• Food Science and Biotechnology
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• v.18 no.5
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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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• v.16 no.2
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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.

• Journal of Food Hygiene and Safety
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• v.33 no.4
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• pp.280-288
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• 2018

In this study, an approach for the analysis of the five cephalopod species (octopus, long-arm octopus, squid, wet-foot octopus, beka squid) consumed in the Republic of Korea is developed. The samples were collected from the Southeast Asian countries Thailand, Indonesia, Vietnam, and China. The SYBR-green-based real-time qPCR method, based on the mitochondrial DNA genome of the five cephalopods was developed and validated. The intergroup variations in the mitochondrial DNA are evident in the bioinformatic analysis of the mitochondrial genomic DNA sequences of the five groups. Some of the highly-conserved and slightly-variated regions are identified in the mitochondrial cytochrome-c-oxidase subunit I (COI) gene, 16s ribosomal RNA (16s rRNA) gene, and 12s ribosomal RNA (12s rRNA) gene of these groups. To specify each five cephalopod groups, specific primer sets were designed from the COI, 16s rRNA and 12s rRNA regions. The specific primer sets amplified the DNA using the SYBR-green-based real-time PCR system and 11 commercially secured animal tissues: Octopus vulgaris, Octopus minor, Todarodes pacificus, Dosidicus gigas, Sepia esculenta, Amphioctopus fangsiao, Amphioctopus aegina, Amphioctopus marginatus, Loliolus beka, Loligo edulis, and Loligo chinensis. The results confirmed by a conveient way to calculate relative amplification levels between different samples in that it directly uses the threshold cycles (Ct)-value range generated by the qPCR system from these samples. This genomic DNA-based molecular technique provides a quick, accurate, and reliable method for the taxonomic classification of the animal tissues using the real-time qPCR.

• Journal of Life Science
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• v.31 no.2
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• pp.115-125
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• 2021

Calcium-dependent protein kinases (CDPKs) are known to be involved in regulating plant responses to abiotic stresses such as salinity, cold temperature and dehydration,. Although CDPKs constitute a large multigene family consisting of 31 genes in rice, only a few rice CDPKs' functions have been identified. Therefore, in order to elucidate the functions of OsCPK11 in rice, this study was intended to focus on the expression pattern analysis of OsCPK11 in wild type and ND0001 oscpk11 mutant plants under these abiotic stresses. For the salt, cold and drought stress treatment, seedlings were exposed to 200 mM NaCl, 4℃ and 20% PEG 6,000, respectively. RT-PCR and quantitative real-time PCR were performed to determine the expression patterns of OsCPK11 in wild type and ND0001 mutant plants. RT-PCR results showed that OsCPK11 transcripts in the wild type and heterozygous mutant were detected, but not in the homozygous mutant. Real-time PCR results showed that relative expression of OsCPK11 of wild type plants was increased and reached to the highest level at 24 hr, at 6 hr and at 24 hr under salt, cold and drought stress conditions, respectively. Relative expression of OsCPK11 of ND0001 homozygous plant was significantly reduced compared to that of wild type. These results suggested that oscpk11 homozygous mutant knocks out OsCPK11 and OsCPK11 might be involved in salt, cold and drought stress signaling by regulating its gene expression.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.1
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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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• v.21 no.1
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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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• v.13 no.5
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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.

• Journal of Dental Rehabilitation and Applied Science
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• v.34 no.3
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• pp.186-195
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• 2018

Purpose: We performed quantitative and qualitative analysis of typical periodontal bacteria using real time PCR method to investigate the microbiological difference according to the severity of peri-implant disease in Koreans. Materials and Methods: Total of 60 implants were divided into three groups (healthy group, peri-implant mucositis group, peri-implantitis group) through periapical radiographs and clinical indices. The evaluated clinical parameters were pocket depth, plaque index, suppuration and bleeding on probing. Using a sterilized curette instrument, microbial samples were collected from the subgingival plaque and real-time PCR was performed on five periodontal bacteria. The relative expression levels of microorganisms were compared by comparative delta-CT method. Results: The relative expression levels of E. corrodens and T. denticola were significantly higher in the peri-implantitis group (P < 0.017). On the other hand, the relative expression level of F. nucleatum and P. gingivalis was relatively high in the healthy implant group regardless of the severity of disease. P. intermedia was significantly lower in the healthy implant group (P < 0.017). Conclusion: Periodontal bacteria were detected in Koreans with peri-implant diseases, but there was no microbiological distribution similar to periodontitis.

• Food Science of Animal Resources
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• v.31 no.6
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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.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.225-230
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• 2009

Sex-sorting of sperm is an assisted reproductive technology (ART) used by the livestock industry for the mass production of animals of a desired sex. The standard method for sorting sperm is the detection of DNA content differences between X and Y chromosome-bearing sperm by flow cytometry. However, this method has variable efficiency and therefore requires verification by a second method. We have developed a sex determination method based on quantitative real-time polymerase chain reaction (qPCR) of the porcine amelogenin (AMEL) gene. The AMEL gene is present on both the X and the Y chromosome, but the length and sequence of its noncoding regions differ between the X and Y chromosomes. By measuring the threshold cycle (Ct) of qPCR, we were able to calculate the relative frequency of X chromosome. Two sets of AMEL primers were used in these studies. One set (AME) targeted AMEL gene sequences present in both X and Y chromosome, but produced PCR products of different lengths for each chromosome. The other set (AXR) bound to AMEL gene sequences present on the X chromosome but absent esholthe Y-chromosome. Relative product levels were calculated by normalizing the AXR fluorescence to the AME fluorescence. The AMEL method accurately predicted the sex ratios of boar sperm, demonstrating that it has potential value as a sex determination method.