THE REAL-TIME PCR FOR DETECTION OF EQUINE ARTERITIS VIRUS

Abstract

The aim of this study was to develop and validate the real-time RT-PCR method in detection of the equine arteritis virus in the nasal swabs, semen plasma and whole blood samples from horses with clinical signs of the disease. The 66 samples - 28 nasal swab samples from mares, 23 semen plasma samples from stallions, 6 whole blood samples from stallions and mares, 7 samples - 10% internal organ tissues suspensions from aborted foetus were used in investigation. Two reference strains „ARVAC” and “Bucyrus” were used as the positive controls. RNA was isolated from cell culture supernatant fluid which was infected with reference viruses and whole blood from infected animals by commercial kit Viral RNA Mini Kit (Qiagen, Germany). The real-time RT-PCR - one step protocol, developed by Balasuriya et al. (2002) was used for the amplification of the 204 bp ORF7 segment of the EAV genome. The 96% of samples showed the positive results by real time RT-PCR. The 3.5% we investigated again and only 0.5 % of the samples were negative. The controls tested positive and this contributed for precise interpretation of the obtained results. The samples which show high CT values were amplified again only with primers and the products were visualized in 2% agarose gel. The positive reaction products show the DNA fragments of about 200 bp.