Event-specific detection of transgenic potato AV43-6-G7 using real-time and digital PCR methods

Abstract

BackgroundThe isolation of unknown DNA sequences flanked by known sequences is an important task in the event-specific detection of GMOs. None of event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic potato AV43-6-G7.ResultsThe flanking sequence between the exogenous fragment and recombinant chromosome of this potato was successfully acquired through exogenous gene 5′-RACE. The event-specific primers and Taqman probe were designed to amplify fragments spanning the exogenous DNA and potato genomic DNA. The specific real-time PCR and digital PCR detection methods for AV43-6-G7 potato were established based on primers designed according to the flanking sequences. The detection limit of the qualitative PCR assay was 0.01 % for AV43-6-G7 potato in 100 ng of potato genomic DNA, corresponding to approximately 11.6 copies of the potato haploid genome. The ddPCR assays for Potato AV43-6-G7 achieved a limit of quantification of approximately 58 target copies, with RSD ≤ 25 %. The aLOQ of this system was approximately 1.2 copies.ConclusionsThese results indicated that these event-specific methods would be useful for the identification of potato AV43-6-G7.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0303-8) contains supplementary material, which is available to authorized users.

Development and Application of Droplet Digital PCR Tools for the Detection of Transgenes in Pastures and Pasture-Based Products

“…GM detection methods has depended on qPCR technologies, using either SYBR Green I or probe based fluorescence (Gao et al, 2016). The SYBR Green I assay showed ability to detect both 1SST-6G-FFT and hph in a range of agricultural products (Figure 2A and Supplementary Figure S4A).…”

“…Fluorescent probe-based qPCR is the preferred method when a quantitative analysis is required, since it reflects high accuracy, specificity and sensitivity (Gao et al, 2016). The evaluation of different agricultural products in this study, allowed to distinguish between different amplification products in the same reaction.…”

“…The minimum labeling threshold on GMO content in feed and foodstuffs are 0.9% in European Union (EU), 1% in New Zealand and Australia, 3% in Korea, and 5% in the United States (US) and Japan (Gao et al, 2016). Although, qPCR showed to be effective, ddPCR achieved a reliable detection of both exogenous constructs below the threshold of all jurisdictions.…”

Detection method for genetically modified potato using an ultra-fast PCR system

“…Several studies have been conducted to develop methods to identify GM potatoes. Detection methods for NewLeaf, NewLeaf Y, NewLeaf Plus, PH05-026-0048, EH92-527-1, AM04-1020, SPS-E12, AV43-6-G7, and insertion Cry3A have been developed using conventional PCR (PCR), real-time PCR (qPCR) (Cote et al, 2005 ; Roh et al, 2004 ; Song et al, 2017 ; Yoo et al, 2013 ), loop-mediated isothermal amplification (Tu et al, 2020 ), and droplet digital PCR (Gao et al, 2016 ). Recent gene detection assays are directed towards simpler and more cost-effective methods.…”

Detection of unapproved genetically modified potatoes in Korea using multiplex polymerase chain reaction

“…EH92-527-1 (Amflora) and AM04-1020, developed by the BASF Plant Science Company GmbH (Ludwigshafen, Germany), are transgenic lines that exhibit increased levels of amylopectin in potato starch through silencing of the expression of granule-bound starch synthase (GBSS) for industrial applications (Gao et al, 2016;Tilocca et al, 2014). PH05-026-0048 (Fortuna), which exhibits increased resistance to Phytophthora infestans, a pathogen causing late blight and tolerance to imidazolinone herbicides, was also developed by BASF (Storck, Bohme, & Schultheiss, 2012).…”