Loop-mediated isothermal amplification assays for detection of Equid herpesvirus 1 and 4 and differentiating a gene-deleted candidate vaccine strain from wild-type Equid herpesvirus 1 strains.

摘要

Loop-mediated isothermal amplification (LAMP) is a novel method for the rapid and sensitive detection of DNA without the need for expensive equipment. In the present study, LAMP assays were developed for the specific detection of Equid herpesvirus 1 and 4 (EHV-1 and EHV-4, respectively) and for the differentiation of glycoprotein E (gE)-deleted EHV-1 ( Delta gE) strain, a candidate strain for a live vaccine, from field EHV-1 strains. Specific primer sets were designed for the gC and gE genes of EHV-1 and for the gC gene of EHV-4. The analytical sensitivities of the LAMP assays were compared with those of polymerase chain reaction (PCR). The detection limits of LAMP for EHV-1 gC and gE and PCR for EHV-1 gC were 1 plaque-forming unit (PFU)/tube, and those of LAMP and PCR for EHV-4 gC were 0.1 PFU/tube. The Delta gE strain could be differentiated from wild-type EHV-1 strains based on the reactivity in the LAMP for EHV-1 gC in combination with the LAMP for EHV-1 gE. The analytical specificities of the LAMP for EHV-1 and EHV-4 were examined by using several equine pathogens, and no cross-reactions were observed. The LAMP detection abilities for EHV-1 and EHV-4 on nasal swab samples collected from experimentally infected horses were in good agreement with that of PCR for EHV-1 and EHV-4, respectively. The LAMP assays developed in the current study were sensitive and specific for EHV-1 and EHV-4, and should provide a valuable alternative to PCR for use in clinical laboratories in the field.