Epsilon Archive for Student Projects

Abstract

The aim of the present work was to develop a sensitive and specific real-time PCR diagnostic system for the rapid detection of bovine adenovirus in cattle. A TaqMan real-time PCR assay capable of simultaneous detection of nine out of ten species of bovine adenovirus 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 species 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 BAV-8 cloned PCR product and the detection limit was five copies of viral genome equivalents. The efficiency of PCR assay was 97%. Only BAdV-2 serotype was not possible to detect and BAdV-7 has shown low amplification rates and fluorescence, but anyway detected, due to four mismatches in the probe region. In conclusion, the TaqMan real-time PCR assay presented here is expected to be a good alternative tool to the traditional diagnostic methods like virus isolation, ELISA or conventional PCR in case of sporadic disease outbreak in which rapid, sensitive and specific diagnosis is required and when appropriate measures should be taken.