• Korean Journal of Veterinary Research
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• v.39 no.3
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• pp.513-522
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• 1999

DNA hybridization assay using probes prepared from liver was carried out to identify species characterization of the domestic animals. Gel electrophoresis showed that the target DNA extracted from raw muscle were 1kb and uniform pattern while fragments size of heated muscle were irregular. Hybridization was performed by adding 200ng/ml probe in hybridization solution and incubating for 12 hours at $68^{\circ}C$. To obtain good discrimination, applied washing buffer and washing step differently depending on the species. The probes of pig, horse and dog formed the specific hybrids with each target DNA respectively. Although cross reaction was detected in cattle, goat and sheep but signal intensity among these species made the discrimination possible each other. Such pattern was the same in the cases of chicken, turkey and duck. The hybridization pattern of heated muscle was similar to that of raw muscle in general, but the signal intensity was inferior to that of raw muscle. Species identification between closely related animal species, hybridized using the target DNA of such closely related animal species as a blocking agent, remarkable increase of discrimination from the evident decrease of non specific reaction compared with the control group. In addition, in the admixture where certain meat was included in the beef, pork, chicken meat, we could find whether any unjust meat was admixed or not. In this case, detection limit of certain meat in admixture was 1%.

• Korean Journal Plant Pathology
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• v.14 no.2
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• pp.177-183
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• 1998

A DNA fragment which is involved in tolassin production was cloned to obtain a molecular marker of Pseudomonas tolaasii, a casual agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). Tolaasin is a lipodepsipeptide toxin and known as a primary disease determinant of the P. tolaasii. It is responsible for formation of white line in agar when P. tolaasii were cultured against white line reacting organisms (WLROs). White line negative mutants (WL-) were generated by conjugation between rifampicin resistant strain of P. tolaasii and E. coli carrying suicidal plasmid pSUP2021 : : Tn5. The ability of tolaasin production of the WL- mutants was examined by hemolysis test, pathogenicity test, and high pressure liquid chromatography (HPLC) analysis of culture filtrate. All of the WL- mutants were lost the ability of tolaasin production (Tol-). Genomic library of the Tol- mutant was constructed in pLAFR3 and the cosmid clone containing Tn5 was selected. DNA fragment fro franking region of Tn5 was cloned from the plasmid and used as a probe in Southern blot. DNA-DNA hybridization with the probe to total DNA from group of bacteria ecologically similar to P. tolaasii including WLORs, fluorescent Pseudomonads isolated from oyster mushroom, P. agarici, P. gingeri, and some of other species of Psedomonas showed that some of the tested bacteria do not have any hybridized band and others have bands sowing RFLP. The cloned DNA fragment or its nucleotide sequence will be useful in detection and identification of the P. tolaasii.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.2
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• pp.93-99
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• 2004

Pathogens pose a significant threat to humans, animals, and plants. Consequently, a considerable effort has been devoted to developing rapid, convenient, and accurate assays for the detection of these unfavorable organisms. Recently, DNA-microarray based technology is receiving much attention as a powerful tool for pathogen detection. After the target gene is first selected for the unique identification of microorganisms, species-specific probes are designed through bioinformatic analysis of the sequences, which uses the info rmation present in the databases. DNA samples, which were obtained from reference and/or clinical isolates, are properly processed and hybridized with species-specific probes that are immobilized on the surface of the microarray for fluorescent detection. In this study, we review the methods and strategies for the development of DNA microarray for pathogen detection, with the focus on probe design.

• Journal of Periodontal and Implant Science
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• v.28 no.4
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• pp.659-678
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• 1998

In the present study, oligonucleotide probes based on 16S rRNA were taken to investigate the diversity of oral spirochetes without culture method. This is the first study that revealed oral spirochetes of both presently cultivable and uncultured oral spirochetes in Korean adult periodontitis patients. Subgingival plaque samples were taken from diseased sites(probing depth ${\geq}6\;mm$, experimental group, n=116) and healthy sites(probing depth${\leq}3mm$, control 1 group, n=28) in 29 patients with adult periodontitis, and from 20 periodontally healthy subjects(probing depth${\leq}3mm$, control 2 group, n=100). Following being examined under phase-contrast microscope, all samples were submitted to dot-blot hybridization after polymerase chain reacton with eubacterial primers. 5 species-specific probes(TVIN, TDEN, TMAL, TSOC, and TPEC) and 7 group-specific probes(TRE I, TRE II, TRE III, TRE IV, TRE V, TRE VI, and TRE VII) were used one by one for the identification of both cultivable and so far uncultivable oral spirochetes. All probes were labeled with digoxigenin(DIG)-ddUTP and detected by chemilumininescence. The following results were obtained. 1. Under phase-contrast microscope, 91.37% and 14.28% of oral spirochetes were observed in the experimental and control 1 groups, respectively. None of oral spirochetes were observed in control 2 group. 2. With universal probe, 98.27%, 46.42%, and 22.0% of oral spirochetes were observed in experimental, control 1, and control 2 groups, respectively. 3. With specific probe, 95.68%, 35.71%, and 19.0% of oral spirochetes were observed in experimental, control 1, and control 2 groups, respectively. 4. With species-specific probes, T. socranskii were recovered in a high percentage of sites(81.89%) examined, followed by T. maltophilum(50.0%), T. vincentii(36.20%), T. denticola(13.79%), respectively. With group- specific probes, TRE IV was recovered in a high percentage of sites(85.34%) examined, followed by TRE II(77.58%), TRE I(56.89%), TRE III(25.86%), TRE VI(5.17%), and TRE V(2.58%), respectively. 5. T. vincentii were only observed in the diseased sites, not in the healthy sites. 6. Neither T. pectinovorum nor group VII oral spirochetes were observed in any sites. The findings warrant further investgations of the recovered spirochetes to elucidate the possible associations of oral spirochetal prevalence in race and types of periodontitis, pathogenesis of T. vincentii and the possible distributional change of oral spirochetes before and after treatments.

• Annals of Clinical Microbiology
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• v.18 no.2
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• pp.37-43
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• 2015

Background: Tuberculosis is globally the most important cause of death from single pathogen. Rapid and accurate identification of mycobacteria is essential for the control of tuberculosis. We evaluated a fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for the differentiation of Mycobacterium tuberculosis complex (MTB) and nontuberculous mycobacteria (NTM) in direct smears of sputum specimens. Methods: The cross-reactivity of MTB- and NTM-specific PNA probes was examined with reference strains of M. tuberculosis ATCC 13950, Mycobacterium kansasii ATCC 12479, Mycobacterium fortuitum ATCC 6841, several clinical isolates of mycobacteria (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium gordonae and Mycobacterium chelonae), and 11 frequently isolated respiratory bacterial species other than mycobacteria. A series of 128 sputa (89 MTB culture positive, 29 NTM culture positive, and 10 under treatment culture negative) with grades of trace to 4+ were used to evaluate the performance of the method. Results: The MTB- and NTM-specific PNA probes showed specific reactions with the reference strains of MTB and M. kansasii and clinical isolates of mycobacteria except M. fortuitum ATCC 6841, and no cross-reactivity with other tested bacteria. The PNA probe-based FISH assay for detection of MTB had a sensitivity and specificity of 100%, respectively. The sensitivity and specificity of the NTM-specific PNA probe was 100%. The smear grades of the PNA FISH test were same as with those of the fluorescence AFB stain in 2+ or higher grade. Conclusion: Detection and differentiation based on PNA FISH is sensitive and accurate for detecting mycobacteria and for differentiating MTB from NTM in clinical sputum smears.

• Restorative Dentistry and Endodontics
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• v.21 no.1
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• pp.1-18
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• 1996

There are many advantages when using IIF and DNA probe methods over anaerobic culture method in that they are time-and effort-saving, more precise and more sensitive. Furthermore, in IIF and DNA probe methods, the detection is possible only with small amount of bacteria, the quantitative analysis is possible, and the cell viability is not necessary. The purpose of this study is to observe the incidence of P.endodontalis by carrying out anaerobic culture, IIF and colony lift using DNA probe method respectively, and to compare these 3 methods in terms of effectiveness and sensitivity in order to identify the most effective detection method. 30 teeth with at least one clinical symptoms, with single canal, and with pulp necrosis were sampled. For sampling bacteria, access cavity was prepared after disinfecting tooth and its surroundings. Then the paper point was inserted up to the periapical area, leave there for a while, and finally it was placed into PRAS Ringer's sol. and PBS sol. In anaerobic culture method, P.endodontalis was identified by biochemical tests after subculturing black and brown colonies which were produced after 7 days of incubation on BAP and Brucella BAP in anaerobic chamber. To identify P.endodontalis in IIF method, species-specific polyclonal rabbit-antisera of P.endodontalis(ATCC 35406) was reacted with sampled PBS sol. dispensed onto glass slide, and then P.endodontalis was examined by phase contrast microscopy after incubating with Goat anti-rabbit lgG conjugated to Fluorescein isothiocyanate. For colony lift using DNA probe method, membranes were laid over colonies on the surface of BAP and were hybridized with cloned DNA probe of P.endodontalis. The existence of P.endodontalis was then identified by the methods of chemiluminescent detection and color metric detection. Black colony was found in 11 teeth out of 30 teeth and P.endodontalis was detected in 6 teeth (20 %) by anaerobic culture method, 16 teeth (53 %) by IIF method, and 7 teeth (23 %) by DNA probe method. IIF method is significantly better in detecting P.endodontalis than DNA probe method and anaerobic culture method. There was no significant differences between DNA probe method and anaerobic culture method. There was significant correlation between the formation of black colony and the existence of P.endodontalis. The probability of detecting P.endodontalis when black colony being present is 2.89 times higher than when not being present. There was significant relationship between the foul odor of clinical symptoms and P.endodontalis. The sensitivity of existing P.endodontalis when foul odor being present was 93.75 %, while the specificity of not existing P.endodontalis when foul odor not being present was 28.57 %. These results suggested that the probes of P.endodontalis will be used to decide the method and prognosis in endodontic treatments.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.284-289
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• 2004

A PCR assay for the rapid detection of Vibrio vulnificus strains was developed using a virulence gene for elastase found in various Vibrio species. The DNA sequences in the elastase gene facilitated the identification of a species-specific probe for pathogenic V. vulnificus strains from both clinical and environmental sources. Using an elastase gene-based PCR reaction, a species-specific 507-bp PCR product was visualized by agarose gel electrophoresis. Three different DNA extraction methods were then compared to improve the simplicity and rapidity of detection. A PCR assay using the conventional DNA extraction or boiling method was able to detect as few as 25 V. vulnificus cells, making the detection limits at least 1-log-scale lower than that for the EDT A-treated DNA extraction method. In particular, the boiling method, which does not require purification of the chromosomal DNA, was very effective in terms of simple and rapid detection. Meanwhile, the detection limit in a mixed bacterial culture that included other bacteria, such as Escherichia coli or Bacillus subtilis, was two V. vulnificus cells, which was 1-log-scale lower than that for the control. Accordingly, when coupled with a new DNA extraction method, the elastase gene-based PCR can provide a rapid, specific, and sensitive method for identifying V. vulnificus in clinical and environmental samples.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1778-1783
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• 2006

Event-specific PCR detection methods are the primary trend in genetically modified (GM) plant detection owing to their high specificity based on the flanking sequence of the exogenous integrant. Therefore, this study describes a real-time PCR system for event-specific GM canola GT73, consisting of a set of primers, TaqMan probe, and single target standard plasmid. For the specific detection of GT73 canola, the 3'-integration junction sequence between the host plant DNA and the integrated specific border was targeted. To validate the proposed method, test samples of 0, 1, 3, 5, and 10% GT73 canola were quantified. The method was also assayed with 15 different plants, and no amplification signal was observed in a real-time PCR assay with any of the species tested, other than GT73 canola.

• The Plant Pathology Journal
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• v.15 no.1
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• pp.57-61
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• 1999

Xanthomonas campestris pv. glycines causes bacterial pustule disease on susceptible soybean leaves and produces a bacteriocin, named glycinecinA, against most xanthomonads including Xanthomonas campestris pv. vesicatoria. One of the 5 isolated DNA regions responsible for bacteriocin production, a 1.7 kb DNA region for the glycinecinA gene, was used as a probe to detect the presence of the homolog DNA in other bacterial strains. Among 55 bacterial strains tested, only X. campestris pv. glycines showed the positive signal with glycinecinA DNA. Two oligomers, heu2 and heu4, derived from a glycinecinA DNA were used to carry out the polymerase chain reaction (PCR) analysis with chromosomal DNA from 55 different bacterial strains including 24 different strains of X. campestris pv. glycines, 9 different pathovars of xanthomonads, and other 22 bacterial strains of different genus and species. By separation of the PCR products on agarose gel, a 0.86 kb DNA fragment was specifically detected when X. campestris pv. glycines was present in the amplification assay. The 0.86 kb fragment was not amplified when DNA from other bacteria was used for the assay. Southern analysis with glycinecinA DNA showed that the PCR signal was obtained with X. campestris pv. glycines isolates from various geographic regions and soybean cultivars. Therefore, the 1.7 kb DNA region for the glycinecinA gene can be used for the pathovar-specific probe for the DNA hybridization and the primers heu2 and heu4 can be used for the pathovar-specific primers for the PCR analysis to detect X. campestris pv. glycines.

• Genomics & Informatics
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• v.21 no.1
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• pp.13.1-13.8
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• 2023

Importance of accurate molecular diagnosis and quantification of particular disease-related pathogenic microorganisms is highlighted as an introductory step to prevent and care for diseases. In this study, we designed a primer/probe set for quantitative real-time polymerase chain reaction (qRT-PCR) targeting rgpA gene, known as the specific virulence factor of periodontitis-related pathogenic bacteria 'Porphyromonas gingivalis', and evaluated its diagnostic efficiency by detecting and quantifying relative bacterial load of P. gingivalis within saliva samples collected from clinical subjects. As a result of qRT-PCR, we confirmed that relative bacterial load of P. gingivalis was detected and quantified within all samples of positive control and periodontitis groups. On the contrary, negative results were confirmed in both negative control and healthy groups. Additionally, as a result of comparison with next-generation sequencing (NGS)-based 16S metagenome profiling data, we confirmed relative bacterial load of P. gingivalis, which was not identified on bacterial classification table created through 16S microbiome analysis, in qRT-PCR results. It showed that an approach to quantifying specific microorganisms by applying qRT-PCR method could solve microbial misclassification issues at species level of an NGS-based 16S microbiome study. In this respect, we suggest that P. gingivalis-specific primer/probe set introduced in present study has efficient applicability in various oral healthcare industries, including periodontitis-related microbial molecular diagnosis field.