A statistical approach for evaluation of PCR results to improve the practical limit of quantification (LOQ) of GMO analyses (SIMQUANT) |
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Authors: | Knut G Berdal Charlotte Bøydler Torstein Tengs Arne Holst-Jensen |
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Affiliation: | 1. Section of Food and Feed Microbiology, National Veterinary Institute, Ullev?lsveien 68, P.O. Box 8156 Dep, 0033, Oslo, Norway
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Abstract: | The predominant approach for quantification of genetically modified organisms (GMO) is the application of quantitative real-time
PCR. However, for a large number of processed food and feed products, this approach is unsuitable, because they contain low
amounts (mass) of amplifiable DNA. Here we present a novel approach, “Single molecule quantification” (SIMQUANT) for GMO quantification
of samples with extremely low amounts of DNA. The approach is based on statistics and application of multiple qualitative
parallel PCRs. Here the qualitative PCRs were done using real-time PCR setup, but this is not a requirement. The difference
is that the quantitative real-time PCR requires that the target copy number exceeds the absolute limit of quantification (LOQabs) and provides quantity estimates by extrapolation from a linear regressional relationship between an observed cycle threshold
(Ct) value and copy numbers, while with SIMQUANT the template DNA typically contains very few, e.g., one target copy per PCR
volume and the quantity is estimated on the basis of observed ratio between positive and negative individual PCRs. The components
of this analysis are the numbers of test samples, the size of each sample and the outcome in number and relative ratio of
positive and negative test results. The approach results in a statistical estimate of the relative GM concentration based
on the probability that one or more amplifiable GM template copies are present in discrete volumes. Thus, the approach is
based on the ratio of discrete volumes without or with one or more PCR-amplifiable GM target copies. The approach described
here can be used reliably with more than a 100-fold improvement of the practical LOQ (LOQpract) compared to real-time quantitative PCR based on standard curves. |
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Keywords: | MPN Most probable number quantification GMO Limit of quantification (LOQ) Event Specific PCR Limiting dilution PCR |
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