Brucella Identification and Speciation by Luminex Bead-based Suspension Array

Introduction: Globally, unpasteurized milk and milk products are vehicles for the transmission of brucellosis, a prevalent zoonosis worldwide and foodborne illness in the United States. The intentional contamination of food with Brucella, a bio-threat agent, is also of serious concern. Although PCR assays do exist for the presence of Brucella spp., they are restricted by the resolution of band sizes on a gel or the number of fluorescent channels in a single real-time system. The Luminex bead-based suspension array is performed in a 96-well plate allowing for high throughput screening in minutes of up to 100 targets in one sample with easily discernible results.

Purpose: The purpose of this study was to develop a Luminex bead-based suspension array to identify Brucella at the genus level as well as B. abortus, B. melitensis, and B. suis.

Methods: A multiplex conventional PCR assay was designed to amplify Brucella DNA. The suspension array consists of uniquely colored fluorescent microspheres conjugated to specific probe DNA sequences for Brucella at both the genus and species level. Labeled PCR amplicons are coupled to the microspheres and analyzed by fluorescence readings through a flow cytometer (Bio-Plex 200 system). Signal-to-background ratios are calculated and samples were considered to be positive when signal to background ratio was greater than 6.0.

Results: The specificity of the assay was tested using at least 5 strains per target species. Signal-to-background ratios of 6 or greater (indicative of a positive sample) were achieved for all targets across all strains tested. Ratios ranged from 50 for most B. abortus strains tested to 26 for a genus-level target (16S FL). All probes showed high specificity, with no false positives (cross-reaction with non-target strains).

Significance: This high throughput assay provides a rapid (3-4 hours including PCR) and accurate sample screening tool for the detection of Brucella spp.