Establishment and application of visual RT-LAMP technology for detecting bovine ephemeral fever virus

Objective  To develop a rapid, convenient and visual molecular assay for detecting bovine ephemeral fever virus (BEFV).

Method  Two pairs of primers based on six conserved regions in G protein gene of BEFV were designed, and the visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting BEFV was developed. The reaction conditions of RT-LAMP assay were optimized, and the assay was compared with PCR.

Result  The reaction ladder bands from the RT-LAMP assay were most obvious when the RT-LAMP reaction system contained 3 mmol·L^-1 Mg²⁺, 0.4 mol·L^-1 betaine and 1.2 μmol·L^-1 dNTPs mix, the ratio of inner to outer primers was 8:1, and the reaction temperature was 63 ℃. Clear reaction ladder bands were observed after 40 min of reaction. The established RT-LAMP assay had excellent specificity with only BEFV being amplified. The sensitivity was 10 times higher than that of ordinary PCR.

Conclusion  The visual RT-LAMP assay is easy to operate and highly specific, and the results can be conveniently determined. This method can be used for the rapid detection of bovine epidemic fever.