KR102383881B1 - Simultaneous detection method for genetically modified maize - Google Patents

Abstract

The present invention relates to a simultaneous detection method of genetically modified maize. More particularly, related is a simultaneous detection method of four kinds of event sites in genetically modified maize by using a multiplex PCR method.

Description

Simultaneous detection method for genetically modified maize

The present invention relates to a method for simultaneous detection of genetically modified maize. More particularly, it relates to a method for simultaneous detection of four event sites in genetically modified maize using a multiplex PCR method.

Since genetically modified (GM) crops were first commercialized in the mid-1990s, genetically modified crops have been widely cultivated and distributed worldwide as feed and food sources due to advances in modern biotechnology. Recently, 24 countries were planting 189.8 million hectares of GM crops, an increase of 1% over 2016 area. To overcome the agricultural challenges posed by pest emergence and climate change, GM crop developers are stacking traits to provide widespread benefits to producers.

Corn (Zea mays L.), a widely used crop, is a source of food for humans and feed for livestock. GM maize is the world's most grown crop and has been grown commercially on 640 million hectares. To detect and identify GM maize, laboratories are using PCR-based methods, especially real-time PCR (qPCR), and detection methods using qPCR validated by the European Union Laboratories for Genetically Modified Foods and Feeds (EURL-GMFF) are widely used. has been used Although qPCR has many advantages for the validation and quantification of GMO assays, this method also has various disadvantages due to the presence of inhibitors and the high cost of constructing the assay platform.

A broad spectrum of GMO detection can be achieved by targeting universal transgene elements such as p35S and tNOS. Despite the wide application of these detection methods, these methods have limitations in specifying which GMO events a sample is.

To efficiently identify approved GMOs, event-specific PCR methods targeting the unique junction region between the plant genome and the transformation cassette within the GMO genome should be applied. At this time, various advanced PCR methods such as real-time PCR, visualized DNA microarray analysis, fluorescent capillary gel electrophoresis, LAMP PCR, and digital PCR can be applied to identify GM corn.

However, these GMO detection methods differ depending on the equipment, and there are clear limitations such as the use of real-time PCR or digital PCR alone, which requires excessively expensive equipment and reagents to qualitatively detect GMO.

Accordingly, the present inventor has developed an improved multiplex PCR technique for the detection of genetically modified crops such as corn, soybean, and canola and a primer set for PCR amplification, which is 'Development of a detection technique for 6 genetically modified crops approved for new import' as J. Plant Biotechnol (2017) 44: 97-106. In addition, in the above paper, the present inventors have disclosed a multiplex PCR technique for amplifying the DAS-40278-9 event in the case of genetically modified corn.

By further improving the multiplex PCR technique disclosed in the above paper, the present inventors multiplexed four MON87708, DAS-68416-4, DAS-44406-6, MON87769 event sites that are unique junction regions between the maize genome and the transformation cassette. The present invention was completed by developing a multiplex PCR method for simultaneous detection of four types of transgenic maize currently recognized in Korea by amplification.

In addition, in order to confirm the specificity and efficiency of the multiplex PCR method of the present invention, the limit of detection (LOD) was evaluated, a mixed authentication reference material (CRM) was identified, and a GMO monitoring sample was analyzed.

The problem to be solved by the present invention is to further improve the PCR technique to target the unique junction region between the maize genomic DNA and the transgene cassette, resulting in four GMO maize events 5307, DAS-40278-9, MON87460, MON87427 This is to develop a multiplex PCR method that can detect events with a single analysis.

It is an object of the present invention to provide a method for simultaneously detecting genetically modified corn by targeting an event site that is a junction site between corn genomic DNA and a transgene cassette in four types of genetically modified corn, wherein the four types of target sites are 5307 (183 bp), DAS-40278-9 (215 bp), MON87460 (319 bp), MON87427 (363 bp) event sites. It is to provide a method for simultaneous detection of genetically modified maize, characterized in that it is detected later.

In this case, the pair of primers specific to the event site is a forward primer pair of SEQ ID NO: 1 and a reverse primer pair of SEQ ID NO: 2 specific to the event 5307 site, and a forward primer of SEQ ID NO: 3 specific to the event DAS-40278-9 site. and a pair of reverse primers of SEQ ID NO: 4, a forward primer of SEQ ID NO: 5 and a reverse primer pair of SEQ ID NO: 6 specific for the MON87460 site, and a forward primer of SEQ ID NO: 7 and a forward primer of SEQ ID NO: 8 specific for the MON87427 site It is characterized as a reverse primer pair.

In addition, in the method for target site amplification, four primer pairs, EF-Taq PCR pre-mix and genomic DNA are added to the sample, and for preliminary denaturation, 1 cycle at 94°C for 5 minutes, denaturation at 94°C, binding at 59°C It is characterized in that the cycle of 72 °C extension is repeated for 33 cycles, and the target site is amplified with 1 cycle of final extension at 72 °C.

The effect of the present invention is to further improve the PCR technique and amplify the four 5307, DAS-40278-9, MON87460, MON87427 event sites, which are unique junction regions between corn genomic DNA and the transformation cassette, to be used in Korea. To provide a multiplex PCR method for the simultaneous detection of four approved genetically modified maize.

1 is a diagram showing a schematic diagram of a transgene structure and event-specific primers of four types of GM corn. The position of each primer is indicated by an arrow. Structural genes are indicated by open squares and filled pentagons and squares are promoters and terminators. The solid line indicates the sequence of the DNA junction region in maize.
2 is a diagram showing the results of event-specific multiplex PCR for four types of GM corn, and is an agarose gel image of multiplex PCR using each genomic DNA and a multiplex primer mixed reaction solution. Each lane is M: 100 bp DNA size marker, lane 1: 5307, lane 2: DAS-40278-9, lane 3: MON87427, lane 4: MON87460, lane 5: unmodified GM, lane 6: 4 standards extracted DNA shows the results of amplification as a template.
3 is a diagram showing the results of multiplex PCR using four types of unmodified corn DNA and 14 other types of GM corn DNA as templates in order to verify the detection specificity of the multiplex PCR method of the present invention. PCR template DNA was indicated at the top of the gel image. 4 positive controls; 5307, DAS-40278-9, MON87460, MON87427; Unmodified maize genomic DNA: 0411-C2, ERM-BF-433a, 0406-A, 0407-A; 14 GM maize event genomic DNA: BT11, NK603, GA21, MON863, 59122, MIR604, MON88017, 98410, MON89034, BT176, MIR162, 1507, 3272, VCO01981 and Adhl genes were used as PCR positive controls. M represents a 100 bp DNA size marker.
4 is a diagram showing the limit of detection (LOD) of multiplex PCR. The results of multiplex PCR using 4 types of mixed standard genomic DNA as a template are shown by diluting them by concentration. Dilution concentrations are indicated at the top of the gel image (100, 50, 25, 12.5, 6.6, 3.1, 1.6 and 0.8 ng/ul DNA, respectively). M represents a 100 bp DNA size marker.
5 is a diagram showing the efficiency test of multiplex PCR using a random standard genomic DNA mixture. The total genomic DNA concentration used for each test was 50 ng/ul for each multiplex PCR reaction. Each lane represents a random mix of lane 1: unmodified GM genomic DNA (negative control), lanes 2-16: GM maize standard genomic DNA. M represents a 100 bp DNA size marker.

Advances in biotechnology have led to advances in crop genetic engineering with the goal of improving agricultural productivity. The application of genetically modified (GM) crops has increased, which has led to increased consumer and regulatory concerns about the safety of GM crops for human health and biodiversity.

The labeling of GM crops is therefore essential and its detection and identification is an important issue for developers and distributors. A multiplex PCR technique has been developed and validated for use in the detection, identification and screening of GM crops in quarantine. Recently, progenitor hybrid GM crops made by crossing multiple events have been developed. Therefore, efforts to identify GM crops with a simple detection technique are required.

Therefore, the present inventors established a simultaneous detection method to simultaneously identify four GM maize traits.

In order to develop a method capable of simultaneously detecting four GM maize events, 5307, DAS-40278-9, MON87460 and MON87427, the specific primers of the present invention were prepared by combining four transgene cassettes and each maize genomic DNA. It was designed based on PCR that can specifically amplify the junction region.

To confirm the efficiency and accuracy of multiplex PCR, specificity analysis, detection limit evaluation, and hybrid authentication reference material identification were performed. To analyze the genetically modified maize, a multiplex PCR technique was performed to identify events, and the results were confirmed by simple PCR using each trait-specific primer.

(1) establishment of multiplex PCR

Although GM crops are grown worldwide, and despite several scientific reports on the risk assessment of GMOs, GMOs have been approved for use as food and feed and for processing due to safety concerns for human health and the environment, although some countries have grown GMO crops. is prohibited According to these concerns, the identification of GMOs is also an important issue at the regulatory level and for consumers. The GM crop detection strategy is divided into two methods: inserted gene detection (DNA-based) and expressed protein detection (protein-based).

Since GMO raw materials and processed products contain the introduced gene fragment, the inserted DNA can be detected and identified. Although these DNA-based methods are generally sensitive, specific, and widely applicable, they require long analysis times and special equipment. Protein-based methods are less sensitive than DNA-based methods and may not be suitable for samples containing denatured proteins, such as processed products. Therefore, a high-throughput DNA-based detection method has recently been developed and applied.

Among them, the multiplex PCR technique, which amplifies multiple DNA targets in a single reaction, is the optimal method for analyzing and screening multiple GM crop samples. Therefore, a multiplex PCR technique for detecting genetically modified gene sites in four types of GM corn can be easily applied to the identification analysis of GM corn at low cost.

In addition, to identify the GM transgene, specific primers for each event were designed by targeting the transformed cassette and the junction sequence adjacent to the crop genome gene (Fig. 1, Table 2). This event-specific multiplex PCR method can detect and identify GMOs in one reaction. For simultaneous detection, multiplex tandem PCR for 35S promoter or T-NOS terminator has been developed and used to screen GM maize, but this method could not be applied to the identification of stacked progenitor GM events due to duplication of gene cassettes.

In the present invention, using event-specific PCR primers, a multiplex PCR method was developed to identify four types of events in GM corn, MON87708, DAS-68416-4, DAS-44406-6, MON87769, and simultaneous detection was effective in PCR. made it possible to increase This newly developed multiplex PCR method is suitable for qualitative analysis of GM corn ( FIG. 2 ).

The development of multiplex PCR is more complex than simple PCR, and to design multiplex assays, many PCR conditions, including the concentration of each primer and reaction component (eg MgCl ₂ ) and annealing temperature, are adjusted to combine all DNA targets within a single reaction tube. should be amplified at the same time. To facilitate the preparation of the PCR reaction mixture, a master mix with a single concentration of PCR primers (0.16 μM) was used, differentiated from the conventional simultaneous detection method, and general PCR conditions (step 3) to simplify the multiplex PCR conditions. ; denaturation-binding-synthesis) was performed in three steps.

The data analysis method is simple by isolating the PCR product on a 2.5% single agarose gel (Fig. 2). These results are applicable to the multiplex PCR method of the present invention to detect GMO in samples in all laboratories with general PCR equipment for the purpose of GMO qualitative testing.

(2) specificity

In order to confirm the specificity of the multiplex PCR method of the present invention, including four unmodified GMO Certified Reference Material (CRM) and four corn CRM (5307, DAS-40278-9, MON87460 and MON87427) Eighteen GM CRMs were tested. In order to evaluate the reliability, four types of corn CRM, a negative control (NTC; No Template Control) and a positive control, were tested.

All four positive control CRMs were amplified successfully while the other 18 maize GM CRMs, including NTC and four unmodified GMO CRMs, did not. These results confirmed the specificity of 4 sets of multiplex PCR primers for amplifying 5307, DAS-40278-9, MON87460 and MON87427 event sites, indicating that the multiplex PCR method can be applied to the analysis of GM corn samples.

From IRMM and AOCS, CRMs with various GMO contents from unmodified GMO to 100% GMO are provided, and each purity is prepared by mixing GMO CRM with unmodified GM CRM. GMO CRM with different mixing ratios is a very important CRM used in quantitative as well as qualitative analysis, and such purity is an important factor in the development of a simultaneous detection method. In the present invention, 10-100% pure (95% confidence level) GM maize CRM (Table 1) was used, and the efficiency of PCR amplification was different for each CRM template.

Compared with the DAS-40278-9 genomic DNA template (10% purity), 5307, MON87460 and MON87427 show stronger amplification efficiency due to the 100% purification purity of CRM (Fig. 2). These varying DNA purities can affect the uneven amplification of multiplex PCR.

Most GM maize samples show an increased DNA copy number of each sample, at least as homozygotes or heterozygotes. Multiplex PCR also produced weak and faint bands (Figure 3). However, due to differences in amplification and amplicon size, these were non-specific bands. These results indicate that the amplification efficiency and non-specific amplification did not affect the PCR results.

(3) Limit of detection (LOD)

Validation of the developed GMO co-detection method is very complex and depends on the availability, type and quality of template DNA. In general, the quantity and quality of genomic DNA obtained from GMO monitoring samples are often not sufficient for analysis. Therefore, the efficiency of the multiplex PCR method developed by limit of detection (LOD) and random CRM mixture analysis was confirmed. As a result of analyzing the serially diluted CRM template DNA mixture by the developed simultaneous detection method, it was confirmed that the PCR band could be detected up to a DNA concentration of 25 ng/μl (FIG. 4).

Random CRM mixture analysis also verified that the multiplex PCR method can detect progenitor GM maize events without non-specific amplification ( FIG. 5 ). These results demonstrated that this co-detection method was effective for the analysis and identification of four types of GM corn by corn multiplex PCR.

(4) Application of multiplex PCR

To apply the corn multiplex PCR technique of the present invention, samples collected from the GMO monitoring project from 2018 to 2019 in Korea were analyzed. Most of the GM maize runoff samples were found primarily in the main transportation routes of food and feed plants or at the final point of consumption. To confirm GM corn, the present inventors analyzed 29 samples of GM corn samples using multiplex PCR, and verified the results using single detection PCR for each event in order to double-check the results.

As a result, multiplex and simple PCR did not detect positivity in 29 GM maize samples. These data imply that 29 GM maize samples did not contain genetic material from 4 GM maize in the stacked trait. However, despite these results, LOD and random CRM mixture analyzes suggest that the multiplex PCR method for event 5307, DAS-40278-9, MON87460 and MON87427 in maize can be used for GM maize identification.

GM corn was approved and imported as feed and food. Appropriate detection methods must be developed to detect and identify newly approved GM maize incidents. The results of the present invention showed that the newly developed maize multiplex PCR method was successfully confirmed for 4 GM maize.

Specificity analysis, LOD and random CRM mixture analysis were performed to validate the multiplex PCR technique. From 2018 to 2019, 29 kinds of GM corn samples were analyzed using multiplex PCR to apply the multiplex PCR technique for domestic GM corn samples. All these results indicate that the newly developed multiplex PCR technique is suitable for the analysis of GM maize.

Hereinafter, the present invention will be described in more detail through examples. However, the scope of the present invention is not limited to the following examples.

(Example 1) Extraction of plant material and DNA

GM maize (Table 1) CRM was obtained from the Institute for Reference Material and Measurement (IRMM, Geel, Belgium) and the American Oil Chemists' Society (AOCS, Urbana, Illinois, USA). 29 GM corn samples were collected from 2018 to 2019 in Korea, dried with silica gel after sampling and stored frozen until gene extraction. Genomic DNA was extracted from the samples using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The extracted genomic DNA concentration was quantified with a spectrophotometer ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA). For multiplex PCR analysis, the final DNA concentration was diluted to 50 ng/μl and stored frozen at -20°C until use.

(Example 2) Primer design

As a positive control for all PCRs, alcohol dehydrogenase 1 (Adh1, GenBank accession number AF123535), an endogenous maize gene, was used. To design four pairs of GM maize event-specific primers, genetic information of GM events was obtained from the Joint Research Center-European Commission (JRC-EC) and the Center for Environmental Risk Assessment (CERA).

The primers shown in Table 2 were synthesized by Macrogen (Seoul, Korea), and then diluted with sterile water to 100 pmol/l and used.

(Example 3) PCR conditions and detection of amplification products

PCR conditions were as follows: 1 cycle at 94 °C for 5 min for pre-denaturation, 33 cycles at 94 °C for denaturation, 30 s at 59 °C for primer annealing, 1 min at 72 °C for tensile, and final extension. for 7 min at 72 °C. 2x EF-Taq PCR Pre-Mix (Solgent, Seoul, Korea) and 50 ng/μl genomic cDNA and 0.16 μM of each primer were used in 30 μl final PCR reaction volume. Gel electrophoresis was performed on a 2.5% agarose gel run at 135V for 25 min, and gel images were captured using ChemiDoc™ XRS + (Bio-Rad, Hercules, CA, USA).

(Example 4) Specificity, Sensitivity and GM Sample Analysis

To determine the specificity of the multiplex PCR method, we used genomic DNA extracted from four unmodified GM corn samples and 18 GM corn samples, including 5307, DAS-40278-9, MON87460 and MON87427. LOD and random mixed sample DNA were analyzed to investigate the efficiency of maize multiplex PCR.

For LOD analysis, four mixed maize CRM genomic DNAs were serially diluted 2-fold with distilled water to concentrations of 50, 25, 12.5, 6.6, 3.1, 1.6, 0.8 and 0 ng/μl. A random mixture of CRM was diluted with unmodified GM maize genomic DNA to control the total DNA used in the reaction. In order to apply the corn multiplex PCR method to GMO monitoring sample analysis, multiplex PCR was performed using 29 GM corn samples collected in Korea from 2018 to 2019.

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Claims (3)

A method for simultaneously detecting genetically modified corn by targeting an event site that is a junction site of four types of corn genomic DNA and a transgene cassette in the genetically modified corn,
The four target sites are 5307 (183 bp), DAS-40278-9 (215 bp), MON87460 (319 bp), and MON87427 (363 bp) event sites, using primer pairs specific for each event site. The target site is amplified, and it is detected after electrophoresis,
The event site-specific primer pair includes a forward primer pair of SEQ ID NO: 1 and a reverse primer pair of SEQ ID NO: 2 specific for the event 5307 site, a forward primer pair of SEQ ID NO: 3 specific for the event DAS-40278-9 site, and A pair of reverse primers of SEQ ID NO: 4, a forward primer of SEQ ID NO: 5 and a reverse primer pair of SEQ ID NO: 6 specific for the event MON87460 site and a forward primer of SEQ ID NO: 7 and a forward primer of SEQ ID NO: 8 specific for the event MON87427 site reverse primer pair of
Event site-specific simultaneous detection method of genetically modified corn, characterized in that
delete The method of claim 1, wherein the method for target site amplification includes adding four primer pairs, EF-Taq PCR pre-mix, and genomic DNA to the sample, and for preliminary denaturation, 1 cycle at 94°C for 5 minutes, 94°C A method for simultaneous detection of genetically modified corn, comprising repeating 33 cycles of denaturation, binding at 59°C, and elongation at 72°C, and amplifying the target site with one cycle of final extension at 72°C.

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