Highly Sensitive Bacteriophage-Based Detection of Brucella abortus in Mixed Culture and Spiked Blood.

For decades, bacteriophages (phages) have been used for species identification in the diagnosis and epidemiology of brucellosis. Traditional phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter) within 72 h without DNA extraction and purification steps. The technique was equally efficient with pure culture and with mixed cultures of . and α-proteobacterial near neighbors that can be misidentified as spp., and . The addition of a simple short sample preparation step enabled the indirect phage-based detection of . in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live . in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types.