{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T05:15:36Z","timestamp":1787030136332,"version":"build-2736575974"},"reference-count":50,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2023,11,27]],"date-time":"2023-11-27T00:00:00Z","timestamp":1701043200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The 2022 global Mpox outbreak swiftly introduced unforeseen diversity in the monkeypox virus (MPXV) population, resulting in numerous Clade IIb sublineages. This propagation of new MPXV mutations warrants the thorough re-investigation of previously recommended or validated primers designed to target MPXV genomes. In this study, we explored 18 PCR primer sets and examined their binding specificity against 5210 MPXV genomes, representing all the established MPXV lineages. Our results indicated that only five primer sets resulted in almost all perfect matches against the targeted MPXV lineages, and the remaining primer sets all contained 1\u20132 mismatches against almost all the MPXV lineages. We further investigated the mismatched primer-genome pairs and discovered that some of the primers overlapped with poorly sequenced and assembled regions of the MPXV genomes, which are consistent across multiple lineages. However, we identified 173 99% genome-wide conserved regions across all 5210 MPXV genomes, representing 30 lineages\/clades with at least 80% lineage-specific consensus for future primer development and primer binding evaluation. This exercise is crucial to ensure that the current detection schemes are robust and serve as a framework for primer evaluation in clinical testing development for other infectious diseases.<\/jats:p>","DOI":"10.3390\/v15122327","type":"journal-article","created":{"date-parts":[[2023,11,27]],"date-time":"2023-11-27T03:33:56Z","timestamp":1701056036000},"page":"2327","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["An In Silico Analysis of PCR-Based Monkeypox Virus Detection Assays: A Case Study for Ongoing Clinical Surveillance"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8680-1970","authenticated-orcid":false,"given":"Kuncheng","family":"Song","sequence":"first","affiliation":[{"name":"Center of Excellence for Bioinformatics, Data Science and AI, Laboratory Corporation of America Holdings (Labcorp), Burlington, NC 27215, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hayden N.","family":"Brochu","sequence":"additional","affiliation":[{"name":"Center of Excellence for Bioinformatics, Data Science and AI, Laboratory Corporation of America Holdings (Labcorp), Burlington, NC 27215, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qimin","family":"Zhang","sequence":"additional","affiliation":[{"name":"Center of Excellence for Bioinformatics, Data Science and AI, Laboratory Corporation of America Holdings (Labcorp), Burlington, NC 27215, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan D.","family":"Williams","sequence":"additional","affiliation":[{"name":"Labcorp Research and Development, Laboratory Corporation of America Holdings (Labcorp), Burlington, NC 27215, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lakshmanan K.","family":"Iyer","sequence":"additional","affiliation":[{"name":"Center of Excellence for Bioinformatics, Data Science and AI, Laboratory Corporation of America Holdings (Labcorp), Burlington, NC 27215, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,11,27]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2023, June 18). Mpox (Monkeypox). Available online: https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/monkeypox."},{"key":"ref_2","first-page":"593","article-title":"A Human Infection Caused by Monkeypox Virus in Basankusu Territory, Democratic Republic of the Congo","volume":"46","author":"Ladnyj","year":"1972","journal-title":"Bull. World Health Organ."},{"key":"ref_3","unstructured":"World Health Organization (2022). WHO Recommends New Name for Monkeypox Disease, World Health Organization."},{"key":"ref_4","unstructured":"Centers for Disease Control and Prevention (CDC) (2003). Update: Multistate Outbreak of Monkeypox\u2014Illinois, Indiana, Kansas, Missouri, Ohio, and Wisconsin, 2003. Morb. Mortal. Wkly. Rep., 52, 561\u2013564."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"980","DOI":"10.3201\/eid2505.190076","article-title":"Diagnosis of Imported Monkeypox, Israel, 2018","volume":"25","author":"Erez","year":"2019","journal-title":"Emerg. Infect. Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1826","DOI":"10.3201\/eid2608.191387","article-title":"Imported Monkeypox, Singapore","volume":"26","author":"Yong","year":"2020","journal-title":"Emerg. Infect. Dis."},{"key":"ref_7","unstructured":"(2023, June 24). World Health Organization Multi-Country Monkeypox Outbreak in Non-Endemic Countries. Available online: https:\/\/www.who.int\/emergencies\/disease-outbreak-news\/item\/2022-DON385."},{"key":"ref_8","unstructured":"(2023, June 18). World Health Organization 2022\u20132023 Mpox (Monkeypox) Outbreak: Global Trends. Available online: https:\/\/worldhealthorg.shinyapps.io\/mpx_global\/."},{"key":"ref_9","unstructured":"(2023, June 07). Centers for Disease Control and Prevention 2022 Mpox Outbreak Global Map, Available online: https:\/\/www.cdc.gov\/poxvirus\/mpox\/response\/2022\/world-map.html."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e01031-17","DOI":"10.1128\/JVI.01031-17","article-title":"Complexities of Viral Mutation Rates","volume":"92","author":"Peck","year":"2018","journal-title":"J. Virol."},{"key":"ref_11","unstructured":"(2023, June 18). American Society for Microbiology Mpox vs. COVID-19. Available online: https:\/\/asm.org\/Articles\/2022\/August\/Monkeypox-vs-COVID-19."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"E28322","DOI":"10.1002\/jmv.28322","article-title":"Inverted Repeats in the Monkeypox Virus Genome Are Hot Spots for Mutation","volume":"95","author":"Warner","year":"2023","journal-title":"J. Med. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1569","DOI":"10.1038\/s41591-022-01907-y","article-title":"Phylogenomic Characterization and Signs of Microevolution in the 2022 Multi-Country Outbreak of Monkeypox Virus","volume":"28","author":"Isidro","year":"2022","journal-title":"Nat. Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4121","DOI":"10.1093\/bioinformatics\/bty407","article-title":"Nextstrain: Real-Time Tracking of Pathogen Evolution","volume":"34","author":"Hadfield","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3773","DOI":"10.21105\/joss.03773","article-title":"Nextclade: Clade Assignment, Mutation Calling and Quality Control for Viral Genomes","volume":"6","author":"Aksamentov","year":"2021","journal-title":"J. Open Source Softw."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Happi, C., Adetifa, I., Mbala, P., Njouom, R., Nakoune, E., Happi, A., Ndodo, N., Ayansola, O., Mboowa, G., and Bedford, T. (2022). Urgent Need for a Non-Discriminatory and Non-Stigmatizing Nomenclature for Monkeypox Virus. PLoS Biol., 20.","DOI":"10.1371\/journal.pbio.3001769"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"194","DOI":"10.1016\/j.jcv.2006.03.012","article-title":"Detection of Monkeypox Virus with Real-Time PCR Assays","volume":"36","author":"Li","year":"2006","journal-title":"J. Clin. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1016\/j.jviromet.2010.07.012","article-title":"Real-Time PCR Assays for the Specific Detection of Monkeypox Virus West African and Congo Basin Strain DNA","volume":"169","author":"Li","year":"2010","journal-title":"J. Virol. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1016\/j.jviromet.2011.05.002","article-title":"Species-Specific Identification of Variola, Monkeypox, Cowpox, and Vaccinia Viruses by Multiplex Real-Time PCR Assay","volume":"175","author":"Shchelkunov","year":"2011","journal-title":"J. Virol. Methods"},{"key":"ref_20","unstructured":"Wawina-Bokalanga, T., Sklenovska, N., Vanmechelen, B., Bloemen, M., Vergote, V., Laenen, L., Andr\u00e9, E., Van Ranst, M., Muyembe-Tamfum, J.-J., and Maes, P. (2023, June 02). Available online: https:\/\/www.medrxiv.org\/content\/10.1101\/2022.06.23.22276033v1."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1016\/j.jviromet.2016.07.024","article-title":"Species-Specific Differentiation of Variola, Monkeypox, and Varicella-Zoster Viruses by Multiplex Real-Time PCR Assay","volume":"236","author":"Maksyutov","year":"2016","journal-title":"J. Virol. Methods"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1177\/153567601502000405","article-title":"Multiplex Real-Time PCR Assay for the Detection and Differentiation of Poxviruses and Poxvirus Vectors","volume":"20","author":"Stellberger","year":"2015","journal-title":"Appl. Biosaf."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1016\/j.mcp.2009.10.008","article-title":"Multicolour, Multiplex Real-Time PCR Assay for the Detection of Human-Pathogenic Poxviruses","volume":"24","author":"Schroeder","year":"2010","journal-title":"Mol. Cell Probe"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1200","DOI":"10.1038\/labinvest.3700143","article-title":"Monkeypox Virus Detection in Rodents Using Real-Time 3\u2032-Minor Groove Binder TaqMan\u00ae Assays on the Roche LightCycler","volume":"84","author":"Kulesh","year":"2004","journal-title":"Lab. Investig."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"746","DOI":"10.4269\/ajtmh.2010.09-0716","article-title":"A Silent Enzootic of an Orthopoxvirus in Ghana, West Africa: Evidence for Multi-Species Involvement in the Absence of Widespread Human Disease","volume":"82","author":"Reynolds","year":"2010","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_26","first-page":"293","article-title":"Identification of Pan-Orthopoxvirus, Monkeypox-Specific and Smallpox-Specific DNAs by Real-Time PCR Assay","volume":"33","author":"Kuo","year":"2013","journal-title":"J. Med. Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2069","DOI":"10.1128\/jcm.33.8.2069-2076.1995","article-title":"PCR Strategy for Identification and Differentiation of Small Pox and Other Orthopoxviruses","volume":"33","author":"Ropp","year":"1995","journal-title":"J. Clin. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/S0166-0934(96)02155-6","article-title":"Gene for A-Type Inclusion Body Protein Is Useful for a Polymerase Chain Reaction Assay to Differentiate Orthopoxviruses","volume":"64","author":"Meyer","year":"1997","journal-title":"J. Virol. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Boyle, B., Dallaire, N., and MacKay, J. (2009). Evaluation of the Impact of Single Nucleotide Polymorphisms and Primer Mismatches on Quantitative PCR. BMC Biotechnol., 9.","DOI":"10.1186\/1472-6750-9-75"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1660","DOI":"10.1128\/AEM.02403-07","article-title":"Quantification of the Detrimental Effect of a Single Primer-Template Mismatch by Real-Time PCR Using the 16S rRNA Gene as an Example","volume":"74","author":"Bru","year":"2008","journal-title":"Appl. Environ. Microb."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e28395","DOI":"10.1002\/jmv.28395","article-title":"Wide Mismatches in the Sequences of Primers and Probes for Monkeypox Virus Diagnostic Assays","volume":"95","author":"Wu","year":"2023","journal-title":"J. Med. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1016\/j.mimet.2013.06.013","article-title":"The Effect of Multiple Primer\u2013Template Mismatches on Quantitative PCR Accuracy and Development of a Multi-Primer Set Assay for Accurate Quantification of pcrA Gene Sequence Variants","volume":"94","author":"Ledeker","year":"2013","journal-title":"J. Microbiol. Meth."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1111\/j.1574-6941.2007.00283.x","article-title":"Effect of Primer Mismatch, Annealing Temperature and PCR Cycle Number on 16S rRNA Gene-Targetting Bacterial Community Analysis: PCR Parameters Influencing Quantitative Bias","volume":"60","author":"Sipos","year":"2007","journal-title":"FEMS Microbiol. Ecol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"D571","DOI":"10.1093\/nar\/gku1207","article-title":"NCBI Viral Genomes Resource","volume":"43","author":"Brister","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e224","DOI":"10.3346\/jkms.2022.37.e224","article-title":"The First Case of Monkeypox in the Republic of Korea","volume":"37","author":"Jang","year":"2022","journal-title":"J. Korean Med. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1006\/viro.2000.0603","article-title":"An Emergent Poxvirus from Humans and Cattle in Rio de Janeiro State: Cantagalo Virus May Derive from Brazilian Smallpox Vaccine","volume":"277","author":"Damaso","year":"2000","journal-title":"Virology"},{"key":"ref_37","unstructured":"Pan American Health Organization, and World Health Organization (2023, June 02). Available online: https:\/\/www.paho.org\/en\/documents\/laboratory-guidelines-detection-and-diagnosis-monkeypox-virus-infection-2-september-2022."},{"key":"ref_38","unstructured":"Centers for Disease Control & Prevention Poxvirus & Rabies Branch (PRB) (2023, July 01). Test Procedure: Monkeypox Virus Generic Real-Time PCR Test, Available online: https:\/\/www.cdc.gov\/poxvirus\/mpox\/pdf\/PCR-Diagnostic-Protocol-508.pdf."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e2584","DOI":"10.7717\/peerj.2584","article-title":"VSEARCH: A Versatile Open Source Tool for Metagenomics","volume":"4","author":"Rognes","year":"2016","journal-title":"PeerJ"},{"key":"ref_40","unstructured":"Team, R.D.C. (2022, October 21). A Language and Environment for Statistical Computing. Available online: http:\/\/www.r-project.org."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1739","DOI":"10.1016\/j.bbrc.2016.09.015","article-title":"Palindrome Analyser\u2014A New Web-Based Server for Predicting and Evaluating Inverted Repeats in Nucleotide Sequences","volume":"478","author":"Coufal","year":"2016","journal-title":"Biochem. Bioph. Res. Commu."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3687","DOI":"10.1093\/bioinformatics\/btaa222","article-title":"GenMap: Ultra-Fast Computation of Genome Mappability","volume":"36","author":"Pockrandt","year":"2020","journal-title":"Bioinformatics"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Li, K., Venter, E., Yooseph, S., Stockwell, T.B., Eckerle, L.D., Denison, M.R., Spiro, D.J., and Meth\u00e9, B.A. (2010). ANDES: Statistical Tools for the ANalyses of DEep Sequencing. BMC Res. Notes, 3.","DOI":"10.1186\/1756-0500-3-199"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1016\/0042-6822(85)90111-4","article-title":"Orthopoxvirus DNA: A Comparison of Restriction Profiles and Maps","volume":"143","author":"Esposito","year":"1985","journal-title":"Virology"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2168","DOI":"10.3390\/v7042168","article-title":"A Phylogeographic Investigation of African Monkeypox","volume":"7","author":"Nakazawa","year":"2015","journal-title":"Viruses"},{"key":"ref_46","unstructured":"Gener, A.R. (2023, July 02). Available online: https:\/\/www.medrxiv.org\/content\/10.1101\/2022.09.06.22279633v1."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"855","DOI":"10.1099\/0022-1317-83-4-855","article-title":"The Sequence of Camelpox Virus Shows It Is Most Closely Related to Variola Virus, the Cause of Smallpox","volume":"83","author":"Gubser","year":"2002","journal-title":"J. Gen. Virol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"e01655-22","DOI":"10.1128\/jcm.01655-22","article-title":"Identification of Human Monkeypox Virus Genome Deletions That Impact Diagnostic Assays","volume":"60","author":"Garrigues","year":"2022","journal-title":"J. Clin. Microbiol."},{"key":"ref_49","first-page":"2200503","article-title":"Frequent Detection of Monkeypox Virus DNA in Saliva, Semen, and Other Clinical Samples from 12 Patients, Barcelona, Spain, May to June 2022","volume":"27","author":"Fuertes","year":"2022","journal-title":"Eurosurveillance"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Gigante, C.M., Plumb, M., Ruprecht, A., Zhao, H., Wicker, V., Wilkins, K., Matheny, A., Khan, T., Davidson, W., and Sheth, M. (2023, July 03). Genomic Deletions and Rearrangements in Monkeypox Virus from the 2022 Outbreak, USA. Available online: https:\/\/www.biorxiv.org\/content\/10.1101\/2022.09.16.508251v1.","DOI":"10.1101\/2022.09.16.508251"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/12\/2327\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:31:05Z","timestamp":1760131865000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/15\/12\/2327"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,11,27]]},"references-count":50,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2023,12]]}},"alternative-id":["v15122327"],"URL":"https:\/\/doi.org\/10.3390\/v15122327","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,11,27]]}}}