• Korean Journal of Food Science and Technology
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• v.31 no.6
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• pp.1628-1634
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• 1999

The polymerase chain reaction (PCR) for the sensitive and specific detection of Listeria monocytogenes was employed by using LM 1 and LM 2 primers which were based on the listeriolysin O gene. The direct use of cell suspension as DNA template, without DNA extraction or lysis step, was suitable and specific enough to detect L. monocytogenes at the level of $10^2$ CFU or less per PCR for the pure culture and milk sample, however, the detection sensitivity became blunt for other food samples such as kimchi and chicken. The nested PCR, in which L-1 and L-2 (both designed from listeriolysin O gene) were employed as inner primers, was specific for detecting L. monocytogenes and enhanced the detection limit by 10 times. The PCR using LM 1 and LM 2 primers was very effective to detect L. monocytogenes from foods in terms of the specificity and time consumed, i. e. within $4{\sim}8\;hrs$ (nested PCR).

• Korean Journal of Environmental Agriculture
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• v.30 no.4
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• pp.367-376
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• 2011

BACKGROUND: In order to develop effective assessment method for Korean paddy soil microbial community structure, reliable genomic DNA extraction method from paddy soil and quantitative real-time PCR (qRT-PCR) method are needed to establish METHODS AND RESULTS: Out of six conventional soil genomic DNA extraction methods, anion exchange resin purification method was turn to be the most reliable. Various PCR primers for distinguishing five bacterial phylum (${\alpha}$-Proteobacteria, ${\beta}$-Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes), all bacteria, and all fungi were tested. Various qRT-PCR temperature conditions were also tested by repeating experiment. Finally, both genomic DNA extraction and qRT-PCR methods for paddy soil were well established. CONCLUSION: Quantitative real-time PCR (qRT-PCR) method to assess paddy soil microbial community was established.

• Journal of Animal Science and Technology
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• v.46 no.5
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• pp.841-848
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• 2004

Rapid detection of viable Salmonella in pasteurized milk is important to protect public health from food poisoning. Reverse transcriptase-polymerase chain reaction(RT-PCR) is recognized as a molecular genetical method to differentiate between live and dead bacteria The RT-PCR in this study was designed to detect specifically viable Salmonella in milk by using the primers whose nucleotide sequences were determined based on fimA gene which encodes the submit of type 1 fimbriae. Treatment of RNA preparation with RNase-free DNase was adequate enough to destroy DNA, which may otherwise be amplified in the RT PCR Seven strains of Salmonella were detected in the RT-PCR but Escherichia coli, Shigella sonnei, Citrobacter freundii, and Klebsiella pneumoniae were not. $10^7/ml$ and $10^6/ml$ of dead Salmonella which were heat-treated in milk were detectable by using the RT-PCR but $10^5{\sim}10/ml$ of the dead bacteria were not. The sensitivity of the RT-PCR in detecting viable Salmonella was 100 cells/ml.

• Korean Journal of Plant Resources
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• v.14 no.3
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• pp.235-240
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• 2001

Polymerase chain reaction (PCR) is used in a wide array of researches in plant molecular genetics and breeding. However, considerable time and cost are still required for the preparation of DNA suitable for reliable PCR results, especially in plant species containing high amounts of polyphenols. To reduce time and effort for PCR-based analysis, a simplified but reliable method was developed by a combinational employment of a simple and fast DNA extraction procedure and BLOTTO (Bovine Lacto Transfer Technique Optimizer) in reaction mixture. Genomic DNAs prepared by one-step extraction method from recalcitrant plant species such as Rubus coreanus, apple, grape and lettuce were successfully amplified by random primers in the reaction mixture containing 2 to 4% BLOTTO. Successful amplification of ${\gamma}$-TMT transgene in lettuce transformants by the specific primers was also achieved in the same condition, making rapid screening of positive transformants possible. Our results suggest that use of a simple DNA extraction procedure and incorporation of BLOTTO in reaction mixture in combination can reduce time and effort required for the analyses of a large number of germplasms and transformants by PCR-based techniques.

• The Korean Journal of Mycology
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• v.49 no.4
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• pp.455-467
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• 2021

Macrofungi are valuable resources as novel drug candidates, new biomaterials, and edible materials. Recently, genetic approaches pertaining to macrofungi have been continuously growing for their identification, molecular breeding, and genetic engineering. However, purification and amplification of fungal DNA is challenging because of the rigid cell wall and presence of PCR inhibitory metabolites. Here, we established a direct PCR method to provide a rapid and efficient method for PCR-grade macrofungal DNA preparation applicable to both conventional PCR and real-time PCR. We first optimized the procedure of lysis and PCR using the mycelia of Lentinula edodes, one of the most widely consumed macrofungal species. Lysates prepared by neutralizing with (NH₄)₂SO₄ after heating the mycelia in a mixture of TE buffer and KOH at 65℃ for 10 min showed successful amplification in both conventional and real-time PCR. Moreover, the addition of bovine serum albumin to the PCR mixture enhanced the amplification in conventional PCR. Using this method, we successfully amplified not only internal transcribed spacer fragments but also low-copy genes ranging in length from 500 to 3,000 bp. Next, we applied this method to 62 different species (54 genera) of macrofungi, including edible mushrooms, such as Pleurotus ostreatus, and medicinal mushrooms such as Cordyceps militaris. It was found that our method is widely applicable to both ascomycetes and basidiomycetes. We expect that our method will contribute to accelerating PCR-based approaches, such as molecular identification, DNA marker typing, gene cloning, and transformant screening, in macrofungal studies.

• Journal of fish pathology
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• v.37 no.1
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• pp.147-153
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• 2024

The World Organization for Animal Health (WOAH) recommends two protocols (ITS and COI) for conventional PCR of G. salaris diagnosis. However, ITS PCR protocol may yield false-positive results, leading to unnecessary countermeasures. It's difficult to distinguish between G. salaris and false-positive by similar amplicon size of PCR, since the amplicon size of ITS PCR in G. salaris and false-positive was 1,300 and 1,187 bp, respectively. The nucleotide sequences of ITS false-positive in rainbow trout is 99.7% identical to previously reported host genome sequences of rainbow trout (Oncorhynchus mykiss) and 95.3 to 89.1% identical to those of other salmonid fish species. To reduce false-positive PCR band, PCR was performed by the different annealing temperature, but PCR bands were still detected. In RFLP analysis by HaeIII, the PCR product of G. salaris was digested into four bands of 512, 399, 234 and 154 bp, while the false-positive was digested into seven bands of 297, 263, 242, 144, 93, 80 and 68 bp. In the RFLP patterns digested by HindIII, G. salaris showed two bands of 659 and 640 bp, while false-positive had one fragment of 1,187 bp without any digestion. Therefore, the RFLP method of ITS PCR with HaeIII and HindIII can be used for differentiation between G. salaris and false-positive. These results might provide important information on the improvement of PCR diagnostic method of G. salaris.

• Applied Biological Chemistry
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• v.50 no.4
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• pp.245-252
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• 2007

FTA (fault tree analysis), an analytical method for system failure management, was employed in the management of faults in running PCR analysis. PCR is executed through several processes, in which the process of PCR machine operation was selected for the analysis by FTA. The reason for choosing the simplest process in the PCR analysis was to adopt it as a first trial to test a feasibility of the FTA approach. First, fault events-top event, intermediate event, basic events-were identified by survey on expert knowledge of PCR. Then those events were correlated deductively to build a fault tree in hierarchical structure. The fault tree was evaluated qualitatively and quantitatively, yielding minimal cut sets, structural importance, common cause vulnerability, simulation of probability of occurrence of top event, cut set importance, item importance and sensitivity. The top event was 'errors in the step of PCR machine operation in running PCR analysis'. The major intermediate events were 'failures in instrument' and 'errors in actions in experiment'. The basic events were four events, one event and one event based on human errors, instrument failure and energy source failure, respectively. Those events were combined with Boolean logic gates-AND or OR, constructing a fault tree. In the qualitative evaluation of the tree, the basic events-'errors in preparing the reaction mixture', 'errors in setting temperature and time of PCR machine', 'failure of electrical power during running PCR machine', 'errors in selecting adequate PCR machine'-proved the most critical in the occurrence of the fault of the top event. In the quantitative evaluation, the list of the critical events were not the same as that from the qualitative evaluation. It was because the probability value of PCR machine failure, not on the list above though, increased with used time, and the probability of the events of electricity failure and defective of PCR machine were given zero due to rare likelihood of the events in general. It was concluded that this feasibility study is worth being a means to introduce the novel technique, FTA, to the management of faults in running PCR analysis.

• Tuberculosis and Respiratory Diseases
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• v.45 no.3
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• pp.519-528
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• 1998

Background: Diagnosis of pulmonary tuberculosis is not easy when the sputum smear for Mycobacterium tuberculosis(M. Tb) is negative. We evaluated the clinical utility of polymerase chain reaction(PCR) for detecting M. Tb in bronchoalveolar lavage(BAL) samples. Methods: We recruited 84 patients whose sputum smear for M. Tb were negative or not available due to no production of sputum. We performed bronchoalveolar lavage for acid-fast stain, culture of mycobacteria, and PCR assay of BAL fluid. We analyzed the results of microbiologic examination. Results: The sensitivity of BAL fluid smear, culture, and PCR were 20%, 38%, and 40%, respectively. The specificity of BAL fluid PCR was 95%. The positive predictive value of PCR was 89%. The smear of BAL fluid was positive in 17%. The PCR of BAL fluid was the only diagnostic test in 17%. Therefore, the BAL fluid analysis including smear and PCR was diagnostic in 34 % within 24 hours. The BAL fluid analysis including smear, PCR, and culture was diagnostic in 55% within 2 month. Conclusion: The BAL fluid PCR was valuable method in the diagnosis of pulmonary tuberculosis in patients whose sputa were not available or reveal negative smear.

• Research in Plant Disease
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• v.12 no.2
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• pp.139-143
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• 2006

Virion captured reverse transcription polymerase chain reaction (VC/RT-PCR) could detect plant virus quickly and accurately. In the VC/RT-PCR, no antibody is needed unlike immuno-captured RT-PCR (IC/RT-PCR) which had been improved method of RT-PCR for plant viruses, and virus nucleic acids can be obtained easily within 30minutes by property of polypropylene PCR tube which is hold and immobilized viral particles on its surface. For the virion capture of Tomato spotted wilt virus (TSWV), the extraction buffer was tested. The optimum macerating buffer for TSWV was 0.01M potassium phosphate buffer, pH 7.0, containing 0.5% sodium sulfite. The viral crude sap was incubated for 30 min at $4^{\circ}C$. The virions in the PCR tubes were washed two times with 0.01M PBS containing 0.05% Tween-20. The washed virions were treated at $95^{\circ}C$ immediately for 1 min containing RNase free water and chilled quickly in the ice. Disclosed virions' RNAs by heat treatment were used for RT-PCR. Dilution end point of $10^{-5}$ from plant's crude sap infected with TSWV showed relatively higher detection sensitivity for VC/RT-PCR. During multiple detection using two or more primers, interference was arisen by interactions between primer-primer and plant species. The result of multiplex RT-PCR was influenced by combinations of primers and the kind of plant, and the optimum extraction buffer for the multiplex detection by VC/RT-PCR should be developed.

• 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.