Development and utility of an internal threshold control (ITC) real-time PCR assay for exogenous DNA detection.

PloS One
Weiyi NiRichard O Snyder

Abstract

Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC) has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to o...Continue Reading

Associated Clinical Trials

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Aug 6, 2013·Analytical and Bioanalytical Chemistry·Irene C PerezRichard O Snyder
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May 8, 2021·Drug Testing and Analysis·Alexandre MarchandMagnus Ericsson

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Datasets Mentioned

BETA
AY315197
AY446894
GQ221975
GQ396662
GU179289

Methods Mentioned

BETA
PCR
sedation
electrophoresis
by

Software Mentioned

SAS
ABI StepOne
ABI Primer Express

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