The development of a competitive PCR-ELISA for the detection of equine herpesvirus-1.
Equine herpesvirus-1 (EHV-1) infection is of significant animal welfare and economic importance. Yet, no standardised molecular techniques are available for diagnosis or confirmation of viral infection. The purpose of this study was to develop a standardised and quantitative assay system for the reliable detection of EHV-1 infection which was capable of eliminating the likelihood of false negative results. A region within the EHV-1 glycoprotein B gene was amplified by polymerase chain reaction (PCR), cloned and subjected to site-directed mutagenesis to generate a control plasmid, amplifiable by identical primers to wild type EHV-1, yet capable of detection by an alternate dinitrophenylated oligonucleotide probe in a PCR-ELISA system. A competitive PCR-ELISA system which can control for the presence of PCR inhibitors and which is capable of detecting 63 genome equivalents of EHV-1 has been developed. EHV-1 presence in infected equine tissue and cell culture material was demonstrated using this system. The entire assay can be completed within one working day and facilitates multiple sample analysis. The availability of a robust, competitive PCR-ELISA system for the detection of EHV-1 will facilitate the rapid and sensitive detection of EHV-1 and offers the potential for eliminating the occurrence of abortion storms in stud farms.
Daly
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P
National Institute for Cellular Biotechnology, Department of Biology, National University of Ireland, Maynooth Co., Kildare, Ireland.
Doyle
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S
D000034:Abortion, Veterinary / Q000175:diagnosis* / Q000821:virology; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006566:Herpesviridae Infections / Q000175:diagnosis* / Q000821:virology; D004861:Herpesvirus 1, Equid / Q000235:genetics / Q000302:isolation & purification*; D006734:Horse Diseases / Q000175:diagnosis* / Q000821:virology; D006736:Horses; D008297:Male; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D012680:Sensitivity and Specificity; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000235:genetics
D015345:Oligonucleotide Probes; D014759:Viral Envelope Proteins