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

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.