Development of a PCR amplification assay as a screening test using bulk milk samples for identifying dairy herds...

The approach of cDNA synthesis followed by polymerase chain reaction (PCR) amplification was used to develop a rapid screening test for the detection of bovine viral diarrhea virus (BVDV) in bulk tank milk samples. The initial development of this detection method was done using lactating Holstein cows; 1 acutely infected with BVDV following experimental inoculation and 2 persistently infected (PI) with BVDV. Viral RNA was extracted from somatic cells purified from whole milk using a guanidinium isothiocyanate and phenol/chloroform extraction method. Oligonucleotide primers were selected from the 5'untranslated region (5'UTR) and p80 region of BVDV genome. In the acutely infected cow, BVDV RNA was identified from days 6 to 10 postinoculation. Viral RNA extracted from somatic cells of milk from PI cows was detected by PCR using both 5'UTR and p80 primer sets. The sensitivity of PCR detection was determined by preparing dilutions of whole milk obtained from the BVDV persistently infected animals with milk from a BVDV-negative cow followed by purification of somatic cells and RNA extraction. BVDV was detected in milk serially diluted to 1:640 using PCR amplification. In addition, PCR amplification was 14.6 times more sensitive than virus isolation in detecting BVDV RNA in purified milk somatic cells. PCR detected BVDV RNA from a minimum of 580 somatic cells while the detection limit of virus isolation was 8500 cells. The sensitivity and specificity of BVDV amplification were confirmed by Southern hybridization analysis. BVDV RNA was detected using PCR in 33 out of 136 bulk milk samples collected from 124 individual herds using the 5'UTR primer set. These results indicate that PCR analysis of bulk tank milk samples may provide a rapid and sensitive method of screening herds for the presence of BVDV infections.