• The Korea Journal of Herbology
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• 제34권6호
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• pp.91-97
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• 2019

Objective : To establish a reliable tool between for the distinction of original plants of Sanguisorbae Radix, we analyzed the complete chloroplast genome sequence of Sanguisorbae Radix and identified single nucleotide polymorphisms (SNPs). Materials and methods : The chloroplast genome sequence of Sanguisorba officinalis, Sanguisorba tenuifolia f. alba (Trautv. & Mey.) Kitam and Sanguisorba tenuifolia Fisch. ex Link obtained using next-generation sequencing technology were described and compared with those of other species to develop specific markers. Candidate genetic markers were identified to distinguish species from the chloroplast sequences of each species using Modified Phred Phrap Consed and CLC Genomics Workbench programs. Results : The structure of the chloroplast genome of each sample that had been assembled and verified was circular, and the length was about 155 kbp. Through comparative analysis of the chloroplast sequences, we found 220 nucleotides, 158 SNPs, and 62 Indel (insertion and/or deletion), to distinguish Sanguisorba officinalis, Sanguisorba tenuifolia f. alba (Trautv. & Mey.) Kitam and Sanguisorba tenuifolia Fisch. ex Link. Finally, 15 specific SNP genetic markers were selected for the verification at positions. Avaliable primers for the dried herb, which is used as medicine, were used to develop the PCR amplification product of Sanguisorbae Radix to assess the applicability of PCR analysis. Conclusion : In this study, we found that Fendel-qPCR analysis based on the chloroplast DNA sequences can be an efficient tool for discrimination of Sanguisorba officinalis, Sanguisorba tenuifolia f. alba (Trautv. & Mey.) Kitam and Sanguisorba tenuifolia Fisch. ex Link.

• Journal of Genetic Medicine
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• 제19권2호
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• pp.63-75
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• 2022

Purpose: Osteoporosis is a common calcium and metabolic skeletal disease which is characterized by decreased bone mass, microarchitectural deterioration of bone tissue and impaired bone strength, thereby leading to enhanced risk of bone fragility. In this study, we aimed to identify novel genes for susceptibility to osteoporosis and/or bone density. Materials and Methods: To identify differentially expressed genes (DEGs) between control and osteoporosis-induced cells, annealing control primer-based differential display reverse-transcription polymerase chain reaction (RT-PCR) was carried out in pre-osteoblast MC3T3-E1 cells. Expression levels of the identified DEGs were evaluated by quantitative RT-PCR. Association studies for the quantitative bone density analysis and osteoporosis case-control analysis of single nucleotide polymorphism (SNPs) were performed in Korean women (3,570 subjects) from the Korean Association REsource (KARE) study cohort. Results: Comparison analysis of expression levels of the identified DEGs by quantitative RT-PCR found seven genes, Anxa6, Col5a1, Col6a2, Eno1, Myof, Nfib, and Scara5, that showed significantly different expression between the dexamethason-treated and untreated MC3T3-E1 cells and between the ovariectomized osteoporosis-induced mice and sham mice. Association studies revealed that there was a significant association between the SNPs in the five genes, ANXA6, COL5A1, ENO1, MYOF, and SCARA5, and bone density and/or osteoporosis. Conclusion: Using a whole-genome comparative expression analysis, gene expression evaluation analysis, and association analysis, we found five genes that were significantly associated with bone density and/or osteoporosis. Notably, the association P-values of the SNPs in the ANXA6 and COL5A1 genes were below the Bonferroni-corrected significance level.

• Korean Journal of Poultry Science
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• 제44권2호
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• pp.93-102
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• 2017

A duplex RT-PCR (dRT-PCR) assay was developed for the simultaneous detection and discrimination of non-virulent and virulent Newcastle disease virus (NDV) in a single PCR tube. Primers targeting the large polymerase protein (L) gene and the fusion protein (F) gene of NDV were designed to detect all NDVs (by common type PCR primers) and virulent NDVs (by pathotype PCR primers), respectively and evaluated experimentally with reference NDV strains and other poultry viral pathogens. PCR products of the expected size of 386 bp were amplified from all NDV samples whereas PCR products of the expected size of 229 bp were amplified from virulent NDV samples alone. Cross reaction was not observed with other avian viral pathogens. The detection limit of NDV by the dRT-PCR was estimated to be $10^3$ 50% egg infectious dose/0.1 mL. In the dRT-PCR using field isolates of NDV, the pathotype PCR primers detected specifically all of virulent field isolates of NDV from Malaysia, Pakistan and China whereas common type PCR primers detected 94.4% (51/54) of field isolates of NDV from China. Three Chinese NDV isolates with false negative result were non-virulent viruses. Our results indicate that the dRT-PCR might provide a rapid and simple tool for rapid simultaneous detection and discrimination of non-virulent and virulent NDVs. Therefore the developed dRT-PCR assay provides a powerful novel means for the rapid diagnosis of Newcastle disease.

• Korean Journal of Microbiology
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• 제51권2호
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• pp.115-125
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• 2015

A monoclonal antibody AP64 IgM binds to human acute nonlymphocytic leukemia (ANLL) cell line HL60 and also cross-reacts with the homologous antigen in a rat ANLL cell. This antibody mediated by complement, has leukemia a suppression effect. In this study, we generated a recombinant single-chain variable domain fragment (ScFv) which were derived from $V_H$ and $V_L$ cDNA of AP64 IgM-secreting hybridoma by RT-PCR. The two variable regions were joined with a single 15 amino acid linker $(G_4S)_3$. This recombinant ScFv was expressed as a single polypeptide chain from Escherichia coli BMH 71-18. The recombinant ScFv was purified by applying the periplasmic extract to $Ni^+$-NTA-agarose affinity column and detected with westernblot. The purified recombinant ScFv recognized a surface antigen (about 30 kDa) of HL60 cell line which is the same antigen detected by parental AP64 IgM. But the affinity of ScFv for a surface antigen of HL60 was lower than that of the parental AP64 IgM, which needs to be further improved. Overall, the recombinant ScFv specific to HL60 might be a useful bioreagent for either diagnostic or therapeutic purposes.

• Journal of Plant Biotechnology
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• 제45권1호
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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 fish pathology
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• 제28권1호
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• pp.27-35
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• 2015

VHSV is a major viral agent that affects freshwater and marine fish, causing serious economic losses in aquaculture in the world. Due to their filter-feeding activity, bivalve mollusks may act as viral transmitters after accumulation of the fish viruses released into seawater from infected fish. Amplification by RT-PCR was carried out to investigate the presence of VHSV in pacific oysters (Crassotrea gigas) and blue mussels (Mytilus edulis), inhabiting regions around aquatic farms in Korea. Primers designed from conserved regions of VHSVs allowed us to detect four different types of VHSV in a single PCR. Twenty two of the eighty four samples showed positive results of VHSV in a 2-step RT-PCR. Using six positive samples from three different regions in Korea, we cloned and sequenced the glycoprotein (G) gene (467-bp long) of VHSVs. Genetic analysis of the VHSVs detected in shellfish in various geographical areas of Korea showed highly restricted results to VHSV type Iva. This was in agreement with the reports showing only a single genotype of VHSV (Iva genotype) in outbreaks in cultured or wild fish in Korea. Consequently, we investigated VHSVs carried by bivalve mollusks inhabiting the vicinity of aquatic farms, and revealed correlationship between the type of viral accumulated in shellfish by filter-feeding, and those detected in disease outbreaks in fish.

• Korean Journal of Microbiology
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• 제42권4호
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• pp.277-285
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• 2006

The most biofilm forming bacteria in catheter, Esctherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were isolated and identified from a patient's catheter occuring catheter-associated urinary tract infection (CA-UTI). We examined mRNA expression and its quantification of AIs synthetic genes encoding signal substance of quorum sensing from each bacterial species in order to elucidated quorum sensing mechanism. Both pure cultures for each bacterial strains and a mixed cultures with three were grown for 24 hr and 30 days. Initial densities to be able to detect mRNA expression oil single strains culture were shown at $2.4{\times}10^5$ CFU/ml, $5.4{\times}10^6$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $6.9{\times}10^4$ CFU/ml of P. aeruginosa for rhlI and lasI. Also, in mixed culture of three, initial cell densities of mRNA expression were appear to at $7.3{\times}10^5$ CFU/ml, $1.6{\times}10^7$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $2.1{\times}10^5$ CFU/ml of P. aeruginosa for rhlI and lasI. Each AIs synthetic gene was expressed in initial cell density and the mRNA expression of the genes were detected continously during 30 days. And then, the quantification of mRNA expression level of ygaG, rhlI, last, and luxS which were related AIs synthesis was done each time point by real-time RT-PCR. Interestingly, the mRNA levels of ygaG, rhlI, lasI, and luxS from the mixed culture was higher than those from each single strain culture. In the case of E. coli ygaG, the amount of transcript from the mixed culture was at least 30 times for that from single culture. In the case of P. aeruginosa rhlI and lasI, the amount of transcript from the mixed culture was at least 40 times and 250 times for that from single strain culture. In the case of S. aureus luxS, the amount of transcript from the mixed culture was at least 5 times for that from single strain culture. And specially, the mRNA expression of rhlI and lasI of P. aeruginosa showed the highest efficency among four AIs synthetic genes.

• Korean Journal of Plant Taxonomy
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• 제34권1호
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• pp.43-61
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• 2004

Molecular taxonomic studies were conducted to evaluate interspecific relationships in Korean Viola 34 taxa including two Japanese populations using RAPD(randornly amplified polymorphic DNA), ISSR(inter simple sequence repeat) and PCR-RFLP(restriction fragment length polymorphism) analysis. Only six and four primers out of 40 arbitrary and 12 ISSR primers were screened for 34 taxa, and were revealed 70 (98.6%) and 28 (96.6%) polymorphic bands, respectively. Fifteen restriction endonucleases produced 80 restriction sites and size variations from the large single copy region of cpDNA, 16 (20%) of which were polymorphic. The separate analyses from the RAPD, ISSR and PCR-RFLP data were incongruent in the relationships among 34 taxa, but combined data was in accordance with previous infrageneric classification system based on morphological characters, especially the subsection and series level. Section Chamaemelanium placed between subsect. Patellares and Vagimtae of section Nomimium was not formed as a distinct group. Viola alb ida complex including three very closely related taxa was recognized independent group within subsect. Patellares in combined data tree. This result strongly suggested that they should be treated to series Pinmtae. RAPD analysis was very useful to clarify the interspecific relationships among the species of Korean Viola than ISSH and PCR-RFLP analyses.

• Toxicological Research
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• 제20권2호
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• pp.101-108
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• 2004

The different social reflections about the benefits and the potential risks of genetically modified (GM) crops have evolved with .different reactions in different countries. Many countries including Korea are working toward setting down new guidelines. Korea requires companies to label all food that contains more than 3% GM ingredients. One of the rapid and convenient detection methods of GM ingredients is amplification of the introduced DNAs by polymerase chain reaction (PCR). Many PCR kits for this purpose are commercially available. The objective of this study was to evaluate performance of commercialized GM crop detection kits. The results showed that 6 out of 15 kits tested did not meet the requirements even purposed by the manufacturers themselves in terms of stability, reproducibility, and detection limits, suggesting a potential quality control problem in their design stage or production line. The evaluation also suggests that, although the duplex and triplex detection kits allowed unambiguous detection in a single PCR reaction, the monoplex detection kits were the most sensitive to the detection of GM ingredients. The detection limits also differ between soybean and corn. Results from this study will be useful in the development of sound qualitative tracking systems of GM ingredients for monitoring throughout the cultivation of GM crops, their trans-boundary movement, and food production using GM grains as well as for complying with government guidelines associated with GM crops.

• Journal of Life Science
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• 제24권8호
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• pp.910-914
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• 2014

For the identification of the Jeju black cattle, Hanwoo and imported beef, we performed a multiplex polymerase chain reaction (PCR) associated with microsatellite (MS) and melanocortin 1 receptor (MC1R) gene. The MC1R gene plays an important role in regulation of the melanin synthesis within mammalian melanocytes. MC1R encoded by extension (E) locus was almost fixed with recessive red e allele in the Hanwoo. We estimated that the specific genotypes ($E^+/E^+$, $E^+/e$) of MC1R gene were characteristic genotypes of Jeju black cattle. But the PCR products resulted from using the MC1R gene derived primers only are not sufficient to identify Jeju black cattle from other relatives. We performed two times of successive multiplex PCR to provide a more accurate result for the identification of Jeju black cattle. The results suggest that two types of successive multiplex PCR methods using MC1R gene and Microsatellite derived primer set will be more useful to identification of Jeju black cattle, Hanwoo and imported beef.