• Journal of Korean society of Dental Hygiene
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• v.17 no.2
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• pp.319-330
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• 2017

Objectives: The purpose of this study was to compare SYBR Green qPCR, TaqMan, and bacterial selective medium cultures for accurate quantitative analysis of oral microorganisms. Methods: The SYBR Green method is widely used to analyze the total amount of oral microorganisms in oral saliva. However, in this study, MTR-PCR method based on TaqMan method was performed using newly developed primers and probes. In addition, it was designed to confirm the detection agreement of bacteria among bacteria detection method. Results: As a result of MRT-PCR and SYBR Green qPCR analysis, more than 40 times (0.9-362.9 times) bacterium was detected by MRT-PCR. In addition, more bacteria were detected in saliva in the order of MRT-PCR, SYBR Green qPCR, and bacterium culture, and the results of MRB-PCR and SYBR Green qPCR showed the highest agreement. The agreement between the three methods for detecting P. intermedia was similar between 71.4 and 88.6%, but the agreement between MRT-PCR and SYBR Green qPCR was 80% for S. mutans. Among them, the number of total bacteria, P. intermedia and S. mutans bacteria in saliva was higher than that of SYBR Green qPCR method, and bacterium culture method by MRT-PCR method. P. intermedia and S. mutans in saliva were detected by MRT-PCR and MRT-PCR in 88.6% of cases, followed by the SYBR Green qPCR method (80.0%). Conclusions: The SYBR Green qPCR method is the same molecular biology method, but it can not analyze the germs at the same time. Bacterial culturing takes a lot of time if there is no selective culture medium. Therefore, the MRT-PCR method using newly developed primers and probes is considered to be the best method.

• Journal of Environmental Health Sciences
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• v.28 no.2
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• pp.193-202
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• 2002

The polymerase chain reaction(PCR) of target lacZ and uidA genes were used to detect total coliform and Escherichia coli for determining water quality, respectively. Of 109 spring waters, coliform were detected from 38 spring waters by lacZ PCR method but 21 spring waters by culture method accepted by the Ministry of Environment for water quality monitoring. The lacz PCR method gave the results statistically equivalent to those of the culture method(kappa=0.62, McNemar=17.00). The uidA PCR method gave the same results to those of the culture method. The sensitivity and specificity of coliform and E. coli by PCR method were 100% and 80.7%, respectively. Therefore, PCR can be used for the rapid identification of Escherichia coli and coliform in potable water using uidA and lacZ.

• Journal of Korean Society on Water Environment
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• v.24 no.6
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• pp.817-822
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• 2008

In order to identify various Cryptosporidium species in environment, nested PCR-RFLP and DNA sequencing method were used. The sensitivity of nested PCR-RFLP based on 18s rRNA gene was shown to 1 oocyst. Therefore, we applied nested PCR-RFLP method to environmental samples. As a result, only 4 samples out of 8 samples confirmed as Cryptosporidium parvum by standard method of Cryptosporidium were identified as Cryptosporidium parvum by nested PCR-RFLP and DNA sequencing method. The rest of 4 samples among 8 samples were identified as Cryptosporidium muris, Cryptosporidium bailey. Therefore, in addition to standard method of Cryptosporidium, supplementary verification through nested PCR-RFLP and DNA sequencing should be needed to give more accurate information about risk of Cryptosporidium.

• Journal of Genetic Medicine
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• v.9 no.1
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• pp.11-16
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• 2012

Purpose: To determine a method to improve the efficacy and accuracy of preimplantation genetic diagnosis (PGD) - polymerase chain reaction (PCR), we compared hot start PCR and conventional multiplex nested PCR. Materials and Methods: This study was performed with single lymphocyte isolated from whole blood samples that were obtained from two couples with osteogenesis imperfecta (OI). We proceeded with conventional multiplex nested PCR and hot start PCR in which essential reaction components were physically removed, and we compared the amplification rate, allele dropout rate and nonspecific products. Afterward, we used selective method for PGD. Results: In the two couples, the respective amplification rate were 93.5% and 80.0% using conventional multiplex nested PCR and 95.5% and 92.0% using hot start PCR. The respective mean allele dropout rates for the two couples were 42.0% and 14.0% with conventional multiplex nested PCR and 36.0% and 6.0% with hot start PCR. Conclusion: The results demonstrate that the hot start PCR procedure provides higher amplification rates and lower allele dropout rate than the conventional method and that it decreased the nonspecific band in multiplex nested PCR. The hot start method is more efficient for analyzing a single blastomere in clinical PGD.

• Tuberculosis and Respiratory Diseases
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• v.42 no.5
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• pp.695-702
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• 1995

Background: Since polymerase chain reaction(PCR) was devised by Saiki in 1985, it has been used extensively in various fields of molecular biology. Clinically, PCR is especially useful in situation when microbiological or serological diagnosis is limited by scanty amount of causative agents. Thus, PCR can provide rapid and sensitive way of detecting M. tuberculosis in tuberculosis pleurisy which is diagnosed in only about 60 % of cases by conventional method. Method: To evaluate the diagnostic usefulness of PCR in tuberculosis pleurisy, The results of PCR was compared with those of conventional method, including pleural biopsy. The pleural effusion fluid was collected from 7 proven patients, 7 clinically suspected patients and control group(7 patients with malignant effusion). We extracted DNA from pleural fluid by modified method of Eisennach method(1991). The amplification target for PCR was 123 base pair DNA, a part of IS6110. Result: 1) Sensitivity of PCR: We detected upto 50fg DNA. 2) In patients with pleural effusion of proven tuberculosis, the positive rate of PCR was 85.7%(6/7). In patients with pleural effusion of clinically suspected tuberculosis, the positive rate was 71.5%(5/7). In control group, positive rate was 0%(0/7). Conclusion: We concluded that PCR method could be a very rapid, sensitive and specific one for diagnosis of M tuberculosis in pleural effusion. Further studies should be followed for the development of easier method.

• 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.

• Food Science and Biotechnology
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• v.17 no.3
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• pp.651-654
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• 2008

In order to enhance the efficacy of norovirus detection by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR, this study developed a norovirus mRNA concentration method using poly oligo dT-conjugated magnetic beads. An efficient norovirus detection protocol was performed on commercial ham using 2 viral elution buffers (glycine buffer and Tris beef extract buffer) and 2 concentration solutions [polyethylene glycol (PEG) and zirconium hydroxide]. The different approaches were verified by RT-PCR and nested PCR. This method was performed on ham in less than 8 hr by artificial inoculation of serial dilutions of the virus ranging from 1,000 to 1 RT-PCR unit/mL. The viral extraction and concentration method had 10-fold higher sensitivity using the combination of Tris beef extract buffer and PEG as compared to glycine buffer and zirconium hydroxide. This method proved that RT-PCR and nested PCR have the sensitive ability to detect norovirus in commercial ham, in that norovirus was successfully detected in artificially contaminated samples at a detection level as low as 1-10 RT-PCR unit/mL. Overall, such a detection limit suggests this protocol is both quick and efficient in terms of its potential use for detecting norovirus in meat products.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1464-1469
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• 2009

To achieve more accurate and rapid detection of macrolide-lincosamide-streptogramin B resistance genes, erm(A), erm(B), and erm(C), we developed a TaqMan probe-based real-time PCR (Q-PCR) method and compared it with conventional PCR (C-PCR), which is the most widely using erm gene identification method. The detection limit of Q-PCR was 5 fg of genomic DNA or 5-8 CFU of bacterial cells of Staphylococcus aureus. The utilization of Q-PCR might shorten the time to erm detection from 3-4 h to about 50 min. These data indicated that Q-PCR assay appears to be not only highly sensitive and specific, but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay will permit rapid and accurate identification of erm genes from clinical and other samples.

• 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.

• Journal of Microbiology
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• v.38 no.4
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• pp.260-264
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• 2000

Reverse transcription (RT)-PCR and nested PCR methods (2-step PCR) were tested for their ability to detect marine birnavirus (MBV) in cultured rockfish, Sebastes schlegeli. One set of primers for RT-PCR was designed, based on a gene of infectious pancreatic necrosis virus (IPNV), and another set of primers for nested PCR was designed based on the VP2/NS junction region of MBV. This 2-step PCR method was specific for MBV and sensitivity was heightened when nested PCR was combined to RT-PCR. This 2-step PCR method was useful for detecting MBV not only in diseased fish, but also in asymptomatic fish. These results indicate that this 2-step PCR method is useful for detecting MBV in rockfish.