APPLICATION OF POLYMERASE CHAIN REACTION FOR DIAGNOSIS OF INFECTIOUS BOVINE RHINOTRACHEITIS | Приложение на полимеразно-верижна реакция за диагностика на заразния ринотрахеит
2010
Peshev, R., National Diagnostic and Research Veterinary Institute, Sofia (Bulgaria) | Khrisyova, L., Institute of Biophysics, BAS, Sofia (Bulgaria)
In present paper are described data from application of polymerase chain reaction - PCR for detection of BHV 1 strains isolated in Bulgaria. Viral DNA are obtained by commercial DNA genome kits GFX, GiAmp, Fermentas, Ilustra, Chelex 100, fenol chloroform isoamyl alcohol. It was determined that the highest yield of DNA ng/µl after spectrofotometric investigation are obtained with 10% and 20% Chelex 100 followed from Fermentas, fenol chloroform isoamyl alcohol, GFX, GiAmp, Ilustra kits. For performance of PCR are used primers according to published DNA sequences in genome bank proving gВ - M21474 gE - NU06934 and gС - M27491 genes coding for BHV 1 glycoproteins. These primers produced DNA products with size 527 bp for gС gen, 478 bp for gB gen and 265 bp for gE genes. To prove DNA of IBR virus two commercial kits Genekam Biotechnology Germany and InGene IBR 12.IBR.K5, Ingenase Spain are used. By using InGene IBR PCR kit were amplified PCR products with size 390 bp and 281 bp for gC and gE genes respectively. After using reagents from Genekam Biotechnology Germany amplicons with 173 bp size for the gC gene were obtained. After investigation for PCR specificity were established that DNA obtained from other herpesviruses, adeno and control non infected cell culture MDBK were not amplified. Serial ten times dilution of DNA were performed and amplified with the same primers and conditions as described for gC gene for determination of PCR analytical sensitivity. By PCR are obtained a specific amplification of DNA fragments at dilutions up 7.6х103 to 0.0766 TCID 50/ml while on cell culture viral growth were at dilution 7.66 TCID 50/ml. This is evidence for higher PCR sensitivity in comparison with isolation on cell culture. The genes gB, gC and gE coding BHV 1 glycoproteins can be successfully demonstrated by PCR.
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