• The Plant Pathology Journal
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• 제34권6호
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• pp.575-579
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• 2018

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as $4.7ng/{\mu}l$ of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of $42^{\circ}C$. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.

• Journal of Korean Neurosurgical Society
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• 제40권5호
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• pp.363-368
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• 2006

Objective : Little is known about the comprehensive molecular and biological mechanism on the development of the degeneration of the intervertebral disc. Many kinds of matrix metalloproteinase[MMP] initiate the degradation of the extracellular matrix including several kinds of collagens and proteoglycans. We compared molecular and immunohistochemical features of degenerated intervertebral disc and normal counterparts in order to investigate the role of MMP-1, 2, 3, 9. Methods : We have evaluated MMP-1, 2, 3, 9 expression in 30 surgically resected lumbar disc from degenerative disc disease patients and 5 normal control cases. RT-PCR[reverse transcriptase-polymerase chain reaction] and immunohistochemistry were performed. Results : By RT-PCR, normal tissue samples showed merely scant expression of MMP-1, 2, 3, 9 mRNA, but degenerated disc samples revealed more pronounced expression. mRNA amplifications were detected in 60%, 63.3%, 70%, 53.3% cases By immunohistochemistry, normal tissue samples showed minimal protein expression of MMP-1, 2, 3, 9, but degenerated disc samples revealed more pronounced expression. Protein expressions were detected in 73.3%, 63.3%, 76.7%, 63.3% cases. Both the mRNA amplification and protein overexpression rates were significantly higher in degenerated disc than in the normal tissue. Concordance between both the mRNA amplification and protein expressions of MMP-1, 3, 9 were not observed, but there is well correlation in MMP-2 expression. Conclusion : We concluded that the over-expressions of the MMP-1, 2, 3, 9 may contribute to the development of degeneration of the intervertebral disc.

• 대한수의학회지
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• 제42권2호
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• pp.225-229
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• 2002

We report that mycoplasma organisms from lung tissues of slaughter pigs were identified to genes fragments with references use of nested-PCR technique(nPCR). Seven strains of mycoplasma species were isolated from 70 lung tissues. The organisms were detected by in vitro amplification of 16S rRNA and 23S rRNA genes. Nucleotide sequences of the spacer between 16S and 23S in the ribosomal RNA operons of mycoplasma were identified by the analysis of products from the nested PCR. Four common PCR primers, MhF1, MhF2 MhR1 and MhR2, were designed by analysis between these sequences by first amplified with F1, R1 and second with F2, R2, respectively. Specific amplification of the spacer region for reference strains of M. hyopneumoniae, M. hyorhinis, M. flocculare were confirmed by first round of PCR in which the traduced fragments of 690bp, 460bp, 630bp. But amplications of second round was changed to 240bp, 210bp, 230bp, respectively. Three different strains (M. hyopneumoniae:4, M. hyorhinis:2, M. flocculare:1) were detected by the nested-PCR technique. The results suggest that the detection of swine mycoplasma by n-PCR can be analyzed the nucleotide sequences between rRNA operons and homology study.

• 한국균학회지
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• 제47권4호
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• pp.437-441
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• 2019

국화에 발생하는 반쪽시들음병은 Veriticillium dahliae에 의해 발생하는 진균병으로 국화 재배농가에 상당한 경제적 손실을 야기한다. 일반 식물병원균을 동정하는 방법으로는 병원균을 진단하기까지 상당한 시간이 소요된다. 본 연구에서는V. dahliae를 신속하고 특이적으로 진단하기 위하여 등온증폭기술 (Loop-mediated isothermal amplification, LAMP)을 적용한 검출법을 개발하였다. 이 방법은 반쪽시들음병균의 cellulose-growth-specific protein partial mRNA 유전자 염기서열을 이용하여 4개의 특이 프라이머 세트를 제작하였다. 최적 반응조건 및 시간은 60℃ 내외의 온도조건에서 60분 이내에서 가장 효율이 좋은 것으로 나타났다. 이 등온증폭 검출법은 4종의 토양전염성 병원균과 기주식물의 DNA에는 반응하지 않았다. 따라서 반쪽시들음병균 등온증폭법을 활용한다면 병원균의 감염 유무를 조기에 신속하게 진단할 수 있고, 반쪽시들음병을 효율적으로 모니터링하고 방제할 수 있을 것으로 기대한다.

• 대한임상검사과학회지
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• 제40권2호
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• pp.113-117
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• 2008

It designed a study to examine the efficiency of DNA and RNA extraction from archival formalin-fixed, paraffin-embedded tissues using an non-heating and heating method. Archival paraffin blocks of liver, kidney, colon were randomly selected. Each paraffin block was prepared in 20 microtubes. For each paraffin blocks were tested non-heating DNA extraction to 10 microtubes and heating protocol under pH 7.0 and $100^{\circ}C$ to 10 microtubes. Evaluation of the results of DNA extraction was carried out by measuring concentration by UV spectrophotometry and then PCR amplification. DNA extraction content that non-heating method was liver $5{\pm}0.7{\mu}g/mL$, kidney $2{\pm}0.3{\mu}g/mL$, colon $6{\pm}0.4{\mu}g/mL$ and heating method was liver $12{\pm}0.6{\mu}g/mL$, kidney $7{\pm}0.5{\mu}g/mL$, colon $10.{\pm}0.3{\mu}g/mL$. Successful RNA extraction was observed, by ${\beta}$-actin amplification, in 46.7% sections for samples treated by the heating method versus 30.0% using non-heating DNA extraction. The extracted nucleic acid showed better values for samples heated at $100^{\circ}C$. Therefore heating extraction of nucleic acid is reliable, quick and efficiency.

• 생약학회지
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• 제48권3호
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• pp.173-178
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• 2017

We investigated the efficacy of single compound of plant extract in coxsackievirus B3 (CVB3) infection. CVB3 is a main cause of Hand-foot-mouth diseases (HFMD) and viral myocarditis in children and adult. Several single compounds of plant extract were purified by HPLC and tested as antiviral drug candidate. Among them, kaempferol was selected to effective anti-enterovirus compound by HeLa cells survival assay. CVB3 infected HeLa cells were treated with kaempferol ($100{\mu}g/ml-100ng/ml$) and their antiviral effect was confirmed. After 16 hours of treatment, HeLa cells were lysed and proteins were extracted for western blot analysis. CVB3 viral capsid protein VP1 production and transcription factor eIF4G-1 cleavage was significantly decreased in $100{\mu}g/ml$ kaempferol treatment. Virus replication was observed by virus RNA amplification. Kaempferol strongly reduced virus positive and negative strand RNA amplification. Moreover, MAPK signal induced by CVB3 infection, pERK and pmTOR, kaempferol treatment significantly inhibited the activity. Plant extract single compound, kaempferol, is a strong candidate to be developed non-toxic anti-enterovirus treatment agent.

• 한국토양비료학회지
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• 제44권1호
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• pp.127-145
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• 2011

Various environmental ecosystems are valuable sources for microbial ecology studies, and their analyses using recently developed molecular ecological approaches have drawn significant attention within the scientific community. Changes in the microbial community structures due to various anthropogenic activities can be evaluated by various culture-independent methods e.g. ARISA, DGGE, SSCP, T-RFLP, clone library, pyrosequencing, etc. Direct amplification of total community DNA and amplification of most conserved region (16S rRNA) are common initial steps, followed by either fingerprinting or sequencing analysis. Fingerprinting methods are relatively quicker than sequencing analysis in evaluating the changes in the microbial community. Being an efficient, sensitive and time- and cost effective method, T-RFLP is regularly used by many researchers to access the microbial diversity. Among various fingerprinting methods T-RFLP became an important tool in studying the microbial community structure because of its sensitivity and reproducibility. In this present review, we will discuss the important developments in T-RFLP methodology to distinguish the total microbial diversity and community composition in the various ecosystems.

• 한국산학기술학회논문지
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• 제21권5호
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• pp.216-222
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• 2020

Tumor necrosis factor receptor associated factor 4 (TRAF4)는 사람의 유방암에서 과발현 되며, 암세포전이, ROS 및 세포 극성 형성 등에 관여하는 것으로 알려져 있다. 그러나 돼지에서는 아직까지 그 기능과 특성에 대한 연구가 보고 된 바 없다. 따라서 본 연구에서는 돼지 TRAF4의 mRNA 전장서열을 분석하고, 그 기능과 특성을 알아보고자 수행되었다. TRAF4의 전장서열을 밝히기 위해 돼지 신장유래세포(pK15)에서 total RNA 추출하여 RACE (Rapid Amplification of cDNA ends) PCR을 수행하였다. 2,030 염기쌍의 mRNA 전장서열을 분석하였고, 470개의 아미노산으로 구성 되어 있는 것을 확인하였다. 사람과 쥐의 Homology를 분석한 결과 각각 93 % 그리고 90 %의 유사도를 가지며, 사람과는 8개, 쥐와는 12개의 아미노산 차이가 있음을 확인하였다. qPCR을 통하여 TRAF4, CLDN4, OCLN 그리고 TJP1의 발현을 분석한 결과 세포의 confluency 정도에 따라 발현이 다르게 나타남을 확인하였고, 세포가 40% 증식한 그룹 보다 60 %와 80 % 이상 증식 한 그룹에서 유의적으로 높게 나타났다. 또한 TRAF4의 기능을 확인하기 위하여 TRAF4 siRNA 처리 한 결과 TRAF4와 tight junction 관련 유전자가 낮게 발현됨을 관찰하였다. 따라서 사람과 마우스와 같이 돼지에서도 TRAF4가 발현되며, 세포-세포 간 중요한 역할을 하는 tight junction에 관여하는 것으로 사료된다.

• The Plant Pathology Journal
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• 제36권5호
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• pp.459-467
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• 2020

Areca palm yellow leaf (AYL) disease caused by the 16SrI group phytoplasma is a serious threat to the development of the Areca palm industry in China. The 16S rRNA gene sequence was utilized to establish a rapid and efficient detection system efficient for the 16SrI-B subgroup AYL phytoplasma in China by loop-mediated isothermal amplification (LAMP). The results showed that two sets of LAMP detection primers, 16SrDNA-2 and 16SrDNA-3, were efficient for 16SrI-B subgroup AYL phytoplasma in China, with positive results appearing under reaction conditions of 64℃ for 40 min. The lowest detection limit for the two LAMP detection assays was the same at 200 ag/μl, namely approximately 53 copies/μl of the target fragments. Phytoplasma was detected in all AYL disease samples from Baoting, Tunchang, and Wanning counties in Hainan province using the two sets of LAMP primers 16SrDNA-2 and 16SrDNA-3, whereas no phytoplasma was detected in the negative control. The LAMP method established in this study with comparatively high sensitivity and stability, provides reliable results that could be visually detected, making it suitable for application and research in rapid diagnosis of AYL disease, detection of seedlings with the pathogen and breeding of disease-resistant Areca palm varieties.

• Genomics & Informatics
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• 제10권4호
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• pp.249-255
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• 2012

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular- weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.