Citation

Wilson WJ, Erler AM, Nasarabadi SL, Skowronski EW, Imbro PM. Mol. Cell. Probes 2005; 19(2): 137-144.

Affiliation

Lawrence Livermore National Laboratory, 7000 East Ave, P.O. Box 808, L-369, Livermore, CA 94550, USA. wilson69@llnl.gov

Copyright

(Copyright © 2005, Elsevier Publishing)

DOI

10.1016/j.mcp.2004.10.005

PMID

15680215

Abstract

We have developed a 10-plexed PCR assay coupled to a 12-plexed liquid bead array to rapidly screen environmental samples for B. anthracis, Y. pestis, F. tularensis, and B. melitensis. Highly validated species-specific primer sets were used to simultaneously amplify multiple diagnostic regions unique to each individual pathogen. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The hybridized beads were processed through a flow cytometer, which detected presence and quantity of each PCR product. The assay was optimized to allow for maximum sensitivity in a multiplexed format. A high-throughput demonstration was performed where 384 simulated environmental samples were spiked with different amounts of B. thuringensis spores and pathogen DNA. The samples were robotically processed to extract DNA and arrayed for multiplexed PCR-liquid bead detection. The assay correctly identified the presence or absence of each pathogen and collected over 3000 individual data points within a single 8-h shift for approximately $4.00 material costs per environmental sample in a 10-plexed assay.

Language: en