Absolute quantification of genetically engineered traits with droplet digital PCR: Effect of DNA treatments and spiking with non-target DNA

摘要

Low level presence of unapproved genetically engineered (GE) materials in non-GE grains has been a challenge for grain importers and exporters and many countries also have regulatory requirements for use and cultivation of GE crops. Digital PCR has been used for absolute quantification of GE traits. The need for DNA pre-treatment for droplet digital PCR (ddPCR) has not been well assessed. The effect of non-shearing, QIAshredding and hydroshearing of genomic DNA (300 ng) on quantitative analysis of low concentrations of OXY235 canola, FP967 flax and DP305423 soybean samples was assessed using RainDance RainDrop (TM) Digital PCR system. 1000 ng DNA was also used for ddPCR to determine if pretreatment is required for high DNA concentration. The measured percentage values were close to the expected 0.01, 0.1 and 1% values for non-sheared, QIAshredded and hydrosheared samples for three GE traits using 300 ng DNA. The ddPCR results were also similar to the expected 0.01, 0.1 and 1% values for non-sheared and QIAshredded samples for the three GE traits using 1000 ng DNA. The feasibility of absolute quantification of low levels of OXY235 canola, FP967 flax and DP305423 soybean samples spiked in non-GE barley and wheat samples was investigated using ddPCR. Successful quantification of OXY235 canola, FP967 flax and DP305423 soybean DNA samples spiked in barley and wheat DNA samples was achieved at 0.01, 0.1 and 1% levels. ddPCR carried out with DNA extracted from ground DP305423 soybean samples spiked in wheat and barley samples resulted in percentage values close to the expected 0.01, 0.1 and 1% values with some exceptions. The result shows the feasibility of using ddPCR for absolute quantification of low level presence of GE materials in other grain samples. (c) 2016 Published by Elsevier Ltd.