Effect of Amount of DNA on Digital PCR Assessment of Genetically Engineered Canola and Soybean Events
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Effect of Amount of DNA on Digital PCR Assessment of Genetically Engineered Canola and Soybean Events Tigst Demeke 1
&
Monika Eng 1 & Michelle Holigroski 1 & Sung-Jong Lee 1
Received: 12 August 2020 / Accepted: 12 October 2020 # The Author(s) 2020
Abstract Low-level detection and quantification of genetically engineered (GE) traits with polymerase chain reaction (PCR) is challenging. For unapproved GE events, any level of detection is not acceptable in some countries because of zero tolerance. Droplet digital PCR (ddPCR) has been successfully used for absolute quantification of GE events. In this study, reliability of low level quantification of GE events with ddPCR was assessed using a total of 50, 100, 200, 400, and 600 ng DNA spiked at 0.01% and 0.1% concentration levels. Genetically engineered canola (GT73 and MON88302 events) and soybean (A2704-12 and DP305423 events) events were used for the study. For samples spiked at 0.1% level, reliable quantification was achieved for the four GE events using 50 or 100 ng DNA. Few target droplets were generated for 0.01% spiked GE samples using 50 and 100 ng DNA. Increasing the amount of DNA for ddPCR generated more number of target droplets. For GE canola events, the use of 400 and 600 ng DNA for ddPCR resulted in saturation. The use of multiple wells of 200 ng DNA (instead of 400 and 600 ng per well) helped to overcome the saturation problem. Overall, the use of high amount of DNA for ddPCR was helpful for the detection and quantification of 0.01% GE samples. Keywords Genetically engineered . Droplet digital PCR . Low-level detection . Amount of DNA . Canola and soybean events
Introduction Accuracy of genetically engineered (GE) trait identification and quantification in non-processed and processed food samples is affected by factors such as sampling/sub-sampling, DNA extraction, DNA quality/purity, DNA amount, and PCR conditions (Alarcon et al. 2019; Demeke and Dobnik 2018). The minimum required performance limit (MRPL), which is the lowest level of GE material for satisfactorily reproducible validation of quantitative methods, has been set at 0.1% (European Commission Regulation No. 619/2011). However, for some countries, any level of detection of unapproved GE events is not acceptable. Unapproved GE events on the European market are reported through the Rapid Alert System for Food and Feed (European Commission, RASFF Portal). For unauthorized FP967 GE flax, sampling and testing protocol was set to achieve a level of detection of 0.01% (Canadian Grain Commission 2018).
One of the challenges for accurate quantification of GE events below 0.1% is obtaining repeatable or reliable results. Transgenic concentrations of 0.1% (m/m) certified reference materials resulted in bias and uncertainty (Cottenet et al. 2019). Concentrations such as 1% and 10% resulted in less bias. Use of high amount of DNA for real-time quantitative PCR enabled reliable detection of GE events at low concentration levels (Mano et al. 2018). Effect of amount of DNA on quantitative detection of G
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