Original Scientific Article
RNASE A enzyme modification of optimized SDS protocol for DNA extraction suitable for real-time PCR screening of GMOs
Arita Sabriu-Haxhijaha*,
Velimir Stojkovski,
Gordana Ilievska,
Dean Jankuloski,
Katerina Blagoevska

Mac Vet Rev 2022; 45 (1): 17 - 25

10.2478/macvetrev-2021-0028

Received: 16 September 2021

Received in revised form: 01 November 2021

Accepted: 09 November 2021

Available Online First: 14 December 2021

Published on: 15 March 2022

Correspondence: Arita Sabriu-Haxhijaha, aritasabriu@hotmail.com

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.

Keywords: 35S, Tnos, DNA purity, yield, real-time PCR


References

  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. 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. 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:PMC4920734
  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:30560659
  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/i6/1000-1009
  6. 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. https://doi.org/10.1016/j.foodchem.2018.09.050 PMid:30373005
  7. 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. https://doi.org/10.5504/BBEQ.2013.0026
  8. Schrader, C., Schielke, A., Ellerbroek, L., Johne, R. (2012). PCR inhibitors-occurrence, properties and removal. J Appl Microbiol. 113(5): 1014-1026. https://doi.org/10.1111/j.1365-2672.2012.05384.x PMid:22747964
  9. Sidstedt, M., Rådström, P., Hedman, J. (2020). PCR inhibition in qPCR, dPCR and MPS-mechanisms and solutions. Anal Bioanal Chem. 412(9): 2009-2023. https://doi.org/10.1007/s00216-020-02490-2 PMid:32052066 PMCid:PMC7072044
  10. 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. https://doi.org/10.1007/s00216-009-3343-2 PMid:20012944
  11. 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. https://doi.org/10.2144/97223st01 PMid:9067025
  12. 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. https://doi.org/10.2478/macvetrev-2020-0017
  13. 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. https://doi.org/10.1186/1746-4811-10-21 PMid:25053969 PMCid:PMC4105509
  14. Abdel-Latif, A., Osman, G. (2017). Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize. Plant Methods 13, 1. https://doi.org/10.1186/s13007-016-0152-4 PMid:28053646 PMCid:PMC5209869
  15. 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
  16. 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. https://doi.org/10.1007/s00217-007-0748-z
  17. 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. https://doi.org/10.1007/s002170100415
  18. 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. https://doi.org/10.1155/2019/5657640
  19. El-Ashram, S., Al Nasr, I., Suo, X. (2016). Nucleic acid protocols: Extraction and optimization. Biotechnol Rep (Amst). 12, 33-39. https://doi.org/10.1016/j.btre.2016.10.001 PMid:28352552 PMCid:PMC5361071
  20. 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. https://doi.org/10.1371/journal.pone.0226818 PMid:31891602 PMCid:PMC6938366
  21. 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. https://doi.org/10.1104/pp.18.00967 PMid:30301776 PMCid:PMC6324247
  22. Lorenz, T.C. (2012). Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp. 63, e3998. https://doi.org/10.3791/3998 PMid:22664923 PMCid:PMC4846334


Copyright

© 2021 Sabriu-Haxhijaha A. This is an open-access article published under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Conflict of Interest Statement

he authors have declared that no competing interests exist.

Citation Information

Macedonian Veterinary Review. Volume 45, Issue 1, Pages 17-25, e-ISSN 1857-7415, p-ISSN 1409-7621, DOI: 10.2478/macvetrev-2021-0028, 2022