Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis

Real-time PCR-based assays specific for Brucella abortus, Brucella melitens is and Brucella suis were developed. The assays utilize an upstream primer that is derived from 3' end of the genetic element IS711, whereas the downs tream primers and probes are designed from signature sequences specific to a species or a biovar; The PCR reactions were monitored for fluorescence, r esonance energy transfer by including two adjacent labeled probes that hybr idize to the amplicons as they are formed. The upstream probes were labeled with fluorescein at 3' end while Cy5 was attached to the 5' end of the dow nstream probes. An increase in the ratio of fluorescein to Cy5 fluorescence during the cycling was indicative of positive amplification event. The ass ays were accomplished in less than 30 min using a LightCycler in real-time mode. The assays were tested on known strains as well as field isolates and were found to be specific for all known biovars of BI abortus, B. melitens is and biovar 1 of B. suis. Therefore, specificity, sensitivity, speed and real-time detection make these assays attractive for use in epidemiological and ecological studies. (C) 2001 Academic Press.