• Genomics & Informatics
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• v.19 no.3
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• pp.34.1-34.6
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• 2021

Digital PCR (dPCR) is the third-generation PCR that enables real-time absolute quantification without reference materials. Recently, global diagnosis companies have developed new dPCR equipment. In line with the development, the Lab On An Array (LOAA) dPCR analyzer (Optolane) was launched last year. The LOAA dPCR is a semiconductor chip-based separation PCR type equipment. The LOAA dPCR includes Micro Electro Mechanical System that can be injected by partitioning the target gene into 56 to 20,000 wells. The amount of target gene per wells is digitized to 0 or 1 as the number of well gradually increases to 20,000 wells because its principle follows Poisson distribution, which allows the LOAA dPCR to perform precise absolute quantification. LOAA determined region of interest first prior to dPCR operation. To exclude invalid wells for the quantification, the LOAA dPCR has applied various filtering methods using brightness, slope, baseline, and noise filters. As the coronavirus disease 2019 has now spread around the world, needs for diagnostic equipment of point of care testing (POCT) are increasing. The LOAA dPCR is expected to be suitable for POCT diagnosis due to its compact size and high accuracy. Here, we describe the quantitative principle of the LOAA dPCR and suggest that it can be applied to various fields.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.167-171
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• 2004

Reduction in the leakage of the amplified PCR product out of cell is required for effective in situ PCR. For this purpose, primers with complementary tail sequences at their 5' sides were utilized to synthesize high molecular weight PCR products, but it is time-consuming and causes deterioration of cellular appearance with many PCR cycles. Therefore, it is required to optimize the PCR condition with minimal PCR cycles. To achieve the pur-pose, primer polymers were made without the target DNA in tube from nonspecific amplification with tailed primers and treated onto the fixed Molt/LAV cells on the glass slide for the 20 cycle-in situ PCR, in which the appropriate target signals were observed for the possible use of primer polymers in in situ PCR.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.198-204
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• 2002

Poliovirus is a member of enterovirus which causes paralytic poliomyelitis, encephalitis and aseptic meningitis. Since poliovirus is spread by the fecal-oral route and poliovirus-contaminated water could be a potential threat for public health, detection of poliovirus in drinking water resource is important. Infectious poliovirus and poliovirus inactivated by heat or UV were used to test three detection methods such as cell culture method, reverse transcription-polymerase chain reaction (RT-PCR) and integrated cell culture (ICC)-PCR. Infectious poliovirus was detected by all three methods and ICC-PCR was the most sensitive and fast in detecting poliovirus. Inactivated polioviruses could not be detected by cell culture or ICC-PCR methods. On the other hand, heat- inactivated viruses could be detected by RT-PCR. Thus it is suggested that ICC-PCR method is the most sensitive and effective in detecting infectious polioviruses in water sample.

• Tuberculosis and Respiratory Diseases
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• v.49 no.3
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• pp.281-289
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• 2000

Background : Mycobacterial culture is a confirmatory test to detect. M. tuberculosis, but it takes at least 6 weeks to diagnose. PCR is a rapid and sensitive method, but it is known that PCR has a high false positive rate due to contamination, and a high false negative rate due to inhibitors. It is also known that LCR and PCR-Hybridization, recently developed methods, are more specific methods than PCR in terms of detecting M. tuberculosis. In this study, we estimated the clinical utility of in house PCR, LCR and PCR-Hybridization for the detection of M. tuberculosis. Methods : We evaluated 75 specimens, upon which M. tuberculosis culture based testing was requested, by PCR LCR, and PCR-Hybridization and compared results. Mycobacterial culture was performed on 3% Ogawa media for 8 weeks, and an in house PCR, LCx Mycobacterium tuberculosis assay kit (Abbott Laboratories, North Chicago, III) and the AMPLICOR M. tuberculosis test kit (Roche Molecular Systems, Inc. Branchburg, NJ. USA). Results : In the view of the culture results, the sensitivities of the three tests were 40%, 80%, and 100% and their specificities were 98.6%, 94.3%, and 94.3%. Conclusion : LCR and PCR-Hybridization are rapid and sensitive methods for detecting M. tuberculosis in clinical laboratories.

• Tuberculosis and Respiratory Diseases
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• v.44 no.5
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• pp.1001-1010
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• 1997

Background : PCR technique is useful in diagnosis of pulmonary tuberculosis. But, its sensitivity and specificity is some different among several studies. Our aim is compare our PCR results with other's previous PCR results in AFB smear negative patients. Methods : PCR were performed in patients that their disease were suspected as active pulmonary tuberculosis and that their initial serial sputum AFB smear results were negative. Total number of patients studied by PCR technique was 177. Also, we analyzed the data only in patients whose bronchial washing fluid AFB smear was negative. And the primer had been used was IS 6110. Results : In our retrograde study, the number of patients who are diagnosed as having active pulmonary tuberculosis, inactive pulmonary tuberculosis and nontuberculous pulmonary disease was 99, 28, 50, respectively. In the sputum study, the sensitivity of PCR is 41.5% (27 PCR positive cases/65 active TBc cases). And the sensitivity of TB culture is 53.8% (35 TB culture positive cases/65 active TBc cases). In the bronchial washing specimen study, the sensitivity of PCR is 53.8% (21 PCR positive cases/39 active TBc cases). And the sensitivity of TB culture is 43.6% (17 TB culture Positive cases/39 active TBc cases). The specificity of PCR in our study is 94.9%. (74 PCR negative cases/78 inactive TBc or nontubereulous cases) In the cases of patients who were never takened anti-TBc medication, the sensitivity of PCR (45.6%--25 positive cases/55 cases) is some lower than culture (58.2%--32 positive cases/55 cases). In the cases of patients who had been takened anti-TBC medication. the sensitivity of PCR (60%--18 positive cases/30 cases) is some superior than culture (50%--15 positive cases/30 cases). Conclusion : We think that PCR results in cases of sputum AFB smear negative patients is nearly same as culture. And PCR is especially useful in patients who had been takened anti-TBc medication on admission.

• Biomedical Science Letters
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• v.24 no.4
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• pp.390-397
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• 2018

For the specific detection of human Parvovirus B19 (HuPaV-B19), we designed ten specific PCR primers from 3,800~4,500 nucleotides of HuPaV-B19 complete genome (NC_000883.2). Seventeen candidate PCR primer sets for specific detecting HuPaV-B19 were constructed. In specific reaction of HuPaV-B19, seventeen PCR primer sets showed specific band, however five PCR primer sets were selected basis of band intensity, amplicon size and location. In non-specific reaction with seven reference viruses, four PCR primer sets showed non-specific band, however one PCR primer set is not. Detection sensitivity of final selective PCR primer set was $100fg/{\mu}L$ for 112 minute, and PCR amplicon is 539 base pairs (bp). In addition, nested PCR primer set was developed, for detection HuPaV-B19 from a low concentration of contaminated samples. Selection of nested PCR primer set was basis of sensitivity and groundwater sample tests. Detection sensitivity of final selective PCR and nested PCR primer sets for the detection of HuPaV-B19 were $100fg/{\mu}L$ and $100ag/{\mu}L$ basis of HuPaV-B19 plasmid, it was able to rapid and highly sensitive detection of HuPaV-B19 than previous reports. We expect developed PCR primer set in this study will used for detection of HuPaV-B19 in various samples.

• Genomics & Informatics
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• v.21 no.2
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• pp.24.1-24.7
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• 2023

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.

• Korean Journal of Veterinary Research
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• v.44 no.1
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• pp.99-103
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• 2004

The aim of the present study was to develop a sensitive and specific assay for the diagnosis of alcelaphine herpesvirus 1 (AlHV-1) which is a cause agent of malignant catarrhal fever in ruminants. A1HV-1 is a gamma herpesvirus, which is frequent latent, and it is often difficult to detect its antigens or specific nucleic acids because of its low genomic copies in the infected tissues. In this study, polymerase chain reaction (PCR)-dot blot hybridization (DBH) assay for detecting AlHV-1 DNA was developed and evaluated for its sensitivity and specificity as comparison with PCR and DBH alone. The developed PCR-DBH was more sensitive than PCR or DBH alone and also very specific. The results showed that the sensitivity of PCR-DBH were higher and stronger than those of PCR and DBH alone. This PCR-DBH assay can be applied efficiently to confirm the presence of AlHV-1 virus on clinical samples and to differentiate specifically between AlHV-1 infection and other viral infections.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.4
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• pp.213-220
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• 2003

Polymerase chain reaction (PCR) is one of the most widely used analytical tool and is an important module that would benefit from being miniaturized and integrated onto diagnostic or analytical chips. There are potentially two different approaches for the miniaturization of the PCR module: chamber-type and flow-type micro-PCR. These miniaturized PCRs have distinct characteristics and advantages. In this article, we review the necessity of micro-PCR, the materials for the chip fabrication, the surface modification, and characteristics of the two types of micro-PCR. The motivation underlying the development of micro-PCR, the advantages and disadvantages of the various materials used in fabrication and the surface modification methods will be discussed. And finally, the precise features of the two different types of micro-PCR will be compared.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.204-205
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• 2001

Milk is easily contaminated by pathogenic microorganisms and contains many ingredients that inhibit normal PCR. In this study, we developed a detection mothed for pathogenic microorganisms existing in milk by usting PCR. 'Sample pretreatment prior to PCR were compared to overcome the inhibition. A high PCR efficiency was achieved by SDS lysis pretreatment. without further purification of DNA for PCR.