.2722.56ND33.81
      Liege-3019.7621.72ND28.32
      Liege-3120.3723.92ND31.79
      Liege-3220.5825.41ND29.82
      Liege-3321.7323.53ND32.87
      Liege-3422.3126.11ND27.43
      Liege-3523.0127.22ND27.32
      Liege-3623.6027.62ND24.54
      Liege-3723.8426.98ND26.27
      Liege-3829.1633.18ND29.60
      Liege-39B.1.22110.8713.8412.6426.47
      Liege-40B.1.22112.6615.7914.4932.66
      Liege-41B.1.22112.8216.2615.2730.33
      Liege-42B.1.22113.5716.3314.9726.81
      Liege-43R.113.7416.9515.4331.34
      Liege-44B.1.214.214.1819.1616.7327.58
      Liege-45B.1.22115.1919.0616.5334.03
      Liege-46B.1.22118.6921.2218.8130.62
      Liege-47B.1.22118.7521.5918.8728.73
      Liege-48B.1.22119.4121.9520.5433.49
      Liege-49B.1.22119.7522.5921.5528.8
      Liege-50B.1.22121.2924.9822.5828.77
      Liege-51B.1.16021.824.1722.5129.61
      Liege-52B.1.22122.5126.6624.5327.41
      Liege-53B.1.17722.9827.2524.5731.07
      Liege-54B.1.22124.9327.6726.4425.68
      Liege-55B.125.0529.8428.5127.44
      Liege-56B.1.22125.5528.626.8224.72
      Liege-57B.1.17727.5532.2127.3521.38
      Liege-58B.1.22131.2535.9232.6535.65
      Our multiplex RT-qPCRcan thus serve as a screening assay to prioritize samples presenting the Δ242/244 LLA deletion for sequencing and to confirm their identification as the emerging 501 V.V2 lineage.

      Declaration of Competing Interest

      None

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        View in Article
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        • Fauver J.
        • Grubaugh N.
        Multiplexed RT-qPCR to screen for SARS-COV-2 B1.1.7, B.1.351, and P.1 variants of concern V.3 Coronavirus Method Development Community.
        medrxiv. 2021; https://doi.org/10.17504/protocols.io.br9vm966
        View in Article
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        • Göbbels P.
        • Franckh M.
        • Maes P.
        • Boreux R.
        • Meex C.
        • Melin P.
        • Hayette M.P.
        • Bours V.
        • Durkin K.
        A recurrent mutation at position 26340 of SARS-CoV-2 is associated with failure of the E Gene quantitative reverse transcription-PCR utilized in a commercial dual-target diagnostic assay.
        J. Clin. Microbiol. 2020; 58https://doi.org/10.1128/JCM.01598-20
        View in Article

      Article info

      Publication history

      Published online: May 03, 2021
      Accepted: April 27, 2021
      Received: April 26, 2021

      Identification

      DOI: https://doi.org/10.1016/j.jinf.2021.04.035

      Copyright

      © 2021 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

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