Development of a new primer-probe energy transfer method for the differentiation of neuropathogenic and non-neuropathogenic strains of equine herpesvirus-1.
Equine herpesvirus-1 (EHV-1) is a major pathogen of horses with worldwide distribution that can cause various clinical signs ranged from mild respiratory disease to neurological symptoms. Comparison of neuropathogenic and non-neuropathogenic EHV-1 strains revealed that a single non-synonymous nucleotide substitution (A/G2254) in the ORF30 region is associated with the altered functions of the viral DNA polymerase and therefore the neuropathogenicity of EHV-1 virus strains. The aim of the present study was the development of a new differentiation method of this particular single nucleotide polymorphism on the basis of the primer-probe energy transfer (PriProET) technique that has been successfully applied for the detection and classification of various DNA and RNA viruses. The results of melting temperature analysis showed an exact correlation with the sequence variations of the targeted region of ORF30, and the two genotypes (A/G2254) could be easily identified by the different peaks of melting temperatures. The new method is simple, fast, specific and robust as well as more flexible than the previous tests.
Malik
Péter
P
Department of Virology, Central Agricultural Office, Veterinary Diagnostic Directorate, Tábornok u. 2, H-1149 Budapest, Hungary. malikp@oai.hu
Pálfi
Vilmos
V
Bálint
Adám
A
D000818:Animals; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D004735:Energy Transfer; D005838:Genotype; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D004861:Herpesvirus 1, Equid / Q000145:classification* / Q000235:genetics / Q000472:pathogenicity; D006734:Horse Diseases / Q000175:diagnosis* / Q000821:virology; D006736:Horses; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D014764:Viral Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D015345:Oligonucleotide Probes; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase