ORIGINAL_ARTICLE RNASE A enzyme modification of optimized SDS protocol for DNA extraction suitable for real-time PCR screening of GMOs 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. https://macvetrev.mk/Files/Article/2021/macvetrev-2021-0028/macvetrev-2021-0028.pdf 2022-03-15T09:00:00 17 25 10.2478/macvetrev-2021-0028 35S Tnos DNA purity yield real-time PCR Arita Sabriu-Haxhijaha aritasabriu@hotmail.com false 1 Faculty of Technological Sciences, “Mother Teresa” University – Skopje, Mirche Acev, No. 4, 1000 Skopje, R. of North Macedonia LEAD_AUTHOR Velimir Stojkovski false 2 Food Institute, Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, R. of North Macedonia AUTHOR Gordana Ilievska false 3 Food Institute, Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, R. of North Macedonia AUTHOR Dean Jankuloski false 4 Food Institute, Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, R. of North Macedonia AUTHOR Katerina Blagoevska false 5 Food Institute, Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, R. of North Macedonia AUTHOR 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. 1 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. 2 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. PMid:27386337 PMCid:PMC4920734 3 10.1186/s40064-016-2395-y 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. PMid:30560659 4 10.1021/acs.jafc.8b05157 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. 5 10.18520/cs/v110/i6/1000-1009 Cottenet, G., Blancpain, C., Sonnard, V., Chuah, P.F. (2019). Two FAST multiplex real-time PCR reactions to assess the presence of genetically modified organisms in food. Food Chem. 274, 760-765. PMid:30373005 6 10.1016/j.foodchem.2018.09.050 Stefanova, P., Taseva, M., Georgieva, T., Gotcheva, V., Angelov, A. (2013). A Modified CTAB method for DNA extraction from soybean and meat products. Biotechnol & Biotechnol Eq. 27(3): 3803-3810. 7 10.5504/BBEQ.2013.0026 Schrader, C., Schielke, A., Ellerbroek, L., Johne, R. (2012). PCR inhibitors-occurrence, properties and removal. J Appl Microbiol. 113(5): 1014-1026. PMid:22747964 8 10.1111/j.1365-2672.2012.05384.x Sidstedt, M., Rådström, P., Hedman, J. (2020). PCR inhibition in qPCR, dPCR and MPS-mechanisms and solutions. Anal Bioanal Chem. 412(9): 2009-2023. PMid:32052066 PMCid:PMC7072044 9 10.1007/s00216-020-02490-2 Gryson, N. (2010). Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review. Anal Bioanal Chem. 396(6): 2003-2022. PMid:20012944 10 10.1007/s00216-009-3343-2 Wilfinger, W.W., Mackey, K., Chomczynski, P. (1997). Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques. 22(3): 474-476, 478-481. PMid:9067025 11 10.2144/97223st01 Sabriu-Haxhijaha, A., Ilievska, G., Stojkovski, V., Blagoevska, K. (2020). A modified SDS - based method applied for extraction of high-quality DNA from raw corn and roasted soybean. Mac Vet Rev. 43(1): 61-67. 12 10.2478/macvetrev-2020-0017 Healey, A., Furtado, A., Cooper, T., Henry, R.J. (2014). Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species. Plant Methods 10, 21. PMid:25053969 PMCid:PMC4105509 13 10.1186/1746-4811-10-21 Abdel-Latif, A., Osman, G. (2017). Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize. Plant Methods 13, 1. PMid:28053646 PMCid:PMC5209869 14 10.1186/s13007-016-0152-4 Hougs, L., Gatto, F., Goerlich, O., Grohmann, L., Lieske, K., Mazzara, M., Narendja, F., Ovesna, J., Papazova, N., Scholtens, I., Žel, J. (2017). Verification of analytical methods for GMO testing when implementing interlaboratory validated methods. EUR 29015 EN, Publication Office of the European Union, Luxembourg 15 Waiblinger, H.U., Ernst, B., Anderson, A., Pietsch, K. (2007). Validation and collaborative study of a P35S and T-nos duplex real-time PCR screening method to detect genetically modified organisms in food products. Eur Food Res and Technol. 226, 1221-1228. 16 10.1007/s00217-007-0748-z Anklam, E., Gadani, F., Heinze, P. et al. (2002). Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. Eur Food Res Technol. 214, 3-26. 17 10.1007/s002170100415 Bitskinashvili, K., Gabriadze, I., Kutateladze, T., Vishnepolsky, B., Mikeladze, D., Datukishvili, N. (2019). Influence of heat processing on DNA degradation and PCR-based detection of wild-type and transgenic maize. J Food Qual. 2019(3): 1-11. 18 10.1155/2019/5657640 El-Ashram, S., Al Nasr, I., Suo, X. (2016). Nucleic acid protocols: Extraction and optimization. Biotechnol Rep (Amst). 12, 33-39. PMid:28352552 PMCid:PMC5361071 19 10.1016/j.btre.2016.10.001 Wang, Y.S., Dai, T.M., Tian, H., Wan, F.H., Zhang, G.F. (2019). Comparative analysis of eight DNA extraction methods for molecular research in mealybugs. PLoS One 14(12): e0226818. PMid:31891602 PMCid:PMC6938366 20 10.1371/journal.pone.0226818 Corneillie, S., De Storme, N., Van Acker, R., Fangel, J.U., De Bruyne, M., De Rycke, R., Geelen, D., Willats, W., Vanholme, B., Boerjan, W. (2019). Polyploidy affects plant growth and alters cell wall composition. Plant Physiol. 179(1): 74-87. PMid:30301776 PMCid:PMC6324247 21 10.1104/pp.18.00967 Lorenz, T.C. (2012). Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp. 63, e3998. PMid:22664923 PMCid:PMC4846334 22 10.3791/3998