Transferring qPCR methods into a droplet digital PCR format – Experiences from Official Food and Feed Control in GMO analysis

Sven Pecoraro 1,
1Bavarian Health and Food Safety Authority, Germany; 

According to Regulation (EC) No 1829/2003 genetically modi- fied organisms (GMO) have to be approved to be put on the market in the EU. Food or feed that contain, consist or are produced from GMO have to be properly labelled. This provision shall not apply up to a proportion of 0.9% of a given GMO material, provided that the presence of such material is adventitious or technically unavoid- able. From this it follows for official food and feed control, that if GMO material is qualitatively detected in a given sample the pro- portion of this material (taxon related) has to be quantified with qPCR. Quantification in GMO analysis consists of two independent qPCR reactions. In one reaction the amount of taxon (plant) spe- cific DNA is measured relative to a standard curve with known DNA copy numbers. The other reaction measures the DNA copy numbers of the specific GMO. These two DNA copy numbers are divided (cp gmo/cp reference gene) and multiplied by 100% to give the final GMO percentage (cp/cp). Typically the limit of quantifica- tion (LOQ) of qPCR in GMO analysis is 0.1%. According to Regulation (EU) No 619/2011 certain GMOs have to be accurately quantified at a GMO proportion of 0.1%. However, quantifying at the lower end of the dynamic range of qPCR can be inaccurate. Digital droplet PCR (ddPCR) offers the advantage that DNA copy numbers can be abso- lutely quantified without the need for standard curves. Additionally ddPCR is capable to accurately measure minor quantities of DNA. Furthermore ddPCR is considered as less susceptible to inhibitory effects which is relevant when analyzing DNA that derives from complex matrices like food or feed. Official control laboratories have to apply validated methods. In GMO analysis predominantly methods published by the Joint Research Centre (JRC) are used. When such qPCR methods are transferred into a ddPCR format several aspects should be considered. As usually the master mix is different in ddPCR than for qPCR, a selected set of GMO should be tested in order to assess the specificity of the reaction. In gen- eral the reaction conditions like annealing temperature, primer and probe concentrations should be taken as they were validated. If results are not satisfying, e.g. because the separation between posi- tive and negative signals is not optimal (intermediate signals = rain) then a temperature gradient can be performed to evaluate the most appropriate annealing temperature. One has to consider that if the annealing temperature is altered more than 1 ◦ C to 2 ◦ C (robustness range) then the method has possibly to be re-validated. Increased primer and/or probe concentrations can also lead to better signal separation. The experience of the GMO laboratory of the Bavarian Health and Food Safety Authority (LGL) with ddPCR is very promis- ing. The procedure is accurate even with minor quantities of DNA and serves as an alternative reference method to standard qPCR when quantifying GMO DNA derived from complex matrices.

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