Multiplex PCR for detection and quantification of GM potato event EH92-527-1 in food

Voprosy pitaniia
N V TyshkoM V Sukhacheva

Abstract

Aim - the elaboration of the protocol for the quantitative detection of genetically modified (GM) potato event EH92-527-1 in the format of duplex real-time polimerase chain reaction (RT-PCR) with TaqMan® PCR technology. Material and methods. The duplex system included two types of specific DNA primers and fluorescent probes: the 1st was for identifying of the event-specific EH92-527-1 DNA, the 2nd was for identifying of the taxon-specific potato gene Stp23. The selection of PCR parameters was carried out by empirical assortment of primers and probes, Mg2+-ions, deoxyribonucleotides and polymerase stabilizing agent, primers annealing temperature and incubation time for the each cycle stage. The results of this research was the optimization of the reaction mixture composition for EH92-527-1 event and Stp23 gene fragment identification in the duplex system: 2.5× RT-PCR buffer in the presence of ROX, primers specific for the GM potato (EH92- f/EH92-r) and target taxon (GРF3/GРR3) in the amount of 250/250 and 100/100 nM, probes - 200 and 200 nM, respectively; bovine serum albumin - 0.04%; MgCl2 - 3.5 mM, deoxynucleoside triphosphates - 0.3 mM, as well as the temperature-time reaction profile (initial denaturation at 95°C - 5 minutes...Continue Reading

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