• 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.

• Asian Pacific Journal of Cancer Prevention
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• 제15권20호
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• pp.8883-8886
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• 2014

Background: Tetracycline is an antibiotic widely used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing due to increasing bacterial resistance. The aim of this study was to investigate the occurrence of 16S rRNA mutations associated with resistance or reduced susceptibility to tetracycline ofHelicobacter pylori by real-time PCR (RT-PCR) assays from culture. Materials and Methods: Tetracycline susceptibility and minimal inhibition concentration (MIC) was determined by the Epsilometer test (Etest) method. A LightCycler assay developed to detect these mutations was applied to DNA extracted from culture. The 16S rRNA of these isolates was sequenced and resistance-associated mutations were identified. From 104 isolates of H. pylori examined, 11 showed resistance to tetracycline. Results: LightCycler assay was applied to DNA extracted from 11 tetracycline-susceptible and 11 tetracycline resistance H. pylori isolates. In our study the sequencing of the H. pylori wild types in 16 s rRNA gene were AGA 926-928 with MIC (0.016 to $0.5{\mu}g/ml$), while the sequencing and MIC for resistant were GGA and AGC, (0.75 to $1.5{\mu}g/ml$), respectively. Also we found a novel mutation in 2 strains with $84^{\circ}C$ as their melting temperatures and exhibition of an A939C mutation. Conclusions: We conclude that real-time PCR is an excellent method for determination of H. pylori tetracycline resistance related mutations that could be used directly on biopsy specimens.

• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.312-317
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• 2002

Indigenous lactobacilli were isolated from vaginas of Korean women for possible use in ecological treatment of bacterial vaginosis. Vaginal swab samples were obtained from a gynecological clinic and streaked on Rogosa SL agar plates to select the most predominant lactobacilli in each sample. The preliminary identification of the isolates as lactobacilli was based on microscopic observation of Gram-positive rod-shaped cell morphology. The initial characterization was performed on 108 isolates in terms of their cell surface hydrophobicity (CSH), antimicrobial activity, and hydrogen peroxide (H₂O₂) production capability, and 10 isolates were then selected for further molecular identification. For a rapid procedure to identify lactobacilli, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of the l6S rRNA genes were applied. The 10 selected lactobacilli and 9 different reference strains of Lactobacillus spp. were characterized by PCR-RFLP where the amplified l6S rDNA was digested with 7 different restriction endonucleases prior to analysis. DNA sequencing of the 16S rRNA gene of one particular isolate, KLB 46, that had been identified as L. crispatus by the PCR-RFLP analysis, further confirmed its identity as L. crispatus.

• 미생물학회지
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• 제25권4호
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• pp.282-289
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• 1987

Pseudomonas putida KU816에서 분리한 플라스미드 pKU10의 여러가지 특성을 조사하고 그 제한 효소 지도를 작성하였다. pKU 10은 암펴설런, 테트라사이클린, 클로람페니콜에 대한 내성 유전자를 갖는 작은 R factor로서 마이토마이신 C에 의하여 큐어링 된다. 플라스미드의 크기는 9.4Kb로 측정되었다. pKU 10은 Pseudomonas와 E.coli블 숙주로 하였을 때 안정하게 형질 발현이 되다. 또한 pKU 10의 불화합성균은 IncP-I으로 조사되었다. Eco RI, Xho I. SaiI, BglII, SmaI은 pKU 10 DNA를 한 부위에서 자르고, Pst I은 두 부위, Hind Ill는 여섯 부위에서 자른다. 제한 효소 지도는 제한 효소를 이중, 삼중으로 완전 소화시키거나, 부분 소화시켜서 얻었다. pKU 10은 Pseudomonas속에서 유용한 클로닝 벡터호 이용된 것으로 기대된다.

• IMMUNE NETWORK
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• 제24권1호
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• pp.1.1-1.6
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• 2024

IL-18 binding protein (IL-18BP) was originally discovered in 1999 while attempting to identify an IL-18 receptor ligand binding chain (also known as IL-18Rα) by subjecting concentrated human urine to an IL-18 ligand affinity column. The IL-18 ligand chromatography purified molecule was analyzed by protein microsequencing. The result revealed a novel 40 amino acid polypeptide. To isolate the complete open reading frame (ORF), various human and mouse cDNA libraries were screened using cDNA probe derived from the novel IL-18 affinity column bound molecule. The identified entire ORF gene was thought to be an IL-18Rα gene. However, IL-18BP has been proven to be a unique soluble antagonist that shares homology with a variety of viral proteins that are distinct from the IL-18Rα and IL-18Rβ chains. The IL-18BP cDNA was used to generate recombinant IL-18BP (rIL-18BP), which was indispensable for characterizing the role of IL-18BP in vitro and in vivo. Mammalian cell lines were used to produce rIL-18BP due to its glycosylation-dependent activity of IL-18BP (approximately 20 kDa). Various forms of rIL-18BP, intact, C-terminal his-tag, and Fc fusion proteins were produced for in vitro and in vivo experiments. Data showed potent neutralization of IL-18 activity, which seems promising for clinical application in immune diseases involving IL-18. However, it was a long journey from discovery to clinical use although there have been various clinical trials since IL-18BP was discovered in 1999. This review primarily covers the discovery of IL-18BP along with how basic research influences the clinical development of IL-18BP.

• 한국미생물·생명공학회지
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• 제41권4호
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• pp.383-390
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• 2013

국내 야생화들로부터 효모를 분리하여 이들을 고부가가치의 의약이나 건강 식품산업에 응용하기 위한 연구의 일환으로 경기도와 제주도 일부 지역의 야생화들을 채집하여 효모들을 분리, 동정하고 이들 가운데 항통풍성 xanthine oxidase 저해물질 생산 균주를 선발한 후 생산 조건을 검토하였다. 서울 구로동과 일부 경기도 지역 야생화 31점에서 26종, 54균주의 효모들을 분리, 동정하였고 이들 중 Pseudozyma sp.가 13종으로 가장 많이 분리되었다. 제주도 송악산과 성산 일출봉 지역의 야생화 21점에서 모두 14종, 32균주의 효모들을 분리, 동정하였고, 이들 균 중에서 Sporobolomyces ruberrimus가 6종으로 가장 많이 분리되었다. 86효모 균주들을 YEPD 배지에서 2일간 배양하여 각각의 배양 상등액과 무세포 추출물을 제조한 후 이들의 항통풍성 Xanthine oxidase 저해활성을 측정한 결과, Pseudozyma hubeiensis 228-S-1의 무세포 추출물이 19.5%의 가장 높은 Xanthine oxidase 저해활성을 보였다. 또한 Pseudozyma hubeiensis 228-S-1을 YEPD 배지에 접종하여 $30^{\circ}C$에서 36시간 배양하였을 때, 세포내 항통풍성 xanthine oxidase 저해물질을 가장 많이 생산하였다.

• 한국식품과학회지
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• 제38권3호
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• pp.419-426
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• 2006

전국 각지에서 채집한 토양에서 분리한 25종의 내열성 균주 중 내열성 단백질 기수분해효소 활성을 갖는 균주 strain JE 375를 선별하였다. 본 균주는 gram 양성 간균의 특징을 나타냈으며 Bergey's Manual of Systematic Bacteriology와 Biochemical Tests for Identification of Medical Bacteria에 준하여 생화학적 특성을 검토한 결과 catalase 양성, 포자형성, motility 양성, glucose 발효, mannitol 발효, xylose 산화, hemolysis ${\beta}$균임을 보아 Bacillus sp.으로 추정 되었다. Strain JE 375의 whole cell fatty acid를 gas chromatography로 분석한 결과 $C_{15:0}$ iso 26.17%, $C_{16:0}$ iso 13.01%, $C_{17:0}$ iso 30.19%로 분석되어 Bacillus 계열로 동정되었다. 16S rDNA sequence분석 결과 strain JE 375는 Bacillus caldoxylolyticus와 sequence가 97.6% 일치하는 유사성을 보였으나 부분적으로 sequence의 차이가 있고 gene bank data base상에서 16S rDNA sequence가 일치하는 균주는 검색되지 않았다. 이 같은 실험 결과에 따라 strain JE 375는 기존에 발표되지 않은 새로운 균주로 판단되어 Bacillus sp. JE 375로 명명하였다. Bacillus sp. JE 375은 tryptone 1%, yeast extract 0.5%, NaCl 1%, maltose 1%의 배지조성분과 배양 온도 $65^{\circ}C$에서 20시간 동안 배양하였을 때 최대의 단백질 분해 효소를 생산하였다. Bacillus sp. JE 375로부터 단백질 분해 효소를 acetone으로 침전시키고 DEAE-sepharose column chromatography를 통하여 효소를 정제하여 SDS-PAGE를 통해 확인한 결과 55 kDa 크기의 band를 확인할 수 있었다. 이 효소의 최적 배양 온도는 $65^{\circ}C$이었으며 배지의 최적 pH는 6.5로 나타났다. pH에 대한 안정성은 중성 부근의 pH에서 효소 활성의 안정성이 높게 나타났다. 본 효소의 반응 조건을 검토한 결과 중성 조건에서 안정하였으며, $60^{\circ}C$의 고온에서 활성을 가졌다. 효소 활성은 1 mM $CaCl_2$ 첨가에 의해 증가하였다.

• Journal of Ginseng Research
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• 제41권4호
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• pp.469-476
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• 2017

Background: Panax ginseng Meyer (Asian ginseng) has a large nuclear genome size of > 3.5 Gbp in haploid genome equivalent of 24 chromosomes. Tandem repeats (TRs) occupy significant portions of the genome in many plants and are often found in specific genomic loci, making them a valuable molecular cytogenetic tool in discriminating chromosomes. In an effort to understand the P. ginseng genome structure, we characterized an ultrahigh copy 167-bp TR (Pg167TR) and explored its chromosomal distribution as well as its utility for chromosome identification. Methods: Polymerase chain reaction amplicons of Pg167TR were labeled, along with 5S and 45S rDNA amplicons, using a direct nick-translation method. Direct fluorescence in situ hybridization (FISH) was used to analyze the chromosomal distribution of Pg167TR. Results: Recently, we reported a method of karyotyping the 24 chromosome pairs of P. ginseng using rDNA and DAPI (4',6-diamidino-2-phenylindole) bands. Here, a unique distribution of Pg167TR in all 24 P. ginseng chromosomes was observed, allowing easy identification of individual homologous chromosomes. Additionally, direct labeling of 5S and 45S rDNA probes allowed the identification of two additional 5S rDNA loci not previously reported, enabling the refinement of the P. ginseng karyotype. Conclusion: Identification of individual P. ginseng chromosomes was achieved using Pg167TR-FISH. Chromosome identification is important in understanding the P. ginseng genome structure, and our method will be useful for future integration of genetic linkage maps and genome scaffold anchoring. Additionally, it is a good tool for comparative studies with related species in efforts to understand the evolution of P. ginseng.

• 대한환경공학회지
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• 제30권4호
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• pp.393-400
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• 2008

유기용제 안정제로 사용되는 1,4-다이옥산($C_4H_8O_2$)은 높은 용해도와 독성으로 인해 생태계에 유해하며 미국 EPA 의해 발암가능성이 있는 물질로 분류되어 있다. 국내에서도 환경부에 따르면 2011년부터 수계로의 배출허용기준이 5 mg/L로 추진될 예정에 있다. 따라서 구미의 폴리에스테르 제조 공정에서 발생되는 현재 운전 중인 활성슬러지가 1,4-다이옥산을 기준에 적합하도록 적절하게 처리할 수 있는지를 조사하였다. 이와 같은 목적으로 일부 회사(K, H 및 T)를 대상으로 1,4-다이옥산의 제거율 및 미생물학적 속성이 평가되었다. 처리효율은 H사에서 98%로 가장 높았으며 K사는 77%로 두 개 사 모두 유출농도가 기준에 부합하였다. 그러나 T사 유출수의 1,4-다이옥산 농도는 23 mg/L로 기준보다 높았다. 한편, 각 업체의 활성슬러지를 100 ppm의 1,4-다이옥산이 포함된 BSM(Basal salt medium)에 식종하여 생물학적 분해실험을 수행하였다. 7일간의 운전 후, H사의 슬러지를 이용한 시험에서 1,4-다이옥산이 완전히 제거되었으며 T사는 67%, K사는 52%로 이 처리효율의 차이는 1,4-다이옥산의 양이 아닌 주어진 활성 슬러지의 생분해능이 서로 다른 것에 의한 것임을 확인할 수 있었다. 결과적으로 각 산업체의 미생물 다양성이 16s rDNA cloning 방법을 통해 조사되었으며 Methylibium petroleiphilum PM1이 H사에서 가장 많이 발견되었으며 K사에서 적은 양이, 그리고 T사에서는 발견되지 않았다. Methylibium petroleiphilum PM1은 methyl tertiary-butyl ether(MTBE)와 같은 에테르 물질을 효과적으로 제거하는 것으로 알려져 있다. 이는 산업분야의 관점에서 H사의 활성 슬러지가 1,4-다이옥산의 생분해에 가장 효과적으로 적용될 수 있다는 것을 나타낸다.

• Journal of Microbiology and Biotechnology
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• 제18권9호
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• pp.1496-1499
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• 2008

A Gram-negative, aerobic, rod-shaped, nonspore-forming bacterial strain, designated Gsoil $357^T$ was isolated from soil sample of a ginseng field in Pocheon Province (South Korea). The isolate contained Q-8 as the predominant ubiquinone and iso-$C_{16:0}$, iso-$C_{17:1}$ ${\omega}9c$, and iso-$C_{15:0}$ as the major fatty acids. The G+C content of the genomic DNA was 69.3 mol%. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Gsoil $357^T$ was most closely related to Lysobacter gummosus (97.6%) and Lysobacter antibioticus (97.6%). However, the DNA-DNA relatedness value between strain Gsoil $357^T$ and its phylogenetically closest neighbors was less than 17%. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 357T should be classified as representing a novel species in the genus Lysobacter, for which the name Lysobacter ginsengisoli sp. novo is proposed. The type strain is Gsoil $357^T$ (=KCTC $12602^T$=DSM $18420^T$).