Loop-mediated isothermal amplification-fluorescent loop primer assay for the genotyping of a single nucleotide polymorphism at position 2254 in the viral DNA polymerase gene of equid alphaherpesvirus 1

We developed a loop-mediated isothermal amplification (LAMP)-fluorescent loop primer (FLP) assay for genotyping the A/G(2254) single nucleotide polymorphism (SNP) in the viral DNA polymerase gene of species Equid alphaherpesvirus 1 (EHV-1), which is associated with the neuropathogenic potential of this virus. In addition to the use of regular LAMP primers to amplify the target region, a 5'-FAM-labeled backward loop primer (FLB) and 3'-dabcyl-labeled quencher probe (QP) were designed for annealing curve analysis of the amplification product. The QP, which contacts the FLB, is located at the SNP site and has the A(2254) allele. LAMP reactions were performed at 63 degrees C for 40 min, and the subsequent annealing curve analyses were accomplished within 20 min. The LAMP-FLP assay could clearly differentiate A(2254) and G(2254) genotypes according to the difference in the annealing temperature of the QP between the 2 genotypes. Good agreement between the LAMP-FLP and the real-time PCR for genotyping of this SNP was observed in the detection of EHV-1 in equine clinical samples. The newly developed assay is a simple and rapid method for detecting and differentiating EHV-1 strains with A(2254) and G(2254) polymorphisms and would be suitable for clinical use.