Low concentrations of microbial pathogens in pure and mixed samples were detected using a bead-based, liquid array technology. A 20-bp sequence in the 23S rRNA gene, rrl, was amplified in four microorganisms: Bacillus cereus, Escherichia coli, Salmonella enterica and Staphylococcus aureus. PCR products were positively identified with the Luminex(®) 100™ system. The system could detect very low amounts of DNA and the instrument response was proportional to the input concentration. The lower limit of detection (LLD) was determined to be 0.5 ng for B. cereus and E. coli and 2 ng for S. enterica. The LLD for S. aureus was not determined as the instrument response was still above the threshold when quantities of DNA as low as 0.25 ng were used. The platform positively identified organisms present in mixed samples even when the minor component was overshadowed by a 10-fold excess of the major component.

A bead-based liquid hybridization assay, Luminex(®) 100™, was used to identify four pathogenic bacteria, Bacillus anthracis, Clostridium botulinum, Francisella tularensis subsp. tularensis, and Yersinia pestis, and several close relatives. Hybridization between PCR-amplified target sequences and probe sequences (located within the 23S ribosomal RNA gene rrl and the genes related to the toxicity of each bacterium) was detected in single-probe or multiple-probe assays, depending on the organism. The lower limits of detection (LLDs) for the probes ranged from 0.1 to 10 ng. Sensitivity was improved using lambda exonuclease to digest the noncomplementary target strand. All contributors in 33 binary, ternary, and quaternary mixtures in which all components were present in a 1:1 ratio were identified with an 80% success rate. Twenty-eight binary mixtures in which the two components were combined in various ratios were further studied. All target sequences were detected, even when the minor component was overshadowed by a tenfold excess of the major component.