• Journal of Microbiology
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• v.44 no.4
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• pp.403-408
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• 2006

The purpose of this study was to determine the efficacy of a nucleic acid sequence-based amplification (NASBA) method of detecting noroviruses in artificially and naturally contaminated shellfish. We used 58 fecal samples that tested positive for noroviruses with electron microscopy (EM) to develop an NASBA assay for these viruses. Oligonucleotide primers targeting the polymerase coding region were used to amplify the viral RNA in an isothermal process that resulted in the accumulation of RNA amplicons. These amplicons were detected by hybridization with digoxigenin-labeled oligonucleotide probes that were highly specific for genogroup I (GI) and genogroup II (GII) of noroviruses. The expected band of 327bp appeared in denaturing agarose gel without any nonspecific band. The specific signal for each amplicon was obtained through Northern blotting in many repeats. All fecal samples of which 46(79.3%) belonged to GII and 12(20.6%) belonged to GI were positive for noroviruses by EM and by NASBA. Target RNA concentrations as low as 5pg/ml were detected in fecal specimens using NASBA. When the assay was applied to artificially contaminated shellfish, the sensitivity to nucleic acid was 100pg/1.5g shellfish tissue. The potential use of this assay was also confirmed in naturally contaminated shellfish collected from different ponds in Guangzhou city of China, of which 24 (18.76%) out of 128 samples were positive for noroviruses; of these, 19 (79.6%) belonged to GII and 5 (20.4%) belonged to GI. The NASBA assay provided a more rapid and efficient way of detecting noroviruses in fecal samples and demonstrated its potential for detecting noroviruses in food and environmental samples with high specificity and sensitivity.

• Journal of the Korean Chemical Society
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• v.30 no.1
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• pp.90-100
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• 1986

Along with synthesizing a model oligodeoxyribonuclotide GpApApTpTpCp which had the EcoRI recognition site according to "Phosphotriester Method", reported methods for protecting functions, condensation, deprotection, purifying products, quantification and identification were evaluated and modified. It was realized that oligomers were efficiently synthesized by elongating by dimer unit per step of the condensation and the condensation yields were decreased with increased numbers of the residues. The problems of quantification/identification involved in the oligonucleotide synthesis using small amount of the reactants were solved by employing UV/IR spectrophotometry and hplc/tlc. It was also proved that a nucleotide having the exposed 5'-OH function was well synthesized by condensing a 5'-OH nucleotide and a phosphodiester nucleotide whose molar ratio was intendedly made to be 1 : 1.2 and then detritylating followed by washing with an aqueous solution of sodium bicarbonate. Spectrophotometric and chromatographic data of the nucleotides and their derivatives concerned in this synthetic work were prepared.

• Korean Journal of Veterinary Research
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• v.41 no.2
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• pp.189-195
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• 2001

Single base substitution and deletion mutation have been introducted into the verotoxin 2 (VT2)A subunit gene from O157:H7 isolates to reduce cytotoxicity of VT2 and the cytotoxicity between wild type toxin and mutant toxoid were compared. A M13-derived recombinant plasmid pEP19RF containing a 940bp EcoRI-PstI fragment of VT2A gene was constructed for oligonucleotide-directed mutagenesis. The duoble mutant pDOEX was constructed by point and deletion mutation of two different highly conserved regions of VT2A encoding active site cleft of enzymatic domain. The key residue, Glu 167(GAA) and the pentamer(WGRIS) consisting of the enzymatic domain were replaced by ASP(GAC) and completely deleted in nucleotide sequence analysis of mutant, respectively. In the comparision of vero cell cytotoxicity between wide type toxin and toxoid from mutant, the wild type toxin expressed cytotoxicity in dilution of $10^{-6}$, but the toxid from mutant did not show cytotoxicity to vero cells.

• Korean Journal of Microbiology
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• v.36 no.2
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• pp.136-141
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• 2000

The temporal variation of bacterial community and environmental factors, affecting on bacterial community structure were estimated monthly kom April, 1998 to May, 1999. Bacterial community structures were determined by in situ hyblidization with rRNA-targeted fluorescently labeled oligonucleotide probes (FISH) and epifluorescence microscopy; and the statistical analysis was done by SPSS program. The oligonucleotide probes used in this study were EUB338, ALFlb, GAM42a, and CF. In surface water, $\alpha$-group was related to only DOC (-0.538, p<0.05) and Chlorophyll a concentration was related to y-group (-0.630, p$\beta$-group and Cytophaga-Flavobacterium group were related to water temperature as 0.665, and 0.685 @<0.05). Between pH and $\beta$-group, there was a positive relationship (0.541, p<0.05), and Cytophaga-Flavobactevizim group was represent to correlation (0.672, p

• Microbiology and Biotechnology Letters
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• v.20 no.3
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• pp.301-310
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• 1992

This work was performed to investigate the possibility of using J96 hemolysin(Hly, Hly A) vaccine against urinary tract infecting Escherichia coli. Based on the known sequence of J96 hemolysin which was originally isolated from a pyelonephritis patient, ten 20-mer oligonucleotide probes were synthesized. Radioactive labelled 8 probes showed positive colony blots against most of the hemolysin producing wild type E. coli, while HA484 and HA661 showed 28.3, 71.7% positive blots, respectively. This result means that hemolysin genes are highly conserved. Also, 12 anti-Hly MABs(monoclonal antibodies) showed more than 90% positive immunoblots against secreted hemolysin from wild type E. coli. Especially, the result that MAB132 neutralized hemolysin from all of the wild type E. coli augments the idea that hemolysin will be effective as a vaccine.

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1637-1646
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• 2010

The bacterium Deinococcus radiodurans is one of the most resistant organisms to ionizing radiation and other DNA-damaging agents. Although, at present, 30 Deinococcus species have been identified, the whole-genome sequences of most species remain unknown, with the exception of D. radiodurans (DRD), D. geothermalis, and D. deserti. In this study, comparative genomic hybridization (CGH) microarray analysis of three Deinococcus species, D. radiopugnans (DRP), D. proteolyticus (DPL), and D. radiophilus (DRPH), was performed using oligonucleotide arrays based on DRD. Approximately 28%, 14%, and 15% of 3,128 open reading frames (ORFs) of DRD were absent in the genomes of DRP, DPL, and DRPH, respectively. In addition, 162 DRD ORFs were absent in all three species. The absence of 17 randomly selected ORFs was confirmed by a Southern blot. Functional classification showed that the absent genes spanned a variety of functional categories: some genes involved in amino acid biosynthesis, cell envelope, cellular processes, central intermediary metabolism, and DNA metabolism were not present in any of the three deinococcal species tested. Finally, comparative genomic data showed that 120 genes were Deinococcus-specific, not the 230 reported previously. Specifically, ddrD, ddrO, and ddrH genes, previously identified as Deinococcus-specific, were not present in DRP, DPL, or DRPH, suggesting that only a portion of ddr genes are shared by all members of the genus Deinococcus.

• Archives of Pharmacal Research
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• v.29 no.10
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• pp.890-897
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• 2006

In inflammatory responses, induction of cytokines and other immune regulator genes in macrophages by pathogen-associated signal such as lipopolysaccharide (LPS) plays a crucial role. In this study, the gene expression profile changes by LPS treatment in the macrophage/monocyte lineage cell line RAW264.7 was investigated. A 60-mer oligonucleotide microarray of which probes target 32381 mouse genes was used. A reverse transcription-in vitro translation labeling protocol and a chemileuminescence detection system were employed. The mRNA expression levels in RAW264.7 cells treated for 6 h with LPS and the control vehicle were compared. 747 genes were up-regulated and 523 genes were down-regulated by more than 2 folds. 320 genes showing more than 4-fold change by LPS treatment were further classified for the biological process, molecular function, and signaling pathway. The biological process categories that showed high number of increased genes include the immunity and defense, the nucleic acid metabolism, the protein metabolism and modification, and the signal transduction process. The chemokine-cytokine signaling, interleukin signaling, Toll receptor signaling, and apoptosis signaling pathways involved high number of genes differentially expressed in response to LPS. These expression profile data provide more comprehensive information on LPS-target genes in RAW264.7 cells, which will be useful in comparing gene expression changes induced by extracts and compounds from anti-inflammatory medicinal herbs.

• Genomics & Informatics
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• v.2 no.2
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• pp.75-80
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• 2004

In a variety of animal species, the perinatal exposure of experimental animals to the 2,3,7,8-tetrachlorodibenzo­p-dioxin (TCDD) leads to the immune dysfunction, which is more severe and persistent than that caused by adult exposure. We report here the changes of gene expression and the identification of the marker-genes representing the dioxin exposure. The expressions of the transcripts were analyzed using the 11 K oligonucleotide­microarray from the bone marrow cells of male C57BL/6J mice after an intraperitoneal injection of $1{\mu}g$ TCDD/kg body weight at various time intervals: gestational 6.5 day(G6.5), 13.5 day(G13.5), 18.5 day(G18.5), and postnatal 3 (P3W)and 6 week (P6W). The type of self-organizing maps(SOM) representing the specific exposure dioxin could be identified as follows; G6.5D(C14), G13.5D(C0, C5, C10, C18), G18.5D(7): P3W(C2, C21), and P6W(C4, C15, C20). The candidate marker-genes were restricted to the transcripts, which could be consistently expressed greater than $\pm$2-fold in three experiments. The resulting candidates were 85 genes, the characteristics of that were involved in cell physiology and cell functions such as cell proliferation and immune function. We identified the biomarker-genes for dioxin exposure: smc -like 2 from SOM C14 for the dioxin exposure at G6.5D, focal adhesion kinase and 6 other genes from C0, and protein tyrosine phosphatase 4a2 and 3 other genes from C5 for G13.5D, platelet factor 4 from C7 for G18.5D, fos from C2 for P3W.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.186-191
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• 2002

The usefulness of oligonucleotide DNA chip was evaluated for detection and primary level identification of major waterborne viruses in environmental samples. The enteric waterborne viruses included enterovirus, adenovirus, and rotavirus. Total intracellular RNA of 10 BGM cell plates showing virus-specific cytopathic effects was extracted at the third day after inoculation. The intracellular RNA was then subjected to either enterovirus-specific RT-PCR followed by sequencing analysis, or the DNA chip. Seven out of 10 positive samples in cell culture were positive but the other three sample were turned out to be negative by both RT-PCR and DNA chip analyses. Nucleotide sequencing results and the DNA chip hybridization results of the RT-PCR product were in complete agreement in the identification of the 7 positive samples as enteroviruses. Using the DNA chip, it took only 3∼4 hr to complete detection and primary level identification of target viruses and additional procedures such as gel electrophoresis or nucleotide sequencing were not necessary. We believe that the DNA chip system can be employed as a highly effective and new detection methodology for environmental viruses.

• Gut and Liver
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• v.12 no.6
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• pp.648-654
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• 2018

Background/Aims: Dual priming oligonucleotide-based multiplex polymerase chain reaction (DPO-based PCR) can detect the presence of clarithromycin resistance without culture. The aim of this study was to investigate the cost-effectiveness of DPO-based PCR for Helicobacter pylori eradication. Methods: From 2015 to 2016, medical records of patients who received H. pylori eradication therapy were analyzed. Patients were divided into two groups: tailored group patients who were treated based on DPO-based PCR and empirical group patients. Eradication rate and medical cost, including diagnostic tests, eradication regimens, and $^{13}C$-urea breath tests, were compared between the two groups. Cost for one successful eradication was calculated in each group. The expected cost of eradication for empirical treatment was investigated by varying the treatment duration and eradication rate. Results: A total of 527 patients were analyzed (tailored group 208, empirical group 319). The eradication success rate of the first-line therapy was higher in the tailored group compared to that in the empirical group (91.8% vs 72.1%, p<0.01). The total medical cost for each group was $114.8{\pm}14.1U.S.$ dollars (USD) and $85.8{\pm}24.4USD$, respectively (p<0.01). The total medical costs for each ultimately successful eradication in the tailored group and in the empirical group were 120.0 USD and 92.4 USD, respectively. The economic modeling expected cost of a successful eradication after a 7- or 14-day empirical treatment was 93.8 to 111.4 USD and 126.3 to 149.9 USD, respectively. Conclusions: Based on economic modeling, the cost for a successful eradication using DPO-based PCR would be similar or superior to the expected cost of a successful eradication with a 14-day empirical treatment when the first-line eradication rate is ${\leq}80%$.