• Journal of Ginseng Research
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• 제43권1호
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• pp.1-9
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• 2019

Background: Korean ginseng is an important cash crop in Asian countries. However, plant yield is reduced by pathogens. Among the Ilyonectria radicicola-species complex, I. mors-panacis is responsible for root-rot and replant failure of ginseng in Asia. The development of new methods to reveal the existence of the pathogen before cultivation is started is essential. Therefore, a quantitative real-time polymerase chain reaction method was developed to detect and quantify the pathogen in ginseng soils. Methods: In this study, a species-specific histone H3 primer set was developed for the quantification of I. mors-panacis. The primer set was used on DNA from other microbes to evaluate its sensitivity and selectivity for I. mors-panacis DNA. Sterilized soil samples artificially infected with the pathogen at different concentrations were used to evaluate the ability of the primer set to detect the pathogen population in the soil DNA. Finally, the pathogen was quantified in many natural soil samples. Results: The designed primer set was found to be sensitive and selective for I. mors-panacis DNA. In artificially infected sterilized soil samples, using quantitative real-time polymerase chain reaction the estimated amount of template was positively correlated with the pathogen concentration in soil samples ($R^2=0.95$), disease severity index ($R^2=0.99$), and colony-forming units ($R^2=0.87$). In natural soils, the pathogen was recorded in most fields producing bad yields at a range of $5.82{\pm}2.35pg/g$ to $892.34{\pm}103.70pg/g$ of soil. Conclusion: According to these results, the proposed primer set is applicable for estimating soil quality before ginseng cultivation. This will contribute to disease management and crop protection in the future.

• 대한의생명과학회지
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• 제28권1호
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• pp.42-49
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• 2022

Tetrodotoxin (TTX) is a natural neurotoxin found in several species of puffer fish belonging to Tetraodon fugu genus and has been reported to affect processes such as proliferation, metastasis and invasion of various cancer cells. However, it was not revealed which genes were influenced by these reactions. In this experiment, it was examined in human SW620 colorectal cancer cells. The proliferation of SW620 cells was significantly reduced when treated with 0, 1, 10 and 100 μM TTX for 48 h. It was confirmed using Annexin V-propidium iodide staining that some apoptosis was induced. Differentially expressed genes (DEGs) affecting cell proliferation through RNA sequencing (RNA-seq) were selected. The expression change of DEGs was confirmed by conducting quantitative real-time polymerase chain reaction (qRT-PCR). As a result, the mRNA expression of FOS and WDR48 genes was found to be increased in the 100 μM TTX treatment group compared to the control group. On the other hand, the mRNA expression of ALKBH7, NDUFA13, RIPPLY3 and SELENOM genes was found to be reduced, and in the case of the ALKBH7 gene was identified to show significant differences. This experiment suggests that TTX can be used as an important fundamental data to elucidate the mechanism that inhibits the proliferation of SW620 cells.

• 한국동물생명공학회지
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• 제37권2호
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• pp.96-105
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• 2022

The ovary undergoes substantial physiological changes along with estrus phase to mediate negative/positive feedback to the upstream reproductive tissues and to play a role in producing a fertilizable oocyte in the developing follicles. However, the disorder of estrus cycle in female can lead to diseases, such as cystic ovary which is directly associated with decline of overall reproductive performance. In gene expression studies of ovaries, quantitative reverse transcription polymerase chain reaction (qPCR) assay has been widely applied. During this assay, although normalization of target genes against reference genes (RGs) has been indispensably conducted, the expression of RGs is also variable in each experimental condition which can result in false conclusion. Because the understanding for stable RG in porcine ovaries was still limited, we attempted to assess the stability of RGs from the pool of ten commonly used RGs (18S, B2M, PPIA, RPL4, SDHA, ACTB, GAPDH, HPRT1, YWHAZ, and TBP) in the porcine ovaries under different estrus phase (follicular and luteal phase) and cystic condition, using stable RG-finding programs (geNorm, Normfinder, and BestKeeper). The significant (p < 0.01) differences in Ct values of RGs in the porcine ovaries under different conditions were identified. In assessing the stability of RGs, three programs comprehensively agreed that TBP and YWHAZ were suitable RGs to study porcine ovaries under different conditions but ACTB and GAPDH were inappropriate RGs in this experimental condition. We hope that these results contribute to plan the experiment design in the field of reproductive physiology in pigs as reference data.

• Journal of Veterinary Science
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• 제25권2호
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• pp.28.1-28.11
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• 2024

Background: Porcine circovirus type 2 (PCV2) infection is ubiquitous around the world. Diagnosis of the porcine circovirus-associated disease requires clinic-pathological elements together with the quantification of viral loads. Furthermore, given pig farms in regions lacking access to sufficient laboratory equipment, developing diagnostic devices with high accuracy, accessibility, and affordability is a necessity. Objectives: This study aims to investigate two newly developed diagnostic tools that may satisfy these criteria. Methods: We collected 250 specimens, including 170 PCV2-positive and 80 PCV2-negative samples. The standard diagnosis and cycle threshold (Ct) values were determined by quantitative polymerase chain reaction (qPCR). Then, two point-of-care (POC) diagnostic platforms, convective polymerase chain reaction (cPCR, qualitative assay: positive or negative results are shown) and EZtargex (quantitative assay: Ct values are shown), were examined and analyzed. Results: The sensitivity and specificity of cPCR were 88.23% and 100%, respectively; the sensitivity and specificity of EZtargex were 87.65% and 100%, respectively. These assays also showed excellent concordance compared with the qPCR assay (κ = 0.828 for cPCR and κ = 0.820 for EZtargex). The statistical analysis showed a great diagnostic power of the EZtargex assay to discriminate between samples with different levels of positivity. Conclusions: The two point-of-care diagnostic platforms are accurate, rapid, convenient and require little training for PCV2 diagnosis. These POC platforms can discriminate viral loads to predict the clinical status of the animals. The current study provided evidence that these diagnostics were applicable with high sensitivity and specificity in the diagnosis of PCV2 infection in the field.

• 한국동물위생학회지
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• 제45권4호
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• pp.305-316
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• 2022

Bordetella (B.) bronchiseptica, Mycoplasma (M.) felis, and Chlamydia (C.) felis are considered as main bacterial pathogens of feline upper respiratory tract disease (URTD). In this study, a new triplex quantitative real-time polymerase chain reaction (tqPCR) assay was developed for the rapid and differential detection of these bacteria in a single reaction. The assay specifically amplified three bacterial genes with the detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 1%. Based on the diagnostic results of the assay using 94 clinical samples obtained from cats with URTD signs, prevalence of B. bronchiseptica, M. felis, or C. felis was 10.6%, 36.2%, or 6.4%, respectively, indicating that the diagnostic sensitivity was comparable to those of previously reported monoplex qPCR assays. The dual infection rates for B. bronchiseptica and M. felis or M. felis and C. felis was 2.1% or 3.2%, respectively. These results indicated that M. felis has been widely spread, and its co-infection with B. bronchiseptica or M. felis has been frequently occurred in Korean cat population. The developed tqPCR assay will serve as a promising tool for etiological and epidemiological studies of these three bacterial pathogens and the prevalence data obtained in this study will contribute to expanding knowledge about the epidemiology of feline URTD in Korea.

• Journal of Microbiology and Biotechnology
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• 제26권8호
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• pp.1398-1403
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• 2016

The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10¹ copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10² copies/20 g fresh lettuce, 9.7 × 10³ copies/20 g frozen strawberries, and 4.1 × 10³ copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

• Journal of Microbiology and Biotechnology
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• 제22권8호
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• pp.1113-1117
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• 2012

Shigella sonnei is a causal agent of fever, nausea, stomach cramps, vomiting, and diarrheal disease. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the specific detection of S. sonnei using a primer pair based on the methylase gene for the amplification of a 325 bp DNA fragment. The qPCR primer set for the accurate diagnosis of Shigella sonnei was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.

• Journal of Microbiology and Biotechnology
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• 제22권11호
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• pp.1575-1579
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• 2012

Paenibacillus polymyxa is known to be a plant-growth-promoting rhizobacterium. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the specific detection and quantitation of P. polymyxa using a primer pair based on the sequence of a membrane-fusion protein for the amplification of a 268 bp DNA fragment. This study reports that the qPCR-based method is applicable for the rapid and sensitive detection of P. polymyxa and can be used as an alternative method for agricultural soil monitoring.

• 농업과학연구
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• 제49권4호
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• pp.795-807
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• 2022

The development and commercialization of industrial genetically modified (GM) organisms is actively progressing worldwide, highlighting an increased need for improved safety management protocols. We sought to establish an environmental monitoring method, using real-time polymerase chain reaction (PCR) and propidium monoazide (PMA) treatment to develop a quantitative detection protocol for living GM microorganisms. We developed a duplex TaqMan quantitative PCR (qPCR) assay to simultaneously detect the selectable antibiotic gene, ampicillin (AmpR), and the single-copy Escherichia coli taxon-specific gene, D-1-deoxyxylulose 5-phosphate synthase (dxs), using a direct cell suspension culture. We identified viable engineered E. coli cells by performing qPCR on PMA-treated cells. The theoretical cell density (true copy numbers) calculated from mean quantification cycle (Cq) values of PMA-qPCR showed a bias of 7.71% from the colony-forming unit (CFU), which was within ±25% of the acceptance criteria of the European Network of GMO Laboratories (ENGL). PMA-qPCR to detect AmpR and dxs was highly sensitive and was able to detect target genes from a 10,000-fold (10^-4) diluted cell suspension, with a limit of detection at 95% confidence (LOD_95%) of 134 viable E. coli cells. Compared to DNA-based qPCR methods, the cell suspension direct PMA-qPCR analysis provides reliable results and is a quick and accurate method to monitor living GM E. coli cells that can potentially be released into the environment.

• 대한임상검사과학회지
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• 제55권3호
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• pp.184-194
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• 2023

골격근은 대사, 열기반 온도 조절, 그리고 전반적인 체내 균형을 위해 필수적인 조직이고 근발생(myogenesis)이라는 다단계 과정을 거쳐서 근관세포를 형성한다. p-아니스알데하이드(p-anisaldehyde, PAA) (4-메톡시벤잘데하이드)는 아니스 씨에서 추출된 에센셜 오일의 주성분이지만, 골격근에서의 기능은 아직까지 알려져 있지 않다. 따라서, 우리는 마우스 C2C12 근육모세포를 이용하여 근육분화가 PAA에 의해 영향을 받는지를 연구하였다. C2C12 근육모세포의 분화를 유도하기 위해 이 세포를 분화배지에서 5일동안 배양하였고, 매일 PAA (50 또는 200 ㎍/mL)를 포함하는 새로운 배지로 교체하였다. 대조군으로서 PAA가 포함되지 않은 배지를 사용하였다. 우리는 분화시작 후 1, 3, 5일째에 근관세포의 길이와 지름을 측정함으로써 PAA가 근관 형성에 미치는 영향을 평가하였고, quantitative real-time polymerase chain reaction 분석을 통해 PAA가 근육 표지인자(myoblast determination protein 1, myogenin, myocyte enhancer factor 2C, muscle creatine kinase, 및 myosin heavy chain)와 근육위축 관련 유전자(atrogin-1과 muscle ring finger-1 [MuRF-1])의 발현에 미치는 영향을 분석하였다. 또한, 주요 근육형성 키나아제인 protein kinase B (Akt)의 인산화를 웨스턴 블롯을 이용해 관찰하였다. 그 결과 PAA가 더 작고 얇은 근관 형성을 유의하게 유발하며 근육 표지인자의 발현을 감소시킨다는 것을 확인하였다. 또한, atrogin-1과 MuRF-1의 발현이 PAA에 의해서 감소하였는데, 이는 Akt 인산화의 감소와 일치하는 결과이다. 결론적으로, 본 연구결과는 PAA가 Akt 인산화와 활성화를 감소시킴으로써 C2C12 세포에서의 근육 분화를 억제하는 역할을 한다는 것을 증명한다.