• Food Science and Biotechnology
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• v.17 no.4
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• pp.829-832
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• 2008

A multiplex polymerase chain reaction (PCR) method was developed to simultaneously detect 4 varieties of genetically modified (GM) cotton. The event-specific primers were used to distinguish the 4 varieties of GM cotton (MON1445, MON15985, MON88913, and LLcotton25) using multiplex PCR. The acyl carrier protein 1 (Acp1) gene was used as an endogenous reference gene of cotton in the PCR detection. The primer pair Acp1-AF/AR containing a 99 bp amplicon was used to amplify the Acp1 gene and no amplified product was observed in any of the 13 different plants used as templates. This multiplex PCR method allowed for the detection of event-specific targets in a genomic DNA mixture of up to 1% GM cotton containing MON1445, MON15985, MON88913, and LLcotton25.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.91-98
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• 2016

The growth area of living modified (LM) cotton has steadily increased every year, since its first commercialization in 1996. Development of environmental risk assessment tools and techniques for LM cotton is required for ecosystem safety. We therefore developed multiplex PCR assays for simultaneous detection of two (MON15985, MON531) and four (GHB614, LLCOTTON25, MON88913 and MON1445) LM cotton events approved in Korea, with event specific primer pairs. The PCR reactions were optimized by using event specific primers of six LM cottons at various concentrations. The reactions allows amplification of estimated amplicons of MON15985 (214 bp), MON531 (270 bp), GHB614 (119 bp), LLCOTTON25 (164 bp), MON88913 (276 bp), and MON1445 (389 bp) from multiplex PCR reactions. The multiplex PCR assay developed allowed that two annealing steps (15 cycles at $55^{\circ}C$ and 25 cycles at $60^{\circ}C$) were performed for amplification of distinguished two LM cottons, and only one annealing step (50 cycles at $60^{\circ}C$) was necessary for tetraplex PCR. Primer extension step of all PCR reactions was skipped for time-effective amplification. Our methods suggest that two multiplex PCR assays can be cost-effective and a rapid diagnostic tool for environmental LMO monitoring of six LM cottons.

• Korean Journal of Environmental Biology
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• v.39 no.4
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• pp.445-451
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• 2021

In this study, we described the production of an antibody to living modified organisms (LMOs) containing the gene encoding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium tumefaciens strain CP4 EPSPS provides resistance to the herbicide glyphosate (N- (phosphonomethyl) glycine). These LMOs were approved and have recently been used in the feed, food production, and processing industries in South Korea. Highly efficient monoclonal antibody (mAb) production is crucial for developing assays that enable the proper detection and quantification of the CP4 EPSPS protein in LMOs. This study describes the purification and characterization of recombinant CP4 EPSPS protein in E. coli BL21 (DE3) based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrixassisted laser desorption/ionization time-of-flight mass spectrometry. The production of mAbs was undertaken based on the standard operating procedure of Abclon, Inc.(South Korea), and the purity of the mAbs was assessed using SDS-PAGE. The following five mAb clones were produced: 2F2, 4B9, 6C11, 10A9, and 10G9. To verify the efficiency and specificity of the five developed mAbs, we performed Western blotting analysis using the LM (living modified) cotton crude extracts. All mAbs could detect the CP4 EPSPS protein in the LM cotton traits MON1445 and MON88913 with high specificity, but not in any other LM cottons or non-LM cottons. These data indicate that these five mAbs to CP4 EPSPS could be successfully used for the further development of antibody-based detection methods to target CP4 EPSPS protein in LMOs.