• 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.

• Fisheries and Aquatic Sciences
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• v.15 no.2
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• pp.157-162
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• 2012

To analyze nucleotide sequence encoding internal transcribed spacer (ITS) regions specific to the Laminariaceae family, genomic DNA was isolated from six brown algae species distributed along the east coast of Korea. These included three species from the Laminariaceae family (Agarum clathratum Dumortier, Costaria costata [C. Agardh] Saunders, and Saccharina japonica Areschoug) and two species from the Alariaceae family (Undaria pinnatifida [Harvey] Suringer and Ecklonia cava Kjellman), both in the order Laminariales, and one species from the family Sargassaceae in the order Fucales (Sargassum serratifolium). Based on a sequence analysis of ITS-1 and ITS-2 for A. clathratum, C. costata, and E. cava, oligonucleotides were designed from the regions that showed sequence conservation in Laminariaceae. Following polymerase chain reaction using three sets of primers, amplification of ITS-1 and ITS-2 was detected in reactions using genomic DNA isolated from the species belonging to Laminariaceae, but not from the species belonging to the other families. The results indicate that this method can be used for the detection and identification of Laminariaceae species.

• Journal of the korean academy of Pediatric Dentistry
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• v.32 no.1
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• pp.75-88
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• 2005

Ameloblastoma is the most commonly occurring odontogenic tumor in oral cavity. Although most are benign epithelial neoplasm, they are generally considered to be locally aggressive and destructive, exhibiting a high rate of recurrence. The biological behavior of this neoplasm is a slowly growing, locally invasive tumor without metastasis, therefore malignant neoplasm, changed its histological appearance to carcinoma or showed distant metastasis, is only defined clinically. In this study, we identified the differentially expressed genes(DEGs) in stages under benign or malignant ameloblastoma compared with normal patient using ordered differential display(ODD) reverse transcription(RT)-PCR and $GeneFishing^{TM}$ technology. ODD RT-PCR is rather effective when the investigation of samples containing very small amounts of total RNA must be accomplished. ODD RT-PCR used the means of amplification with anchored T-primer and adaptor specific primer. bearing definite two bases at their 3' ends and so this method could display differential 3'-expressed sequence taqs(ESTs) patterns without using full-length cDNAs. Compared with standard differential display, ODD RT-PCR is more simple and have enough sensitivity to search for molecular markers by comparing gene expression profiles, However, this method required much effort and skill to perform. $GeneFishing^{TM}$ modified from DD-PCR is an improved method for detecting differentially expressed genes in two or more related samples. This two step RT-PCR method uses a constant reverse primer(anchor ACP-T) to prime the RT reaction and arbitrary primer pairs(annealing control primers, ACPs) during PCR. Because of high annealing specificity of ACPs than ODD RT-PCR, the application of $GeneFishing^{TM}$ to DEG discovery generates reproducible, authentic, and long(100bp to 2kb) PCR products that are detectable on agarose gels. Consequently, various DEGs observed differential expression levels on agarose gels were isolated from normal, benign, and malignant tissues using these methods. The expression patterns of the some isolated DEGs through ODD RT-PCR and $GeneFishing^{TM}$ were confirmed by Northern blot analysis and RT-PCR. The results showed that these identified DEGs were implicated in ameloblastoma neoplasm processes. Therefore, the identified DEGs will be further studied in order to be applied in candidate selection for marker as an early diagnosis during ameloblastoma neoplasm processes.

• Journal of Plant Biotechnology
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• v.45 no.1
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• pp.63-70
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• 2018

Brazzein is the smallest sweet protein and was isolated from the fruit pulp of Pentadiplandra brazzeana Baillon, native to tropical Africa. From ancient times, the indigenous people used this fruit in their diet to add sweetness to their daily food. Brazzein is 500 to 2000 times sweeter than sucrose on a weight basis and 9500 times sweeter on a molar basis. This unique property has led to increasing interest in this protein. However, it is expensive and difficult to produce brazzein other than in its native growing conditions which limits its availability for use as a food additive. In this study, we report high production yields of, brazzein protein in transgenic rice plants. An ORF region encoding brazzein and driven by the $2{\times}CaMV\;35S$ promoter was introduced into rice genome (Oryza sativa Japonica) via Agrobacterium-mediated transformation. After transformation, 17 regenerated plant lines were obtained and these transgene-containing plants were confirmed by PCR analysis. In addition, the selected plant lines were analyzed by Taqman PCR and results showed that 9 T0 lines were found to have a single copy out of 17 transgenic plants. Moreover, high and genetically stable expression of brazzein was confirmed by western blot analysis. These results demonstrate that recombinant brazzein was efficiently expressed in transgenic rice plants, and that we have developed a new rice variety with a natural sweetener.

• Journal of Plant Biotechnology
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• v.48 no.1
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• pp.18-25
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• 2021

Solanum demissum is one of the wild Solanum species originating from Mexico. It has wildly been used for potato breeding due to its resistance to Phytophthora infestans. S. demissum has an EBN value of four, which is same as that of S. tuberosum, so that it is directly crossable for breeding purposes with the cultivated tetraploid potato (S. tuberosum). In this study, the chloroplast genome sequence of S. demissum obtained by next-generation sequencing technology was described and compared with those of seven other Solanum species to develop S. demissum-specific markers. Thetotal sequence length of the chloroplast genome is 155,558 bp, and its structural organization is similar to those of other Solanum species. Phylogenetic analysis with ten other Solanaceae species revealed that S. demissum is most closely grouped with S. hougasii and S. stoloniferum followed by S. berthaultii and S. tuberosum. Additional comparison of the chloroplast genome sequence with those of seven other Solanum species revealed two InDels specific to S. demissum. Based on these InDels, two PCR-based markers for discriminating S. demissum from other Solanum species were developed. The results obtained in this study will provide an opportunity to investigate more detailed evolutionary and breeding aspects in Solanum species.

• Korean Journal of Food Science and Technology
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• v.40 no.1
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• pp.101-105
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• 2008

Enterobacter sakazakii is implicated in severe forms of neonatal infections such as meningitis and sepsis. This organism has been isolated from a wide range of foods, including cheese, vegetables, grains, herbs, and spices, but its primary environment is still unknown. Generally, dried infant milk formula has been epidemiologically identified as the source of E. sakazakii. Sunsik (a powdered mixture of roasted grains and other foodstuffs) is widely consumed in Korea as a side dish or energy supplement. Sunsik is consumed without heat treatment; thus, lacking an additional opportunity to inactivate foodborne pathogens. Therefore, its microbiological safety should be guaranteed. In this study, the prevalence of E. sakazakii was monitored in 23 different sunsik component flours, using FDA recommended methods; but E. sakazakii medium (Neogen) and Chromogenic E. sakazakii medium (Oxoid) were used as the selective media. In total, presumptive E. sakazakii strains were isolated from 8 different sunsik powders. Subsequently, an API 20E test was conducted, and 15 strains from 5 different sunsik flours (sea tangle, brown rice, non-glutinous rice, cheonggukjang, dried anchovy) were confirmed as E. sakazakii. Fifteen strains were again confirmed by PCR amplification, using three different primer sets (tDNA sequence, ITS sequence, 16S rRNA sequence), and compared to ATCC strains (12868, 29004, 29544, 51329). They were once again confirmed by their enzyme production profiles using an API ZYM kit. Finally, RAPD (random amplified polymorphic DNA)-genotyping was carried out as a monitoring tool to determine the contamination route of E. sakazakii during processing.

• Journal of Veterinary Clinics
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• v.39 no.5
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• pp.207-216
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• 2022

Canine babesiosis has been scarcely investigated in the Republic of Korea (ROK). Although it is known that Babesia gibsoni is its primary causative agent, its clinical presentation has not been completely clarified in the ROK. Consequently, the aim of this study was to evaluate the clinical appearance of this parasitic infection based on the anamnesis of the patient and compare of hematological and biochemical test results. Four hundred whole blood samples from patients with a presumptive diagnosis of tick-borne disease were analyzed by polymerase chain reaction (PCR) to amplify the Babesia spp. 18S rRNA gene and by a rapid diagnostic test kit (VetAll Laboratories^®) to detect B. gibsoni seroreactive animals. Thirty-six (9.0%) dogs were PCR-positive but only 24 (6.0%) were seropositive. The investigation revealed that all the courses of the disease are present in the ROK, with the acute course being predominant. The acute course tends to consist of inappetence, lethargy, pyrexia, gastrointestinal symptoms, and occasionally hematuria. It also occurs with common hematological abnormalities, such as thrombocytopenia and anemia, and to a lesser extent biochemical abnormalities, such as hyperbilirubinemia, hypoalbuminemia, and elevated liver enzymes. This research shows that B. gibsoni is an endemic hemoparasite capable of producing a variety of clinical manifestations in dogs. For its accurate diagnosis, a descriptive history of the clinical signs, hematology, and biochemical profile of the patient, along with a well-performing PCR assay, are essential. These findings will help in planning pragmatic preventive strategies against this potent threat in the ROK.

• Korean Journal of Poultry Science
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• v.51 no.2
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• pp.117-125
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• 2024

This study aimed to find genetic markers for breed-independent identification of early- and late-feathering chickens. We explored the novel sequences of the ev21-K locus associated with late-feathering and investigated its characterization. Additionally, the genetic transmission pattern of the identified sequences were investigated to understand its potential application in auto-sexing lines. A total of 707 chickens from 5 chicken breeds were employed for the study. The ev21-K locus was identified through a comparative analysis of the ev21 gene and the K gene related to feather development. For analysis of identified loci, specific primers for the target sequences were prepared and polymerase chain reaction (PCR) was performed to obtain the products, and then their nucleotide sequences were analyzed. Crossbreeding tests of early-feathering and late-feathering chickens were conducted to examine the genetic transmission patterns of the identified sequences. The results showed that the identified 230 bp ev21-K locus, which named as ev21-related K specific sequences were 99% homology with the ev21 gene. PCR analysis confirmed its presence exclusively in late-feathering chickens. Comparative analyses across tissues, breeds, and ages demonstrated the sequences consistency in identifying late-feathering chickens. Genetic transmission patterns were investigated through crossbreeding tests, revealing sex-linked inheritance and consistent segregation with feathering phenotypes. The inheritance patterns of the ev21-related K specific sequences demonstrated that this locus follows the typical Mendelian inheritance pattern as a dominant gene. In conclusion, the novel sequences of ev21-K locus were a reliable molecular marker for identifying early- and late-feathering chickens across breeds.

• Parasites, Hosts and Diseases
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• v.52 no.1
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• pp.9-19
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• 2014

Anisakis spp. (Nematoda: Anisakidae) parasitize a wide range of marine animals, mammals serving as the definitive host and different fish species as intermediate or paratenic hosts. In this study, 18 fish species were investigated for Anisakis infection. Katsuwonus pelamis, Euthynnus affinis, Caranx sp., and Auxis thazard were infected with high prevalence of Anisakis type I, while Cephalopholis cyanostigma and Rastrelliger kanagurta revealed low prevalence. The mean intensity of Anisakis larvae in K. pelamis and A. thazard was 49.7 and 5.6, respectively. A total of 73 Anisakis type I larvae collected from K. pelamis and A. thazard were all identified as Anisakis typica by PCR-RFLP analysis. Five specimens of Anisakis from K. pelamis and 15 specimens from A. thazard were sequenced using ITS1-5.8S-ITS2 region and 6 specimens from A. thazard and 4 specimens from K. pelamis were sequenced in mtDNA cox2 region. Alignments of the samples in the ITS region showed 2 patterns of nucleotides. The first pattern (genotype) of Anisakis from A. thazard had 100% similarity with adult A. typica from dolphins from USA, whereas the second genotype from A. thazard and K. pelamis had 4 base pairs different in ITS1 region with adult A. typica from USA. In the mtDNA cox2 regions, Anisakis type I specimens from A. thazard and K. pelamis showed similarity range from 94% to 99% with A. typica AB517571/DQ116427. The difference of 4 bp nucleotides in ITS1 regions and divergence into 2 subgroups in mtDNA cox2 indicating the existence of A. typica sibling species in the Makassar Strait.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1999.10a
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• pp.59.2-59
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• 1999