Development of a HiFi‐LAMP Assay for Detection of Herpes simplex virus type 1

Development of a HiFi‐LAMP Assay for Detection of Herpes simplex virus type 1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Development of a HiFi‐LAMP Assay for Detection of Herpes simplex virus type 1 Ziyan Wang, Zhenzhou Wan, Yongjuan Zhao, Zhengfang Wang, Shangwen Song, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8728651/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Objective To establish a rapid, sensitive, and highly specific high-fidelity loop-mediated isothermal amplification (HiFi-LAMP) method for detection of herpes simplex virus type 1 (HSV-1). Methods The HiFi-LAMP assay was developed by targeting the highly conserved re-gion of the HSV-1 US4 gene. Analytical sensitivity of the assay was determined using serial dilutions of standard templates, and specificity was assessed against nine com-mon pathogens. Intra- and inter-assay reproducibility were assessed. The assay was assessed using 30 swab samples from skin lesions of 30 patients with suspected HSV-1 infection, and compared with a gold-standard real-time quantitative PCR (qPCR) assay. Results The HSV-1 HiFi-LAMP assay can be completed within 40 minutes, with a limit of detection (LOD) of 61 copies per 25 µL reaction. Specificity testing demonstrated no cross-reactivity with nine other common pathogens, including HSV-2, varicella-zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6B (HHV-6B), Neisseria gonorrhoeae, Treponema pallidum, Ureaplasma urealyticum, and HIV-1. Intra-assay and inter-assay coefficients of variation (CVs) were below 3%, indicating high stability of the assay. Clinical evaluation with 30 clinical samples revealed a spec-ificity of 100% and a sensitivity of 100%. Conclusion This study established a rapid, simple, sensitive, and highly specific HiFi-LAMP assay for detection of HSV-1. The method does not rely on complex in-strumentation and is suitable for point-of-care testing (POCT) in primary healthcare settings and resource-limited regions. Biological sciences/Biological techniques Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research Biological sciences/Microbiology HSV-1 HiFi-LAMP isothermal amplification molecular diagnostics POCT Full Text Additional Declarations No competing interests reported. Supplementary Files HSV1supplementarymaterials.docx Cite Share Download PDF Status: Published Journal Publication published 26 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 24 Mar, 2026 Reviews received at journal 23 Mar, 2026 Reviews received at journal 20 Mar, 2026 Reviewers agreed at journal 09 Mar, 2026 Reviewers agreed at journal 08 Mar, 2026 Reviews received at journal 27 Feb, 2026 Reviewers agreed at journal 17 Feb, 2026 Reviewers invited by journal 12 Feb, 2026 Editor assigned by journal 12 Feb, 2026 Editor invited by journal 04 Feb, 2026 Submission checks completed at journal 02 Feb, 2026 First submitted to journal 02 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8728651","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":592971914,"identity":"9c9df515-7d0d-4ebf-889c-619ec20627ee","order_by":0,"name":"Ziyan Wang","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ziyan","middleName":"","lastName":"Wang","suffix":""},{"id":592971915,"identity":"c274e695-5469-4e11-98e3-2e9c44646c01","order_by":1,"name":"Zhenzhou 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amplification, molecular diagnostics, POCT","lastPublishedDoi":"10.21203/rs.3.rs-8728651/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8728651/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo establish a rapid, sensitive, and highly specific high-fidelity loop-mediated isothermal amplification (HiFi-LAMP) method for detection of herpes simplex virus type 1 (HSV-1).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe HiFi-LAMP assay was developed by targeting the highly conserved re-gion of the HSV-1 US4 gene. Analytical sensitivity of the assay was determined using serial dilutions of standard templates, and specificity was assessed against nine com-mon pathogens. Intra- and inter-assay reproducibility were assessed. The assay was assessed using 30 swab samples from skin lesions of 30 patients with suspected HSV-1 infection, and compared with a gold-standard real-time quantitative PCR (qPCR) assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe HSV-1 HiFi-LAMP assay can be completed within 40 minutes, with a limit of detection (LOD) of 61 copies per 25 \u0026micro;L reaction. Specificity testing demonstrated no cross-reactivity with nine other common pathogens, including HSV-2, varicella-zoster virus (VZV), Epstein\u0026ndash;Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6B (HHV-6B), Neisseria gonorrhoeae, Treponema pallidum, Ureaplasma urealyticum, and HIV-1. Intra-assay and inter-assay coefficients of variation (CVs) were below 3%, indicating high stability of the assay. Clinical evaluation with 30 clinical samples revealed a spec-ificity of 100% and a sensitivity of 100%.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study established a rapid, simple, sensitive, and highly specific HiFi-LAMP assay for detection of HSV-1. 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