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3 . 2020

Multiplex polymerase chain reaction for genetically modified potato event AV43-6-G7 quantification. Proof of efficiency

Abstract

One of the ways to improve the laboratory control methodology of genetically modified organisms of plant origin (GМО^) is to use multiplexing - an approach that allows you to increase the number of targets and enlarge the number of simultaneously processed samples, maximizing the potential of polymerase chain reaction in real time (PCR-RT). The aim of the study is to develop a quantitative identification protocol for genetically engineered (GE) potato event AV43-6-G7 in the format of duplex PCR-RT with the use of TaqMan® PCR technology.

Material and methods. The duplex system included 2 types of specific DNA-primers and fluorescence-labeled probes: the first one is for detection of transformation event AV43-6-G7 DNA sequence, the second is for detection of Stp23 taxon-specific potato gene. PCR parameters were chosen by empirical selection of concentrations of primers and probes, Mg2+ ions, deoxyribonucleotides, stabilizing agent for polymerase, as well as primer annealing temperature and incubation duration for each stage of the cycle.

Results. As a result of these studies, the composition of the reaction mixture was optimized for the detection and quantification of GE potato event AV43-6-G7 in food. Oligonucleotide primers and fluorescent probes were selected. The compositions of reaction mixtures and temperature-time parameters of PCR were tested: 2.5-fold reaction buffer for PCR-RT in the presence of ROX (carboxy-X-rhodamine), specific to the GE component primers (AV43-6-G7-f/AV43-6-G7-r) and target taxon (GRF3/ GRR3) at 300 пМ/300 пМ and 100 пМ/100 пМ, probes at 200 пМ and 200 пМ, respectively; bovine serum albumin - 0.04%; MgCl2 - 3.5 mM, deoxynucleoside triphosphates - 0.3 mM, as well as the temperature-time profile of the reaction (initial denaturation of 95 °C - 5 min, followed by 45 cycles: 95 °C - 20 sec, 58 °C - 20 sec, 62 °C -40 sec).

Conclusion. The validity of the developed method is confirmed by laboratory studies and testifies to its reliability.

Keywords:GM-potato, methods of control, PCR-RT, duplex PCR, transformation event AV43-6-G7

Funding. This work was supported by the Russian Science Foundation (grant No. 16-16-04123).

Conflict of interests. The authors declare no conflict of interests.

For citation: Tyshko N.V., Sadykova E.O., Sukhacheva M.V., Grouzdev D.S. Multiplex polymerase chain reaction for genetically modified potato event AV43-6-G7 quantification. Proof of efficiency. Voprosy pitaniia [Problems of Nutrition]. 2020; 89 (3): 62-70. DOI: 10.24411/0042-8833-2020-10030 (in Russian)

References

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CHIEF EDITOR
CHIEF EDITOR
Viktor A. Tutelyan
Full Member of the Russian Academy of Sciences, Doctor of Medical Sciences, Professor, Scientific Director of the Federal Research Centre of Nutrition, Biotechnology and Food Safety (Moscow, Russia)

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