{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:07:46Z","timestamp":1785434866307,"version":"3.56.0"},"reference-count":31,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T00:00:00Z","timestamp":1777507200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP24fk0108627 and JP25fk0108716"],"award-info":[{"award-number":["JP24fk0108627 and JP25fk0108716"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"award":["JP24fk0108627 and JP25fk0108716"],"award-info":[{"award-number":["JP24fk0108627 and JP25fk0108716"]}],"id":[{"id":"https:\/\/ror.org\/004rtk039","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Molecular typing of enteroviruses (EVs) is essential for surveillance of hand, foot, and mouth disease (HFMD). Conventional reverse-transcription polymerase chain reaction (RT-PCR) targeting the VP4\u2013VP2 region can be insufficiently sensitive, reducing the detectability of Enterovirus A (EV-A). We developed a single-round RT-PCR assay using a modified reverse primer design (C3R) for rapid EV detection and genotyping. Sensitivity was evaluated using EV-A71 and poliovirus type 1 reference strains, across 60 EV-positive clinical specimens. The C3R-based assay showed ~1000-fold higher sensitivity for EV-A71 than for conventional assays (limit of detection: 6.6 copies\/reaction). The assay detected 98.3% (59\/60) of clinical specimens in a single-round format, whereas the conventional assay detected only 45.0% (27\/60) and showed a marked decline in detection at higher Ct values. The C3R-based assay maintained complete detection for clinical specimens with Ct values below 40. The majority of the amplified products yielded high-quality sequences suitable for genotyping. This C3R-based RT-PCR overcomes sensitivity limitations of existing protocols and provides reliable genotyping from low-viral-load specimens, supporting its use in routine diagnostics and large-scale HFMD surveillance.<\/jats:p>","DOI":"10.3390\/v18050527","type":"journal-article","created":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T09:33:17Z","timestamp":1777627997000},"page":"527","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Enterovirus Testing in Hand, Foot, and Mouth Disease and Herpangina: A Highly Sensitive Single-Round VP4\u2013VP2 Reverse-Transcription Polymerase Chain Reaction Assay with a Redesigned Reverse Primer"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4861-4349","authenticated-orcid":false,"given":"Tsuguto","family":"Fujimoto","sequence":"first","affiliation":[{"name":"Department of Fungal Infection, National Institute of Infectious Diseases, Japan Institute for Health Security (JIHS), Tokyo 162-8640, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-5125-4240","authenticated-orcid":false,"given":"Miki","family":"Ogi","sequence":"additional","affiliation":[{"name":"Infectious Disease Research Division, Hyogo Prefectural Institute of Public Health Science, Kakogawa 675-0003, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6967-348X","authenticated-orcid":false,"given":"Kazuhiro","family":"Kitakawa","sequence":"additional","affiliation":[{"name":"Fukushima Prefectural Institute for Public Health, Fukushima 960-8560, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-2575-4902","authenticated-orcid":false,"given":"Takako","family":"Sano","sequence":"additional","affiliation":[{"name":"Microbiology Division, Kanagawa Prefectural Institute of Public Health, Chigasaki 253-0087, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-7561-8509","authenticated-orcid":false,"given":"Yorihiro","family":"Nishimura","sequence":"additional","affiliation":[{"name":"Department of Virology II, National Institute of Infectious Diseases, Japan Institute for Health Security (JIHS), Tokyo 208-0011, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9692-8724","authenticated-orcid":false,"given":"Kouichi","family":"Kitamura","sequence":"additional","affiliation":[{"name":"Department of Virology II, National Institute of Infectious Diseases, Japan Institute for Health Security (JIHS), Tokyo 208-0011, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5852-8738","authenticated-orcid":false,"given":"Minami Kikuchi","family":"Ueno","sequence":"additional","affiliation":[{"name":"Department of Virology II, National Institute of Infectious Diseases, Japan Institute for Health Security (JIHS), Tokyo 208-0011, Japan"},{"name":"Department of Diagnostic Testing and Technology Research, National Institute of Infectious Diseases, Japan Institute for Health Security (JIHS), Tokyo 208-0011, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3314-6626","authenticated-orcid":false,"given":"Minetaro","family":"Arita","sequence":"additional","affiliation":[{"name":"Department of Virology II, National Institute of Infectious Diseases, Japan Institute for Health Security (JIHS), Tokyo 208-0011, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Xie, Z., Khamrin, P., Maneekarn, N., and Kumthip, K. (2024). Epidemiology of Enterovirus Genotypes in Association with Human Diseases. Viruses, 16.","DOI":"10.3390\/v16071165"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1186\/s12929-024-01058-x","article-title":"Enteroviruses: Epidemic potential, challenges and opportunities with vaccines","volume":"31","author":"Jartti","year":"2024","journal-title":"J. Biomed. Sci."},{"key":"ref_3","unstructured":"ICTV (2026, April 28). Family: Picornaviridae Genus: Enterovirus. Available online: https:\/\/ictv.global\/report\/chapter\/picornaviridae\/picornaviridae\/enterovirus."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"20180507","DOI":"10.1098\/rsif.2018.0507","article-title":"Epidemic dynamics, interactions and predictability of enteroviruses associated with hand, foot and mouth disease in Japan","volume":"15","author":"Takahashi","year":"2018","journal-title":"J. R. Soc. Interface"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1007\/s00430-016-0465-y","article-title":"Hand, foot and mouth disease (HFMD): Emerging epidemiology and the need for a vaccine strategy","volume":"205","author":"Aswathyraj","year":"2016","journal-title":"Med. Microbiol. Immunol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1007\/s12519-019-00277-9","article-title":"Diagnosis and treatment of herpangina: Chinese expert consensus","volume":"16","author":"Yu","year":"2020","journal-title":"World J. Pediatr."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"354","DOI":"10.2188\/jea.JE20180060","article-title":"Nationwide Survey of Pediatric Inpatients with Hand, Foot, and Mouth Disease, Herpangina, and Associated Complications During an Epidemic Period in Japan: Estimated Number of Hospitalized Patients and Factors Associated with Severe Cases","volume":"29","author":"Takechi","year":"2019","journal-title":"J. Epidemiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1097","DOI":"10.1016\/S1474-4422(10)70209-X","article-title":"Clinical features, diagnosis, and management of enterovirus 71","volume":"9","author":"Ooi","year":"2010","journal-title":"Lancet Neurol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Nagai, T., Hanaoka, N., Katano, H., Konagaya, M., Tanaka-Taya, K., Shimizu, H., Mukai, T., and Fujimoto, T. (2021). A fatal case of acute encephalopathy in a child due to coxsackievirus A2 infection: A case report. BMC Infect. Dis., 21.","DOI":"10.1186\/s12879-021-06858-2"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e324","DOI":"10.1097\/INF.0000000000004390","article-title":"A Pediatric Case of Encephalopathy with Hypoglycemia Induced by Coxsackievirus A4 Infection","volume":"43","author":"Ishii","year":"2024","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2141","DOI":"10.1099\/0022-1317-71-9-2141","article-title":"Detection and differentiation of picornaviruses in clinical samples following genomic amplification","volume":"71","author":"Olive","year":"1990","journal-title":"J. Gen. Virol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"744","DOI":"10.1086\/339298","article-title":"Molecular diagnosis of human enteroviruses by phylogeny-based classification by use of the VP4 sequence","volume":"185","author":"Ishiko","year":"2002","journal-title":"J. Infect. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2698","DOI":"10.1128\/JCM.00542-06","article-title":"Sensitive, seminested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens","volume":"44","author":"Nix","year":"2006","journal-title":"J. Clin. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"497","DOI":"10.7883\/yoken.JJID.2008.497","article-title":"Detection and quantification of enterovirus 71 genome from cerebrospinal fluid of an encephalitis patient by PCR applications","volume":"61","author":"Fujimoto","year":"2008","journal-title":"Jpn. J. Infect. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Kitamura, K., and Arita, M. (2024). Evaluation of VP4-VP2 sequencing for molecular typing of human enteroviruses. PLoS ONE, 19.","DOI":"10.1371\/journal.pone.0311806"},{"key":"ref_16","unstructured":"National Institute of Infectious Diseases (NIID) (2026, April 28). Hand, Foot, and Mouth Disease Pathogen Detection Manual, Available online: https:\/\/id-info.jihs.go.jp\/manuals\/pathogen-detection\/HFMdis20230704.pdf."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1111\/j.1348-0421.2002.tb02743.x","article-title":"Outbreak of central nervous system disease associated with hand, foot, and mouth disease in Japan during the summer of 2000: Detection and molecular epidemiology of enterovirus 71","volume":"46","author":"Fujimoto","year":"2002","journal-title":"Microbiol. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1128\/jcm.28.3.438-442.1990","article-title":"Enzymatic RNA amplification of the enteroviruses","volume":"28","author":"Rotbart","year":"1990","journal-title":"J. Clin. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1926","DOI":"10.1128\/JCM.01925-10","article-title":"Replacing traditional diagnostics of fecal viral pathogens by a comprehensive panel of real-time PCRs","volume":"49","author":"Wolffs","year":"2011","journal-title":"J. Clin. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Ueno, M.K., Kitamura, K., Nishimura, Y., and Arita, M. (2023). Evaluation of Direct Detection Protocols for Poliovirus from Stool Samples of Acute Flaccid Paralysis Patients. Viruses, 15.","DOI":"10.3390\/v15102113"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"634","DOI":"10.1093\/clinchem\/hvaf043","article-title":"MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines","volume":"71","author":"Bustin","year":"2025","journal-title":"Clin. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1386","DOI":"10.1093\/bioinformatics\/btq098","article-title":"An extended IUPAC nomenclature code for polymorphic nucleic acids","volume":"26","author":"Johnson","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_23","doi-asserted-by":"crossref","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\u2019 nuclease assay","volume":"12","author":"Stadhouders","year":"2010","journal-title":"J. Mol. Diagn."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"801","DOI":"10.1373\/clinchem.2017.282285","article-title":"The Effect of Single Mismatches on Primer Extension","volume":"64","author":"Rejali","year":"2018","journal-title":"Clin. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1941","DOI":"10.1128\/JVI.73.3.1941-1948.1999","article-title":"Molecular evolution of the human enteroviruses: Correlation of serotype with VP1 sequence and application to picornavirus classification","volume":"73","author":"Oberste","year":"1999","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Majumdar, M., Celma, C., Pegg, E., Polra, K., Dunning, J., and Martin, J. (2021). Detection and Typing of Human Enteroviruses from Clinical Samples by Entire-Capsid Next Generation Sequencing. Viruses, 13.","DOI":"10.3390\/v13040641"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"461","DOI":"10.3201\/eid0904.020395","article-title":"Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: Comparative analysis of the VP1 and VP4 genes","volume":"9","author":"Cardosa","year":"2003","journal-title":"Emerg. Infect. Dis."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"963","DOI":"10.1128\/JCM.42.3.963-971.2004","article-title":"Analysis of the serotype and genotype correlation of VP1 and the 5\u2019 noncoding region in an epidemiological survey of the human enterovirus B species","volume":"42","author":"Thoelen","year":"2004","journal-title":"J. Clin. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"veaa048","DOI":"10.1093\/ve\/veaa048","article-title":"Genetic recombination in fast-spreading coxsackievirus A6 variants: A potential role in evolution and pathogenicity","volume":"6","author":"Song","year":"2020","journal-title":"Virus Evol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"70","DOI":"10.15585\/mmwr.mm7304a1","article-title":"Surveillance for Acute Flaccid Myelitis\u2014United States, 2018\u20132022","volume":"73","author":"Whitehouse","year":"2024","journal-title":"MMWR Morb. Mortal. Wkly. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"653","DOI":"10.1093\/cid\/cix860","article-title":"Clinical Features of Acute Flaccid Myelitis Temporally Associated with an Enterovirus D68 Outbreak: Results of a Nationwide Survey of Acute Flaccid Paralysis in Japan, August\u2013December 2015","volume":"66","author":"Chong","year":"2018","journal-title":"Clin. Infect. Dis."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/5\/527\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,16]],"date-time":"2026-05-16T04:28:30Z","timestamp":1778905710000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/5\/527"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,30]]},"references-count":31,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["v18050527"],"URL":"https:\/\/doi.org\/10.3390\/v18050527","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,30]]}}}