A Multiplex qPCR to Differentiate Monkeypox Virus Clades

A Multiplex qPCR to Differentiate Monkeypox Virus Clades | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search A Multiplex qPCR to Differentiate Monkeypox Virus Clades Christopher T. Williams , Alessandra Romero-Ramirez , Adeleye Adesola Semiu , Samuel Oluwafunmbi Ifabumuyi , Susan Gould , Dominic Wooding , Collette Allen , Anushri Somasundaran , Nicodemus Nnabuike Mkpuma , Dorcas Gado , Jolly Amoche Adole , Abdulakeem Eniola Amoo , Abisola Ajoke Adeyemi , Laure Bosquillon de Jarcy , Christine Goffinet , Jake Dunning , View ORCID Profile Malcolm G Semple , ISARIC CCP UK investigators , Esto Bahizire , Afolabi Akinpelu , Thomas E Fletcher , Ana Cubas-Atienzar , Cristina Leggio , Adeyinka Adedeji , Adesuyi A. Omoare , View ORCID Profile Thomas Edwards doi: https://doi.org/10.1101/2025.02.07.25321854 Christopher T. Williams a Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine , Liverpool, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alessandra Romero-Ramirez a Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine , Liverpool, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Adeleye Adesola Semiu b National Reference Laboratory Department of Public Health Laboratory Services (PHLS), Nigeria Centre for Disease Control and Prevention (NCDC) , Gaduwa, Abuja 900110, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site Samuel Oluwafunmbi Ifabumuyi b National Reference Laboratory Department of Public Health Laboratory Services (PHLS), Nigeria Centre for Disease Control and Prevention (NCDC) , Gaduwa, Abuja 900110, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site Susan Gould a Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine , Liverpool, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dominic Wooding a Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine , Liverpool, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Collette Allen c UK Health Security Agency, Porton Down–Salisbury , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anushri Somasundaran a Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine , Liverpool, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicodemus Nnabuike Mkpuma d Department of Infectious and Transboundary animal diseases, National Veterinary Research Institute , Vom, Plateau State, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dorcas Gado d Department of Infectious and Transboundary animal diseases, National Veterinary Research Institute , Vom, Plateau State, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jolly Amoche Adole e Virology Division, National Veterinary Research Institute , Vom, Plateau State, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abdulakeem Eniola Amoo f Central Public Health Laboratory, Yaba Lagos, Nigeria Centre for Disease Control and Prevention (NCDC) Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abisola Ajoke Adeyemi f Central Public Health Laboratory, Yaba Lagos, Nigeria Centre for Disease Control and Prevention (NCDC) Find this author on Google Scholar Find this author on PubMed Search for this author on this site Laure Bosquillon de Jarcy g Tropical Disease Biology, Liverpool School of Tropical Medicine , Liverpool, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christine Goffinet g Tropical Disease Biology, Liverpool School of Tropical Medicine , Liverpool, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jake Dunning h Pandemic Sciences Institute, University of Oxford , UK i Infectious Diseases Department, Royal Free London NHS Foundation Trust , London, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Malcolm G Semple j Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool , Liverpool, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Malcolm G Semple k The International Severe Acute Respiratory and emerging Infection Consortium (ISARIC) Esto Bahizire l Catholic University of Bukavu - Bugabo Campus, Mission Ave, Bukavu, Kinshasa, Democratic Republic of Congo Find this author on Google Scholar Find this author on PubMed Search for this author on this site Afolabi Akinpelu b National Reference Laboratory Department of Public Health Laboratory Services (PHLS), Nigeria Centre for Disease Control and Prevention (NCDC) , Gaduwa, Abuja 900110, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thomas E Fletcher m Clinical Sciences, Liverpool School of Tropical Medicine , Liverpool, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ana Cubas-Atienzar a Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine , Liverpool, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Cristina Leggio c UK Health Security Agency, Porton Down–Salisbury , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Adeyinka Adedeji d Department of Infectious and Transboundary animal diseases, National Veterinary Research Institute , Vom, Plateau State, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site Adesuyi A. Omoare b National Reference Laboratory Department of Public Health Laboratory Services (PHLS), Nigeria Centre for Disease Control and Prevention (NCDC) , Gaduwa, Abuja 900110, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thomas Edwards a Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine , Liverpool, United Kingdom d Department of Infectious and Transboundary animal diseases, National Veterinary Research Institute , Vom, Plateau State, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thomas Edwards For correspondence: Thomas.Edwards{at}lstmed.ac.uk Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract We designed a multiplex qPCR to differentiate monkeypox virus clades. In evaluations using clinical samples collected in the UK and Nigeria, the assay had an overall sensitivity of 78% (95% CI: 67.67% to 86.14%) and specificity of 94% (95% CI: 80.84% to 99.30%); for samples under Ct35 sensitivity was 98% (95% CI: 91.72% to 99.96%) and specificity was 94% (95% CI: 80.84% to 99.30%). Introduction Mpox is a viral zoonosis caused by the monkeypox virus (MPXV). There are two mpox clades with clade I typically associated with a higher disease severity and case fatality ratio compared with clade II [ 1 ]. Clade II is subdivided into IIa and IIb. Lineage B.1, a newly emerged lineage of IIb, was responsible for the 2022 global outbreak. Lineage B.1 is more transmissible than previously observed, potentially driven by human adaptation [ 2 ], with significant human-to-human transmission reported, particularly in men who have sex with men (MSM)[ 3 , 4 ]. Lower mortality rates were recorded during the 2022 outbreak, potentially driven by the population demographics and health systems involved [ 5 ]. Clade I is subdivided into Ia and Ib, with Ib emerging during an outbreak in the Democratic Republic of the Congo (DRC) in 2023. Much like Lineage B.1, clade Ib infection causes lower mortality than previous clade I outbreaks whilst being more infectious, with sustained human-to-human transmission [ 6 ] linked to sexual contact [ 7 ]. A ∼1kb deletion occurs in the OPG032 gene in clade Ib [ 6 ], where the US CDC clade II PCR assay is located. Mpox diagnosis is typically by PCR performed on swabs taken from lesion scabs or respiratory samples [ 8 ]. As mpox presents with similar clinical features between clades, clade identification typically requires sequencing which can be time-consuming, expensive and difficult to implement in low- and middle-income countries (LMICs). Clade/lineage identification is important due to differences in disease severity and for epidemiological purposes, particularly with new outbreak strains. The study For an initial evaluation, we used DNA extracted from cultured Lineage B.1 MPXV (Slovenia_MPXV-1_2022 strain, Clade hMPXV-1, lineage B.1, European Virus Archive), as well as 47 clinical samples from the ISARIC Clinical Characterisation Protocol study (ethical approval; National Research Ethics Service and Health Research Authority IRAS ID:126600, REC 13/SC/0149). Thirty-two samples were MPXV-positive by the US CDC reference PCR [ 9 ] and 15 were negative. Eleven samples were collected from participants with West Africa travel-associated mpox in 2018 and 36 from UK participants infected with Lineage B.1 mpox in 2022. Samples were classified by clade using the geographic source and date of collection; all confirmed UK cases from 2018 were from West Africa and infected with clade II mpox, whilst UKHSA sequencing data revealed that 99% of mpox samples from the 2022 cohort were Lineage B.1. [ 10 ] DNA was extracted using the QIAamp 96 Virus QIAcube HT Kit (Qiagen, UK) on a QIAcube HT (Qiagen, UK) following manufacturer’s instructions. We also tested 54 MPXV-positive samples collected as part of routine mpox diagnostic surveillance at the National Reference Laboratory (NRL) of the Nigerian Centre for Disease Control and Prevention (NCDC) Abuja, Nigeria (approval was granted by the Research Governance Unit at NCDC). Samples were collected in 2017 (n=7), 2018 (n=11), 2019 (n=10), 2021 (n=10), 2023 (n=12) and 2024 (n=4). Twenty Varicella Zoster Virus (VZV) qPCR-positive samples were used as negatives. DNA was extracted using a QIAmp DNA mini kit (Qiagen, UK) following manufacturer’s instructions. Clade-specific SNPs were identified using Nextstrain ( https://nextstrain.org/mpox/all-clades ). Sequences for target genes for each clade/lineage were downloaded from NCBI Genbank and aligned by ClustalW using MEGA 11. Probes were manually designed to contain the SNP(s) in the middles of the probe. Primers were designed using PrimerQuest (IDT DNA). The Clade I probe targets the F3L gene (T46417C, G46421A, A46427C, C46435T), and the lineage B.1 probe targets OPG109 (C84587T). We designed an assay on the OPG210 gene (G183695A, C183696T) to distinguish IIb from ‘non-IIb’; non-IIb includes IIa and Clade I which contain wild-type nucleotides where IIb contains two mutations. An assay targeting the ∼1 kb deletion (Δ19,128–20,270) in the OPG032 gene of clade Ib was designed after assay evaluations. See Table 1 for results interpretation. View this table: View inline View popup Download powerpoint Table 1. Assay interpretation View this table: View inline View popup Table 2. Evaluation results qPCR assays were performed in a 12.5 μl reaction volume, containing TaqPath Fast mix (Applied Biosystems, US), differing concentrations of primers and probes (Supplementary Table 1), nuclease-free H2O and 2.5 μl DNA. To improve specificity for the Lineage B.1 assay we designed a ‘blocker’ oligo, which is identical to the B.1 probe, except that it contains the wild-type nucleotide in place of the SNP, and a phosphate at the 5’ end instead of a fluorophore. All experiments were performed on a Quantstudio 5 (Applied Biosystems, US) using the following thermal profile; 95 °C for 20 seconds, followed by 40 cycles of 95 °C for 1 second and 60 °C for 10 seconds. We used a cutoff of Ct 38 for positivity, and thresholds were set at 10% of the maximum fluorescence of the positive control curve. Standard curves of 500bp synthetic double-stranded DNA containing the amplicons from each target (Twist Bioscience, USA) were performed for clade I, clade Ib, IIb and non-IIb in triplicate from 1×10 7 copies/μl to 1 copy/μl ( Figure 1 ). DNA was extracted from lineage B.1 culture and quantified into copies/μl using the IIb standard curve to make the standard curve for lineage B1, starting at 1×10 5 copies/μl to 1 copy/μl. All targets were successfully amplified in three replicates down to 10 copies/μl. Clade I and IIb assays amplified all three replicates for 1 copy/μl; clade IIa, lineage B.1 and clade Ib amplified 2/3 replicates. Download figure Open in new tab Figure 1. Standard curve of each assay target For the UK evaluation our assay has 97% (95% CI: 83.78% to 99.92%) sensitivity and 87% (95% CI: 59.54% to 98.34%) specificity compared with the US CDC Mpox qPCR. Discrepant samples were tested using the Sansure Monkeypox virus Nucleic Acid Diagnostic Kit (Sansure Biotech, China) The two false positives and single false negative were positive by Sansure, suggesting two false negative results using the US CDC mpox qPCR. Our assay was 100% specific and 80% sensitive for detecting Lineage B.1. The five false negatives were all Ct34 or higher. In the evaluation at the NCDC we had an overall sensitivity of 67% (95% CI: 52.53% to 78.91%) and specificity of 100% (95% CI: 83.16% to 100.00%) compared with the US CDC assay. 20% (11/54) of the NCDC samples were >Ct35; typically, Ct values from lesion swabs in the acute phase would be lower [ 11 ], with samples >Ct 35 predicted to have no or very little infectivity [ 12 ]. When testing samples <Ct35 we observed a sensitivity of 97% (95% CI: 84.24% to 99.92%) and a specificity of 100% (95% CI: 83.16% to 100.00%). All samples were Clade IIb. Conclusions We designed a multiplexed qPCR to identify MPXV clades/lineages in a single reaction and evaluated it on a collection of clade IIb and Lineage B.1 clinical samples, DNA extracted from culture of Lineage B.1, as well as MPXV-negative samples.. Whilst we did not have access to clinical samples containing clade I or clade IIa viruses we are planning further evaluations to address this. Other PCR assays can distinguish between clade I and clade II. Recently, another qPCR assay to detect clade I, clade II, clade IIb and the outbreak strain B.1 was developed [ 13 ], which requires two tubes per sample and does not include clade Ib. Another assay can detect clade Ib in singleplex [ 14 ]. The rapid identification of MPXV clades is becoming ever more necessary with the advent of multiple outbreaks attributed to different clades. Whilst sequencing remains the gold standard, PCR-based methods remain a useful tool when access to sequencing is limited or extra throughput is required. Data Availability All data produced in the present work are contained in the manuscript Funding The UK Public Health Rapid Support Team is funded by UK Aid from the Department of Health and Social Care and is jointly run by the UK Health Security Agency and the London School of Hygiene & Tropical Medicine. The views expressed in this publication are those of the author(s) and not necessarily those of the Department of Health and Social Care. ISARIC4 was funded from the National Institute for Health Research [award CO-CIN-01], the Medical Research Council [grant MC_PC_19059] and by Liverpool Pandemic Institute and the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections at University of Liverpool in partnership with UK Health Security Agency (UK-HSA), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford [NIHR award 200907], Wellcome Trust and Department for International Development [215091/Z/18/Z], and the Bill and Melinda Gates Foundation [OPP1209135], and Liverpool Experimental Cancer Medicine Centre (Grant Reference: C18616/A25153) Acknowledgements ISARIC CCP investigators: Dr Mike Beadsworth, Dr Ingeborg Welters, Dr Lance Turtle, Dr Jane Minton, Karl Ward, Dr Elinor Moore, Dr Elaine Hardy, Dr Mark Nelson, Dr Jane Minton, Karl Ward, Dr David Brealey, Dr Ashley Price, Dr Brian Angus, Dr Graham Cooke and Dr Oliver Koch Footnotes ↵ # Joint last authors References 1. ↵ McCollum , A.M. and I.K. Damon , Human monkeypox . Clin Infect Dis , 2014 . 58 ( 2 ): p. 260 – 7 . OpenUrl CrossRef PubMed 2. ↵ Isidro , J. , et al. , Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus . Nat Med , 2022 . 28 ( 8 ): p. 1569 – 1572 . OpenUrl PubMed 3. ↵ Endo , A. , et al. , Heavy-tailed sexual contact networks and monkeypox epidemiology in the global outbreak, 2022 . Science , 2022 . 378 ( 6615 ): p. 90 – 94 . OpenUrl CrossRef PubMed 4. ↵ Thornhill , J.P. , et al. , Monkeypox Virus Infection in Humans across 16 Countries - April-June 2022 . N Engl J Med , 2022 . 387 ( 8 ): p. 679 – 691 . OpenUrl CrossRef PubMed 5. ↵ Fink , D.L. , et al. , Clinical features and management of individuals admitted to hospital with monkeypox and associated complications across the UK: a retrospective cohort study . The Lancet Infectious Diseases , 2023 . 23 ( 5 ): p. 589 – 597 . OpenUrl CrossRef PubMed 6. ↵ Vakaniaki , E.H. , et al. , Sustained human outbreak of a new MPXV clade I lineage in eastern Democratic Republic of the Congo . Nat Med , 2024 . 7. ↵ Kibungu , E.M. , et al. , Clade I-Associated Mpox Cases Associated with Sexual Contact, the Democratic Republic of the Congo . Emerg Infect Dis , 2024 . 30 ( 1 ): p. 172 – 176 . OpenUrl CrossRef PubMed 8. ↵ Organization, W.H ., Diagnostic testing for the monkeypox virus (MPXV): interim guidance, 9 November 2023, in Diagnostic testing for the monkeypox virus (MPXV): interim guidance , 9 November 2023. 2023 . 9. ↵ CDC . Test Procedure: Monkeypox virus Generic Real-Time PCR Test . [cited 2024 01.05.2024 ]; Mpox CDC PCR] . 10. ↵ Agency, U.H.S . Investigation into monkeypox outbreak in England: technical briefing 8 . 2024 [cited 2024 14/11/2024 ]; Research and analysis]. Available from: https://www.gov.uk/government/publications/monkeypox-outbreak-technical-briefings/investigation-into-monkeypox-outbreak-in-england-technical-briefing-8#part-6genomic-surveillance . 11. ↵ Edman-Wallér , J. , et al. , Results of PCR Analysis of Mpox Clinical Samples, Sweden, 2022 . Emerg Infect Dis , 2023 . 29 ( 6 ): p. 1220 – 1222 . OpenUrl PubMed 12. ↵ Paran , N. , et al. , Monkeypox DNA levels correlate with virus infectivity in clinical samples, Israel, 2022 . Euro Surveill , 2022 . 27 ( 35 ). 13. ↵ Huo , S. , et al. , Development of two multiplex real-time PCR assays for simultaneous detection and differentiation of monkeypox virus IIa, IIb, and I clades and the B.1 lineage . Biosaf Health , 2022 . 14. ↵ Schuele , L. , et al. , Real-time PCR assay to detect the novel Clade Ib monkeypox virus, September 2023 to May 2024 . Euro Surveill , 2024 . 29 ( 32 ). View the discussion thread. Back to top Previous Next Posted February 12, 2025. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about medRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following A Multiplex qPCR to Differentiate Monkeypox Virus Clades Message Subject (Your Name) has forwarded a page to you from medRxiv Message Body (Your Name) thought you would like to see this page from the medRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share A Multiplex qPCR to Differentiate Monkeypox Virus Clades Christopher T. Williams , Alessandra Romero-Ramirez , Adeleye Adesola Semiu , Samuel Oluwafunmbi Ifabumuyi , Susan Gould , Dominic Wooding , Collette Allen , Anushri Somasundaran , Nicodemus Nnabuike Mkpuma , Dorcas Gado , Jolly Amoche Adole , Abdulakeem Eniola Amoo , Abisola Ajoke Adeyemi , Laure Bosquillon de Jarcy , Christine Goffinet , Jake Dunning , Malcolm G Semple , ISARIC CCP UK investigators , Esto Bahizire , Afolabi Akinpelu , Thomas E Fletcher , Ana Cubas-Atienzar , Cristina Leggio , Adeyinka Adedeji , Adesuyi A. Omoare , Thomas Edwards medRxiv 2025.02.07.25321854; doi: https://doi.org/10.1101/2025.02.07.25321854 Share This Article: Copy Citation Tools A Multiplex qPCR to Differentiate Monkeypox Virus Clades Christopher T. Williams , Alessandra Romero-Ramirez , Adeleye Adesola Semiu , Samuel Oluwafunmbi Ifabumuyi , Susan Gould , Dominic Wooding , Collette Allen , Anushri Somasundaran , Nicodemus Nnabuike Mkpuma , Dorcas Gado , Jolly Amoche Adole , Abdulakeem Eniola Amoo , Abisola Ajoke Adeyemi , Laure Bosquillon de Jarcy , Christine Goffinet , Jake Dunning , Malcolm G Semple , ISARIC CCP UK investigators , Esto Bahizire , Afolabi Akinpelu , Thomas E Fletcher , Ana Cubas-Atienzar , Cristina Leggio , Adeyinka Adedeji , Adesuyi A. Omoare , Thomas Edwards medRxiv 2025.02.07.25321854; doi: https://doi.org/10.1101/2025.02.07.25321854 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Genetic and Genomic Medicine Subject Areas All Articles Addiction Medicine (569) Allergy and Immunology (863) Anesthesia (300) Cardiovascular Medicine (4442) Dentistry and Oral Medicine (444) Dermatology (383) Emergency Medicine (609) Endocrinology (including Diabetes Mellitus and Metabolic Disease) (1511) Epidemiology (15230) Forensic Medicine (30) Gastroenterology (1126) Genetic and Genomic Medicine (6610) Geriatric Medicine (668) Health Economics (998) Health Informatics (4542) Health Policy (1370) Health Systems and Quality Improvement (1613) Hematology (543) HIV/AIDS (1266) Infectious Diseases (except HIV/AIDS) (15923) Intensive Care and Critical Care Medicine (1103) Medical Education (623) Medical Ethics (147) Nephrology (668) Neurology (6607) Nursing (346) Nutrition (999) Obstetrics and Gynecology (1146) Occupational and Environmental Health (957) Oncology (3337) Ophthalmology (974) Orthopedics (369) Otolaryngology (420) Pain Medicine (436) Palliative Medicine (130) Pathology (664) Pediatrics (1693) Pharmacology and Therapeutics (692) Primary Care Research (712) Psychiatry and Clinical Psychology (5448) Public and Global Health (9238) Radiology and Imaging (2202) Rehabilitation Medicine and Physical Therapy (1370) Respiratory Medicine (1196) Rheumatology (596) Sexual and Reproductive Health (714) Sports Medicine (530) Surgery (712) Toxicology (99) Transplantation (289) Urology (265) (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'a01bebdb19ec8e2e',t:'MTc3OTc4OTUwNQ=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();