Background: Real-time PCR assay sensitivity is affected by the choice and concentrations of reaction mix constituents among other factors such as primers, probes, and analytical assay platforms. Commercially available reagent mixes facilitate PCR assay set-up with fewer steps and timeliness. However, determination of analytical assay framework is important for ready-to-use real-time PCR reagent systems for rapid, quantitative and accurate detection of bioterror pathogens such as Bacillus anthracis.
Methods: In this study, performance characteristics of five commercially available quantitative PCR reagent mixes were evaluated using TaqMan-based real-time PCR. The reagent systems were tested for compatibility on the ABI 7000 assay platform and compared for their distinctive analytical characteristics using the B. anthracis rpoB and pag gene real-time PCR assays.
Results and conclusions: Knowledge of distinctive assay performance characteristics of commercially available qPCR reagent mixes is critical for carefully designing analytical assay systems. The ABI, ABgene and Eppendorf reagent systems performed consistently overall for the two TaqMan assays for B. anthracis detection that were used in the current study. However, the use of Eppendorf reagent system requires shorter thermal cycling time. In addition, while the ABI and Eppendorf systems have similar assay sensitivity for both the rpoB and pag assays, the Eppendorf system achieves the same with lower C(T) values.