Establishment of a loop-mediated isothermal amplification-lateral flow dipstick assay for the point-of-care testing of feline herpesvirus-1.

Introduction: Feline herpesvirus-1 (FHV-1) is a major pathogen responsible for feline upper respiratory tract disease (URTD), with a global distribution that poses significant threats to both the health of domestic cat and the conservation of wild felids. Thus, the development of a rapid and specific diagnostic method is crucial for controlling FHV-1 infection. In this study, we established a visual detection assay for FHV-1 by combining loop-mediated isothermal amplification (LAMP) with lateral flow dipstick (LFD). Methods: The assay utilized six of primers targeting the highly conserved TK gene of FHV-1. The LAMP amplification was performed using biotin-labeled H-FIP and 6-FAM-labeled H-LF, followed by visual detection using LFD. After optimizing the reaction conditions, the LAMP-LFD assay was established and systematically evaluated for its specificity, sensitivity, and repeatability. Furthermore, the performance of this assay was assessed using nasal swabs from 87 cats suspected with feline URTD. Results: The optimal reaction conditions were determined as follows: 0.2 μM each of outer primers, 1.2 μM each of inner primers, and 0.6 μM each of loop primers, with amplification at 63 °C for 40 min. The assay showed no cross-reactivity with other pathogens caused feline URTD, demonstrating high specificity. The detection limit was 10 Discussion: The LAMP-LFD assay developed in this study is highly sensitive, specific, simple, independent of specialized equipment and easy to perform with results visualized by the naked eye, making it well-suited for point-of-care testing of FHV-1 in primary veterinary hospitals and field setting.