• Research in Plant Disease
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• v.17 no.2
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• pp.169-176
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• 2011

Phytophthora katsurae is the fungus responsible for chestnut ink disease. The objectives of this study were to determine if a single-strand conformation polymorphism (SSCP) analysis of rDNA-ITS region, elongation factor 1 alpha gene and ${\beta}$-tubulin gene could be used for rapid identification and genetic diversity of P. katsurae, and to assess the potential use of the SSCP technique as a diagnostic tool for P. katsurae. Each regions amplified by PCR using primers designed to overlap the genus Phytophthora were characterized for the Phytophthora species. PCR products were denatured and electrophoresed for SSCP analysis. P. katsurae isolates showed an unique pattern in SSCP analysis and were easily distinguished from other Phytophthora species used as the control. This indicates that SSCP analysis is an useful technique for distinguishing Phytophthora species from genetically close relatives, and show that the SSCP analysis of each region is an efficient detection tool for P. katsurae. But PCR-SSCP analysis of single-gene may have difficulty in distinguishing P. katsurae from other Phytophthora species. Therefore, PCR-SSCP analysis of multi-genes can be useful for rapid and effective identification of P. katsurae.

• Journal of Life Science
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• v.14 no.3
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• pp.521-524
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• 2004

This experiment was carried out to analyze genetic characteristics and to develop the breed specific DNA marker for Cheju-native horse. If this marker contains high repetitive sequences, it is possible to convert a RAPD marker of interest into a single-locus PCR marker called a sequence characterized amplified region(SCAR). Twenty six Cheju-native horse and Fifty thoroughbred genomic DNA were pooled and PCR. were accomplished using 800 random primers. Comparing the pooled DNA from Cheju-native horse and thoroughbred, we found 9 primers which identified markers present in the pooled DNA from breed but absent in the other breed. Among 9 random primers, 6 primers were thoroughbred specific and 3 primers were Cheju-native horse specific. Testing individual horse revealed that 5 marker showed the similar band pattern between Cheju-native horse and Thoroughbred. However, 4 marker were wholly absent in breed while present in the other breed. UBC $126_{3500bp}$, UBC $162_{500bp}$, and UBC $244_{1200bp}$ was detected only Thoroughbred and UBC $562_{560bp}$was detected Cheju-native horse, respectively. After determining of the cloned breed-specific fragment sequence, we designed the SCAR-primers and carried out PCR. Compared to random primer, RAPD-SCAR primer didn't show significantly higher specific band. However, RAPD analysis is useful for genetic characterization of Cheju-native horse.

• Korean Journal of Veterinary Research
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• v.42 no.4
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• pp.513-522
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• 2002

Antigenic ompC genes of S. gallinarum, S. pullorum and S. dublin were characterized among Salmonella spp. isolated from chickens and other animals to identify genetic variation. Salmonella ompC gene fragment (1,027 bp) was amplified by PCR and the amplicons were cloned for comparison of nucleotide sequences. The identity of the sequences between S. gallinarum and S. pullorum, S. gallinarum and S. dublin, S. pullorum and S. dublin was 99.8%, 97.6% and 97.8%, respectively. Also, we found that ompC has some diversity between S. gallinarum and S. pullorum, and other Salmonella spp. which may be useful to type the organisms. Similar to diagnosis in other organisms, the TaqMan PCR method can be applied to rapid and accurate diagnosis of salmonellosis in chickens and other animals. We designed PCR primers and TaqMan probe for flagellin gene (fliC) for detection of Salmonella spp. by TaqMan PCR. The TaqMan PCR method was 10,000 times more sensitive than conventional PCR.

• Journal of Korean society of Dental Hygiene
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• v.17 no.1
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• pp.13-25
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• 2017

Objectives: The purpose of this study was to compare colony forming unit (CFU) method and multiplex real-time polymerase chain reaction (MRT-PCR) method for accurate quantitative analysis of bacteria. Methods: We compared the CFU method and the MRT-PCR method, which are still used in Korea, for Prevotella intermedius (P. intermedius), a periodontal disease pathogen selected by MRT-PCR, and Streptococcus mutans (S. mutans), a dental caries causative organism. The subjects of this study were 30 patients who visited the C dental hospital. Results: Total microorganisms in MRT-PCR method were significantly higher in both types of bacteria (p<0.05), since DNA of dead bacteria was also analyzed. This was because the periodontal dise(-) anaerobes, and even dead bacteria contain large amounts of toxic substances called LPS in the extracellular membrane, and fimbriae and pili, which are motility structures, still remain as a strong toxic substance in periodontal tissue. Conclusions: Therefore, in terms of the total amount of bacteria found, the MRT-PCR method will be a useful technique for searching all the bacteria in the oral cavity including live bacteria, as well as sterilization.

• Journal of Korean society of Dental Hygiene
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• v.21 no.5
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• pp.555-565
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• 2021

Objectives: This study was conducted to analyze peri-implantitis bacteria and identify their associations with health status and health activities. Methods: Gingival sulcus fluid at the implant's periodontal pockets sampled from the participants were analyzed by multiplex real time PCR. Results: Participants had strains in the order of 100% F. nucleatum, 98.0% E. corrodens, and 96.0% P. micra, and the correlation between C. rectus and E. nodatum was high (p<0.01). Diabetic group (P. gingivalis, P. nigrescens) hypertension (P. nigrescens), group with four or more periodontal pockets (P. gingivalis, T. dentica, P. intermedia, E. nodatum, and C. rectum), smoking (P. micra, E. corrodens), drinking (T. dentola), and scaling groups (C. rectus) were found to have more strains (p<0.05). Conclusions: Representative pathogenic microorganisms detected in periodontal pockets of implants were similar to dental periodontal pockets; however there were differences in the amount and distribution of microorganisms, and they were affected by health status and health behavior.

• Journal of dental hygiene science
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• v.9 no.2
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• pp.249-255
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• 2009

This study was carried out for the purpose of comparing bacterial culture method, single PCR, and multiplex PCR for identification of F. nucleatum and A. actinomycetemcomitans in subgingival plaque of adult periodontitis. Targeting 20 patients with adult periodontitis, the subgingival plaque was collected in teeth, respectively, for #16, #36, #44. A bacillus was cultivated by painting it over the solid selective media of F. nucleatum and A. actinomycetemcomitans. Bacterial species were detected in 0 tooth with 12 pieces, respectively. Through single PCR and multiplex PCR, the positive reaction was indicated in 43 teeth with 45 pieces, respectively, as for F. nucleatum, and in 1 tooth with 4 pieces, respectively, as for A. actinomycetemcomitans. In the comparative analysis between bacterial identification methods. F. nucleatum showed the more statistically significant difference(p=0.0(0) in comparison between single PCR and multiplex PCR. Even A. actinomycetemcomitans was indicated significantly(p=0.067) in a case that is based on 0.1 in significant level in the comparison between single PCR and multiplex PCR. In conclusion, as a result of comparing the bacterial identification methods, the detection frequency was indicated to be higher in PCR than in bacterial culture method. Single PCR and multiplex PCR showed the mutually similar detection frequency. Accordingly, given thinking of economic efficiency, quickness, and reduction in labor force, it is thought to be more efficient method to use single PCR as the bacterial identification method.

• Korean Journal of Microbiology
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• v.50 no.2
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• pp.114-118
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• 2014

A total of 122 ESBL-producing intestinal bacteria were collected from regional hospitals in the Chungcheong area. Combination disk test (CDT) was performed for antimaicrobial susceptability using cefotaxime and cefotaxime/clavulanate according to Clinical Laboratory Standard Institute (CLSI). Mutiplex PCR using specific primers was performed for a detection of ESBL-genotypes and enterobacterial repetitive intergenic consensus (ERIC)-PCR was carried out for the tracking of molecular epidemiology. In the confirmation test using CDT, 73 out of 76 (96.1%) ESBL-producing Escherichia coli and 43 out of 46 (93.4%) ESBL-producing Klebsiella pnemoniae were positive. In the multiplex PCR, 60.5% of E. coil were positive for CTX-M-2 type gene and 56.5% of K. pneumoniae were positive for VEB -1 type gene. In the ERIC-PCR, E. coil isolates formed 5 clusters and K. pneumoniae isolates were grouped into 4 clusters depending on region. Genotypes of clinical isolates are useful for detection and differentiation of ESBL producing intestinal bacteria. The ERIC-PCR method is thought to be helpful for establishing a regional surveillance system for infection due to its formation of different clusters depending on region.

• Korean Journal of Clinical Laboratory Science
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• v.41 no.4
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• pp.153-157
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• 2009

The production of extended-spectrum ${\beta}$-lactamases ($ESBL_S$) of the TEM or SHV type by bacterial pathogens is a major threat to the use of the clinically important expanded-spectrum cephalosporins. The characterization of the SHV ESBLs producing Klebsiella pneumoniae strains present in clinical isolates is time-consuming processes. We describe here in the development of a novel system, which consists of a real time PCR. We found 11 K. pneumoniae strains to be presumptive strains ESBLs producers by clinical and laboratory standards institute (CLSI) guidelines. The double disk synergy test showed 8 ESBL positive and conventional PCR showed 10 SHV ESBL positive, which were K. pneumoniae strains isolates. By real time PCR analysis, SHV gene in 11 of 11 strains were identified. When sequencing analysis was compared with real time PCR, both analysis were presented 99% similarity. In this study, we used a rapid, sensitive, and specific real-time PCR (RT-PCR) method for detection of the assay SHV ESBL producing K. pneumoniae strains in clinical isolates.

• Journal of fish pathology
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• v.31 no.2
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• pp.101-107
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• 2018

Quantitative analysis of a myxosporean parasite, Parvicapsula sp. in internal organs (kidney, intestine, spleen, brain and liver) from non-emaciated (farm-A) or emaciated (farm-B and farm-C) olive flounder Paralichthys olivaceus were performed by real-time PCR. The highest DNA copy number ($1.7{\times}10^7copies/mg$ tissue) was detected in kidney of the emaciated olive flounder from farm-C, while the DNA copy number was below detection limit in all the organs of the olive flounder from farm-B. There was not positive result in all of organs from olive flounder in farm-A. PCR and histopathological analysis were also performed using the same specimen and showed same results as those by real-time PCR.

• Journal of Korean Society of Water and Wastewater
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• v.23 no.2
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• pp.199-205
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• 2009

It is very important to differentiate of DNA derived from live or dead bacteria within mixed microbial communities in aquatic environments. Ethidium monoazide (EMA) is a DNA intercalating agent and the treatment of EMA with strong visible light cleaves the genomic DNA of bacteria. In dead bacterial cells, EMA intercalates into the genomic DNA, induces the cleavage of DNA, and inhibits the PCR amplification. In this study, we developed the EMA-PCR and EMA real-time PCR to detect the DNA derived from viable Escherichia coli (E.coli) in mixed cultures of live and dead E.coli. The treatment of EMA, $50{\mu}g/mL$, and 650 W visible halogen light exposure for 2 minutes cleaved the genomic DNA derived from heat killed E.coli but did not those of live E.coli. EMA-PCR could detect the DNA from live E.coli in mixed culture samples of live and dead E.coli at various ratio and there was no DNA amplification in only dead E.coli cultures. Similar results were observed in EMA real-time PCR. Further studies are needed to develop various EMA-PCR methods to detect viable waterborne pathogens such as Helicobacter pylori, Giardia lamblia, and so on.