15A-16 |
Quantitative, multiplexed detection of foodborne pathogens: protein and DNA applications of the Luminex LabMAP system |
C. A. VANDER ZEE, S. A. Dunbar, K. G. Oliver, and J. W. Jacobson. Research & Development, Luminex Corporation, 12212 Technology Blvd., Austin, TX 78727 Escherichia coli, Salmonella enterica, Listeria monocytogenes, and Campylobacter jejuni are bacterial pathogens commonly implicated in foodborne illnesses. Generally used detection methods (i.e., intracellular staining, ELISA and nucleic acid amplification) can be costly, laborious, time-consuming and require multiple tests to detect all of the pathogens. Rapid identification of pathogens is crucial for patient management and therapeutic decisions. Our objective was to develop rapid assays to simultaneously detect these four organisms through presence of antigen or DNA using the Luminex Corporation LabMAPTM system. For nucleic acid detection, organism-specific capture probes corresponding to the 23S ribosomal RNA gene (rrl) were coupled covalently to LabMAP microspheres. Target molecules included synthetic complementary oligonucleotides and genomic DNA isolated from ATCC type strains or other well-characterized strains of each organism. Universal PCR primers were designed to amplify several variable regions of bacterial 23S ribosomal DNA, yielding biotinylated amplicons of 86 to 170 basepairs in length. Varying quantities of targets were hybridized to the combined microsphere sets, labeled with streptavidin-R-phycoerythrin and analyzed on the Luminex100 system. Capture-sandwich immunoassays were developed with organism-specific monoclonal antibodies coupled to separate microsphere sets. Microspheres were incubated with organism-specific standards and reactivity was assessed with biotinylated detection antibodies and streptavidin-R-phycoerythrin. Results of nucleic acid detection assays, obtained in less than 30 minutes following amplification, correctly and specifically identified each bacterial species with a detection sensitivity of fewer than 1000 genome copies. In the immunoassays, microsphere-associated fluorescence was organism concentration dependent with detectable response at < 1000 organisms/ml and with no apparent cross-reactivity. We have demonstrated that the Luminex LabMAP system is a rapid, flexible platform capable of simultaneous, sensitive and specific detection of pathogens. The practical significance of this multiplexing approach would be to provide more timely and comprehensive diagnostic information for clinically relevant decisions.
Session 15A, Biotechnology
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