Detection of equine herpesvirus type 1 (EHV-1) by recombinase polymerase amplification

Equine herpesvirus type 1 is a major pathogen of horses, which must be detected quickly and effectively to isolate infected animals and prevent transmission to healthy horses. Laboratory diagnosis depends on a combination of serology and molecular biology, making it challenging to establish a definitive field test. The aim of this study was to use recombinase technology for the field detection of this virus, thereby enabling a point-of-care test. Glycoprotein E (gE), one of the primary targets for detecting animal herpesviruses, was chosen for isothermal amplification with a specific recombinase kit. Several primer, time, and temperature combinations were tested. The optimal amplification conditions were 41°C for 25 minutes. This combination proved effective for amplifying the gE fragment (226 bp), even when the template DNA was diluted 100-fold. These parameters were subsequently applied in the point-of-care assay, which used a lateral flow detection device and produced comparable results, with a limit of detection ranging from 5 × 10² to 10¹ viral copies. The point-of-care test was compared to a real-time PCR method reported in the literature for field samples with known positivity/negativity. There was perfect agreement with negative samples (34/34), and 18 of 26 positives were accurately diagnosed. The assay identified EHV-1 DNA in 11/12 highly positive samples (with a low Ct in real-time PCR) and 7/14 weakly positive samples (Ct more than 26). The overall diagnostic specificity was 100%, and the sensitivity was 69.2%, which increased to 91.7% when highly positive samples were used. This work represents the first application of this technology for EHV-1 detection. Although viral load affects accuracy, this device has the potential to be used in the field, as it requires no specialized equipment and yields results rapidly.