1. [Development of a real-time RT-PCR for detection of equine influenza virus].
- Author
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Aeschbacher S, Santschi E, Gerber V, Stalder HP, and Zanoni RG
- Subjects
- Animals, Birds, Dogs, Horse Diseases virology, Horses, Influenza A virus classification, Influenza A virus genetics, Madin Darby Canine Kidney Cells, Orthomyxoviridae Infections diagnosis, Orthomyxoviridae Infections virology, Phylogeny, Reproducibility of Results, Sensitivity and Specificity, Swine, Viral Matrix Proteins genetics, Virus Shedding genetics, Horse Diseases diagnosis, Influenza A virus isolation & purification, Orthomyxoviridae Infections veterinary, Real-Time Polymerase Chain Reaction veterinary
- Abstract
Equine influenza is a highly contagious respiratory disease in horses caused by influenza A viruses. In this work a real-time RT-PCR for fast and sensitive diagnosis of equine influenza viruses (EIV) targeting a highly conserved region of the matrix gene was developed. In addition two RT-PCR methods for the amplification of large parts of the matrix- and HA gene were adapted for molecular-epidemiological characterization of viruses. The primers of the real-time RT-PCR had homologies of 99.4% to EIV- and 97.7% to all influenza A viral sequences, whereas the minor groove binder (MGB) probe showed homologies of 99.3% and 99.6%, respectively. These high values allow application of the assay for influenza viruses in other species. Using 20 equine, 11 porcine and 2 avian samples, diagnostic suitability of the assay was confirmed. High specificity for influenza viruses was shown both experimentally and by software simulation. The assay analytical sensitivity was at 10(2)-10(3) copies of RNA and 10(0)-10(1) copies of DNA, respectively. This allows virus detection also in circumstances of minor viral shedding. All amplified EIV sequences were classified phylogenetically within the known lineages.
- Published
- 2015
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