{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T07:45:58Z","timestamp":1761896758919,"version":"build-2065373602"},"reference-count":42,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T00:00:00Z","timestamp":1761868800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:p>\n                    Molecular diagnostic assays are critical tools to test, diagnose and treat infectious and other diseases. For example, PCR test results have been extremely valuable during the COVID-19 pandemic, not only to provide appropriate health care for infected and symptomatic individuals as needed, but also for implementing public health measures such as test, trace and isolate infected and asymptomatic individuals to prevent further transmission of the virus. Sustained transmission and unhindered proliferation of the pathogen across the population during a continuous, ongoing pandemic such as COVID-19, resulted in many variants with mutations. These mutations may lead to signature erosion, a phenomenon wherein diagnostic tests developed using the genomic sequence of an earlier version of the pathogen, may fail and cause a false negative (FN) result in a sample containing a new variant. We and others have developed applications such as PSET (PCR Signature Erosion Tool) to monitor the performance of diagnostic tests\n                    <jats:italic>in silico<\/jats:italic>\n                    using pathogen genomic sequences. Here, we present and discuss the data on wet lab testing of the\n                    <jats:italic>in silico<\/jats:italic>\n                    predictions to assess assay performance with mismatches in assay signatures. We found that the majority of the assays performed without drastic reduction in assay performance even with mismatches in primer and probe regions as measured by PCR efficiencies and C\n                    <jats:sub>t<\/jats:sub>\n                    value shifts. We identified critical residues and positions and types of changes that may impact assay performance. Despite\u00a0the extensive accumulation of mutations in SARS-CoV-2 variants over the course of the various waves of the pandemic, most PCR assays proved to be extremely robust and continued to perform well even with drastic changes and signature erosion.\n                  <\/jats:p>","DOI":"10.3389\/fcimb.2025.1524025","type":"journal-article","created":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T06:25:44Z","timestamp":1761891944000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Reality check: testing the in silico predictions of false negative results due to mutations in SARS-CoV-2 PCR assays using templates with mismatches in vitro"],"prefix":"10.3389","volume":"15","author":[{"given":"Taylor","family":"Otwell","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brittany","family":"Knight","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael","family":"Coryell","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer","family":"Stone","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Phillip","family":"Davis","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bryan","family":"Necciai","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul","family":"Carlson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shanmuga","family":"Sozhamannan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,10,31]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1016\/0022-2836(73)90184-8","article-title":"Reduction in the rate of DNA reassociation by sequence divergence","volume":"81","author":"Bonner","year":"1973","journal-title":"J. Mol. Biol."},{"key":"B2","doi-asserted-by":"publisher","first-page":"75","DOI":"10.1186\/1472-6750-9-75","article-title":"Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR","volume":"9","author":"Boyle","year":"2009","journal-title":"BMC Biotechnol."},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2022.823306","article-title":"The impact of accumulated mutations in SARS-coV-2 variants on the qPCR detection efficiency","volume":"12","author":"Cao","year":"2022","journal-title":"Front. Cell Infect. Microbiol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"1847","DOI":"10.1002\/bip.1974.360130915","article-title":"Effects of microscopic and macroscopic viscosity on the rate of renaturation of DNA","volume":"13","author":"Chang","year":"1974","journal-title":"Biopolymers"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1128\/jcm.02374-21","article-title":"Impact of SARS-coV-2 variants on the analytical sensitivity of rRT-PCR assays","volume":"60","author":"Chen","year":"2022","journal-title":"J. Clin. Microbiol."},{"key":"B6","doi-asserted-by":"publisher","first-page":"654","DOI":"10.1093\/nar\/25.3.654","article-title":"The effects of internal primer-template mismatches on RT-PCR: HIV-1 model studies","volume":"25","author":"Christopherson","year":"1997","journal-title":"Nucleic Acids Res."},{"key":"B7","doi-asserted-by":"publisher","first-page":"1422","DOI":"10.1093\/bioinformatics\/btp163","article-title":"Biopython: freely available Python tools for computational molecular biology and bioinformatics","volume":"25","author":"Cock","year":"2009","journal-title":"Bioinformatics"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1126\/science.abg3055","article-title":"Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England","volume":"372","author":"Davies","year":"2021","journal-title":"Science"},{"key":"B9","first-page":"702","volume-title":"Enzymology primer for recombinant DNA technology","author":"Eun","year":"1996"},{"key":"B10","volume-title":"Lack of N2-gene amplification on the Cepheid Xpert Xpress SARS-CoV-2 assay and potential novel causative mutations: A case series from Auckland, New Zealand. IDCases. 25. Netherlands: \u00a9 2021 The Authors","author":"Fox-Lewis","year":"2021"},{"key":"B11","doi-asserted-by":"publisher","first-page":"237","DOI":"10.1186\/1471-2105-15-237","article-title":"Simulate_PCR for amplicon prediction and annotation from multiplex, degenerate primers and probes","volume":"15","author":"Gardner","year":"2014","journal-title":"BMC Bioinf."},{"key":"B12","doi-asserted-by":"publisher","first-page":"D482","DOI":"10.1093\/nar\/gkw1065","article-title":"Virus Variation Resource - improved response to emergent viral outbreaks","volume":"45","author":"Hatcher","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"B13","doi-asserted-by":"publisher","first-page":"303","DOI":"10.3390\/pathogens9040303","article-title":"GoPrime: development of an in silico framework to predict the performance of real-time PCR primers and probes using foot-and-mouth disease virus as a model","volume":"9","author":"Howson","year":"2020","journal-title":"Pathogens"},{"key":"B14","doi-asserted-by":"publisher","first-page":"845","DOI":"10.3390\/cimb43020061","article-title":"Next-generation sequencing (NGS) in COVID-19: A tool for SARS-coV-2 diagnosis, monitoring new strains and phylodynamic modeling in molecular epidemiology","volume":"43","author":"John","year":"2021","journal-title":"Curr. Issues Mol. Biol."},{"key":"B15","doi-asserted-by":"publisher","first-page":"200636","DOI":"10.1098\/rsos.200636","article-title":"Presence of mismatches between diagnostic PCR assays and coronavirus SARS-CoV-2 genome","volume":"7","author":"Khan","year":"2020","journal-title":"R. Soc. Open Sci."},{"key":"B16","doi-asserted-by":"publisher","first-page":"1049","DOI":"10.46234\/ccdcw2021.255","article-title":"GISAID's role in pandemic response","volume":"3","author":"Khare","year":"2021","journal-title":"China CDC. Wkly."},{"key":"B17","doi-asserted-by":"publisher","first-page":"16184","DOI":"10.1038\/s41598-025-98444-8","article-title":"Using Machine learning models to predict the impact of template mismatches on polymerase chain reaction (PCR) assay performance","volume":"15","author":"Knight","year":"2025","journal-title":"Sci. Rep."},{"key":"B18","doi-asserted-by":"publisher","first-page":"1470","DOI":"10.1373\/clinchem.2013.203653","article-title":"Single-nucleotide polymorphisms and other mismatches reduce performance of quantitative PCR assays","volume":"59","author":"Lefever","year":"2013","journal-title":"Clin. Chem."},{"key":"B19","doi-asserted-by":"publisher","first-page":"3094","DOI":"10.1093\/bioinformatics\/bty191","article-title":"Minimap2: pairwise alignment for nucleotide sequences","volume":"34","author":"Li","year":"2018","journal-title":"Bioinformatics"},{"key":"B20","doi-asserted-by":"publisher","first-page":"18651","DOI":"10.1038\/s41598-022-21953-3","article-title":"Identification of mutations in SARS-CoV-2 PCR primer regions","volume":"12","author":"Mentes","year":"2022","journal-title":"Sci. Rep."},{"key":"B21","doi-asserted-by":"publisher","first-page":"101707","DOI":"10.1016\/j.mcp.2021.101707","article-title":"Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes","volume":"56","author":"Miranda","year":"2021","journal-title":"Mol. Cell Probes"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.3389\/fpubh.2022.889973","article-title":"Impact of SARS-coV-2 mutations on PCR assay sequence alignment","volume":"10","author":"Negr\u00f3n","year":"2022","journal-title":"Front. Public Health"},{"key":"B23","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1016\/0378-1119(86)90004-1","article-title":"Discrimination among the transcripts of the allelic human beta-globin genes beta A, beta S and beta C using oligodeoxynucleotide hybridization probes","volume":"43","author":"Nozari","year":"1986","journal-title":"Gene"},{"key":"B24","doi-asserted-by":"publisher","first-page":"297","DOI":"10.1007\/s12560-019-09387-z","article-title":"Missing the match might not cost you the game: primer-template mismatches studied in different hepatitis A virus variants","volume":"11","author":"Persson","year":"2019","journal-title":"Food Environ. Virol."},{"key":"B25","unstructured":"Investigation of novel SARS-CoV-2 variant 202012\/01: technical briefing 1\n          \n          \n          \n          2020"},{"key":"B26","doi-asserted-by":"publisher","first-page":"669","DOI":"10.1016\/j.jiac.2022.01.019","article-title":"A SARS-CoV-2 Delta variant containing mutation in the probe binding region used for RT-qPCR test in Japan exhibited atypical PCR amplification and might induce false negative result","volume":"28","author":"Rajib","year":"2022","journal-title":"J. Infect. Chemother."},{"key":"B27","doi-asserted-by":"publisher","first-page":"1403","DOI":"10.1038\/s41564-020-0770-5","article-title":"A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology","volume":"5","author":"Rambaut","year":"2020","journal-title":"Nat. Microbiol."},{"key":"B28","doi-asserted-by":"publisher","first-page":"2965666","DOI":"10.1155\/2022\/2965666","article-title":"Rational primer and probe construction in PCR-based assays for the efficient diagnosis of drifting variants of SARS-coV-2","volume":"2022","author":"Rana","year":"2022","journal-title":"Adv. Virol."},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1002\/ecs2.3193","article-title":"Pitfalls during in silico prediction of primer specificity for eDNA surveillance","volume":"11","author":"So","year":"2020","journal-title":"Ecosphere"},{"key":"B30","doi-asserted-by":"publisher","first-page":"3130","DOI":"10.3390\/v7062763","article-title":"Evaluation of signature erosion in ebola virus due to genomic drift and its impact on the performance of diagnostic assays","volume":"7","author":"Sozhamannan","year":"2015","journal-title":"Viruses"},{"key":"B31","doi-asserted-by":"publisher","first-page":"109","DOI":"10.2353\/jmoldx.2010.090035","article-title":"The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5' nuclease assay","volume":"12","author":"Stadhouders","year":"2010","journal-title":"J. Mol. Diagn."},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.3389\/fpubh.2022.1042647","article-title":"Development, testing and validation of a SARS-CoV-2 multiplex panel for detection of the five major variants of concern on a portable PCR platform","volume":"10","author":"Stanhope","year":"2022","journal-title":"Front. Public Health"},{"key":"B33","doi-asserted-by":"publisher","first-page":"100037","DOI":"10.1016\/j.jcvp.2021.100037","article-title":"Single base mutations in the nucleocapsid gene of SARS-CoV-2 affects amplification efficiency of sequence variants and may lead to assay failure","volume":"1","author":"Storey","year":"2021","journal-title":"J. Clin. Virol. Plus."},{"key":"B34","doi-asserted-by":"publisher","first-page":"316","DOI":"10.1016\/j.mimet.2008.12.003","article-title":"Studying the effect of single mismatches in primer and probe binding regions on amplification curves and quantification in real-time PCR","volume":"76","author":"S\u00fcss","year":"2009","journal-title":"J. Microbiol. Methods"},{"key":"B35","unstructured":"In Vitro Diagnostics EUAs - Molecular Diagnostic Tests for SARS-CoV-2"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.3001236","article-title":"Multiplex qPCR discriminates variants of concern to enhance global\u00a0surveillance of SARS-CoV-2","volume":"19","author":"Vogels","year":"2021","journal-title":"PloS Biol."},{"key":"B37","doi-asserted-by":"publisher","first-page":"1299","DOI":"10.1038\/s41564-020-0761-6","article-title":"Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets","volume":"5","author":"Vogels","year":"2020","journal-title":"Nat. Microbiol."},{"key":"B38","doi-asserted-by":"publisher","first-page":"3543","DOI":"10.1093\/nar\/6.11.3543","article-title":"Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch","volume":"6","author":"Wallace","year":"1979","journal-title":"Nucleic Acids Res."},{"key":"B39","doi-asserted-by":"publisher","first-page":"227","DOI":"10.3109\/10409239109114069","article-title":"DNA probes: applications of the principles of nucleic acid hybridization","volume":"26","author":"Wetmur","year":"1991","journal-title":"Crit. Rev. Biochem. Mol. Biol."},{"key":"B40","doi-asserted-by":"publisher","first-page":"1354","DOI":"10.1111\/1462-2920.12259","article-title":"Exploiting extension bias in polymerase chain reaction to improve primer specificity in ensembles of nearly identical DNA templates","volume":"16","author":"Wright","year":"2014","journal-title":"Environ. Microbiol."},{"key":"B41","doi-asserted-by":"publisher","first-page":"267","DOI":"10.1016\/j.mimet.2009.03.001","article-title":"Quantitative effects of position and type of single mismatch on single base primer extension","volume":"77","author":"Wu","year":"2009","journal-title":"J. Microbiol. Methods"},{"key":"B42","doi-asserted-by":"publisher","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."}],"container-title":["Frontiers in Cellular and Infection Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2025.1524025\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T06:25:46Z","timestamp":1761891946000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2025.1524025\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,31]]},"references-count":42,"alternative-id":["10.3389\/fcimb.2025.1524025"],"URL":"https:\/\/doi.org\/10.3389\/fcimb.2025.1524025","relation":{},"ISSN":["2235-2988"],"issn-type":[{"value":"2235-2988","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,31]]},"article-number":"1524025"}}