Development and Analytical Characterization of a Sequence-Traceable Positive Control Plasmid for PCR Detection of the Edited SlGAD3 Allele in GABA-Enriched Tomato

Yu Kyong Hu; Chea Yeon Kim; Byoung-Eun Min; Sung Don Lim

doi:10.22698/jales.20260026

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2026 Vol.38, Issue 3 Preview Page Next Page

Research Article

Development and Analytical Characterization of a Sequence-Traceable Positive Control Plasmid for PCR Detection of the Edited SlGAD3 Allele in GABA-Enriched Tomato

30 September 2026. pp. 374-391

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Abstract

GABA-enriched tomato developed by targeted mutagenesis contains a single thymine insertion in the C-terminal autoinhibitory region of the endogenous SlGAD3 gene. Because the analytical target is a short endogenous sequence variant, conventional screening assays targeting common transgenic elements are unsuitable for direct detection of the edited SlGAD3 allele. Detection of this allele does not by itself establish that the sequence variant originated from CRISPR/Cas9-mediated genome editing. This study developed a sequence-traceable positive-control plasmid for conventional PCR and TaqMan real-time PCR detection of the edited SlGAD3 allele. The assay used the allele-specific forward primer SlGAD3_var1_F, the common reverse primer SlGAD3_WT_R, and a FAM-labeled GABA-TaqMan probe. A 120-bp synthetic insert containing the primer- and probe-binding sites and two plasmid-specific spacer sequences was cloned into the pMG-Kan vector. Sanger sequencing confirmed the integrity of the insert and the presence of the intended spacer sequences. Conventional PCR generated the expected 120-bp amplicon across a 10-fold plasmid dilution series, with decreasing band intensity at lower template concentrations. A copy-number-based TaqMan real-time PCR standard curve covering 10¹－10⁶ nominal plasmid copies per reaction yielded an R² value of 0.9969 and an amplification efficiency of 93.5%. The expected amplicon was detected in the positive-control plasmid and GABA-enriched tomato but not in any of the 11 conventional commercial tomato samples. Sequence analysis further distinguished plasmid-derived amplicons from the corresponding tomato genomic target. These results demonstrate that the developed plasmid is suitable as a sequence-defined qualitative positive control for the SlGAD3_var1 assay and for investigating suspected positive-control carryover.

Keywords

Allele-specific PCR

Edited SlGAD3 allele

GABA-enriched tomato

Positive control plasmid

Sequence traceability

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Information

Publisher :Agriculture and Life Sciences Research Institute, Kangwon National University
Publisher(Ko) :None
Journal Title :Journal of Agricultural, Life and Environmental Sciences
Volume : 38
No :3
Pages :374-391
Received Date : 2026-08-07
Revised Date : 2026-09-01
Accepted Date : 2026-09-02
DOI :https://doi.org/10.22698/jales.20260026

Journal of Agricultural, Life and Environmental Sciences ISSN:2233-8322(Print) 2508-870X(Online)

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