Overcoming PCR inhibition: Next Generation qPCR and RT-qPCR ToughMix® Reagents for High Sensitivity Quantification and Accurate Genotyping from Crude Samples.

David Mark Schuster Quanta BioSciences, United States of America

Abstract

Naturally occurring PCR-inhibitory components can reduce the sensitivity, efficiency or specificity of PCR assays and gives rise to false negatives and allele dropouts. Reported PCR inhibitors include compounds such as hemin (or its derivatives), bile salts, vitreous fluid, aromatic halides, melanin, complex carbohydrates, polyphenolic compounds from plant extracts, and humic substances produced during the decomposition of organic matter. While the most effective approach to prevent PCR inhibition is to remove the inhibitor from the sample, this is not always certain. Often inhibitors may co-purify with the DNA sample. Effective nucleic acid extraction/purification methods are expensive, time consuming, and often result in a loss of material. Implementation of high throughput, cost effective DNA analysis by PCR therefore demands robust amplification reagents that can relieve PCR inhibition. This talk will present the development of new qPCR reagents that overcome many reported PCR inhibitors: PerfeCta qPCR ToughMix, AccuStart Genotyping ToughMix, and qScript XLT One-Step RT-qPCR ToughMix.