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Suitability of ear notch biopsy (EN) and serum pairs (n= 307) collected from 10 Holstein dairy herds located in Charlottetown, Canada was evaluated for simultaneous detection, nature of bovine viral diarrhea virus (BVDV) infection and genotype of the prevailing BVDV through Real time RT-PCR. Depending upon vaccination status and age, the sampled animals were categorized into two groups, A (n=123, ≤ 6 month of age) and B (n=184, ≥ 6 months of age) originating from 4 vaccinated (n=108) and 3 non-vaccinated (n=76) animal herds. On first round of testing a discrepancy between ear notch biopsies and sera pairs (3.25 and 6.50%; P<0.05) of groups A was observed, however, a complete harmony (50% for EN and sera each, P<0.01 was found on second round of testing that confirmed the presence of 4 persistent infection (PI) animals harboring genotype 1 of BVDV. Complete concordance between EN and sera pairs (P<0.01) on first and follow up testing in group B was observed (2.77%, each), depicting 3 PI animals with the same genotype as in group A. In the study, ear notch biopsies did not detect any transient infection (TI) but sera samples detected 3.25% transiently infected animals in group A that was 1.30 % among all the test samples (n=307) while no TI animal was found in group B. It may be concluded that both the serum and ear notch biopsy can be used to detect PI animals and that, serum samples are more sensitive than ear notch (P < 0.05) for detection of TI using real time RT-PCR.