An amount of 5 ml of 2.0 nM synthetic biotin-labeled target was mixed with hybridization buffer consisting of 33 ml 3 M TMAC buffer (3 M tetramethylammonium chloride, 0.1% Sarkosyl, 50 mM Tris-HCl, pH 8.0, 4 mM EDTA, pH 8.0; Sigma) and 12 ml 1Â TE-buffer pH 8.0 and 0.05 ml ($2500 microspheres) for each probe-coupled Luminex microsphere. The mixture was heated at 95 C for 2 min to denature the DNA targets and probes, followed by hybridization at 45 or 55 C for 30 min while shaking on the Thermostar (BMG LabTech; Offenburg, Germany) microplate incubator. An amount of 2 ml (0.05 mg/ml) of streptavidin-R-phycoerythrin (QIAGEN, Hilden, Germany) was added to the mixture, which was further incubated at 45 or 55 C for 15 min before analysis for internal microsphere and R-phycoerythrin reporter fluorescence on the Luminex Õ 200 TM system (Luminex corporation, Austin, TX). The amount of biotinylated target that hybridizes to the microsphere-bound probes is directly proportional to the Median Fluorescence Intensity (MFI) reported by the instrument (in all experiments, fluorescence was measured from a minimum number of each type of microsphere: a set of 100 beads). The term total MFI describes the hybridization signal from a perfectly matching probetarget duplex, while the percentage (%) of the total MFI describes the ratio between the hybridization signal of a mismatching probe-target duplex and the total MFI of that particular probe. Titration experiments established that $60% of the maximum hybridization capacity of the microsphere-bound probes was reached using the conditions under which total MFI was measured (i.e. microsphere-bound probes were not saturated). An MFI of 100 was used as the lower limit of detection (LLOD).