• Proceedings of the Korean Society of Crop Science Conference
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• 2020.06a
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• pp.158-158
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• 2020

• Genomics & Informatics
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• v.19 no.3
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• pp.34.1-34.6
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• 2021

Digital PCR (dPCR) is the third-generation PCR that enables real-time absolute quantification without reference materials. Recently, global diagnosis companies have developed new dPCR equipment. In line with the development, the Lab On An Array (LOAA) dPCR analyzer (Optolane) was launched last year. The LOAA dPCR is a semiconductor chip-based separation PCR type equipment. The LOAA dPCR includes Micro Electro Mechanical System that can be injected by partitioning the target gene into 56 to 20,000 wells. The amount of target gene per wells is digitized to 0 or 1 as the number of well gradually increases to 20,000 wells because its principle follows Poisson distribution, which allows the LOAA dPCR to perform precise absolute quantification. LOAA determined region of interest first prior to dPCR operation. To exclude invalid wells for the quantification, the LOAA dPCR has applied various filtering methods using brightness, slope, baseline, and noise filters. As the coronavirus disease 2019 has now spread around the world, needs for diagnostic equipment of point of care testing (POCT) are increasing. The LOAA dPCR is expected to be suitable for POCT diagnosis due to its compact size and high accuracy. Here, we describe the quantitative principle of the LOAA dPCR and suggest that it can be applied to various fields.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.2
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• pp.88-93
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• 2016

Dirofilaria immitis is a filarial nematode parasite that causes cardiopulmonary dirofilariasis in dogs. The purpose of this study was the development of real-time PCR assays for efficient detection of D. immitis. The D. immitis-specific primers confirmed in our previous study and a newly designed TaqMan probe were used for quantitative diagnostics. First, SYBR Green real-time PCR was performed using the specific primers and serially diluted genomic DNA or plasmid DNA, and melting curve analyses were performed after amplification. The melting curve showed one specific peak in each of the genomic and plasmid DNA reactions, suggesting that the primers specifically amplify the D. immitis cytochrome c oxidase subunit I gene. Comparison of SYBR Green and TaqMan real-time PCR using serially diluted plasmid DNA showed higher efficiency and specificity with TaqMan real-time PCR. The real-time PCR assays developed in this study will provide improved diagnostic methods to overcome the limitations of conventional diagnostic tools and facilitate more rapid and accurate diagnoses.

• Journal of Life Science
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• v.33 no.12
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• pp.1025-1035
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• 2023

In this study, we analyzed SNPs that appear between Korean and Chinese Scapharca subcrenata using the nucleotide sequence data of S. subcrenata analyzed by genotyping by sequencing (GBS). To distinguish the country of origin for S. subcrenata in Korean and Chinese, we developed a primer set as single nucleotide polymorphism (SNP) markers for quantitative real-time PCR (qPCR) analysis and validated by sequencing SNPs. A total of 180 samples of S. subcrenata were analyzed by genotyping by sequencing, and 15 candidate SNPs were selected. SNP marker selection for country of origin were identified through real-time qPCR. Insertion 1 and SNP 21 markers showed the most distinct separation between the sequence types as well as the country of origin through qPCR, with the observed amplification patterns matching the expected outcomes.. Additionally, in a blind test conducted by mixing samples of S. subcrenata at random, Insertion 1 showed 74% accuracy, 52% sensitivity, and 96% specificity, and SNP 21 showed 86% accuracy, 79% sensitivity, and 93% specificity. Therefore, the two SNP markers developed are expected to be useful in verifying the authenticity of the country of origin of S. subcrenata when used independently or in combination.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.39 no.4
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• pp.281-288
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• 2013

Hyaluronic acid (HA) is one of the major extracellular matrix components in skin. The HA content is reported to decline with age, which may contribute to decrease in skin moisture, wrinkle formation and the decrease in elasticity of the skin. Among the family of HA synthase genes (HAS-1, 2, 3) identified so far, HAS-2 plays crucial roles in the regulation of HA synthesis in human skin fibroblasts. In this study, we elucidated the effects of ferulic acid isolated from Cnidium officinale on HA production. Semi-quantitative RT-PCR and quantitative real-time PCR showed that ferulic acid increased mRNA level of HAS-2 gene and ELISA assay also revealed that ferulic acid increased HA production in human skin fibroblasts. Our study suggests that ferulic acid might prevent age-dependent skin deteriorations such as wrinkles, dryness and elasticity decrease, all of which could be ascribed to the reduction of the HA content in human skin.

• Biomedical Science Letters
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• v.29 no.4
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• pp.363-370
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• 2023

Human respiratory viral infections such as COVID-19 are highly contagious, so continuous management of airborne viruses is essential. In particular, indoor air monitoring is necessary because the risk of infection increases in poorly ventilated indoors. However, the current method of detecting airborne viruses requires a lot of time from sample collection to confirmation of results. In this study, we proposed a system that can monitor airborne viruses in real time to solve the deficiency of the present method. Air samples were collected in liquid form through a bio sampler, in which case the virus is present in low concentrations. To detect viruses from low-concentration samples, viral RNA was concentrated and extracted using silica-magnetic beads. RNA binds to silica under certain conditions, and by repeating this binding reaction, bulk samples collected from the air can be concentrated. After concentration and extraction, viral RNA is specifically detected through real-time qPCR (quantitative polymerase chain reaction). In addition, based on liquid handling technology, we have developed an automatic machine that automatically performs the entire testing process and can be easily used even by non-experts. To evaluate the system, we performed air sample collection and automated testing using bacteriophage MS2 as a model virus. As a result, the air-collected samples concentrated by 45 times then initial volume, and the detection sensitivity of PCR also confirmed a corresponding improvement.

• Parasites, Hosts and Diseases
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• v.48 no.3
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• pp.195-201
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• 2010

We studied on the proteomic characteristics of Toxoplasma gondii KI-1 tachyzoites which were originally isolated from a Korean patient, and compared with those of the well-known virulent RH strain using 2-dimensional electrophoresis (2-DE), mass spectrometry, and quantitative real-time PCR. Two-dimensional separation of the total proteins isolated from KI-1 tachyzoites revealed up to 150 spots, of which 121 were consistent with those of RH tachyzoites. Of the remaining 29 spots, 14 showed greater than 5-fold difference in density between the KI-1 and RH tachyzoites at a pH of 5.0-8.0. Among the 14 spots, 5 from the KI-1 isolate and 7 from the RH strain were identified using MALDI-TOF mass spectrometry and database searches. The spots from the KI-1 tachyzoties were dense granule proteins (GRA 2,3,6, and 7), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGRPTase), and uracil phosphoribosyltransferase (UPRTase). The spots from the RH strain were surface antigen 1 (SAG 1), L-lactate dehydrogenase (LDH), actin, chorismate synthase, peroximal catalase, hexokinase, bifunctional dihydrofolate reductase-thymidylate synthase (DHTR-TS), and nucleosidetriphosphatases (NTPases). Quantitative real-time PCR supported our mass spectrometric results by showing the elevated expression of the genes encoding GRA 2,3, and 6 and UPRTase in the KI-1 tachyzoites and those encoding GRA 7, SAG 1, NTPase, and chorismate synthase in the RH tachyzoites. These observations demonstrate that the protein compositions of KI-1 and RH tachyzoites are similar but differential protein expression is involved in virulence.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.12
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• pp.1665-1671
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• 2013

Real-time quantitative PCR (qRT-PCR) is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2) in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.9
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• pp.1097-1105
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• 2017

This study examined how much chicken meat was in sausage made with pork. Both real-time polymerase chain reaction (PCR) and internal standard addition were used. Fifty ng of chicken DNA was added to the sausages as an internal standard. The addition of standard DNA increased the amplification efficiency of PCR and confirmed the possibility of quantitative analysis. A QIAamp DNA Micro Kit was used to improve the DNA recovery and amplification efficiency. The density of template DNA and primer were suitable for $3.0{\sim}5.0{\mu}L$ and $0.5{\mu}L$, respectively. Each DNA of pig and chicken was diluted in 10-fold from steps 50 ng to 0.05 ng. The detection limit of both pig and chicken meat was more than 0.05 ng and the correlation coefficient of the standard curve was at least 0.98. The result of the quantitative analysis after heat treatment of 3 samples of pigs and chickens mixed at 70:30 showed a 5.7% difference (64.3:35.7) between the expected value and measured value. The quantitative value was changed by affecting the DNA according to the heat treatment ($70^{\circ}C$, 10 min). An analysis of the pork and chicken content in sausages showed that it was difficult to detect chicken meat and the quantitative value of DNA according to the Ct value was very low. On the other hand, when adding standard material (50 ng of chicken DNA) to the sausages, the Ct value decreased gradually with increasing chicken mixing ratio. Thus, the mixing ratio of chicken in sausages could be estimated.

• The Plant Pathology Journal
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• v.27 no.4
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• pp.301-309
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• 2011

The filamentous fungus Fusarium graminearum is an important cereal pathogen. Although quantitative realtime PCR (qRT-PCR) is commonly used to analyze the expression of important fungal genes, no detailed validation of reference genes for the normalization of qRT-PCR data has been performed in this fungus. Here, we evaluated 15 candidate genes as references, including those previously described as housekeeping genes and those selected from the whole transcriptome sequencing data. By a combination of three statistical algorithms (BestKeeper, geNorm, and NormFinder), the variation in the expression of these genes was assessed under different culture conditions that favored mycelial growth, sexual development, and trichothecene mycotoxin production. When favoring mycelial growth, GzFLO and GzUBH expression were most stable in complete medium. Both EF1A and GzRPS16 expression were relatively stable under all conditions on carrot agar, including mycelial growth and the subsequent perithecial induction stage. These two genes were also most stable during trichothecene production. For the combined data set, GzUBH and EF1A were selected as the most stable. Thus, these genes are suitable reference genes for accurate normalization of qRT-PCR data for gene expression analyses of F. graminearum and other related fungi.