A Multiplex PCR-coupled Liquid Bead Array for the Simultaneous Detection of Four Biothreat Agents
Journal Article
·
· Published in: Molecular and Cellular Probes, vol. 19, no. 2, May 1, 2005, pp. 137-144
OSTI ID:15014375
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 3,000 individual data points within a single 8-hour shift for approximately $1.20 per sample in a 10-plexed assay.
- Research Organization:
- Lawrence Livermore National Laboratory (LLNL), Livermore, CA
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- W-7405-ENG-48
- OSTI ID:
- 15014375
- Report Number(s):
- UCRL-JRNL-202231
- Journal Information:
- Published in: Molecular and Cellular Probes, vol. 19, no. 2, May 1, 2005, pp. 137-144, Journal Name: Published in: Molecular and Cellular Probes, vol. 19, no. 2, May 1, 2005, pp. 137-144 Journal Issue: 2 Vol. 19
- Country of Publication:
- United States
- Language:
- English
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