• Applied Biological Chemistry
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• v.49 no.3
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• pp.192-195
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• 2006

For the development of a qualitative PCR detection method for genetically modified perilla (Perilla frutescens), perilla species-specific gene, KAS-I (Beta-ketoacyl-ACP synthase I), was selected and validated as suitable for the use as an endogenous reference gene in perilla. Primer specificity was first tested by the means of qualitative PCR analysis. The primer pair Pfru3-F/R amplifying the perilla endogenous gene, KAS-I, gave rise to an amplicon 95 bp. No amplified product was observed when DNA samples from 15 different plants were used as templates. Qualitative PCR detection method was assayed with vitamin E-enriched GM Perilla developed in Korea. For the qualitative PCR detection method, the construct-specific detection primer pairs were constructed. The primer pair TMTO-F/R amplifying the junction region of TMT (${\gamma}$-tocopherol methyltransferase) gene and OCS (Octopine synthase) terminator introduced in GM perilla gave rise to an amplicon 148 bp.

• Applied Biological Chemistry
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• v.48 no.4
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• pp.335-338
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• 2005

For the development of qualitative PCR detection method of genetically modified rice (Oryza sativa L.), rice species specific gene, SAMDC1 (S-adenosylmethionine decarboxylase), was selected and validated as suitable for use as an endogenous reference gene in rice. The primer pair OsSAMDC1-5'/3' with 110 bp amplicon was used for amplification of the rice endogenous gene, SAMDC1 and no amplified product was observed from 19 different plants as templates. Qualitative PCR method was assayed with 2 different GM rices (Milyang 204 and Iksan 483) developed in Korea. For the qualitative PCRs, the construct-specific detection primer pairs were constructed. Os204-5'/OsNOS-3' amplifying the junction region of GUS gene and NOS terminator introduced in Milyang 204 gave rise to an amplicon 172 bp; also, Os483-5'/OsNOS-3' amplifying the junction region of Bar gene and NOS terminator introduced in Iksan 483 gave rise to an amplicon 161 bp.

• Journal of Applied Biological Chemistry
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• v.52 no.1
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• pp.1-7
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• 2009

For the development of qualitative PCR detection method of genetically modified (CM) rice, rice species-specific gene, OsCc-1 (rice cytochrome c gene), was selected as suitable far use as an endogenous gene in rice. The primer pair OsCytC-5'/3'with 111 bp amplicon was used for PCR amplification of the rice endogenous gene, OsCc-1 and no amplified product was observed from 8 different crops as templates. Qualitative PCR method was carried out with stack traits of L528$\times$CryIAc1 GM rice developed in Korea. For the qualitative PCRs, some primer pairs were designed with a construct-specific and event-specific type based on T-DNA and junction sequences of T-DNA in GM rice. Actck-5'/3' amplifying between actin promoter and OsCK1 gene introduced in LS28 gave rise to an amplicon 306 bp; also, CrLB-5'/3' from CryIAcl and CKRB-5'/3'amplifying the junction region of T-DNA and genome sequence from LS28 as event-specific primers gave rise to an amplicon 142 bp and 91 bp, respectively. These primer pairs for the detection of event-specific targets not produced PCR amplicons on non-CM rice and various crops in contrast to event lines. Therefore, in this study we verified that event-specific primers were effective to specifically detect stack trait lines and demonstrated that this method presented could be provided with the detection-method data for risk assessment analysis of GM rice to be commercialized.

• Journal of Food Hygiene and Safety
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• v.25 no.3
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• pp.278-280
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• 2010

This study was aimed to develop a novel qualitative multiplex polymerase chain reaction (PCR) for simultaneous detection of genetically modified (GM) soy and maize within a single reaction. The specific primers designed to detect four respective GM events (A2704-12, MON88017, Bt11, and MON863) were included in the tetraplex PCR system. Each of PCR products for four GM events could be distinguished by agarose gel based on their different lengths. The specificity and reproducibility of this multiplex PCR were evaluated. This multiplex PCR consistently amplified only a fragment corresponding to a specific inserted gene in each of the four GM events and also amplified all four of the PCR products in the simulated GM mixture. These results indicate that this multiplex PCR method could be an effective qualitative detection method for screening GM soy and maize in a single reaction.

• Journal of Plant Biotechnology
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• v.40 no.1
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• pp.18-26
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• 2013

Genetically modified (GM) rice Agb0101, which expresses the insecticidal toxin modified cry1Ac (mcry1Ac1) gene, was developed by the Rural Development Administration in Korea. To monitor the probable release of Agb0101 in the future, it is necessary to develop a reliable detection method. Here, we developed the PCR detection method for monitoring and tracing of GM rice. The primer pair (RBEgh-1/-2) from a starch branching enzyme (RBE4) gene was designed as an endogenous reference, giving rise to an expected PCR amplicon of 101 bp. For the qualitative PCR detection, construct- and event-specific primers were designed on the basis of integration sequence of T-DNA. Event-specific PCRs amplified specifically 5'- or 3'-junction region spanning the native genome DNA and the integrated gene construct, while none of amplified product was shown on crops, rice varieties, and other insect-resistant transgenic rice lines. The event-specific real-time PCR method was performed using TaqMan probe and plasmid pRBECrR containing both rice endogenous gene RBE4 sequence and 5'-junction sequence as the reference molecule. The absolute limit of quantification (LOQ) of real-time PCR was established with around 10 copies for one plasmid molecule pRBECrR. Thereafter, the different amounts of transgenic rice (1, 3, 5, and 10%, respectively) were quantified by using the established real-time PCR method, with a range below 19.55% of the accuracy expressed as bias, 0.06-0.40 of standard deviation (SD) and 3.80-7.01% of relative standard deviations (RSD), respectively. These results indicate that the qualitative and quantitative PCR methods could be used effectively to detect the event Agb0101 in monitoring and traceability.

• Journal of Microbiology and Biotechnology
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• v.17 no.2
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• pp.335-341
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• 2007

For the development of qualitative and quantitative PCR methods of genetically modified (GM) pepper developed in Korea, a capsanthin-capsorubin synthase (CCS) gene was used as the endogenous reference gene. The primer pair ccs-F/R amplifying the pepper endogenous gene gave rise to an amplicon of 102 bp. No amplified product was observed when DNA samples from 16 different plants were used as templates. The construct-specific primer pairs amplifying the junction region of the bar gene and Ti7 introduced in GM pepper gave rise to an amplicon of 182 bp. Quantitative PCR assay was performed using a TaqMan probe and a standard plasmid as a reference molecule, which contained both an endogenous and event-specific sequence. For the validation of this method, the test samples containing 0.1, 1, 3, 5, and 10% GM pepper were quantified.

• Journal of Life Science
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• v.16 no.5
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• pp.806-811
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• 2006

The regulation of labelling criterion for genetically modified (GM) foods has been enforced since 2001 in Korea. Therefore, GM soybean (GMS) or GM maize (GMM) processed foods must be labeled as GMO derived. We surveyed to see whether this regulation is kept relevantly or not and the distributive statue of GM processed foods. Using the method of polymerase chain reaction (PCR) based on endogenous gene (Le1n, SSIIb), promoter gene (P35S), terminator gene (NOS) and transgenic gene (RRS, Bt11, Bt176, GA21, T25, Mon810), we detected GMS and GMM processed foods circulating at the market in Busan area. Out of total 100 samples, 38 items were showed to be contaminated with recombinant gene by qualitative PCR. Among 82 domestic and 18 imported items, 32 (39.0%) and 6 (33.3%) items were detected with GM ingredients respectively. Also among the 80 soybean and 20 maize processed foods, 23 (28.7%) and 15 (75.0%) foods were sensitive to detect GMS and GMM ingredients respectively. For the qualitative PCR positive foods, we chased identity preservation (IP) certificates. And we verified that the PCR positive crops were grown up, harvested and shipped separately from GMO but just mixed with GMO in the threshold of the non attentional contamination levels (3%). Thus we can not find out any regulation-violent case at all. The results of this study will help to keep the regulations of GM labelling and be informative to consumers who want to know the laboratory results of GMO testing.

• Korean Journal of Microbiology
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• v.49 no.3
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• pp.292-296
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• 2013

This study utilized an analysis method for detecting six microorganisms, such as Actinobacillus actinomycetemcomitans, Campylobacter rectus, Porphyromonas gingivalis, Tannerella forsythus, Treponema denticola, and Prevotella intermedia, triggering periodontal disease, using multiplex real-time polymerase chain reaction (PCR). The analysis including internal control was made by dividing the six species into two groups using four fluorescence dyes, and it was verified that there was no interference or cross-reaction between the target species and different kinds of oral microbial species. Qualitative and quantitative analyses were conducted on each microorganism in various samples, such as saliva and the plaque, using the multiplex real-time PCR and comparative analysis between periodontitis patients and healthy people, revealing obvious differences between them.

• Journal of Applied Biological Chemistry
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• v.49 no.4
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• pp.143-147
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• 2006

A rapid PCR-based assay for detecting hepatitis B viral DNA(HBV DNA) in serum and plasma was developed using a new PCR instrument named GenSpector(TMC-1000, Samsung electronics). PCR was carried out using a chip-based platform, which enabled 50 PCR cycles with internal controls, and melting-curve analysis in 30 minutes. Verification of the amplified HBV DNA product and the internal control was based on specific melting temperatures(Tm) analysis, executed by the GenSpector software. Primers were designed within the region conserved through HBV genotypes A to F. The lower limit of detection was 840 copies/ml serum, conducted with serial dilutions of a HBV DNA positive control(ACCURUN 325 series 700, Boston Biomedica Inc.). The assay was also compared to another assay for HBV DNA(Versant HBV DNA 3.0 assay, Bayer HealthCare) for 200 samples(each 100 clinical negative and positive samples). The sensitivity and specificity were 100% matched. This rapid PCR-based assay is specific, reproducible, and enables qualitative detection of HBV DNA.

• Applied Biological Chemistry
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• v.45 no.4
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• pp.185-189
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• 2002

Along with the worldwide rapid increase of the cultivation area and commercial production of genetically modified (GM) crops, the amount of GM grains imported to Korea has also been increasing. Roundup-Ready soybean (RRS) was introduced with 5-enolpyruvyl shikimate-3-photphate synthase (EPSPS) gene derived from Agrobacterium CP4 to confer the resistance to herbicide, glyphosate. In this study, we tried to develop PCR-based analytical method to detection the presence of RRS among non-GM soybeans. In order to detect RRS specifically, oligonucleotide primers were specifically designed based on the nucleotide sequence of EPSPS transgene. Qualitative PCR method was established and its specificity and accuracy were confirmed by analysing the nucleotide sequence of PCR DNA fragments. Bioassay was also conducted by spraying glyphosate at seedling stage. Survived individuals showed obvious resistance to Roundup Ready, however all of non-GM seedlings died in two weeks after spray. Conclusively, the highly selective detection systems for RRS were successfully established by both PCR using specific primers to EPSPS transgene and bioassay using the herbicide resistance of RRS. In addition to, the imported soybean showed to be mixed to several varieties regarding to 100-seed weight and hilum color.