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. 2023 Jun 30;33(3):637-643.
doi: 10.1007/s10068-023-01377-z. eCollection 2024 Feb.

Multiplex PCR detection method of genetically modified canola event (MON94100, LBFLFK, and NS-B50027-4) combined with capillary electrophoresis

Seung-Man Suh  1 , Hyun-Jae Kim  1 , Min-Ki Shin  2 , Seung-Jin Hong  2 , Jae-Eun Cha  1 , Hae-Yeong Kim  1
Affiliations

Multiplex PCR detection method of genetically modified canola event (MON94100, LBFLFK, and NS-B50027-4) combined with capillary electrophoresis

Seung-Man Suh et al. Food Sci Biotechnol. .

Abstract

Genetically modified organisms (GMOs) have been continuously developed for their convenience and productivity. In the past three years, three new GM canola events (MON94100, LBFLFK, and NS-B50027-4) have been developed. To efficiently control these GM canola events, the detection methods were needed. Therefore, the multiplex PCR method combined with capillary electrophoresis was developed for three GM canola events. Ten GM canola, eighteen GM soybean, thirty-two GM maize, and ten non-GM crops were used to evaluate the specificity of the method. The detection limit of the multiplex PCR assay was determined to be 0.005 ng in the DNA mixture and 0.1% in the spiked sample. The aim of this study was to establish multiplex PCR coupled with capillary electrophoresis for the newly produced three GM canola events. The developed method is expected to contribute to monitor the commercially available GM canola events.

Supplementary information: The online version contains supplementary material available at 10.1007/s10068-023-01377-z.

Keywords: Capillary electrophoresis; Event-specific primer; Genetically modified canola; Multiplex PCR.

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Conflict of interest statement

Conflict of interestOn behalf of all authors, the corresponding author states that there is no conflict of interest.

Figures

Fig. 1
Fig. 1
Schematic diagrams of event-specific primers designed to detect three GM canola events. The locations of the primers used in the multiplex PCR assays are indicated by arrows. All primer pairs were designed based on sequences of the transferred DNA flanking region and the inserted region of the GMO genome. P, promoter; T, terminator; RB, right border region; LB, left border region
Fig. 2
Fig. 2
Specificity analysis of multiplex PCR results (MON94100, LBFLFK, and NS-B50027-4). Lane M, 100 bp ladder; lane P, positive control; lane 1, NS-B50027; lane 2, LBFLFK; lane 3, MON94100; lane4, GT73; lane 5, RF3; lane 6, Ms8; lane 7, T45; lane 8, MON88302; lane 9, Ms11; lane 10, DP-073496–4 lane 11, NGM canola; lane 12, GM soy mix; lane 13, GM maize mix; lane 14, Non-GM soybean; lane 15, Non-GM maize; lane 16, Non-GM cotton; lane 17, Non-GM rice; lane 18, Non-GM wheat; lane 19, Non-GM barley; lane 20, Non-GM potato; lane 21, Non-GM sugar beet; lane 22, Non-GM alfalfa; lane N, no template
Fig. 3
Fig. 3
Sensitivity analysis of the multiplex PCR detection method (MON94100, LBFLFK, and NS-B50027-4) using gDNA mixtures. A gel, Lane M, 100 bp ladder; lane 1–6; 50, 5, 0.5, 0.05, 0.005, 0.0005 ng of genomic DNA of GM canola (MON94100, LBFLFK, and NS-B50027-4); lane N, no template. B peak, a: Endogenous, b: MON94100, c: LBFLFK, d: NS-B50027-4
Fig. 4
Fig. 4
Sensitivity analysis of the multiplex PCR detection method (MON94100, LBFLFK, and NS-B50027-4) using spiked samples. A gel, Lane M, 100 bp ladder; lane 1–6; 10, 5, 1, 0.5, 0.1, 0.05 of spiked samples of GM canola (MON94100, LBFLFK, and NS-B50027-4); lane N, no template. B peak, a: Endogenous, b: MON94100, c: LBFLFK, d: NS-B50027-4
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