Development of a TaqMan Real-Time PCR Assay for the Rapid Detection of Bovine Adenovirus Serotypes in Cattle

The aim of this work was to develop a sensitive and specific real time PCR diagnostic assay for the rapid detection and identification of bovine adenoviruses (BAdVs) in cattle. A TaqMan real time PCR assay, capable of simultaneous detection of nine out of ten BAdV serotypes (species) belonging to the genera Atadenovirus and Mastadenovirus, was developed. The oligonucleotide primers and probe were selected from conserved sequences flanking the genome region coding for the hexon protein. DNA from BAdV serotypes 1 to 10 was isolated by standard phenol chloroform extraction. Standard curve using a ten fold dilution series of BAdV-8 positive DNA template was generated. The sensitivity of assay was determined using BAdV-8 cloned PCR product and the detection limit was five copies of viral genome equivalents. The efficiency of PCR assay was 97%. BAdV-2 was the only serotype, which was not detected. BAdV-7 has shown low amplification rates and fluorescence due to four mismatches in the probe region. In conclusion, the real time PCR assay presented here is expected to be a useful and practical alternative tool to the traditional diagnostic methods like virus isolation, ELISA or conventional PCR for the simple and rapid diagnosis of adenovirus infections in Bovidae.