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

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). 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. . 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, Mg ions, deoxyribonucleotides, stabilizing agent for polymerase, as well as primer annealing temperature and incubation duration for each stage of the cycle. . 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%; MgCl - 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). . The validity of the developed method is confirmed by laboratory studies and testifies to its reliability.