• Restorative Dentistry and Endodontics
• /
• 제24권1호
• /
• pp.40-48
• /
• 1999

This study was designed to examine the specificities of the designed primers for F. nucleatum and F. necrophorum and to compare the PCR results using clinical samples with those of the anaerobic culture method. F. nucleatum and F. necrophorum spp. are frequently isolated in infected root canals, and they are related to periapical diseases. F. nucleatum(VPI 10197) and F. necrophorum(ATCC 25286) were used as references for PCR reaction, and thirty five teeth with one canal and periapical lesion were used. The samples were cultured anaerobically and identified using Rapid ID 32A(BioMerieux Vitek, Inc., France) as biochemical battery. In the GenBank database, species-specific PCR primers(nuc1/nuc2 primers for F. nucleatum and nec1/nec2 primers for F. necrophorum) were designed from the 16S ribosomal DNA(rDNA) sequences of F. nucleatum(accession number M58683) and F. necrophorum(accession number AF044948). PCR procedures of F. nucleatum(VPI 10197) and F. necrophorum (ATCC 25286) were simulated on a computer software. Amplify(v.1.2${\beta}$ for Macintosh). 820 bps and 817 bps of nucleotides were expected, respectively. Using extracted DNAs with QiaAmp tissue kit(Qiagen co., Germany), PCR was done. The results were as follows : 1. The nuc1/nuc2 primers produced an amplicon of 820 bps and the nec1/nec2 primers produced an amplicon of 817 bps. 2. The nuc1/nuc2 primers and the nec1/nec2 primers were specific and did not react with species other than the designated ones(i.e. nuc1/nuc2 primers did not produce amplicons for F. necrophorum, and vice versa.). And the PCR products of Porphyromonas endodontalis(ATCC 35406), Porphyromonas gingivalis(ATCC 33277), Prevotella intermedia(ATCC 25611), and Prevotella nigrescens(ATCC 33563), frequently isolated in infected root canals and periapical lesions, were not amplified by the primers specific for Fusobacterium nucleatum and Fusobacterium necrophorum. 3. This method utilizing PCR could detect F. nucleatum and F. necrophorum in clinical samples, while anaerobic culture method could detect neither.

• 한국미생물·생명공학회지
• /
• 제43권2호
• /
• pp.97-104
• /
• 2015

식초는 세계적으로 사용되는 조미료로 밀, 과일, 곡물 등을 원료로하여 다양한 방법으로 제조된다. 지금까지 식초에 대한 대부분의 연구들은 항산화활성에 한정된 연구였다. 본 연구에서는 현미를 이용하여 만든 현미 발효 식초의 이화학적 특성과 항균활성에 대해 시험하였으며, 현미발효식초의 항균활성은 paper disc-agar diffusion 방법을 이용하여 조사하였 때, 병원성 박테리아와 효모에 대해 강한 항균활성을 나타내었다. 특히 Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Yersinia enterocolitica, and Lodderomyces elongisporus의 균주에 대해서는 상용되는 항생제인 카베니실린과 테트라사이클린보다 더 높은 항균활성을 보였다. 항산화활성은 2,2-diphenyl-1-picrylhydrazyl (DPPH) 라디칼 소거능을 이용하여 측정하였고, 대표되는 항산화제인 아스코르빅 산과 비슷한 활성을 나타내었다. 현미발효흑초의 발효중에 나타나는 균주를 동정하기 위해 TSB 고체배지와 YPD 고체배지에 현미발효흑초를 도포하였을 때, 분리된 콜로니를 16S rDNA sequence 분석을 통하여, FDVS-1, 2, 3 세가지 균주를 분리하였으며, phylogenic tree 분석법을 이용하여 조사하였을 때, 각각 Acetobacter papayae, Acetobacter pasteuranus, Acetobacter peroxidans와 유사하였다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제14권6호
• /
• pp.880-884
• /
• 2001

Rumen microbiology research has undergone several evolutionary steps: the isolation and nutritional characterization of readily cultivated microbes; followed by the cloning and sequence analysis of individual genes relevant to key digestive processes; through to the use of small subunit ribosomal RNA (SSU rRNA) sequences for a cultivation-independent examination of microbial diversity. Our knowledge of rumen microbiology has expanded as a result, but the translation of this information into productive alterations of ruminal function has been rather limited. For instance, the cloning and characterization of cellulase genes in Escherichia coli has yielded some valuable information about this complex enzyme system in ruminal bacteria. SSU rRNA analyses have also confirmed that a considerable amount of the microbial diversity in the rumen is not represented in existing culture collections. However, we still have little idea of whether the key, and potentially rate-limiting, gene products and (or) microbial interactions have been identified. Technologies allowing high throughput nucleotide and protein sequence analysis have led to the emergence of two new fields of investigation, genomics and proteomics. Both disciplines can be further subdivided into functional and comparative lines of investigation. The massive accumulation of microbial DNA and protein sequence data, including complete genome sequences, is revolutionizing the way we examine microbial physiology and diversity. We describe here some examples of our use of genomics- and proteomics-based methods, to analyze the cellulase system of Ruminococcus flavefaciens FD-1 and explore the genome of Ruminococcus albus 8. At Illinois, we are using bacterial artificial chromosome (BAC) vectors to create libraries containing large (>75 kbases), contiguous segments of DNA from R. flavefaciens FD-1. Considering that every bacterium is not a candidate for whole genome sequencing, BAC libraries offer an attractive, alternative method to perform physical and functional analyses of a bacterium's genome. Our first plan is to use these BAC clones to determine whether or not cellulases and accessory genes in R. flavefaciens exist in clusters of orthologous genes (COGs). Proteomics is also being used to complement the BAC library/DNA sequencing approach. Proteins differentially expressed in response to carbon source are being identified by 2-D SDS-PAGE, followed by in-gel-digests and peptide mass mapping by MALDI-TOF Mass Spectrometry, as well as peptide sequencing by Edman degradation. At Ohio State, we have used a combination of functional proteomics, mutational analysis and differential display RT-PCR to obtain evidence suggesting that in addition to a cellulosome-like mechanism, R. albus 8 possesses other mechanisms for adhesion to plant surfaces. Genome walking on either side of these differentially expressed transcripts has also resulted in two interesting observations: i) a relatively large number of genes with no matches in the current databases and; ii) the identification of genes with a high level of sequence identity to those identified, until now, in the archaebacteria. Genomics and proteomics will also accelerate our understanding of microbial interactions, and allow a greater degree of in situ analyses in the future. The challenge is to utilize genomics and proteomics to improve our fundamental understanding of microbial physiology, diversity and ecology, and overcome constraints to ruminal function.

• Journal of Periodontal and Implant Science
• /
• 제30권2호
• /
• pp.419-429
• /
• 2000

Prevotella intermedia has been implicated as a potent pathogen in many kinds of periodontal, pulpal and periapical diseases. However, it has been isolated from periodontally healthy adults and from edentulous children as well. The intraspecies heterogeneity of Prevotella intermedia has been demonstrated in early studies and finally Shah & Gharbia confirmed the existence of 2 DNA homology groups and proposed dividing Prevotella intermedia into 2 species, Prevotella intermedia and Prevotella nigrescens. This study was designed to examine the frequency of Prevotella intermedia and Prevotella nigrescens in diseased periodontal pockets and healthy gingival sulcus of Korean people by PCR based on 16s ribosomal DNA sequence. One hundred adults who had adult periodontitis but not taken any periodontal treatment or antibiotics during previous 6 months and 50 adults who had healthy periodontal tissue were selected for this study. The sulcular fluid was collected into VMGA by sterilized paper point and diluted to 1,000 times in anaerobic chamber. $100{\mu}{\ell}$ of sample was cultured in $37^{\circ}C$ for 10 days. Among the bacterial colonies, BPB were selected and cultured in BHI broth and then Prevotella intermedia was identified through Gram staining and biochemical test. Identified Prevotella intermedia was cultured again and centrifuged. DNA was extracted from the pellet using several reagents. PCR was performed by previously designed primer. The results were followed. 1. BPB were isolated from 39 of 100 samples of diseased periodontal pockets(39%). 2. Prevotella intermedia was identified from 24 of 39 BPB samples. 3. Among 24 Prevotella intermedia, 21 were confirmed as Prevotella inter - media(87.5) and 2 were confirmed as Prevotella nigrescens(8.33%). 4. BPB were isolated from 9 of 50 samples of periodontally healthy patients. Among them only two were identified as Prevotella intermedia, that is, one was confirmed as Prevotella intermedia and the other was Prevotella nigrescens.

• 한국균학회지
• /
• 제27권4호통권91호
• /
• pp.274-279
• /
• 1999

먹물버섯속 및 눈물버섯속 균류의 계통학적 유연관계 분석을 위해 rDNA의 cluster 중 ITS 영역을 Polymerase Chain Reaction(PCR)로 증폭하여 염기서열을 밝혔다. 이들의 ITS I, II 영역은 각각 $258{\sim}301\;bp,\;253{\sim}275\;bp$의 염기쌍으로 이루어져 있었으며 균주간 ITS 염기서열의 상동성은 $43.9{\sim}96%$로 나타났다. 이들의 염기서열을 이용해 분류도(tree)를 작성한 결과, Singer의 형태적 분류체계와 거의 유사한 결과를 보였으나 눈물버섯속 균류들은 먹물버섯속 내 그룹 II의 종들과 함께 분지를 형성하였고 먹물버섯속의 의기준종(type species)인 C. comatus는 조사된 다른 동일 속의 종들과의 유사도에서 50%수준의 저조한 상동성을 보여주었다. 이와 같은 결과로 볼 때 먹물버섯류가 기존의 다수 학자들이 보고한 단일 계통진화(monophyletic evolution)를 해온 속이란 점에서 의문점을 남기었고 좀더 많은 조사가 필요하겠으나 눈물버섯속 균류도 먹물버섯속 균류와 분리시키는 것보다는 동일 속으로 분류하는 것이 바람직 할 것으로 사료되었다.

• 식물병연구
• /
• 제18권2호
• /
• pp.133-138
• /
• 2012

2009년 11월에 서귀포에서 꽃댕강나무 흰가루병을 발견하였다. 이어 제주, 부산, 통영 등 남부지방에서도 추가적으로 발견되었다. 흰색의 균체가 잎, 어린 줄기, 꽃을 감염하여 관상가치를 떨어뜨렸으며, 발병이 지속되면 잎 앞면의 병환부는 적자색으로 변하였다. 이 흰가루병균의 형태적 특징과 분자적 분석을 통하여 이 곰팡이는 Erysiphe abeliicola U. Braun & S. Takam.로 동정되었다. 무성세대의 분류학적 특징은 이 연구를 통하여 처음으로 기재되는데, 균사의 굴곡형 부착기와 분생포자경의 짧은 기부세포가 특징적이었다. 성숙한 자낭구의 분류학적 특징은 앞선 일본의 기재와 거의 일치하였다. 우리나라 시료에서 rDNA ITS 영역의 염기서열을 처음으로 분석하여 이 종이 Erysiphe속의 Microsphaera절에 속함을 밝혔으며, 이는 형태적 특징과 상응하는 결과였다. 이로써 우리나라에서 E. abeliicola에 의한 꽃댕강나무 흰가루병을 처음으로 보고하고, 꽃댕강나무가 이 흰가루병균의 기주로 확인된 것은 세계적으로 처음이다.

• 생명과학회지
• /
• 제29권5호
• /
• pp.524-529
• /
• 2019

엉겅퀴는 대표적인 다년생의 약용식물이다. 최근 국제적 추세에 따라 자국의 유전자원의 발굴, 보존 등이 강화 됨에 따라 인접국가와 국내 자생 엉겅퀴 계통을 판별 할 수 있는 기준 설정에 관한 연구의 필요성이 대두되고 있지만, 분자생물학적 판별 기술의 개발은 아직 미흡한 실정이다. 본 연구에서는 국내 토종과 해외 유래 엉겅퀴종의 기원을 판별하기 위해 엽록체에 존재하는 trnL-trnF와 MatK 유전자단편에서 SNP를 이용한 판별 프라이머를 확보하였으며 이를 보완하여 보다 신속하게 판별하기 위하여 HRM 분석 기술을 이용한 판별 마커와 그 조건을 확립하였다. 그러므로, 본 연구에서 개발된 SNP 마커는 다양한 지역 또는 국가에서 서식하는 엉겅퀴 종들의 신속한 확인을 위해 매우 유용하게 이용될 것으로 생각된다.

• Journal of Microbiology and Biotechnology
• /
• 제27권3호
• /
• pp.561-572
• /
• 2017

The global commercial cultivation of transgenic crops, including glyphosate-tolerant soybean, has increased widely in recent decades with potential impact on the environment. The bulk of previous studies showed different results on the effects of the release of transgenic plants on the soil microbial community, especially rhizosphere bacteria. In this study, comparative analyses of the bacterial communities in the rhizosphere soils and surrounding soils were performed between the glyphosate-tolerant soybean line NZL06-698 (or simply N698), containing a glyphosate-insensitive EPSPS gene, and its control cultivar Mengdou12 (or simply MD12), by a 16S ribosomal RNA gene (16S rDNA) amplicon sequencing-based Illumina MiSeq platform. No statistically significant difference was found in the overall alpha diversity of the rhizosphere bacterial communities, although the species richness and evenness of the bacteria increased in the rhizosphere of N698 compared with that of MD12. Some influence on phylogenetic diversity of the rhizosphere bacterial communities was found between N698 and MD12 by beta diversity analysis based on weighted UniFrac distance. Furthermore, the relative abundances of part rhizosphere bacterial phyla and genera, which included some nitrogen-fixing bacteria, were significantly different between N698 and MD12. Our present results indicate some impact of the glyphosate-tolerant soybean line N698 on the phylogenetic diversity of rhizosphere bacterial communities together with a significant difference in the relative abundances of part rhizosphere bacteria at different classification levels as compared with its control cultivar MD12, when a comparative analysis of surrounding soils between N698 and MD12 was used as a systematic contrast study.

• 한국해양학회지:바다
• /
• 제20권3호
• /
• pp.141-150
• /
• 2015

와편모류는 해양의 주요 일차 생산자로서, 독립영양성, 종속영양성 및 혼합영양성 등의 다양한 영양방식을 가지는 생물군으로서 해양 미소먹이망에서 중요한 생태학적 역할을 담당하고 있다. 그러나 와편모류에 대한 대부분의 연구가 연안이나 외해의 표영생태계에 국한되어 수행되었으며, 사질조간대에 서식하는 저서성 와편모류에 대한 연구는 매우 미흡하며, 국내에서는 제주연안을 제외하고는 거의 전무하다. 본 연구는 전라북도 서해안에 위치한 동호의 사질조간대에서 2012년 11월부터 2014년 2월까지 매월 간조기에 저서성 와편모류의 출현양상을 조사하고 주요 출현종의 28S rDNA 염기서열을 획득하여 분자계통학적 분석을 실시하였다. 연구기간동안 Gymnodiniales, Gonyaulacales, Peridiniales, Prorocentrales의 4개 목에 속하는 총 13속 27종의 저서성 와편모류가 출현하였고, Amphidinium 속이 9종으로 가장 다양한 종이 출현하였다. 동호를 비롯하여 서해안에 위치한 모항, 송호, 가마미와 제주 협재에서 분리한 저서성 와편모류 총 16종, 34개 종주에서 28S rDNA 염기서열 정보를 성공적으로 확보하였으며, 분자계통학적 분석에 이용하였다. Amphidinium에 속하는 종들은 Gymodiniales 목의 4개의 분기군 가운데 3개의 분기군에 걸쳐서 분지하여, 다계통학적(polyphyletic) 특성을 나타내었다. 28S rDNA염기서열을 이용한 유전적 거리를 비교한 결과 국내에서 출현한 Amphidinium mootonorum 종주들은 캐나다에서 분리한 종주와 19.2%의 유전적 차이를 보여 종내 변이가 출현 종들 가운데 가장 큰 것으로 나타났다. 본 연구 해역에서 독성 와편모류인 Amphidinium carterae와 A. operculatum이 간헐적으로 출현하여 이들의 독성과 정량적인 모니터링이 추후에 필요하다.

• Parasites, Hosts and Diseases
• /
• 제54권2호
• /
• pp.173-179
• /
• 2016

Echinostoma hortense (Digenea: Echinostomatidae) is one of the intestinal flukes with medical importance in humans. However, the mitochondrial (mt) genome of this fluke has not been known yet. The present study has determined the complete mt genome sequences of E. hortense and assessed the phylogenetic relationships with other digenean species for which the complete mt genome sequences are available in GenBank using concatenated amino acid sequences inferred from 12 protein-coding genes. The mt genome of E. hortense contained 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 non-coding region. The length of the mt genome of E. hortense was 14,994 bp, which was somewhat smaller than those of other trematode species. Phylogenetic analyses based on concatenated nucleotide sequence datasets for all 12 protein-coding genes using maximum parsimony (MP) method showed that E. hortense and Hypoderaeum conoideum gathered together, and they were closer to each other than to Fasciolidae and other echinostomatid trematodes. The availability of the complete mt genome sequences of E. hortense provides important genetic markers for diagnostics, population genetics, and evolutionary studies of digeneans.