• Food Science and Biotechnology
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• 제18권5호
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• pp.1118-1123
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• 2009

Event-specific real-time polymerase chain reaction (PCR) detection method for genetically modified (GM) maize MIR604 was developed based on integration junction sequences between the host plant genome and the integrated transgene. In this study, 2 primer pairs and probes were designed for specific amplification of 100 and 111 bp DNA fragments from the zSSIIb gene (the maize endogenous reference gene) and MIR604. The quantitative method was validated using 3 certified reference materials (CRMs) with levels of 0.1, 1, and 10% MIR604. The method was also assayed with 14 different plants and other GM maize. No amplification signal was observed in real-time PCR assays with any of the species tested other than MIR604 maize. As a result, the bias from the true value and the relative deviation for MIR604 was within the range from 0 to 9%. Precision, expressed as relative standard deviation (RSD), varied from 2.7 to 10% for MIR604. Limits of detections (LODs) of qualitative and quantitative methods were all 0.1%. These results indicated that the event-specific quantitative PCR detection system for MIR604 is accurate and useful.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.205-211
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• 2006

A quantitative analytical method to detect new lines of genetically modified (GM) maize, NK603 and TC1507, has been developed by using a real-time polymerase chain reaction (PCR). To detect these GM lines, two specific primer pairs and probes were designed. A plasmid as a reference molecule was constructed from an endogenous DNA sequence of maize, a universal sequence of a cauliflower mosaic virus (CaMV) 35S promoter used in most GMOs, and each DNA sequence specific to the NK603 and TC1507 lines. For the validation of this method, the test samples of 0, 0.1, 0.5, 1.0, 3.0, 5.0, and 10.0% each of the NK603 and TC1507 GM maize were quantitated. At the 3.0% level, the biases (mean vs. true value) for the NK603 and TC1507 lines were 3.3% and 15.7%, respectively, and their relative standard deviations were 7.2% and 5.5%, respectively. These results indicate that the PCR method developed in this study can be used to quantitatively detect the NK603 and TC1507 lines of GM maize.

• 한국식품위생안전성학회지
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• 제33권4호
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• pp.280-288
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• 2018

본 연구는 국내에서 생산되거나 해외에서 수입되어 국내에서 유통되는 수산물 중에서 두족류를 문어류, 낙지류, 오징어류, 주꾸미류, 꼴뚜기류의 5개 그룹으로 구분하여 분석하였다. 두족류 5개 그룹을 판별을 하기 위해 미토콘드리아에 존재하는 유전자를 분석하였고, 그 중에서 COI (mitochondrial cytochrome C oxidase subunit I), 16s rRNA (16s ribosomal RNA), 12s rRNA (12s ribosomal RNA) 내에서 상당히 유사한 DNA 서열 부분과 일부 서열 변화 부분이 확인되었다. 명확하게 두족류 5개 그룹 판별을 하기 위해 COI, 16s rRNA, 12s rRNA 유전자의 일부 서열 변화 부분에서 그룹 특이적 프라이머 세트를 디자인하였다. 국내 외에서 확보한 두족류 시료(참문어, 낙지, 살오징어, 아메리카 대왕오징어, 갑오징어, 주꾸미, 모래주꾸미, 하이야주꾸미, 참꼴뚜기, 창꼴뚜기, 한치꼴뚜기)의 genomic DNA을 추출하여 각 그룹의 특이적 프라이머를 이용하여 SYBR 기반의 real-time PCR 시스템에 의해 분석되었고, threshold cycle (Ct) value와 같은 real-time PCR 결과 분석에 의해 두족류 내 그룹 판별이 가능하였다(Table 3).

• 생명과학회지
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• 제31권2호
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• pp.115-125
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• 2021

칼슘-의존성 단백질 카이네이즈(CDPK)는 식물의 Ca2+ 매개 신호 전달에 필수적인 역할을 한다. CDPK는 염분, 저온, 가뭄 등과 같은 비생물적 스트레스에 대한 식물의 반응을 조절하는 데 관여하는 것으로 알려져 있다. 벼의 CDPK는 31개의 유전자로 구성된 거대 다유전자군으로 되어 있지만, 단지 일부 벼의 CDPK 기능만이 확인되었다. 따라서, 벼에서 OsCPK11의 기능을 알아보기 위해, 이 연구는 염분, 저온 및 가뭄과 같은 비생물적 스트레스 조건에서 벼의 야생형과 ND0001 oscpk11 돌연변이체의 OsCPK11 발현 분석에 초점을 맞추었다. 염분, 저온, 가뭄 스트레스 처치를 위해 유식물을 각각 200 mM NaCl, 4℃, 20% PEG 6,000에 노출시켰다. 야생형과 ND0001 돌연변이체에서 OsCPK11의 발현을 확인하기 위해 RT-PCR과 quantitative real-time PCR을 수행하였다. RT-PCR 결과에 의하면, 야생형과 이형접합성 돌연변이체에서는 OsCPK11 전사체가 검출되었지만, 동형접합성 돌연변이체에서는 검출되지 않았다. Quantitative real-time PCR 결과에 의하면 야생형에서 염분, 저온, 가뭄 스트레스에 의해 OsCPK11의 상대적인 발현이 증가하였으며, 각각 24시간, 6시간, 24시간 후 최대 수준에 도달하였다. ND0001 동형접합성 돌연변이체의 OsCPK11의 상대적 발현은 야생형에 비해 현저히 감소하였다. 이러한 결과는 oscpk11 동형접합성 돌연변이체에서는 OsCPK11발현을 완전히 저해하며, OsCPK11유전자 발현 조절이 염분, 저온 및 가뭄 스트레스 신호 전달 과정에 관여할 수 있음을 의미한다.

• Asian-Australasian Journal of Animal Sciences
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• 제32권1호
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• pp.92-102
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• 2019

Objective: To investigate changes in rumen fermentation characteristics and bacterial community by a sudden change to a high concentrate diet (HC) in Korean domestic ruminants. Methods: Major Korean domestic ruminants (each of four Hanwoo cows; $545.5{\pm}33.6kg$, Holstein cows; $516.3{\pm}42.7kg$, and Korean native goats; $19.1{\pm}1.4kg$) were used in this experiment. They were housed individually and were fed ad libitum with a same TMR (800 g/kg timothy hay and 200 g/kg concentrate mix) twice daily. After two-week feeding, only the concentrate mix was offered for one week in order to induce rapid rumen acidosis. The rumen fluid was collected from each animals twice (on week 2 and week 3) at 2 h after morning feeding using an oral stomach tube. Each collected rumen fluid was analyzed for pH, volatile fatty acid (VFA), and $NH_3-N$. In addition, differences in microbial community among ruminant species and between normal and an acidosis condition were assessed using two culture-independent 16S polymerase chain reaction (PCR)-based techniques (terminal restriction fragment length polymorphism and quantitative real-time PCR). Results: The HC decreased ruminal pH and altered relative concentrations of ruminal VFA (p<0.01). Total VFA concentration increased in Holstein cows only (p<0.01). Terminal restriction fragment length polymorphism and real-time quantitative PCR analysis using culture-independent 16S PCR-based techniques, revealed rumen bacterial diversity differed by species but not by HC (p<0.01); bacterial diversity was higher in Korean native goats than that in Holstein cows. HC changed the relative populations of rumen bacterial species. Specifically, the abundance of Fibrobacter succinogenes was decreased while Lactobacillus spp. and Megasphaera elsdenii were increased (p<0.01). Conclusion: The HC altered the relative populations, but not diversity, of the ruminal bacterial community, which differed by ruminant species.

• Genomics & Informatics
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• 제21권1호
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• pp.13.1-13.8
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• 2023

Importance of accurate molecular diagnosis and quantification of particular disease-related pathogenic microorganisms is highlighted as an introductory step to prevent and care for diseases. In this study, we designed a primer/probe set for quantitative real-time polymerase chain reaction (qRT-PCR) targeting rgpA gene, known as the specific virulence factor of periodontitis-related pathogenic bacteria 'Porphyromonas gingivalis', and evaluated its diagnostic efficiency by detecting and quantifying relative bacterial load of P. gingivalis within saliva samples collected from clinical subjects. As a result of qRT-PCR, we confirmed that relative bacterial load of P. gingivalis was detected and quantified within all samples of positive control and periodontitis groups. On the contrary, negative results were confirmed in both negative control and healthy groups. Additionally, as a result of comparison with next-generation sequencing (NGS)-based 16S metagenome profiling data, we confirmed relative bacterial load of P. gingivalis, which was not identified on bacterial classification table created through 16S microbiome analysis, in qRT-PCR results. It showed that an approach to quantifying specific microorganisms by applying qRT-PCR method could solve microbial misclassification issues at species level of an NGS-based 16S microbiome study. In this respect, we suggest that P. gingivalis-specific primer/probe set introduced in present study has efficient applicability in various oral healthcare industries, including periodontitis-related microbial molecular diagnosis field.

• Asian Pacific Journal of Cancer Prevention
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• 제13권5호
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• pp.2305-2309
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• 2012

Crocin, the main pigment of Crocus sativus L., has been shown to have antiproliferative effects on cancer cells, but the involved mechanisms are only poor understood. This study focused on probable effect of crocin on the immortality of hepatic cancer cells. Cytotoxicity of crocin ($IC_{50}$ 3 mg/ml) in hepatocarcinoma HepG2 cells was determined after 48 h by neutral red uptake assay and MTT test. Immortality was investigated through quantification of relative telomerase activity with a quantitative real-time PCR-based telomerase repeat amplification protocol (qTRAP). Telomerase activity in 0.5 ${\mu}g$ protein extract of HepG2 cells treated with 3 mg/ml crocin was reduced to about 51% as compared to untreated control cells. Two mechanisms of inhibition, i.e. interaction of crocin with telomeric quadruplex sequences and down regulation of hTERT expression, were examined using FRET analysis to measure melting temperature of a synthetic telomeric oligonucleotide in the presence of crocin and quantitative real-time RT-PCR, respectively. No significant changes were observed in the $T_m$ telomeric oligonucleotides, while the relative expression level of the catalytic subunit of telomerase (hTERT) gene showed a 60% decrease as compared to untreated control cells. In conclusion, telomerase activity of HepG2 cells decreases after treatment with crocin, which is probably caused by down-regulation of the expression of the catalytic subunit of the enzyme.

• 구강회복응용과학지
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• 제34권3호
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• pp.186-195
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• 2018

목적: 한국인에서 임플란트 주위 질환의 심도에 따른 미생물학적 차이를 알아보기 위해 real time Polymerase Chain Reaction(real-time PCR)법을 이용하여 5종의 치주세균의 정량적, 정성적 분석을 시행하였다. 연구 재료 및 방법: 임플란트가 식립된 총 60명의 환자를 치근단 방사선 사진 및 임상지수 검사를 통해 3군(건강군, 임플란트 주위 점막염군, 임플란트 주위염군)으로 나누었다. 멸균된 curette기구를 이용해 치은연하에서 미생물 샘플을 채취한 후 치주세균 5종에 관해 real time PCR을 시행하였고 comparative delta-CT method를 이용하여 분석한 후 미생물의 상대적 발현량을 비교하였다. 결과: Eikenella corrodens, Treponema denticola의 상대적 발현량은 임플란트 주위염 그룹에서 유의하게 높게 나타났다(P < 0.017). 반면 Fusobacterium nucleatum, Porphyromonas gingivalis의 상대적 발현량은 질환의 심도와는 관련 없이 건강한 임플란트 그룹에서 가장 높게 나타났다. Prevotella intermedia의 상대적 발현량은 건강한 임플란트 그룹에서 유의하게 낮게 나타났다(P < 0.017). 결론: 한국인의 임플란트 주위질환에서 대표적인 치주염 세균이 검출되었으나 치주염과 유사한 미생물학적 분포를 보이지는 않았다.

• 한국축산식품학회지
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• 제31권6호
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• pp.827-833
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• 2011

The objective of this study was to develop a fluorogenic real-time PCR-based assay for detecting and quantifying amounts of cow milk in cow/goat milk mixtures or goat milk products. In order to quantify the exact amount of cow milk in cow/goat raw milk mixtures and commercial goat milk products, it was necessary to achieve quantitative extraction of total genomic DNA from the raw milk matrix. Both mammalian-specific PCR and cow-specific PCR were performed. A cow-specific 252 bp band obtained from the raw cow milk and raw goat milk mixtures, commercial goat milk, and two goat milk powders was identified, along with the relationship between the cow milk amount and band intensity of the electrophoresis image. The detection threshold was found to be 0.1%. The expression of cow's 12S rRNA in the cow/goat milk mixtures, commercial goat milk, and two goat milk powders was identified. The expression quantity of the milk 12S rRNA increased with increasing ratios of the cow/goat milk mixtures. Using these calibrated relative expression levels as a standard curve in the cow/goat raw milk mixtures, the contents of cow milk were 1.8% in the commercial goat milk, 9.6% in goat milk powder A, and 11.6% in goat milk powder C. However, cow milk was not detected in goat milk powder B.

• Genomics & Informatics
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• 제21권4호
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• pp.52.1-52.10
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• 2023

Accurate and efficient microbial diagnosis is crucial for effective molecular diagnostics, especially in the field of human healthcare. The gold standard equipment widely employed for detecting specific microorganisms in molecular diagnosis is quantitative real-time polymerase chain reaction (qRT-PCR). However, its limitations in low metagenomic DNA yield samples necessitate exploring alternative approaches. Digital PCR, by quantifying the number of copies of the target sequence, provides absolute quantification results for the bacterial strain. In this study, we compared the diagnostic efficiency of qRT-PCR and digital PCR in detecting a particular bacterial strain (Staphylococcus aureus), focusing on skin-derived DNA samples. Experimentally, specific primer for S. aureus were designed at transcription elongation factor (greA) gene and the target amplicon were cloned and sequenced to validate efficiency of specificity to the greA gene of S. aureus. To quantify the absolute amount of microorganisms present on the skin, the variable region 5 (V5) of the 16S rRNA gene was used, and primers for S. aureus identification were used to relative their amount in the subject's skin. The findings demonstrate the absolute convenience and efficiency of digital PCR in microbial diagnostics. We suggest that the high sensitivity and precise quantification provided by digital PCR could be a promising tool for detecting specific microorganisms, especially in skin-derived DNA samples with low metagenomic DNA yields, and that further research and implementation is needed to improve medical practice and diagnosis.