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GMO Detection[ PCR Procedures | Qualitative PCR Analysis | Quantitative PCR Analysis ] It has been our experience that analytical reports showing quantitative results for GM products are often misunderstood or misinterpreted. Unlike the reporting of most chemical tests, which are based on an absolute weight percentage, quantitative, real-time PCR results are reported as ratio of target GM DNA relative to the total amount of species DNA present, for example, the number of copies of RUR soy DNA vs. the number of copies of total soy DNA (RUR + non-GM). Quantitative PCR results are reported as a percentage where the amount of species DNA makes up the denominator and GM DNA makes up the numerator. The diagrams below illustrate how a Detection Limit (DL) and a Quantification Limit (QL) are both related to the amount of DNA present and how they differ from one another.
Click on the image to view a larger version. Detection Limit Theoretically, a PCR analysis can detect a single molecule of DNA. In the routine testing in the various GeneScan laboratories, we have proof for each individual sample DNA that the detection limit is ten copies of target DNA or less. However, a specific detection limit, expressed as a percentage, can only be determined by counting the number of copies of DNA molecules present using a real-time quantitative PCR analysis. Daily routine testing in our various laboratories, using real time PCR, has shown that amounts less than 0.1% of GM soy can be reliably detected in soybeans. In some matrices a detection limit in the range of 0.01% can be achieved. The detection limit of a quantitative PCR, given as a percentage of GM material, depends upon the amount of DNA molecules from the plant species present in the sample. In Diagram 1, as an example, if 10,000 copies of DNA had been extracted from the sample, then the theoretical detection limit (a single copy of GM target DNA) would be 0.01%. (In actuality, with real samples, the DL could range from the theoretical 0.01% to 0.1%.) If another sample only had 1000 copies of DNA then the theoretical detection limit would be 0.1% for that sample (again, in reality it could range up to 1%). It is axiomatic to say that the DL, given as a percentage, cannot be stated unless a quantitative PCR is performed on that particular sample since the number of plant species DNA molecules extracted from the sample is not known until the completion of a quantitative analysis. Quantification Limit Similarly, the amount of DNA present is a factor in determining the limit of quantification for each real-time PCR analysis. The QL for a sample is the lowest level at which reproducible results can be calculated. In our experience, over many tens of thousands of samples, real-time PCR, using the TaqMan system, can reliably quantify target DNA molecules down to a lower limit of about ten (10) copies. Although the TaqMan system can easily detect single copies of target DNA, it is virtually impossible to obtain reproducible data in triplicate or quadruplicate with such very low copy numbers. Consequently, in the calculations used in GeneScan labs, if the sample DNA showed 10,000 copies of species DNA in a real-time PCR reaction, then the limit of quantification would be 0.1% (10 molecules of GM DNA out of 10,000 molecules of species DNA). If another sample had only one thousand copies of species DNA, then the QL would be 1% for that sample. In Diagram 1, a sample with 10,000 copies of species DNA the DL would be 0.01% - 0.1% and the QL would be 0.1%. 20 copies of GM target DNA would be reported as 0.2%, which is above the QL for this sample.
In the second diagram, the sample with 1,000 copies would have a DL of 0.1% to 1% and a QL of 1%. Using our 10 copy rule, we could not report a quantified result of less than 1% for this sample. In our experience the ratio of 2 copies of target GM DNA to 1000 copies of plant species DNA would not be reproducible at a statistically valid level. So the 2 molecules of GM DNA found would not be reported as 0.2%; instead they would be reported as belo » Return to PCR Procedures Main Page
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