Affiliation:
1. Faculty of Technological Sciences , Mother Teresa University – Skopje , Mirche Acev No. 4 , Skopje , North Macedonia
2. Food Institute , Faculty of Veterinary Medicine - Skopje , Ss. Cyril and Methodius University in Skopje , North Macedonia
Abstract
Abstract
As the number of genetically modified crops increases rapidly, their accurate detection is significant for labelling and safety assessment. Currently, real-time PCR is the “golden standard” method for GMO detection. Hence, extraction of high quality DNA represents a crucial step for accurate and efficient DNA amplification. For GMO presence evaluation in the extracted DNA from raw corn kernels and roasted soybean, we used real-time PCR method, in consistent with the ISO17025 accreditation standards. As for DNA extraction, modified basic SDS protocol by adding RNase A enzyme in different steps of the protocol, with different time and temperature of incubation was used. The results showed as most suitable, the protocol where 10 µl of RNase A enzyme was added together with the lysis buffer at 65 °C for 30 minutes. Data for DNA yield and purity for roasted soybean was 469.6±3.3 µg/ml with A260/280 absorbance ratio 1.78±0.01. Suitability of DNA extracts for GMO analysis was assessed by screening for the presence of 35S promotor and Tnos terminator. Diluted extracts in concentrations 10, 1, 0.1, 0.01 and 0.0027 ng/µl, were tested in six replicates. Positive signal of amplification (LOD) was detected in all concentrations for both genetic elements in both matrices. The LOQ for 35S and Tnos for both matrices was 0.1 ng, while for Tnos in raw corn kernels was 0.01 ng. This in-house developed DNA extraction method is simple and obtains high-quality DNA suitable for GMO screening of 35S promotor and Tnos terminator in both raw and processed matrices.
Reference22 articles.
1. 1. European Commission Regulation, (EC) No 1829/2003 of the European parliament and of the council of 22 September 2003 of genetically modified food and feed. Off J Eur Union L268, 1-23.
2. 2. Directive 2001/18/EC of the European Parliament and of the Council 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC-Commission Declaration. OJ L 106, 1-39.
3. 3. Peng, C., Wang, P., Xu, X., Wang, X., Wei, W., Chen, X., Xu, J. (2016). Development of a qualitative real-time PCR method to detect 19 targets for identification of genetically modified organisms. Springerplus 5(1): 889. https://doi.org/10.1186/s40064-016-2395-y PMid:27386337 PMCid:PMC492073410.1186/s40064-016-2395-y492073427386337
4. 4. Alarcon, C.M., Shan, G., Layton, D.T., Bell, T.A., Whipkey, S., Shillito, R.D. (2018). Application of DNA-and protein-based detection methods in agricultural biotechnology. J Agric Food Chem. 67(4): 1019-1028. https://doi.org/10.1021/acs.jafc.8b05157 PMid:3056065910.1021/acs.jafc.8b0515730560659
5. 5. Randhawa, G., Singh, M., Sood, P. (2016). DNA-based methods for detection of genetically modified events in food and supply chain. Curr Sci. 110(6): 1000-1009. https://doi.org/10.18520/cs/v110/i6/1000-100910.18520/cs/v110/i6/1000-1009