• Korean Journal of Veterinary Service
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• v.30 no.2
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• pp.251-258
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• 2007

Bovine brucellosis has occurred for years in Gyeonggi province under the national test and slaughter scheme. The serum agglutination test (SAT) is a diagnostic tool to confirm the disease despite the argument on its specificity. We selected 8 farms where only one or two individuals were diagnosed as brucellosis through SAT at the primary regular herd check and isolated the causative organism and characterized the species by species-specific PCR. The pathogen isolation was successful in 6 farms out of 8 farms by microbiological culture, showing the successful rate of 75%. The isolation rate of the causative organism represents 70% from supra-mammary lymph node and 60% from uterine tissues. They were characterized as Brucella abortus biovar 1 after biotyping by PCR, showing the fragment of 498 bp. Five of 8 farms were diagnosed as brucellosis two to four times more over the intervals of two or three months. Here in this study we briefly showed the correlation of the sporadic outbreak of brucellosis tested by SAT and the isolation of the causative organism. Moreover one or two reactors against brucellosis among considerable size of herd may indicate that SAT failed to detect potentially infected individuals in the incubation stage or chronic phase of the disease.

• Mycobiology
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• v.32 no.3
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• pp.119-122
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• 2004

The intergenic spacer(IGS) sequence of Fusarium oxysporum have been reported to provide reliable information concerning intraspecific variation and phylogeny of fungal species. The eleven strains of Fusarium oxysporum and its formae speciales belonging to section Elegans were compared with sequencing analysis. The direct sequencing of partial IGS was carried out using PCR with primer NIGS1(5'-CTTCGCCTCGATTTCCCCAA-3')/NIGS2(5'-TCGTCGCCGACAGTTTTCTG-3') and internal primer NIGS3(5'-TCGAGGATCGATTCGAGG-3')/NIGS4(5'-CCTCGAATCGATCCTCGA-3'). A single PCR product was found for each strain. The PCR fragments were sequenced and revealed a few within species polymorphisms at the sequence level. The size of partial IGS sequencing of F. oxysporum was divided into three groups; $526{\sim}527$ bp including F. o. f. sp. chrysanthemi, cucumerinum, cyclaminis, lycopersici, and fragariae; $514{\sim}516$ bp including F. o. f. sp. lilii, conglutinans, and raphani; 435 bp for F. o. f. sp. cucumerinum from Korea. Sequence analysis of PCR products showed that transitions were more frequent than transversions as well as the average numbers of substitution per site were range 0.41% to 3.54%.

• Korean Journal of Ecology and Environment
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• v.54 no.3
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• pp.209-220
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• 2021

Pacific herring, Clupea pallasii, a keystone species with significant ecological and commercial importance, is declining globally throughout much of its range. While traditional fishing equipment methods remain limited, new sensitive and rapid detection methods should be developed to monitor fisheries resources. To monitor the presence and quantity of C. pallasii from environmental DNA (eDNA) extracted from seawater samples, a pair of primers and a TaqMan^® probe specific to this fish based on mitochondrial cytochrome b (COB) sequences were designed for the real-time PCR (qPCR) assay. The combination of our molecular markers showed high specificity in the qPCR assay, which affirmed the success of presenting a positive signal only in the C. pallasii specimens. The markers also showed a high sensitivity for detecting C. pallasii genomic DNA in the range of 1 pg~100 ng rxn^-1 and its DNA plasmid containing COB amplicon in the range of 1~100,000copies rxn^-1, which produced linear standard calibration curves (r²=0.99). We performed a qPCR assay for environmental water samples obtained from 29 sampling stations in the southeastern coastal regions of South Korea using molecular markers. The assay successfully detected the C. pallasii eDNA from 14 stations (48.2%), with the highest mean concentration in Jinhae Bay with a value of 76.09±18.39 pg L^-1 (246.20±58.58 copies L^-1). Our preliminary application of molecular monitoring of C. pallasii will provide essential information for efficient ecological control and management of this valuable fisheries resource.

• The Korean Journal of Mycology
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• v.32 no.1
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• pp.1-7
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• 2004

To establish the phylogenetic relationships of Dictyophora spp., rDNA-ITS regions of 11 strains of veiled lady mushroom collected from various countries were amplified and sequenced. It was observed that the 11 strains were divided into four groups based on PCR band patterns of each ITS region cleaved by eight different restriction enzymes in cleaved amplified polymorphic sequence analysis (CAPS). The phylogenic relationship of each group by cleaved amplified polymorphic sequence (CAPS) analysis matches well with previously reported morphological phylogeny, such as 5 strains of D. indusiata, 4 strains of D. echinovolvata, and a strain of Phallus rugulosus. Sequence analysis using the cluster V methods showed more detail classification than CAPS analysis. The 5.8S region showed two point nucleotide base exchanges from G to A according to four groups, and four groups were subdivided by sequence variation of ITS I and ITS II regions. But sequence variation of Phallus rugulosus was not showed in full ITS region. This study further delineates the taxonomic level at which ITS sequences, in comparison to ribosomal gene sequence, are most useful in systematics and other mushroom study.

• Journal of Microbiology and Biotechnology
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• v.34 no.5
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• pp.1146-1153
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• 2024

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2% nucleotide sequence identity) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

• Journal of Food Hygiene and Safety
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• v.23 no.2
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• pp.121-128
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• 2008

The aim of this study was to develop a LightCycler-based real time PCR (LC-PCR) assay and to evaluate its diagnostic use for the detection of Staphylococcus aureus in raw milk samples. Following amplification of 113 bp of coa gene encoding an coagulase precursor specific for Staphylococcus aureus, melting curve and DNA sequencing analysis was performed to verify the specificity of the PCR products. Amplification of 209 bp gene encoding an altered penicillin-binding protein, PBP2a (mecA), melting curve analysis and DNA sequencing analysis was performed to verify methicillin resistance Staphylococcus aureus (MRSA). According to this study, 6 of 647 raw milk samples showed S. aureus positive and 2 of them showed a mecA positive and the detection limit was 10 fg of DNA. And we also isolated Staphylococcus chromogenes a causative agent of exudative epidermitis in pigs and cattle from 3 samples.

• Research in Plant Disease
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• v.21 no.4
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• pp.321-325
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• 2015

A rapid and simple RT-PCR diagnosis method for detection of Rice stripe virus (RSV), one of major virus infecting rice, was developed using porous ceramic cubes in this study. The porous ceramic cube can rapidly absorb biological molecules such as small-sized proteins and nucleic acid fragments into its pores. We examined whether this ability of porous ceramic cubes could be applied for isolating viral nucleic acids or particles from the RSV- infected plant tissues. In this study, we found that the porous ceramic cube was capable of absorbing a detection level of viruses from the rice tissues infected with RSV and established RT-PCR-based RNA diagnosis method using porous ceramic cubes.

• Genomics & Informatics
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• v.21 no.4
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• pp.52.1-52.10
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• 2023

Accurate and efficient microbial diagnosis is crucial for effective molecular diagnostics, especially in the field of human healthcare. The gold standard equipment widely employed for detecting specific microorganisms in molecular diagnosis is quantitative real-time polymerase chain reaction (qRT-PCR). However, its limitations in low metagenomic DNA yield samples necessitate exploring alternative approaches. Digital PCR, by quantifying the number of copies of the target sequence, provides absolute quantification results for the bacterial strain. In this study, we compared the diagnostic efficiency of qRT-PCR and digital PCR in detecting a particular bacterial strain (Staphylococcus aureus), focusing on skin-derived DNA samples. Experimentally, specific primer for S. aureus were designed at transcription elongation factor (greA) gene and the target amplicon were cloned and sequenced to validate efficiency of specificity to the greA gene of S. aureus. To quantify the absolute amount of microorganisms present on the skin, the variable region 5 (V5) of the 16S rRNA gene was used, and primers for S. aureus identification were used to relative their amount in the subject's skin. The findings demonstrate the absolute convenience and efficiency of digital PCR in microbial diagnostics. We suggest that the high sensitivity and precise quantification provided by digital PCR could be a promising tool for detecting specific microorganisms, especially in skin-derived DNA samples with low metagenomic DNA yields, and that further research and implementation is needed to improve medical practice and diagnosis.

• Journal of Food Hygiene and Safety
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• v.29 no.1
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• pp.31-39
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• 2014

Norovirus causes acute gastroenteritis in all age groups and its food poisoning outbreaks are rapidly increasing in Korea. Reverse transcription-polymerase chain reaction (RT-PCR) is most widely used for the rapid detection of foodborne viruses due to high sensitivity. However, the false positive results of RT-PCR obtained against already inactivated viruses could be a serious drawbacks in food safety area. In this study, we investigated a method to yield true positive RT-PCR results only with alive viruses. To decompose the RNA genes from dead viruses, the enzymatic treatments composed of proteinse K and Ribonuclease A were applied to the sanitized and inactivated virus particles. Another aim of this study was to quantify the efficiencies of several major sanitizing treatments using real-time RT-PCR. Feline calicivirus (FCV) that belongs to the same Caliciviridae family with norovirus was used as a surrogate model for norovirus. The initial level of virus in control suspension was approximately $10^4$ PFU/mL. Most of inactivated viruses treated with the enzymatic treatment for 30 min at $37^{\circ}C$ were not detected in RT-PCR, Quantification results to verify the inactivation efficiencies of sanitizing treatments using real-time RT-PCR showed no false positive in most cases. We could successfully develope a numerical quantification process for the inactivated viruses after major sanitizing treatments using real-time RT-PCR. The results obtained in this study could provide a novel basis of rapid virus quantification in food safety area.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.4
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• pp.585-588
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• 1997

The polymerase chain reaction (PCR) technique was used to distinguish from two morphologically similar red algal species; Acrosorium polyneurum and A. yendoi. Total DNA was extracted by the LiCl method. The extracted DNA (15 ng) in a $25{\mu}\ell$ reaction volume was amplified by the PCR technique using primers covering with mitochondrial D-loop gene, nuclear rDNA internal transcribed spacer (ITS), and nuclear rDNA external transcribed spacer. A. yendoi could be distinguished from A. polyneurum on the producible basis of amplified ITS fragment of 650 bp.