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JMD 2006, Vol. 8, No. 1
Copyright © 2006 American Society for Investigative Pathology & Association for Molecular Pathology

Multiplexed Detection of Anthrax-Related Toxin Genes

Michael J. Moser*, Deanna R. Christensen{dagger}, David Norwood{dagger} and James R. Prudent*

From the Eragen Biosciences, Inc., * Madison, Wisconsin; and the Diagnostic Systems Division, {dagger} U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland

Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.






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Copyright © 2006 by the American Society for Investigative Pathology and the Association for Molecular Pathology.