• 보존과학연구
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• s.32
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• pp.171-183
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• 2011

In this study molecular genetic analysis was carried out on 4 human skeletal remains from Osuri, Buyeo. We showed that real-time PCR is the method of the choice to assess the initial number of genuine ancient DNA molecules. Human mitochondrial DNA quantification was accomplished by the real-time PCR for the cytochrome b gene of the mitochondria. Histological results proved to be a good potentiality for biochemical analysis using biomolecule. The level of specimen's preservation state was proved that level of quantitative result was BO-04, BO-01, BO-03, BO-02. Continually, we showed that biochemical and biomolecule results for the level of preservation state were similar. This study will be useful to important material for predicting biochemistry and biology analysis of the ancient bone.

• Korean Journal of Microbiology
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• v.43 no.2
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• pp.91-99
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• 2007

For the detection of Human Immunodeficiency Virus (HIV), multiple and ultra-rapid real-time PCR methods were developed. The target DNA sequences were deduced from HIV-1 specific 495bp partial env gene (gi_1184090) and from HIV-2 specific 294 bp partial env gene (gi_1332355), and were synthesized by using PCR-based gene synthesis on the reason of safety. Ultra-rapid real-time PCR was performed by $Genspector^{TM}$ using microchip-based, $1\;{\mu}l$ of reaction volume with extremely short time in each 3 step in PCR. The detection including DNA-amplification and melting temperature analysis was completed inner 15 minutes. The HIV-1 specific 117 bp-long and HIV-2 specific 119 bp-long PCR products were successfully amplified from minimum of template,2.3 molecules of each env gene. This kind of real-time PCR was designated as ultra-rapid real-time PCR in this study and it could be applied not only an alternative detection method against HIV, but also other pathogens using PCR-based detection.

• Korean Journal Plant Pathology
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• v.12 no.4
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• pp.432-436
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• 1996

역전사 중합효소 연쇄반응(RT-PCR)을 이용하여 담배(Nicotiana glutinosa)에 증식시킨 7종의 오이 모자이크 바이러스(CMV)를 검정하였다. RT-PCR을 위한 간단하고 신속한 바이러스 핵산의 조즙액 추출법이 개발되었으며, CMV 외피단백질 유전자 부위를 기초로 하여 제작한 20개의 염기로 구성된 primer를 사용하여 RT-PCR을 실시한 결과, 약 490 염기쌍의 DNA 단편들이 이병식물의 조즙액으로부터 증폭되었다. EcoRI 및 MspI을 이용한 RT-PCR 산물의 분석에 의하여, 공시한 7종의 바이러스는 모두 CMV subgroup I으로 동정되었다. Ouchterlony 한천젤 이중 확산법을 이용한 항혈청 검정에서도 7종의 바이러스 모두 CMV-Y의 항혈청과 단일의 침강선을 형성하였다.

• Korean Journal of Microbiology
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• v.43 no.4
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• pp.264-272
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• 2007

For the detection of human immunodeficiency virus (HIV), ultra-rapid real-time PCR methods were developed. The target DNA sequences were used 495 bp HIV-1-specific env gene (gi_1184090) and 294 bp HIV-2-specific env gene (gi_1332355). Ultra-rapid real-time PCR was peformed by $Genspector^{TM}$ (Samsung, Korea) using microchip-based, $6\;{\mu}l$ of reaction volume with extremely short running time in only 2 steps (denaturation, annealing/extension) in each cycle of PCR. Total reaction for 30 cycled ultra-rapid PCR detection including melting temperature analysis was completed in 7 min and 30 sec. The HIV-1-specific 117 bp-long or HIV-2-spe-cific 119 bp-long PCR products were successfully amplified from the minimum of template, $2.3{\times}10^3$ copies of each euv gene using 30 cycled two-steps ultra-rapid PCR. This kind of ultra-rapid real-time PCR method would be useful not only for the rapid-detection of HIV, but also rapid-detection of other pathogens.

• Korean Journal of Clinical Laboratory Science
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• v.53 no.1
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• pp.49-59
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• 2021

This study investigated the use of real-time PCR melting curves for the diagnosis of blaKPC and blaNDM genes among the most frequently detected carbapenemase-producing Enterobacteriaceae in Korea. As a means of addressing the shortcomings of phenotype tests and conventional PCR. The modified Hodge test confirmed positivity in 25 of 35 strains, and carbapenemase inhibition testing confirmed positivity in 14 strains by meropenem+PBA or meropenem+EDTA. PCR analysis showed amplification products in 25 strains of Klebsiella pneumoniae carbapenemases (KPC), 10 of K. pneumoniae, 5 of E. coli, 5 of A. baumannii, 4 of P. aeruginosa, and 1 of P. putida. New Delhi metallo β-lactamase (NDM) identified amplification products in 8 strains, that is, 2 K. pneumoniae, 3 E. coli, 1 P. aeruginosa, 1 E. cloacae, and 1 P. retgeri strains. Real-time PCR melting curve analysis confirmed amplification in 25 strains of KPC and 8 strains of NDM, and these results were 100% consistent with PCR results. In conclusion, our findings suggest early diagnosis of carbapenem resistant Enterobacteriaceae by real-time PCR offers a potential means of antibacterial management that can prevent and control nosocomial infection spread.

• Journal of Korean Society on Water Environment
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• v.34 no.1
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• pp.46-56
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• 2018

A mcyB-specific Ultra-Rapid quantitative PCR was developed for the quantitative detection of Microcystis aeruginosa, which is often a dominant species in green tide. McyB-specific UR-qPCR was optimized under extremely short times of each step in thermal cycles, based on the specific primers deduced from the mcyB in microcystin synthetase of M. aeruginosa. The M. aeruginosa strain KG07 was used as a standard for quantification, after the microscopic counting and calculation by mcyB-specific UR-qPCR. The water samples from the river water with the Microcystis outbreak were also measured by using both methods. The $1.0{\times}10^8$ molecules of mcyB-specific DNA was recognized inner 4 minutes after beginning of UR-qPCR, while $1.0{\times}10^4$ molecules of mcyB-specific templates was detected inner 7 minutes with quantitative manner. From the range of $1.0{\times}10^2$ to $1.0{\times}10^8$ initial molecules, quantification was well established based on $C_T$ using mcyB-specific UR-qPCR (Regression coefficiency, $R^2=0.9977$). Between the numbers of M. aeruginosa cell counting under microscope and calculated numbers using mcyB-specific UR-qPCR, some differences were often found. The reasons for these differences were discussed; therefore, easy compensation method was proposed that was dependent on the numbers of the cell counting. Additionally, to easily extract the genomic DNA (gDNA) from the samples, a freeze-fracturing of water-sample using liquid nitrogen was tested, by excluding the conventional gDNA extraction method. It was also verified that there were no significant differences using the UR-qPCR with both gDNAs. In conclusion, the mcyB-specific UR-qPCR that we proposed would be expected to be a useful tool for rapid quantification and easy monitoring of M. aeruginosa in environmental water.

• Food Science of Animal Resources
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• v.33 no.5
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• pp.610-616
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• 2013

Loop-mediated isothermal amplification (LAMP) is an emerging detection technology for the amplification of DNA under isothermal conditions. The aim of this study was to develop a rapid and reliable LAMP technique for the detection of Cronobacter sakazakii in milk powder. In order to enhance the sensitivity and specificity, LAMP primers targeting outer membrane protein A (ompA) gene of C. sakazakii were designed using Explorer V4 software. Thirty seven C. sakazakii strains and 13 pathogenic microorganisms were used for comparative detection of C. sakazakii using polymerase chain reaction (PCR), real-time PCR, and LAMP. LAMP developed in this study could specifically detect C. sakazakii strains without cross-reactivity with other foodborne pathogens. LAMP products amplified from ompA gene of C. sakazakii were digested with with HhaI and NruI enzyme. The specificity of LAMP was confirmed by restriction fragment length polymorphism (RFLP) analysis. LAMP could detect C. sakazakii within 1 h without bacterial culture and its detection limit was as low as 1 CFU/mL C. sakazakii in milk. In the comparison of the sensitivity, LAMP showed 10,000- and 100-times higher detection limit than PCR or real-time PCR, respectively. Therefore, this study can conclude that LAMP is a rapid and reliable detection technique for C. sakazakii contaminated in powdered milk.

• Journal of Life Science
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• v.33 no.1
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• pp.25-33
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• 2023

The fermentation process of kimchi, which is a traditional Korean food, influences the resulting compo- sition of microorganisms, such as the genera Leuconostoc, Weissella, and Lactobacillus. In addition, several factors, including the type of kimchi, fermentation conditions, materials, and ingredients, can influence the distribution of the kimchi microbial community. In this study, next-generation sequencing (NGS) of kimchi samples obtained from central (Gangwon-do and Gyeonggi-do) and southern (Jeolla-do and Gyeongsang-do) regions in Korea was performed, and the microbial communities in samples from the two regions were compared. Good's coverage prediction for all samples was higher than 99%, indicating that there was sufficient reliability for comparative analysis. However, in a α -diversity analysis, there was no significant difference in species richness and diversity between samples. The Firmicutes phylum was common in both regions. At the species level, Weissella kandleri dominated in central (46.5%) and southern (30.8%) regions. Linear discriminant analysis effect size (LEfSe) analysis was performed to identify biomarkers representing the microbial community in each region. The LEfSe results pointed to statistically significant differences between the two regions in community composition, with Leuconostocaceae (71.4%) dominating in the central region and Lactobacillaceae (61.0%) dominating in the southern region. Based on these results, it can be concluded that the microbial communities of kimchi are significantly influenced by regional properties and that it can provide more useful scientific data to study the relationship between regional characteristics of kimchi and their microbial distribution.

• Clinical and Experimental Pediatrics
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• v.48 no.2
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• pp.197-203
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• 2005

Purpose : Changes in metalloproteinases(MMP) activity have been demonstrated in several disease states, including rheumatoid arthritis and tumor metastasis. More importantly, increased myocardial MMP activity has been reported to occur in both clinical and experimental forms of dilated cardiomyopathy. There was no report about MMP in adriamycin(ADR)-induced cardiomyopathy. The purpose of this study was to investigate gene expression of MMP and tissue inhibitor of metalloproteinases(TIMP) in ADR-induced cardiomyopathy and clarify the relationship between MMP and cytokines. Methods : Male Sprague-Dawley rats were divided into two groups. The first group was control. The second group was given intraperitoneal injections of ADR(5 mg/kg) twice a week over two weeks. Serum concentrations of MMP, TIMP, interleukin(IL)-6 and tumor necrosis factor(TNF)-${\alpha}$ were measured. RNA extraction was performed from frozen rat hearts. Reverse transcription polymerase chain reaction(RT-PCR) was employed. cDNA Microarray analysis was performed by using a set of 5,184 sequence-verified rat cDNA clones. Results : Serum MMP and TIMP levels were not significantly different between the two groups. IL-6 was $36.8{\pm}2.8pg/mL$ and TNF-${\alpha}$ $2.2{\pm}2.7pg/mL$ in the ADR group. They were significantly higher than in the control group. Serum MMP correlated significantly with TNF-${\alpha}$(r=0.41, P<0.05). There was no gene expression of MMP, IL-6 or TNF-${\alpha}$ in the hearts of both groups. Gene expression of TIMP was significantly depressed in the hearts of the ADR group. Conclusion : These results suggested a potential role for TNF-${\alpha}$ in the regulation of extracellular matrix remodeling in ADR induced cardiomyopathy. Rapid screening of multiple decreased gene expression by DNA chip may be a useful diagnostic test to detect early cardiac injury before developing ADR induced cardiomyopathy.

• Horticultural Science & Technology
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• v.31 no.2
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• pp.246-254
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• 2013

Five isolates of Tomato yellow leaf curl virus (TYLCV) collected from various regions of Korea were amplified using PCR and determined the sequences of full-length genome, respectively. The PCR-amplified DNA of each TYLCV isolate was introduced into a binary vector to construct infectious clone containing 1.9 copies of the corresponding viral genome. Various cultivars and breeding lines of tomato were inoculated with Agrobacterium tumefaciens harboring infectious clone of each TYLCV isolate to assess resistance against TYLCV. Susceptible cultivar 'Super-sunread' revealed typical yellowing and narrowing of the upper leaves. In contrast, breeding linesTY12, GC9, GC171, and GC173, which contained the TY-1 and/or TY-3 genes that confer resistance against TYLCV in nature, were completely symptomless, suggesting that the lines were resistant to challenging TYLCV isolates. Symptoms of TYLCV in susceptible tomato cultivars are significantly different from those of TYLCV in the resistant tomato cultivars at 30 days after agroinfiltration. Although genomic DNAs of TYLCV were detected from the breeding lines TY12, GC9, GC171, and GC173 using real-time PCR analysis with specific primers, levels of TYLCV DNA accumulation in the resistant breeding lines were much lower than those of TYLCV DNA accumulation in susceptible tomato cultivars. Similar symptom severity and levels of TYLCV DNA accumulation were observed from TYLCV infections mediated by Bemisia tabaci in the resistant and susceptible tomato cultivars. Concentration of agrobacterium did not affect the response of tomato cultivars against TYLCV inoculation. Taken together, these results suggest that TYLCV inoculation via agroinfiltration is as effective as inoculation through Bemisia tabaci and is useful for breeding programs of TYLCV-resistant tomato.