• The Plant Pathology Journal
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• v.35 no.6
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• pp.654-661
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• 2019

The soybean cyst nematode, Heterodera glycines, is a major plant-parasitic nematode that has caused important economic losses to Korea's soybean production. Four species of cyst nematodes, H. schachtii, H. glycines, H. trifolii, and H. sojae, all belong to schachtii group are coexist in field soil in Korea. The rapid identification of the nematode is crucial for preventing crop damage and in decision making for controlling this nematode. This study aimed to develop a species-specific primer set for quantitative PCR (qPCR) assay of H. glycines. The specific primer set (HGF1 and HGR1) for H. glycines was designed based on the cytochrome c oxidase subunit I (COI) sequence of mitochondrial DNA. After optimization, it is possible to identify the H. glycines using a qPCR assay with DNA extracted from a single cyst and single second-stage juvenile (J2). The specificity was confirmed by the absence of SYBR fluorescent signals of three other Heterodera species. A serial dilution of DNA extracted from a single cyst was obtained for the sensitivity test. The result showed that the standard curve of the test had a highly significant linearity between DNA concentration and Ct value (R² = 0.996, slope = -3.49) and that the detection limit concentration of DNA of the primer set was 10 pg of DNA per reaction. Our findings suggested that H. glycines could be distinguished from H. sojae and other Heterodera species when a qPCR assay is used with a specific primer set.

• Journal of Mushroom
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• v.22 no.2
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• pp.37-47
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• 2024

Fluorescent bacteria were isolated from sporocarps that browned into various mushrooms during survey at places of the production in Korea. We examined the pathogenicity, biodiversity, and genetic characteristics of the 19 strains identified as Pseudomonas tolaasii by sequence analysis of 16S rRNA and White Line Assay. The results emphasize the importance of rpoB gene system, fatty acid profiles, specific and sensitive PCR assays, and lipopeptide detection for the identification of P. tolaasii. As a result of these various analyses, 17 strains (CHM03~CHM19) were identified as P. tolaasii. The phylogenetic analysis based on the 16S rRNA gene showed that all strains were clustered closest to P. tolaasii lineage, two strains (CHM01, CHM02) were not identified as P. tolaasii and have completely different genetic characteristics as a result of fatty acids profile, specific and sensitive PCR, lipopetide detection, rpoB sequence and REP-PCR analysis. Pathogenicity tests showed 17 strains produce severe brown discolouration symptoms to button mushrooms and watersoaking of sporophore tissue within three days after inoculation. But two strains did not produce discolouration symptoms. Therefore, these two strains will be further investigated for correct species identification by different biological and molecular characteristics.

• Korean Journal of Veterinary Research
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• v.43 no.3
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• pp.463-469
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• 2003

Eight nursery to grower pigs exhibiting weight loss and sudden death were diagnosed as postweaning multisystemic wasting syndrome (PMWS) based on the results of gross findings, histopathology, immunohistochemistry, fluorescent antibody test, virus isolation, PCR, serology, and electron microscopy. Groosly, the pigs had a rough hair coats and were severely emaciated. And moot lymph nodes were pale and enlarged. Lungs were not fully collapsed and exhibited 10 to 40% pale red cranioventral consolidation. Histopathologically, typical lymphohistiocytic interstitial to bronchointerstitial pneumonia, chronic lymphadenitis, severe lymphoid depletion, and basophilic intracytoplasmic inclusions were noted in the most lymphoid tissues. Porcine circovirus panicles were observed in the inguinal lymph node of the pigs by electron microscopy. Porcine circovirus type 2 (PCV2) antigens or viral DNAs were detected in the lesions of all pigs using immunohistochemistry or PCR. Two PCV2 were isolated from a homogenate of pooled lung and lymph node in 2 of the 5 pigs. Additionally, antigens of porcine reproductive and respiratory syndrome virus (PRRSV) and Hemophilus (H.) parasuis were also detected by immunofluorescent antibody test. Serologically, 55% of randomly selected sows and fattening pigs was serum antibody positive to PCV2 by an indirect fluorescent antibody (IFA) test and approximately 18 % of animals in the herd were serologically pooitive by the ELISA kit for PRRSV. To our knowledge, this is the first report of PMWS co-infected with PCV-2, PRRS, and H. parasuis in Korea.

• Korean Journal of Veterinary Service
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• v.27 no.4
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• pp.345-354
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• 2004

Two cases of classical swine fever (CSF) disease have broken out in Cheolwon (7 April, 2002). The suspected pig herds were huddled together because of high fever (over $40^{\circ}C$) and showed remarkable decrease of the leukocytes. The staggering gait related to posterior weakness, constipation and lethargy, hyperemia, hemorrhagic lesions (on the skin, muzzle, ears, limbs, tail and inner part of legs) and conjunctivitis with dirty streaks below the eyes were observed. The inflammation in the lung, infarction in the spleen, swelling and hemorrhage in lymph nodes, kidney, intestine, heart and cheese like purulent inflammation of the tonsil were observed. The ulcers of the colon were also detected. Several clinical and laboratory techniques including blood test, histo-pathological examinations, indirect fluorescent antibody (IFA) test and RT-PCR test were applied to diagnose the disease. Inoculation test on PK-15 cell was also performed. The necrosis of the lymphatic cells and infiltration of the vessel circumferential cells in the brain and lymph organs were commonly viewed. The proliferation of the glia cell (gliosis) in the lymph was particular. Cytopathogenic effect (CPE) and specific fluorescent-bright-green areas (with IFA) appeared in PK-15 cells inoculated with suspected blood plasma. The IFA test on the epithelial and mucous membrane cells of tonsil was positive. RT-PCR technique required more working hours and labor than other techniques in this examination but it was useful because of the sensitivity to the CSF viral gene.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.645-650
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• 2019

Despite differences in virulence between strains of Toxoplasma gondii, rapid and accurate genotyping methods are lacking. In this study, a method was developed to detect and genotype T. gondii in food and environmental samples using PCR and a novel peptide nucleic acid (PNA) melting array. An alignment of genome sequences for T. gondii type I, II, and III obtained from NCBI was generated, and a single nucleotide polymorphism analysis was performed to identify targets for PCR amplification and a PNA melting array. Prior to the PNA melting array, conventional PCR was used to amplify GRA6 of T. gondii. After amplification, the PNA melting array was performed using two different PNA hybridization probes with fluorescent labels (FAM and HEX) and quenchers. Melting curves for each probe were used to determine genotypes and identify mutations. A 214-bp region of the GRA6 gene of T. gondii was successfully amplified by PCR. For all T. gondii strains (type I, II, and III) used to evaluate specificity, the correct genotypes were determined by the PNA melting array. Non-T. gondii strains, including 14 foodborne pathogens and 3 protozoan parasites, such as Giardia lamblia, Cryptosporidium parvum, and Entamoeba histolytica, showed no signal, suggesting that the assay has a high specificity. Although this is only a proof-of-concept study, the assay is promising for the fast and reliable genotyping of T. gondii from food and environmental samples.

• Journal of Physiology & Pathology in Korean Medicine
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• v.26 no.4
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• pp.483-490
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• 2012

This study was designed to identify the effects of Polygoni cuspidati Radix(PCR) on the generation of superoxide anion radicals (${\cdot}O_2{^-}$), nitric oxide (NO), peroxynitrite ($ONOO^-$) in the renal epithelial cells of mouse(LLC-$PK_1$). The effects of PCR on the expression of inflammation-related proteins, IKK-${\alpha}$, phospho-$I{\kappa}B-{\alpha}$, NF-${\kappa}B$ (p50, p65), COX-2, iNOS, IL-$1{\beta}$, VCAM-1, were examined by western blotting. For this study, the fluorescent probes, namely dihydrorhodamine 123 (DHR 123), 2',7'-dichloro dihydrofluorescein diacetate (DCFDA), 4,5-diaminofluorescein (DAF-2) were used. Protein expression levels of IKK-${\alpha}$, phospho-$I{\kappa}B-{\alpha}$, NF-${\kappa}B$ (p50, p65), COX-2, iNOS, IL-$1{\beta}$, VCAM-1 were assayed by western blot. PCR reduced $H_2O_2$-induced cell death dose-dependently. It inhibited the generation of ${\cdot}O_2{^-}$, NO, $ONOO^-$ and $PGE^2$ in the $H_2O_2$-treated LLC-PK1 cells in vitro. PCR inhibited the espression of IKK-${\alpha}$, phospho-$I{\kappa}B-{\alpha}$, COX-2, iNOS, IL-$1{\beta}$ and VCAM-1 genes by means of decreasing the NF-${\kappa}B$ activation. These results suggest that PCR is an effective NO, ${\cdot}O_2{^-}$, $ONOO^-$ scavenger, and this substance recommended to be applied in treatment for the inflammatory process and inflammation-related disease.

• Journal of Veterinary Science
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• v.21 no.2
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• pp.22.1-22.9
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• 2020

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID₅₀/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

• Microbiology and Biotechnology Letters
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• v.48 no.1
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• pp.24-31
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• 2020

Five genes (mxbDM, mxcQE and mxaB) are responsible for the transcription of methanol oxidation genes in Methylobacterium strains. Among these, MxbDM and MxcQE constitute the two-component system (TCS) regulating methanol metabolism. In this study, we integrated the methanol-sensing domain of MxbD and MxcQ with the EnvZ/OmpR from Escherichia coli. The domain-swapping strategy resulted in chimeric histidine kinases (HK's) MxbDZ and MxcQZ AM1 containing recombinant E. coli. Real-time quantitative PCR was used to monitor OmpC expression mediated by the chimeric HK and response regulator (RR) OmpR. Further, an ompC promoter based fluorescent biosensor for sensing methanol was developed. GFP fluorescence was studied both qualitatively and quantitatively in response to environmental methanol. GFP measurement also confirmed ompC expression. Maximum fluorescence was observed at 0.05% methanol and 0.01% methanol using MxbDZ and MxcQZ AM1, respectively. Thus the chimeric HK containing E. coli were found to be highly sensitive to methanol, resulting in a rapid response making them an ideal sensor.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.928-933
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• 2007

Cameleon is a genetically engineered $Ca^{2+}$ sensing molecule consisting of two variants of the green fluorescent protein (GFP), calmodulin and calmodulin-binding protein, M13. HEK-293 cells stably expressing three types of cameleons, yellow cameleon-2, cameleon-3er, and cameleon-2nu, were constructed, and the expression and localization of these cameleons were confirmed by fluorescent imaging. Among the cameleons, the yellow cameleon-2 was selected for analyzing the change in $Ca^{2+}$ induced by the olfactory receptor-mediated signal transduction, because it is localized in the cytosol and binds to cytosolic $Ca^{2+}$ ions. Cells stably expressing yellow cameleon-2 were transfected with each of the test olfactory receptor genes, odr-10 and 17, and the expression of the olfactory receptor genes were examined using immunocytochenmical methods and RT-PCR. Stimulating each olfactory receptor with its specific odorant caused an increase in the intracellular $Ca^{2+}$ level, which was measured using yellow cameleon-2. These results demonstrate that yellow cameleon-2 can be conveniently used to examine the function of the olfactory receptors expressed in heterologous cells.

• The Plant Pathology Journal
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• v.33 no.4
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• pp.429-433
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• 2017

Chrysanthemums (Chrysanthemum morifolium) are susceptible to tobacco mosaic virus (TMV). TMV-based expression vectors have been used in high-throughput experiments for production of foreign protein in plants and also expressing green fluorescent protein (GFP) to allow visualization of TMV movement. Here, we used TMV expressing the GFP to examine the infection of chrysanthemum by a TMV-based expression vector. Viral replication, movement and GFP expression by TMV-GFP were verified in upper leaves of chrysanthemums up to 73 days post inoculation (dpi) by RT-PCR. Neither wild-type TMV nor TMV-GFP induced symptoms. GFP fluorescence was seen in the larger veins of the inoculated leaf, in the stem above the inoculation site and in petioles of upper leaves, although there was no consistent detection of GFP fluorescence in the lamina of upper leaves under UV. Thus, a TMV-based expression vector can infect chrysanthemum and can be used for the in vivo study of gene functions.