Comparison of different protocols for the bovine viral diarrhea virus detection by RT-PCR in pools of whole blood and blood serum artificially contaminated

Abstract

The RT-PCR technique was optimized and evaluated to detect the 5’ untranslated region of bovine viral diarrhea virus (BVDV) from clinical samples that consisted of blood serum and whole blood artificially contaminated with the NADL strain of BVDV. To optimization of technique, the following conditions were evaluated: i) two pairs of primers, 103 / 372 (WEINSTOCK et al., 2001) and 324 / 326 (VILCEK et al., 1994), ii) four methods of nucleic acid extraction (phenol/chloroform/isoamyl alcohol; silica/guanidine isothiocyanate; a combination of the two previous methods; and TRIzol™) and iii) different concentrations and compositions of reagents and time/temperature of the reactions. Between the alternatives tested that resulted in the amplification of the 290 bp product that was easily visualized in ethidium bromide stained 2% agarose gel was that presented the following conditions: i) primers 103 and 372; ii) initial volume and clinical sample: 50 mL of blood serum; iii) extraction of nucleic acid: silica/guanidine isothiocyanate method; iv) reverse transcription: 9 mL extracted nucleic acid, 1xPCR buffer (20 mM Tris-HCl pH 8.4 and 50 mM KCl), 1.5 mM MgCl2; 60 units of reverse transcriptase enzyme M-MLV, RNA denaturation at 97°C / 4 min, and reverse transcription at 42°C / 30 min; v) PCR: primers 103 / 372 with anneling temperature at 59°C. The utilization of RT-PCR within these optimized conditions allowed the amplification of the BVDV NADL strain (103,56 TCID50) from pools of artificially contaminated blood serum until the dilution 1:160.