Development of a HiFi-LAMP assay for the detection of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) has a significant impact on global public health. Developing a rapid, sensitive, and specific diagnostic assay for the detection of HSV-1 is of critical clinical significance for guiding individualized therapy and optimizing outcomes. A high-fidelity loop-mediated isothermal amplification (HiFi-LAMP) assay was developed by targeting the highly conserved region of the HSV-1 US4 gene. The sensitivity of the assay was determined using serial dilutions of standard templates, and specificity was assessed against ten common pathogens. Intra- and inter-assay reproducibility were assessed. The assay was assessed using 30 swab samples from skin lesions of patients with suspected HSV-1 infection and compared with the gold-standard real-time quantitative PCR (qPCR) assay. The HSV-1 HiFi-LAMP assay can be completed within 40 min, with a limit of detection (LOD) of 61 copies per 25 µL reaction. Specificity testing demonstrated no cross-reactivity with ten common pathogens, including HSV-2, varicella-zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6B (HHV-6B), Neisseria gonorrhoeae (NG), Treponema pallidum (TP), Ureaplasma urealyticum (UU), HIV-1, and monkeypox virus (MPXV). 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 specificity of 100% and a sensitivity of 100%. The novel HSV-1 HiFi-LAMP assay does not rely on complex instrumentation and is suitable for point-of-care testing (POCT) in primary healthcare settings and resource-limited regions.