• Fisheries and Aquatic Sciences
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• v.19 no.4
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• pp.21.1-21.11
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• 2016

Background: The evaluation of suitable reference genes as normalization controls is a prerequisite requirement for launching quantitative reverse transcription-PCR (RT-qPCR)-based expression study. In order to select the stable reference genes in abalone Haliotis discus hannai tissues (gill and hepatopancreas) under heavy metal exposure conditions (Cu, Zn, and Cd), 12 potential candidate housekeeping genes were subjected to expression stability based on the comprehensive ranking while integrating four different statistical algorithms (geNorm, NormFinder, BestKeeper, and ${\Delta}CT$ method). Results: Expression stability in the gill subset was determined as RPL7 > RPL8 > ACTB > RPL3 > PPIB > RPL7A > EF1A > RPL4 > GAPDH > RPL5 > UBE2 > B-TU. On the other hand, the ranking in the subset for hepatopancreas was RPL7 > RPL3 > RPL8 > ACTB > RPL4 > EF1A > RPL5 > RPL7A > B-TU > UBE2 > PPIB > GAPDH. The pairwise variation assessed by the geNorm program indicates that two reference genes could be sufficient for accurate normalization in both gill and hepatopancreas subsets. Overall, both gill and hepatopancreas subsets recommended ribosomal protein genes (particularly RPL7) as stable references, whereas traditional housekeepers such as ${\beta}-tubulin$ (B-TU) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were ranked as unstable genes. The validation of reference gene selection was confirmed with the quantitative assay of MT transcripts. Conclusions: The present analysis showed the importance of validating reference genes with multiple algorithmic approaches to select genes that are truly stable. Our results indicate that expression stability of a given reference gene could not always have consensus across tissue types. The data from this study could be a good guide for the future design of RT-qPCR studies with respect to metal regulation/detoxification and other related physiologies in this abalone species.

• Korean Journal of Pharmacognosy
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• v.45 no.2
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• pp.168-173
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• 2014

The aim of this study was to evaluate the anti-helicobacter activity of the ethanol extract of Sohamhyoongtang (Coptidis Rhizoma, Pinelliae Tuber and Trichosanthis Semen) and Coptidis Rhizoma total alkaloids, which is one of the components of Sohamhyoongtang. Crude ethanol extract of Sohamhyoongtang (ESHHT) and Coptidis Rhizoma total alkaloids (CRTA) were used for this experiment. Five different types of H. pylori (including H. pylori 26695) were used as test strain. To determine anti-helicobacter activity, minimum inhibitory concentration (MIC) was determined by agar dilution method. The effect of ESHHT and CRTA on the gene expression of H. pylori was investigated by quantitative realtime-PCR (qRT-PCR). MICs of ESHHT against five H. pylori strains were $250{\sim}500{\mu}g/ml$ and MICs of CRTA against five H. pylori strains were $50{\sim}200{\mu}g/ml$. Four representative virulence genes of H. pylori, cagA, ureA, ureB and ureI were tested as target genes for qRT-PCR. According to the qRT-PCR results, both ESHHT and CRTA markedly repressed the expression of cagA gene of H. pylori 26695 (6.91 and 20 folds respecively). These results showed that the ESHHT and CRTA demonstrated antihelicobacter properties, suggesting their potential use in gastritis or duodenal ulcer.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.51 no.1
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• pp.95-106
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• 2018

To evaluate appropriate reference genes for the normalization of quantitative reverse transcription PCR (RT-qPCR) data with embryonic and larval samples from Russian sturgeon Acipenser gueldenstaedtii, the expression stability of eight candidate housekeeping genes, including beta-actin (ACTB), elongation factor-1A (EF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 2A (H2A), ribosomal protein L5 (RPL5), ribosomal protein L7 (RPL7), succinate dehydrogenase (SDHA), and ubiquitin-conjugating enzyme E2 (UBE2A), were tested using embryonic samples from 12 developmental stages and larval samples from 11 ontogenic stages. Based on the stability rankings from three statistic software packages, geNorm, NormFinder, and BestKeeper, the expression stability of the embryonic subset was ranked as UBE2A>H2A>SDHA>GAPDH>RPL5>EF1A>ACTB>RPL7. On the other hand, the ranking in the larval subset was determined as UBE2A>GAPDH>SDHA>RPL5>RPL7>H2A>EF1A>AC TB. When the two subsets were combined, the overall ranking was UBE2A>SDHA>H2A>RPL5>GAPDH>EF1A>ACTB>RPL7. Taken together, our data suggest that UBE2A and SDHA are recommended as suitable references for developmental and ontogenic samples of this sturgeon species, whereas traditional housekeepers such as ACTB and GAPDH may not be suitable candidates.

• 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.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2020.12a
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• pp.56-56
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• 2020

감초는 다년생 콩과(Leguminocae) 식물로 국내에서 시중가격이 높은 만주감초가 일부 재배되고 있다. 우리나라에서 감초 재배법이 불완전한 상황에서 한반도의 기후변화에 의한 온도 상승은 약용작물의 생산 및 품질에 많은 영향을 미칠 것으로 예상되므로 본 연구에서는 재배환경 중 온도 조건만 조절할 수 있는 온도구배터널(temperature gradient tunnel system)을 이용하여 4개의 T1(외기온도+0.5~1.3℃), T2(+1.3~2.2℃), T3(+2.2~3.2℃), T4(+3.2~4.0℃) 처리로 온도구배 하여 4년생 만주감초(Glycyrrhiza uralensis F.)를 재배하였다. 지하부가 오래된 모주와 신초1의 경우 저온(T1)과 중간구간(T2, T3)에서 초장과 총화수가 우세하였고, 번식이 가장 늦은 신초2의 경우 중간구간(T2, T3)에서의 생육 및 개화반응이 뚜렷했다. 각 온도처리구마다 3개의 감초 개체를 선발하여 모주의 정단으로부터 5개의 성엽을 채취하였다. Reverse transcription quantitative PCR (RT-qPCR)은 AccuPower® GreenStar^TM RT-qPCR Master Mix (Bioneer, Korea)를 이용하여 진행되었다. Primer 디자인은 NCBI Primer-blast 프로그램을 사용해 제작하였고 ABI StepOne real time system (Applied Biosystem)의 melting curve analysis에서 one-peak test를 통해 gene specific primer임을 확인하였다. 각 온도처리구의 감초 잎에서 RNA를 추출하였고, RT-qPCR을 통해 감초의 유전자 발현양상을 비교, 분석하였다. Phytochrome interacting factor 4 (PIF4)는 식물 호르몬을 유발하는 전사조절을 조정함으로써 고온 신호전달에 핵심적인 역할을 수행한다. 활성화된 Phytochrome B(PhyB)는 PIF4의 활성을 억제한다고 알려졌다. Eukaryotic initiation factors(eIFs)는 mRNA 번역 개시인자로 유전자 발현과 특정 단백질 생산을 조절하는 역할을 한다. 본 결과는 온도조건에서 반응하는 생리적 변화를 전사체 수준에서 조사 분석하여 생리해석의 기초자료로 활용, 국내 감초 재배를 위한 환경조건 구명 및 적지 선정 기초자료로서 활용을 기대한다.

• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1280-1286
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• 2011

A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at $3.6{\times}10^6$ CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above $10^5$/ml for approx. 10 days, after which cell numbers decreased to levels of $10^3$ CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca. $1{\times}10^2$ CFU/ml was detected. The minimum detection level for quantitative PCR in this study was $1{\times}10^2$ to $1{\times}10^3$ CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.

• Environmental Engineering Research
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• v.20 no.1
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• pp.51-57
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• 2015

As expected, the expression of denitrifying genes in a Typha wetland (relatively stagnant compared to other ponds), showing higher nitrogen removal efficiency in summer, was affected by temperature. The abundance and gene transcripts of nitrate reductase (narG), nitrite reductase (nirS), nitric oxide reductase (norB), and nitrous oxide reductase (nosZ) genes in seasonal sediment samples taken from the Acorus and Typha ponds of free surface flow constructed wetlands were investigated using quantitative polymerase chain reaction (Q-PCR) and quantitative reverse transcription PCR (Q-RT-PCR). Denitrifying gene copy numbers ($10^5-10^8$ genes $g^{-1}$ sediment) were found to be higher than transcript numbers-($10^3-10^7$ transcripts $g^{-1}$ sediment) of the Acorus and Typha ponds, in both seasons. Transcript numbers of the four functional genes were significantly higher for Typha sediments, in the warm than in the cold season, potentially indicating greater bacterial activity, during the relatively warm season than the cold season. In contrast, copy numbers and expression of denitrifying genes of Acorus did not provide a strong correlation between the different seasons.

• Genomics & Informatics
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• v.21 no.4
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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.

• Genomics & Informatics
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• v.21 no.2
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• pp.24.1-24.7
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• 2023

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.

• The Plant Pathology Journal
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• v.34 no.3
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• pp.163-170
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• 2018

Strawberry Fusarium wilt disease, caused by Fusarium oxysporum f. sp. fragariae, is the most devastating disease in strawberry production. The pathogen produces chlamydospores which tolerate against harsh environment, fungicide and survive for decades in soil. Development of detection and quantification techniques are regarded significantly in many soilborne pathogens to prevent damage from diseases. In this study, we improved specific-quantitative primers for F. oxysporum f. sp. fragariae to reveal correlation between the pathogen density and the disease severity. Standard curve $r^2$ value of the specific-quantitative primers for qRT-PCR and meting curve were over 0.99 and $80.5^{\circ}C$, respectively. Over pathogen $10^5cfu/g$ of soil was required to cause the disease in both lab and field conditions. With the minimum density to develop the wilt disease, the pathogen affected near 60% in nursery plantation. A biological control microbe agent and soil solarization reduced the pathogen population 2-fold and 1.5-fold in soil, respectively. The developed F. oxysporum f. sp. fragariae specific qRT-PCR protocol may contribute to evaluating soil healthiness and appropriate decision making to control the disease.