Detection of Biological Threat Agents by Real-Time PCR: Comparison of Assay Performance on the R.A.I.D., the LightCycler, and the Smart Cycler Platforms
Abstract
Rapid detection of biological threat agents is critical for timely therapeutic administration. Fluorogenic PCR provides a rapid, sensitive, and specific tool for the molecular identification of these agents. A common chemistry that can be used on a variety of rapid, real-time PCR instruments provides the greatest flexibility for assay utilization. Methods: Real-time PCR primers and dual-labeled fluorogenic probes were designed to detect Bacillus anthracis, Brucella species, Clostridium botulinum, Coxiella burnetii, Francisella tularensis, Staphylococcus aureus, and Yersinia pestis. DNA amplification assays were optimized by using Idaho Technology, Inc. buffers and dNTPs supplemented with Invitrogen Platinum Taq DNA polymerase, and were subsequently tested for sensitivity and specificity on the Idaho Technology, Inc. R.A.P.I.D. , the Roche LightCycler , and the Cepheid Smart Cycler . Results: Limit of detection experiments indicated that assay performance was comparable among the platforms tested. Exclusivity and inclusivity testing with a general bacterial nucleic acid cross-reactivity panel containing 60 DNAs and agent-specific panels containing nearest neighbors for the organisms of interest indicated that all assays were specific for their intended targets. Conclusion: With minor supplementation, such as the addition of Smart Cycler Additive Reagent to the Idaho Technology buffers, a common chemistry could be used for DNA templates that resulted in similar performance, sensitivity, and specificity on all three platforms.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 2006
- Accession Number
- ADA443583
Entities
People
- Bonnie M. Loveless
- Carson D. Baldwin
- David A. Kulesh
- David A. Norwood
- Deanna L. Bridge
- Deanna R. Christensen
- Jeffrey Garrison
- Laurie J. Hartman
- Melissa S. Frye
- Michelle A. Shipley
- Michelle J. Richards
- Rebecca S. Kaplan
Organizations
- United States Army Medical Research Institute of Infectious Diseases