• Korean Journal of Veterinary Research
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• v.52 no.2
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• pp.75-82
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• 2012

We have developed and optimized a multiplex polymerase chain reaction (mPCR) for simultaneous detection of Brucella, Salmonella and Leptospira with high sensitivity and specificity. Three pairs of oligonucleotide primers were designed to specifically amplify the targeted genes of Salmonella, Leptospira and Brucella species with sizes of 521, 408 and 223 bp, respectively. The mPCR did not produce any nonspecific amplification products when tested against 15 related species of bacteria. The sensitivity of the mPCR was 100 fg for Brucella and 1 pg for both Salmonella and Leptospira species. In the field application, kidney, liver and spleen were collected from wild rats and stray cats and examined by mPCR. The high specificity and sensitivity of this mPCR assay provide a valuable tool for diagnosis and for the simultaneous and rapid detection of three zoonotic bacteria that cause disease in both humans and animals. Therefore, this assay could be a useful alternative to the conventional method of culture and single PCR for the detection of each pathogen.

• 한국균학회소식:학술대회논문집
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• 2015.11a
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• pp.41-41
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• 2015

Oomycetes belong to the kingdom Straminipila, a remarkably diverse group which includes brown algae and planktonic diatoms, although they have previously been classified under the kingdom Fungi. These organisms have evolved both saprophytic and pathogenic lifestyles, and more than 60% of the known species are pathogens on plants, the majority of which are classified into the order Peronosporales (includes downy mildews, Phytophthora, and Pythium). Recent phylogenetic investigations based on DNA sequences have revealed that the diversity of oomycetes has been largely underestimated. Although morphology is the most valuable criterion for their identification and diversity, morphological species identification is time-consuming and in some groups very difficult, especially for non-taxonomists. DNA barcoding is a fast and reliable tool for identification of species, enabling us to unravel the diversity and distribution of oomycetes. Accurate species determination of plant pathogens is a prerequisite for their control and quarantine, and further for assessing their potential threat to crops. The mitochondrial cox2 gene has been widely used for identification, taxonomy and phylogeny of various oomycete groups. However, recently the cox1 gene was proposed as a DNA barcode marker instead, together with ITS rDNA. To determine which out of cox1 or cox2 is best suited as universal oomycete barcode, we compared these two genes in terms of (1) PCR efficiency for 31 representative genera, as well as for historic herbarium specimens, and (2) in terms of sequence polymorphism, intra- and interspecific divergence. The primer sets for cox2 successfully amplified all oomycete genera tested, while cox1 failed to amplify three genera. In addition, cox2 exhibited higher PCR efficiency for historic herbarium specimens, providing easier access to barcoding type material. In addition, cox2 yielded higher species identification success, with higher interspecific and lower intraspecific divergences than cox1. Therefore, cox2 is suggested as a partner DNA barcode along with ITS rDNA instead of cox1. Including the two barcoding markers, ITS rDNA and cox2 mtDNA, the multi-locus phylogenetic analyses were performed to resolve two complex clades, Bremia lactucae (lettuce downy mildew) and Peronospora effuse (spinach downy mildew) at the species level and to infer evolutionary relationships within them. The approaches discriminated all currently accepted species and revealed several previously unrecognized lineages, which are specific to a host genus or species. The sequence polymorphisms were useful to develop a real-time quantitative PCR (qPCR) assay for detection of airborne inoculum of B. lactucae and P. effusa. Specificity tests revealed that the qPCR assay is specific for detection of each species. This assay is sensitive, enabling detection of very low levels of inoculum that may be present in the field. Early detection of the pathogen, coupled with knowledge of other factors that favor downy mildew outbreaks, may enable disease forecasting for judicious timing of fungicide applications.

• The Plant Pathology Journal
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• v.34 no.6
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• pp.490-498
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• 2018

Panicle blight and seed rot disease caused mainly by Burkholderia glumae and Burkholderia gladioli is threatening rice cultivation worldwide. The bacteria have been reported as seed-borne pathogens from rice. Accurate detection of both pathogens on the seeds is very important for limiting the disease dissemination. Novel primer pairs targeting specific molecular markers were developed for the robust detection of B. glumae and B. gladioli. The designed primers were specific in detecting the target species with no apparent cross-reactions with other related Burkholderia species at the expected product size. Both primer pairs displayed a high degree of sensitivity for detection of B. glumae and B. gladioli separately in monoplex PCR or simultaneously in duplex PCR from both extracted gDNA and directly preheated bacterial cell suspensions. Limit of detection was as low as 0.1 ng of gDNA of both species and $3.86{\times}10^2cells$ for B. glumae and $5.85{\times}10^2cells$ for B. gladioli. On inoculated rice seeds, the designed primers could separately or simultaneously detect B. glumae and B. gladioli with a detection limit as low as $1.86{\times}10^3cells$ per rice seed for B. glumae and $1.04{\times}10^4cells$ per rice seed of B. gladioli. The novel primers maybe valuable as a more sensitive, specific, and robust tool for the efficient simultaneous detection of B. glumae and B. gladioli on rice seeds, which is important in combating rice panicle blight and seed rot by early detection and confirmation of the dissemination of pathogen-free rice seeds.

• Journal of Environmental Impact Assessment
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• v.32 no.2
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• pp.94-111
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• 2023

In order to establish an effective management strategy for invasive species early detection and regular monitoring are required to assess their introduction or dispersal. Environmental DNA (eDNA) is actively applied to evaluate the fauna including the presence of invasive species as it has high detection sensitivity and can detect multiple species simultaneously. In Korea, the applicability evaluation of metabarcoding is being conducted mainly on fish, and research on other taxa is insufficient. Therefore, this study identified the feasibility of detecting invasive species in Korea using eDNA metabarcoding. In addition, to confirm the possibility of detection by taxa, the detection of target species was evaluated using four universal primers (MiFish, MiMammal, Mibird, Amp16S) designed for fish, mammals, birds, and amphibians. As a result, target species (Trachemys scripta, 3 sites; Cervus nippon, 3 sites; Micropterus salmoides, 7 sites; Rana catesbeiana, 4 sites) were detected in 17 of the total 55 sites. Even in the selection of dense sampling sites within the study area, there was a difference in the detection result by reflecting the ecological characteristics of the target species. A comparison of community structures (species richness, abundance and diversity) based on the presence of invasive species focused on M.salmoides and T.scripta, showed higher diversity at the point where invasive species were detected. Also, 1 to 4 more species were detected and abundance was also up to 1.7 times higher. The results of invasive species detection through metabarcoding and the comparison of community structures indicate that the accumulation of large amounts of monitoring data through eDNA can be efficiently utilized for multidimensional ecosystem evaluation. In addition, it suggested that eDNA can be used as major data for evaluation and prediction, such as tracking biological changes caused by artificial and natural factors and environmental impact assessment.

• International Journal of Oral Biology
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• v.36 no.1
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• pp.1-6
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• 2011

The purpose of this study was to develop species-specific real-time quantitative PCR (RT-qPCR) primers for use in the detection of Aggregatibacter actinomycetemcomitans. These primers were designed based on the nucleotide sequences of the RNA polymerase ${\beta}$-subunit gene (rpoB). We assessed the specificity of the primers against nine strains of A. actinomycetemcomitans, eight strains (three species) of the Haemophilus genus, and 40 strains of 40 other oral bacterial species. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC $33384^T$. Our data reveal that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 2 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these qRT-PCR primers are suitable for application in epidemiological studies.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.87-87
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• 2013

The interface between nanomaterials and biosystems is emerging as one of the broadest and most dynamic areas of science and technology, bringing together biology, chemistry, physics and many areas of engineering, biomedicine. The combination of these diverse areas of research promised to yield revolutionary advances in healthcare, medicine, and life science. For example, the creation of new and powerful nanosensors that enable direct, sensitive, and rapid analysis of biological and chemical species can advance the diagnosis and treatment of disease, discovery and screening of new drug molecules. Nanowire based sensors are emerging as a powerful and general platform for ultrasensitive and multiplex detection of biological and chemical species. Here, we present the studies about noble metal nanowire sensors that can be used for sensitive detection of a wide-range of biological and chemical species including nucleic acids, proteins, and toxic metal ions. Moreover, the optical and electrochemical applications of noble metal nanowires are introduced. Noble metal nanowires are successfully used as plasmonic antennas and nanoelectrodes, thereby provide a pathway for a single molecule sensor, in vivo neural recording, and molecular injection and detection in a single living cell.

• Journal of information and communication convergence engineering
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• v.18 no.4
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• pp.267-277
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• 2020

It is important to understand the exact habitat distribution of endangered species because of their decreasing numbers. In this study, we build a system with a deep learning module that collects the image data of endangered animals, processes the data, and saves the data automatically. The system provides a more efficient way than human effort for classifying images and addresses two problems faced in previous studies. First, specious answers were suggested in those studies because the probability distributions of answer candidates were calculated even if the actual answer did not exist within the group. Second, when there were more than two entities in an image, only a single entity was focused on. We applied an object detection algorithm (YOLO) to resolve these problems. Our system has an average precision of 86.79%, a mean recall rate of 93.23%, and a processing speed of 13 frames per second.

• The Korean Journal of Mycology
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• v.44 no.2
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• pp.103-107
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• 2016

To establish a rapid and accurate detection of Phytophthora pinifolia, which is a quarantine pathogenic fungus in Korea, a species-specific primer was developed based on the ras-related protein (Ypt1) gene. Species-specific primer based on the DNA sequences of Ypt1 gene amplified 193 bp polymerase chain reaction (PCR) product for P. pinifolia. The primer pair yielded the predicted PCR product size exactly in testing with target pathogen DNAs, but not from the other 10 species of Phytophthora and 14 species of other phytopathogenic fungi. The primer pair also showed only the species-specific amplification curve on realtime PCR on target pathogen DNA. The detection sensitivity of real time PCR using species-specific primer pair was 10 to 100 times higher than conventional PCR, with 1 to $10pg/{\mu}L$.

• Korean Journal of Veterinary Research
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• v.34 no.1
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• pp.115-125
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• 1994

In this study, we try to establish the rapid and specific detection system for Salmonella species. The PhoE gene of Salmonella species was amplified with two specific primers, ST5 and ST8c, using PCR. The probe prepared from the amplified PhoE gene was sequenced and applied for Southern blot analysis. After PCR with ST5 and ST8c primers for PhoE gene, DNA bands of expected size(365bp) from 7 different Salmonella species were detected, but not from 12 enterobacteriaceae and 3 gram positive bacteria. PCR was highly sensitive to detect up to 10fg of purified DNA template and to identify Salmonella species with only 320 heat-lysed bacterial cells. The inhibition of PCR amplification from stool specimen was occurred with 50-fold dilution but disappeared over 100 fold dilution of samples. It was confirmed that the PhoE genes were amplified and cloned with over 97% nacleotide sequence homology of PCR products compared with that of S. typhfmurium LT2. The DNA probe derived from S. typhimurium TA 3,000 showed highly specific and sensitive reaction with PCR products of all tested Salmonella species. These results indicate that PCR was rapid and sensitive detection method for Salmonella species and DNA probe prepared from S. typhimurium TA 3,000 was specific to identify PCR products of different Salmonella species.

• Journal of Ecology and Environment
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• v.29 no.4
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• pp.331-335
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• 2006

We investigated the distribution of three anuran species, Three-striped pond frogs (Rana nigromaculata), Bullfrogs (Rana catesbeiana), and Narrow-mouthed toads (Kaloula borealis), in an administrative district, Haenam Gun, Junnam Province, Korea using volunteer call surveys. Twenty-eight volunteer call surveyors were assigned to each $2{\times}2km^2$ survey plot. Call surveys on whether the species are present or not were conducted for 5 minutes between 30 minutes after sunset and the midnight on rice fields and ponds from 10 April to 28 August in 2005. Depending on species, call surveys were carried out at seven to 28 plots with average 8.4 to 10.7 visits per the plot. We calculated the detection probabilities and occupancy rates of the three species using four models with three covariates: temperature, humidity, and the amount of water at the habitat. The model average detection probabilities of three anuran species of R. nigromaculata, R. catesbeiana, and K. borealis were 0.53, 0.74, and 0.41 respectively, and the site occupancy rates of them were 0.93, 0.94, and 0.86 respectively. Our results indicate that R. nigromaculata, R. catesbeiana, and K. borealis are common in Haenam Gun.