Contact: Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”
P6-10 Development and validation of a real-time PCR assay for detection of Brucella spp. in samples of animal origin


real-time PCR
Method validation



Brucellosis is a zoonosis with serious implications for human and animal health. Direct diagnosis in domestic ruminants depends on the isolation of Brucella from abortion material, vaginal secretions, milk or from tissues taken after slaughtering. Even if serological methods are sufficient to determine the sanitary status of a herd, bacteriological isolation is still the gold standard to establish a definitive diagnosis of Brucellosis in a single animal. The culture-based method to detect Brucella spp. can include the use of an enrichment medium that could be incubated for up to 6 weeks. It presents several inconveniences such as: managing laboratory spaces, intensive manipulation of potentially infectious material with increased risk of infection for the laboratory personnel. The aim of this study was to develop and validate a robust and reliable real-time PCR designed to amplify a portion of IS711 genetic element which is unique to Brucella species. It includes a chimeric DNA as internal amplification control that is coamplified with the target sequence to monitor the presence of PCR-inhibitory substances in clinical samples. It allows a higher predictivity in identifying the positive samples, or in case of a negative result, it avoids the long-term culture based-methods. To validate the method a total of 335 samples from domestic ruminants and marine mammals were examined and the results were compared with those obtained using the culture method. The Receiver Operating Characteristic (R.O.C.) curve was calculated from the resulting data which allowed us to identify the optimal threshold value (best cut-off) with results of sensitivity, specificity, accuracy of 91.9%, 94.5%, 94.3%, respectively, which places the diagnostic method in the range of values attributable to highly accurate testing. This developed real-time PCR could represent a valid and timely tool for the diagnosis of brucellosis and, therefore, a support to the eradication program of this zoonosis.


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