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 2 copies/μL for recombinant plasmid and 10 1.5 TCID 50 /mL for FHV-1 viral culture. Good repeatability was also confirmed. Clinical testing of 87 nasal swabs revealed positive rates of 49.43% (43/87). Compared to qPCR, the assay exhibited a specificity of 97.56%, sensitivity of 91.30%, and an overall agreement of 94.25%, with a kappa value of 0.89, indicating almost perfect concordance between the two methods. 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.