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

Abstract

We developed a loop-mediated isothermal amplification (LAMP)-fluorescent loop primer (FLP) assay for genotyping the A/G₂₂₅₄ 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 A₂₂₅₄ allele. LAMP reactions were performed at 63°C for 40 min, and the subsequent annealing curve analyses were accomplished within 20 min. The LAMP-FLP assay could clearly differentiate A₂₂₅₄ and G₂₂₅₄ 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 A₂₂₅₄ and G₂₂₅₄ polymorphisms and would be suitable for clinical use.

Keywords: equid herpesvirus 1; genotyping techniques; polymorphism; single nucleotide.

Figure 1.

The results of annealing curve analysis of the loop-mediated isothermal amplification assay using a fluorescent loop primer (LAMP-FLP) of equid alphaherpesvirus 1 strains. The A₂₂₅₄ and G₂₂₅₄ genotypes were clearly differentiated according to their annealing temperature.