TY - JOUR
T1 - Performance evaluation of five commercial real-time PCR reagent systems using TaqMan assays for B. anthracis detection
AU - Sohni, Youvraj
AU - Kanjilal, Sagarika
AU - Kapur, Vivek
N1 - Funding Information:
This research was supported in part by the U.S. Department of Homeland Security (Grant number N-00014-04-1-0659), through a grant awarded to the National Center for Food Protection and Defense at the University of Minnesota. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not represent the policy or position of the Department of Homeland Security.
PY - 2008/5
Y1 - 2008/5
N2 - 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 CT values.
AB - 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 CT values.
UR - http://www.scopus.com/inward/record.url?scp=41849083022&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=41849083022&partnerID=8YFLogxK
U2 - 10.1016/j.clinbiochem.2008.01.007
DO - 10.1016/j.clinbiochem.2008.01.007
M3 - Article
C2 - 18242168
AN - SCOPUS:41849083022
SN - 0009-9120
VL - 41
SP - 640
EP - 644
JO - Clinical Biochemistry
JF - Clinical Biochemistry
IS - 7-8
ER -