[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 (GMO) 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 nM/300 nM and 100 nM/100 nM, probes at 200 nM and 200 nM, 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 °С - 20 sec, 58 °С - 20 sec, 62 °С - 40 sec). Conclusion. The validity of the developed method is confirmed by laboratory studies and testifies to its reliability.

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