Detection of biological threat agents by real-time PCR: comparison of assay performance on the R.A.P.I.D., the LightCycler, and the Smart Cycler platforms.
BACKGROUND
Rapid detection of biological threat agents is critical for timely therapeutic administration. Fluorogenic PCR provides a rapid, sensitive, and specific tool for molecular identification of these agents. We compared the performance of assays for 7 biological threat agents on the Idaho Technology, Inc. R.A.P.I.D., the Roche LightCycler, and the Cepheid Smart Cycler.


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 use of Idaho Technology buffers and deoxynucleotide triphosphates supplemented with Invitrogen Platinum Taq DNA polymerase, and were subsequently tested for sensitivity and specificity on the R.A.P.I.D., the LightCycler, and the 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.


CONCLUSIONS
With minor supplementation, such as the addition of Smart Cycler Additive Reagent to the Idaho Technology buffers, assays for DNA templates from biological threat agents demonstrated similar performance, sensitivity, and specificity on all 3 platforms.
Christensen
Deanna R
DR
United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA.
Hartman
Laurie J
LJ
Loveless
Bonnie M
BM
Frye
Melissa S
MS
Shipley
Michelle A
MA
Bridge
Deanna L
DL
Richards
Michelle J
MJ
Kaplan
Rebecca S
RS
Garrison
Jeffrey
J
Baldwin
Carson D
CD
Kulesh
David A
DA
Norwood
David A
DA
D001419:Bacteria / Q000145:classification* / Q000235:genetics; D001431:Bacteriological Techniques; D001694:Biological Warfare; D004269:DNA, Bacterial / Q000235:genetics; D005470:Fluorometry; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial