Development of a Closed One-Tube Colorimetric and Fluorescent LAMP Assay for the Rapid Detection of Feline...

Feline herpesvirus type 1 (FHV-1) is one of the major pathogens causing upper respiratory tract disease (URTD) in cats, and is widely prevalent among domestic cats and other felids worldwide. Herein, we developed a closed one-tube colorimetric and fluorescent LAMP assay targeting the FHV-1 TK gene, using cresol red as the colorimetric indicator and EvaGreen as the fluorescent dye. The assay was completed within 40 min at 63 °C, with results readable either by colorimetric observation or fluorescence amplification curve. FHV-1-positive reaction could be confirmed by a color change from violet to yellow with the naked eye, and also could be determined by fluorescence amplification curve. The assay showed no cross-reactivity with other related pathogens, and had a detection limit of 100 copies. Among 87 clinical nasal swabs tested, 44 were positive, achieving an overall agreement of 97.7% compared to TaqMan quantitative PCR. The newly developed LAMP assay is simple, efficient, result-visual and reliable, which can be applied in the clinical diagnosis and epidemiological investigation of FHV-1, especially for point-of-care testing of FHV-1 in primary veterinary clinics and field settings.