• Food Science of Animal Resources
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• v.30 no.3
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• pp.410-418
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• 2010

Staphylococcus aureus is one of the major pathogens that can cause staphylococcal infection and food poisoning. In this study, we compared conventional culture methods and real-time PCR for detection of S. aureus in artificially inoculated milk, sausage, raw pork, and vegetable salad. The performance of a coagulase test for confirming S. aureus was also compared with a colony PCR test. Bulk food samples (500 g each) were artificially inoculated with S. aureus and divided into 20 samples (25 g or mL each). All samples were added to tryptic soy broth (225 mL/sample) with 10% NaCl and incubated at $37^{\circ}C$ for 24 h. After the enrichment, broth cultures were streaked onto Baird-Parker (BP) agar with egg yolk tellulite, and incubated at $37^{\circ}C$ for 24 h. In addition, 1 mL of broth cultures was collected to perform real-time PCR. Two suspicious colonies from the BP agar were picked up and plated on nutrient agar and incubated at $37^{\circ}C$ for 24 h followed, by a coagulase confirmation test and a colony PCR analysis. There were no statistical differences between culture methods and realtime PCR in food samples with low background microflora, such as milk and sausage. However, a significant statistical difference was found between the culture methods and real-time PCR for raw pork and vegetable salad. Furthermore, the colony PCR test of the presumptive colonies on BP agar for confirming S. aureus is more accurate and efficient than the coagulase test for unprocessed foods.

• 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.

• Mycobiology
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• v.47 no.3
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• pp.350-354
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• 2019

In the post-genomic era, gene function analysis has attracted much attention. Transformation is often needed to investigate gene function. In this study, an easy, rapid, reliable, and cost-effective colony polymerase chain reaction (PCR) method for screening mushroom transformants was developed: picking up a suitable amount of transformant's tissue ($1-10{\mu}g$) to $20{\mu}l$ 0.25% Lywallzyme solution, and vortexing for 10 s followed by incubation at $34^{\circ}C$ for 15 min. Finally, $2{\mu}l$ of the suspension was used as templates to perform PCR and single target bands were successfully amplified from respective transformants of Tremella fuciformis, Pleurotus ostreatus, and Pleurotus tuber-regium. This procedure could be widely employed for screening transformants in mushroom transformation experiments.

• The Korean Journal of Mycology
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• v.26 no.4 s.87
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• pp.466-477
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• 1998

Twenty-two Phellinus strains were characterized using colony morphologies and polymerase chain reaction (PCR) to divide into Phellinus linteus. There were some differences in mycelial growth and colony shapes among the strains when they were grown on various media such as PDA, MCM, MEA and YM. Phellinus linteus was slowly growing, formed golden-yellow colony, and produced blue pigment on PDA media. When the regions of internal transcribed spacer (ITS) were amplified from ribosomal RNA (rRNA) coding genes of P. igniarius and P. linteus strains by means of PCR, two types of band (700 bp and 800 bp) were appeared, respectively. For the amplified intergenic region I (IGRI), P. igniarius strains showed a different band among 500, 600, 700 and 800 bp according to the strains, whereas P. linteus strains did one specific band of 700 bp. By polymorphism analysis after digesting the amplified products with 6 different restriction enzymes, a band specific to P. linteus was generated when the products for ITS region were digested with HaeIII, suggesting that the enzyme digestion could provide effective method to distinguish between P. igniarius and P. linteus. And also, the analysis of genetic relationship showed that the genetic similarities were 89% and 95% in P. igniarius and P. linteus strains, respectively. Random amplification polymorphic DNA (RAPD) analysis using multiple primer sets and arbitrarily primed PCR (AP-PCR) with ITS3 primer could also result in a reproducible way to identify P. linteus strains.

• Research in Plant Disease
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• v.22 no.4
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• pp.269-275
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• 2016

Black shoot blight disease caused by Erwinia pyrifoliae have damaged economic loss to apple and pear growers until now since it was firstly reported in 1995 in Korea. This study was performed to reduce economic loss by mandatory eradication of all infected trees in case of more 10% disease incidence per orchard as official control. It also aims to set up effective management protocol for this disease by examining how far bacterial pathogen is present from the border of symptomatic and asymptomatic regions in infected apple twigs. Colony-PCR using isolated bacterial cells instead of genomic DNA was used to identify bacterial pathogen, EpSPF/EpSPR primer designed in enterobacterial repetitive intergenic consensus (ERIC) region was selected as specific for E. pyrifoliae. As results of monitoring of this disease during April to October in 2014-2015 by colony-PCR, occurrence of this disease was frequent from mid-May to early-July, when daily average temperature was around $25^{\circ}C$. Moreover, bacterial cells were continuously detected only in symptomatic regions and also asymptomatic regions of less than 20 cm from symptomatic regions. Therefore, we concluded that pruning of infected twigs at the region of more than 20 cm from symptomatic regions might be effective to manage black shoot blight disease in apple trees.

• Journal of Environmental Health Sciences
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• v.42 no.3
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• pp.189-195
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• 2016

Objectives: To ensure the microbiological safety of groundwater, it was confirmed whether waterborne pathogenic bacteria in groundwater samples tested positive for total coliforms in the Chungcheongnam-do Province region. Methods: Total colony counts, total coliforms and fecal coliforms were tested according to the process mandated by the drinking water quality testing standards of Korea. DNA was extracted from the samples, tested positive for total coliforms, and then subjected to real-time PCR to detect waterborne pathogenic bacteria. Results: A total of 115 samples were inadequate for drinking water. Thirty-one cases (27%) showed positive for fecal coliforms and nine cases (7.8%) showed total colony counts exceeding drinking water standards. Twenty-seven cases (23.5%) showed three items (total colony counts, total coliforms and fecal coliforms). Using the real-time PCR method, waterborne pathogens were detected in 57 cases (49.6%) in 115 samples. Seventy-eight cases of waterborne pathogenic bacteria were detected (including duplications): 27 cases of pathogenic E. coli (EPEC (19), ETEC (5), EHEC (1), EAEC (1) and EIEC (1)); 45 of Bacillus cereus; two of Yersinia spp.; two of Salmonella spp.; one of Staphylococcus aureus; one of Clostridium perfringens. Conclusion: The real-time PCR method can offer rapid and accurate detection of waterborne pathogenic bacteria. Therefore, this assay could be an alternative to conventional culture methods and can further ensure the microbiological safety of groundwater.

• Korean Journal of Microbiology
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• v.41 no.4
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• pp.269-274
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• 2005

We evaluated the PCR assay and two commercialized PCR kits for the detection of mycoplasma in veterinary via live vaccines. The PCR assay could specifically detect all the tested Mycoplasma spp. and Acholeplasma spp., whereas two commercialized PCR kits did not. Also, the specificity of the PCR assay showed that 4 reference strains and 7 field isolates belonging to avian mycoplasma species could be all detected. The sensitivity of the PCR assay was determined using pure cultured Mycoplasma spp. and Acholeplasma spp. with a range of 1 to 100 colony forming units/ml in 9 CFR Mycoplasma broth. To test the availability of the PCR assay for veterinary live viral vaccines, A. laidlawii was artificially inoculated into the swine transmissible gastroenteritis-rota virus combined vaccine and canine parvovirus vaccine, respectively and the sensitivity of the PCR assay was similar with the result of cultured samples. In this study, the PCR assays could be used as rapid and sensitive methods for the detection of mycoplasma in veterinary live viral vaccines.

• Korean Journal of Breeding Science
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• v.43 no.5
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• pp.448-456
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• 2011

We used suppression subtractive hybridization (SSH) combined with mirror orientation selection (MOS) method to screen differentially expressed genes from red-fleshed kiwifruit 'Hongyang'. As a result, the 288 clones were obtained by subcloning PCR product and 192 clones that showed positive clones on colony PCR analysis were selected. All the positive clones were sequenced. After comparisons with the NCBI/Genbank database using the BLAST search revealed that 30 clones showed sequence similarity to genes from other organisms; 10 clones showed significant sequence similarity to known genes. Among these clones, 3 clones (AcF21, AcF42 and AcF106) had sequence homology to 1-aminicyclopropane-carboxylic acid (ACC)-oxidase (ACO) that known to be related to fruit ripening. The expression patterns of differentially expressed genes were further investigated to validate the SSH data by reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qReal-time PCR) analysis. All the data from qReal-time PCR analysis coincide with the results obtained from RT-PCR analysis. Three clones were expressed at higher levels in 'Hongyang' than 'Hayward'. AcF21 was highly expressed in the other genes at 120 days after full bloom (DAFB) and 160 DAFB of 'Hongyang'.

• Journal of Food Hygiene and Safety
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• v.37 no.1
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• pp.9-14
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• 2022

The purpose of this study was to analyze the hazard of fungi in Garaetteok (Korean rice cake) by isolating and identifying of fungi contaminated with Garaetteok and investigating the possibility of mycotoxin production. Garaetteok used in this study were the ones that were returned back to the manufacturers in Jeollanam-do due to the presence of foreign matters presumed to be fungi. The fungi foreign matter was collected and inoculated on Potato dextrose agar, Malt extract agar, and Czapek yeast extract agar, and then cultured at 25℃ for 7 days. The micro-structure was observed under an optical microscope for the colonies in which pure isolation was confirmed. The gene sequencing of the product of amplified PCR was analyzed using the ITS primer. Colony-1 and 2 maintained the same properties in each tray, confirming that they were purely isolated. Budding cells were observed from the Colony-1, thus, it was determined to be yeast. Colony-2 was determined to be a fungus that belongs to Fusarium spp. as fusiform conidia were observed. As a result of gene sequencing, a total of 76 cases of fungi of Fusarium spp. were found, among which Fusarium solani was the most observed cases (53 cases). From the morphological and genetic identification, Colony-2 was identified as Fusarium spp., specifically, Fusarium solani. The fungi found in Fusarium spp. produce mycotoxins such as nivalenol, zearalenone, and fumonisin, which may cause vomiting, diarrhea, and cancer. Conclusively, the results confirm the possibility of mycotoxin production by Fusarium spp. isolated from Garaetteok. Consequently, when an unknown fungus was found, it is necessary to isolate and identify the fungus, determine whether it is a mycotoxin producing species, and strengthen relative administrative measures, accordingly.

• Korean Journal of Food Science and Technology
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• v.45 no.6
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• pp.791-795
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• 2013

The aim of this study was to compare the performance of a standard culture method and polymerase chain reaction (PCR) for the detection of Staphylococcus aureus (S. aureus) in milk and meat products. Milk, dried infant formula, sausage and ground beef that had been artificially inoculated with S. aureus were enriched in tryptic soy broth. After the enrichment, a loopful was inoculated onto Baird-Parker agar with egg-yolk-tellurite. In parallel, 23S rRNA was amplified by PCR from samples of the enriched broth. Suspected S. aureus colonies grown on selective agars were finally confirmed by a coagulase test and colony PCR. No significant statistical differences were observed between the incidence of S. aureus detected by the culture method and the incidence detected by PCR, in milk or dried infant formula. However, in sausage and ground beef, the number of positives detected by PCR was significantly higher than by the culture method (p<0.05). Our findings suggest that PCR could be an effective screening tool for the detection of S. aureus compared to the standard culture method.