• 한국환경농학회지
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• 제36권4호
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• pp.299-305
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• 2017

탄저병이 발생한 자두에서 11개 탄저병균을 순수 분리하여 병원성을 검정한 후 PDA에 접종하여 $25^{\circ}C$에서 7-10일 동안 배양하면서 각 균주의 균사 생장속도, colony의 특징과 색, 포자 형태 및 크기를 관찰하였다. 각 균주의 genomic DNA를 추출하여 rDNA-ITS 영역을 증폭한 다음, PCR을 하여 염기서열을 해독하였다. 각 균주의 배양적 특성, 포자의 형태와 크기 및 염기서열을 NCBI GenBank의 염기서열과 상동성을 비교하여 동정한 결과 6개 균주는 Colletotrichum acutatum으로, 5개 균주는 C. gloeosporioides로 동정되었다.

• Genomics & Informatics
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• 제1권1호
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• pp.20-24
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• 2003

The analysis of DNA microarry data is one of the most important things for functional genomics research. The matrix representation of microarray data and its successive 'optimal' incisional hyperplanes is a useful platform for developing optimization algorithms to determine the optimal partitioning of pairwise proximity matrix representing completely connected and weighted graph. We developed Deterministic Annealing (DA) approach to determine the successive optimal binary partitioning. DA algorithm demonstrated good performance with the ability to find the 'globally optimal' binary partitions. In addition, the objects that have not been clustered at small non­zero temperature, are considered to be very sensitive to even small randomness, and can be used to estimate the reliability of the clustering.

• 한국균학회지
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• 제44권1호
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• pp.1-7
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• 2016

우리나라 토양에서 효모 종 분포특성을 조사하기 위해 먼저 대전광역시를 포함하는 충청남도 전 지역의 밭 토양 61점을 2015년 6월부터 8월까지 수집하여 97균주 20종의 야생효모들을 분리하였고, polymerase chain reaction (PCR)을 통해 internal transcribed spacer (ITS) 부위와 26S rDNA의 D1/D2 부위의 염기서열 상동성 비교법을 이용하여 종을 동정하였다. 이들 가운데 Cryptococcus podzolicus가 11균주를 포함하는 Cryptococcus 속 균이 전체 분리 균주 중 16균주(16%)로 가장 많이 분리되었고, 다음으로 Debaryomyces hansenii와 Trichorsporon asahii도 각각 6주씩 분리되었다.

• 한국균학회지
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• 제44권4호
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• pp.365-370
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• 2016

본 연구에서는 잣나무, 눈측백의 침엽과 자란의 뿌리에서 식물에 공생하는 내생균을 분리하여 동정하였다. 이를 위해 specific primer인 ITS1F와 ITS4를 이용하여 internal transcribed spacer (ITS) rDNA영역과 primer LR0R과 LR 16을 이용하여 large subunit rDNA 영역을, primer Bt2a과 Bt2b를 이용하여 ${\beta}$-tubulin 영역을, 그리고 EF-1 primer를 이용하여 translation elongation factor 지역의 염기서열을 동시에 분석하였다. 연구 결과 Colletotrichum simmondsii, Fusarium sterilihyphosum, Diatrypella pulvinata, Ochroconis globalis, Sphaeria chrysosperma 등 국내 미기록 균주 5종을 분리하여 형태적, 분자생물학적 동정 결과를 서술하였다.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2005년도 International Symposium & conference of the Plant Resources Society of Korea
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• pp.183-183
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• 2005

• Journal of Microbiology and Biotechnology
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• 제17권7호
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• pp.1090-1097
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• 2007

Genomic analysis of Thermococcus sp. NA1 revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at pH 7.5 and $75^{\circ}C$. TNA1_pol was highly thermostable, with a half-life of 3.5h at $100^{\circ}C$ and 12.5h at $95^{\circ}C$. Polymerase chain reaction parameters of TNA1_pol such as error-rate, processivity, and extension rate were measured in comparison with rTaq, Pfu, and KOD DNA polymerases. TNA1_pol averaged one incorrect bp every 4.45 kilobases (kb), and had a processivity of 150 nucleotides (nt) and an extension rate of 60 bases/s. Thus, TNA1_pol has a much faster elongation rate than Pfu DNA polymerase with 7-fold higher fidelity than that of rTaq.

• The Plant Pathology Journal
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• 제31권2호
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• pp.123-131
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• 2015

Clavibacter michiganensis subsp. sepedonicus (Cms) multiplies very rapidly, passing through the vascular strands and into the stems and petioles of a diseased potato. Therefore, the rapid and specific detection of this pathogen is highly important for the effective control of the pathogen. Although several PCR assays have been developed for detection, they cannot afford specific detection of Cms. Therefore, in this study, a computational genome analysis was performed to compare the sequenced genomes of the C. michiganensis subspecies and to identify an appropriate gene for the development of a subspecies-specific PCR primer set (Cms89F/R). The specificity of the primer set based on the putative phage-related protein was evaluated using genomic DNA from seven isolates of Cms and 27 other reference strains. The Cms89F/R primer set was more specific and sensitive than the existing assays in detecting Cms in in vitro using Cms cells and its genomic DNA. This assay was also able to detect at least $1.47{\times}10^2copies/{\mu}l$ of cloned-amplified target DNA, 5 fg of DNA using genomic DNA or $10^{-6}$ dilution point of 0.12 at $OD_{600}$ units of cells per reaction using a calibrated cell suspension.

• Journal of Microbiology
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• 제43권6호
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• pp.537-545
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• 2005

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used $H-2K^b$ restricted T-cell epitopes of NP. The NP-specific $CD8^+$ T cell response was analyzed using a $^{51}Cr-release$ assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific $CD8^+$ T cell response at eight days after infection. We also found that several different methods to check the NP-specific $CD8^+$ T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited $2\~4$ weeks after immunization and maximized at $6\~8$ weeks. NP-specific $CD8^+$ T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.

• 한국동물위생학회지
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• 제41권4호
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• pp.257-262
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• 2018

Avian pathogenic E. coli (APEC) causes severe economic losses in the poultry farms, due to systemic infections leading to lethal colisepticemia. It causes a variety of diseases from air sac infection to systemic spread leading to septicemia. Secondary infection contains opportunistic infections due to immunosuppression disease. Collibacillosis causes the great problems in the poultry industry in Korea. Thus, it is necessary to identify and classify the characteristics of E. coli isolate of chicken origin to confirm the diversity of symptoms and whether they are transmitted among the farms. Fragment analysis is identify the difference in the number of Variable-Number Tandem-Repeats (VNTRs) for genotyping. VNTRs have repeating structure (Microsatellite, Short tandem repeats; STR, Simple sequence repeats; SSR) in the chromosome. This region can be used as a genetic marker because of its high mutation rate. And various lengths of the amplified DNA fragment cause the difference in the number of repetition of the DNA specific site. The number of repetition sequences indicates the separated size of fragments, so the each fragments can be distinguished by specific samples. The results of the sample show that there is no difference in six microsatellite loci (yjiD, aidB, molR_1, ftsZ, b1668, yibA). There are differences among the farms in relation of the number of repetitions of other six microsatellite loci (ycgW, yaiN, yiaB, mhpR, b0829, caiF). Four (ycgW, yiaB, b0829, caiF) of these six microsatellite loci show statistically significant differences (P<0.05). It means that the analysis using four microsatellite loci including ycgW, yiaB, b0829, and caiF can confirm among the farms. Five E. coli samples in one farm have same SSR repetition at all markers. But, there are significant differences from other farms at Four (ycgW, yiaB, b0829, caiF) microsatellite loci. These results emphasize again that the four microsatellite loci makes a difference in the amplified DNA fragments, enabling it to be used for E. coli genotyping.

• Genomics & Informatics
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• 제21권4호
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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.