• Journal of Microbiology and Biotechnology
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• v.22 no.7
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• pp.1021-1028
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• 2012

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (NNV) in olive flounder, Paralichthys olivaceus, in Korea. A set of six specific primers was designed to target the RNA 2 gene encoding the coat protein of Korean NNV strains. The RT-LAMP reaction successfully detected NNV after 30 min at $65^{\circ}C$. When the sensitivities among RT-LAMP, RT-PCR, and nested RTPCR were compared, the RT-LAMP was shown to be able to detect the RNA template at $2.58{\times}10^{-2}\;TCID_{50}/ml$, whereas the RT-PCR and nested RT-PCR were only able to detect the RNA template at $2.58{\times}10^2\;TCID_{50}/ml$ and $2.58TCID_{50}/ml$, respectively. Thus, the sensitivity of the RT-LAMP assay was higher than those of the RT-PCR assays. In the specificity test of the RT-LAMP, 2 genotypes of NNVs (SJNNV and RGNNV) were positive; however, no other fish viruses were positive with the primers, indicating that the RT-LAMP assay is only specific to NNV. A total of 102 olive flounder were collected from hatcheries between 2009 and 2011. The occurrence of NNV in olive flounder was determined to be 53.9% (55/102) by the RT-LAMP. On the other hand, the prevalence based on the nested RT-PCR and RT-PCR results was 33.8% (34/102) and 20.6% (21/102), respectively. This result indicates that the RT-LAMP assay developed in this study is suitable for early field diagnosis of NNV with high sensitivity.

• 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.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.54 no.5
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• pp.668-675
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• 2021

The infection status of domestic farmed eels Anguilla japonica, Anguilla bicolor and Anguilla marmorata with Japanese eel endothelial cell-infecting virus (JEECV) and anguillid herpesvirus 1 (AnHV) was examined at the major eel farming areas in Korea. These viruses were detected in all areas examined, regardless of the eel species or age. Any farm with a history of viral infection in adult fish confirmed the infection to be transmitted to stocked fry within 3 to 5 months. It is proposed that both viruses are horizontally transmitted within a given farm. The primary symptoms and histopathological lesions produced by the two viral infections are similar, making it difficult to distinguish the two diseases through clinical symptoms. Both viruses displayed 100% detection in the gills, suggesting that the gills are an optimal tissue for JEECV and AnHV monitoring. This study concluded that JEECV and AnHV were prevalent on eel farms across the country and caused very high mortality when the two viruses co-infected fry. Additional studies, including experimental infections, are needed to clearly understand the pathogenicity of each virus and the risk of co-infection.

• Parasites, Hosts and Diseases
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• v.60 no.4
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• pp.289-293
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• 2022

Blastocystis is a genus of unicellular heterokont parasites belonging to a group of organisms known as Stramenopiles, which includes algae, diatoms, and water molds. Blastocystis includes several species that habitat in the gastrointestinal tracts of organisms as diverse as humans, farm animals, birds, rodents, reptiles, amphibians, fish, and cockroaches. It is important to public health and distributed globally, but its prevalence in dogs in Korea has not been reported to date. Here, we collected 787 canine fecal samples and assessed Blastocystis infection by age, sex, region, season, and diarrhea symptoms. We determined Blastocystis subtypes using phylogenetic analyses based on 18S rRNA gene sequences. We identified, 10 Blastocystis positive samples (1.3%). A higher proportion of infected dogs was asymptomatic; however, infection rates did not significantly differ according to region, age, sex, and season. Phylogenetic analysis showed that the Blastocystis sp. identified belonged to 4 subtypes (STs), ST1, ST5, ST10, and ST14, thus revealed the genetic diversity of Blastocystis sp. in dogs Korean. This is first report on the presence of Blastocystis sp. in dogs Korean. This study revealed a lower infection rate than expected and differed from previous studies in STs. Further studies are warranted to observe the national infection status of Blastocystis in dogs and the genetic characteristics of this genus.

• International Journal of Industrial Entomology and Biomaterials
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• v.47 no.1
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• pp.1-11
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• 2023

The Indian golden muga silkworm, Antheraea assamensis Helfer is an economically important wild silkworm endemic to Northeastern part of India. In recent years, climate change has posed a threat to muga silk production due to the requirement that larvae be reared outdoors. Since the muga silkworm larvae are exposed to the vagaries of nature, the changing climate has increased the incidence of microbial diseases in the rearing fields. Accurate diagnosis of the disease causing pathogens and its associated epidemiology are prerequisites to manage the diseases in the rearing field. Although conventional microbial culturing methods are widely used to identify pathogenic bacteria, they would not provide meaningful information on a wide variety of silkworm pathogens. The information on use of molecular diagnostic tools in detection of microbial pathogens of wild silk moths is very limited. A wide range of molecular and immunodiagnostic techniques including denaturing gradient gel electrophoresis (DGGE), random amplified polymorphism (RAPD), 16S rRNA/ITSA gene sequencing, multiplex polymerase chain reaction (M-PCR), fluorescence in situ hybridization (FISH), immunofluorescence, and repetitive-element PCR (Rep-PCR), have been used for detecting and characterizing the pathogens of insects with economic significance. Nevertheless, the application of these molecular tools for detecting and typing entomopathogens in surveillance studies of muga silkworm rearing is very limited. Here, we discuss the possible application of these molecular techniques, their advantages and major limitations. These methods show promise in better management of diseases in muga ecosystem.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.10
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• pp.1343-1356
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• 2008

The evaluation of microbiological quality for school food samples collected from 19 selected middle and high schools located in Seoul was undertaken. Eighty-nine food samples consisting of 38 non-pretreated vegetables, 13 pre-washed and cut vegetables, 9 meats and poultry, 3 fish and shellfish, 7 dried fish, and shellfish and 20 processed foods were collected. Aerobic plate count, total coliforms, and Escherichia coli (E. coli ) were detected using $Petrifilm^{TM}$, and the food-borne pathogens were screened by multiplex PCR with species-specific primer sets. Sequentially, the quantitative and confirmative test of the food-borne pathogens were carried out with the selective media and biochemical kits. The contamination of coliform counts was observed on the pre-washed vegetables ($3.4{\sim}4.3\;log\;CFU/g$) and meats ($2.2{\sim}4.3\;log\;CFU/g$). Also, the cooked foods were heavily contaminated with coliform, ranging from 1.0 to $5.5\;log\;CFU/g$. E. coli counts were found in 16 raw and cooked food samples, exceeding the microbiological standards for the guideline of safety management for school foods. Through PCR detection, B acillus cereus was detected in 32 raw and cooked foods, and quantitatively found in pre-washed carrot, radish, and pan-broiled dried shrimp and filefish ranging from $2.3{\sim}3.6\;log\;CFU/g$, respectively. E. coli O157:H7 was detected on frozen pork sample and was confirmed with API kit. Campylobacter jejuni was found in 3 ready-to-eat type vegetables. Vibrio parahaemolyticus were found in 4 pre-washed vegetables and 2 cooked foods, indicating unsatisfactory quality based upon the microbiological standards of ready-to-eat vegetables and cooked foods by Korea Food and Drug Administration. Salmonella spp. was detected in frozen chicken sample and confirmed by API kit and latex antisera agglutination.

• Journal of Food Hygiene and Safety
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• v.21 no.3
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• pp.189-195
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• 2006

This research was designed to evaluate the nutritional an microbiol quality assessment of Chungmukimbab purchased from market in Tongyeoung area. Contents of calories, calcium, iron, thiamin and riboflavin in ordinary kimbab and Chungmukimbab were lower than the recommended levels of Korean adult men. So, We suggested that a fruit, beverage and ffod stuff were supplemented to maintain nutritional balance. Total aerobic bacteria and coliform group of just prepared ordinary kimbab and Chungmukimbab samples from market were not significantly different, showing approximately $5.50{\pm}0.38 log_{10} CFU/g,\;2.10{\pm}0.47log_{10}MPN/100g$ in ordinary kimbab, $5.61{\pm}0.42log_{10}CFU/g,\;1.75{\pm}0.34 log_{10} MPN/100g$ in Chungmukimbab, respectively. Total aerobic bacteria of law ingredients of chungmukimbab sample were 3 to $4 log_{10}CFU/g$ in kimbab, seasoning squid and radish roots kimchi, 4 to $5 log_{10}CFU/g$ in boiled fish paste. The coliform groups were 1 to $2 log_{10}$ MPN/100 g in kimbab, seasoning squid and radish roots kimchi, 2 to $3 log_{10}$ MPN/100g in boiled fish paste. Detection rate of E. coli and Staphylococcus aureus counts were 10.0, 12.5% in Chungmu-kimbab, 15.0, 10.0% in seasoning squid, 0, 10.0% in radish roots kimchi respectively, not detected in boiled fish paste samples. During storage at $15^{\circ}C$ for 24 hours, total aerobic counts and coliform groups in ordinary kimbab and Chungmukimbab were increased by the 1.94, $0.97log_{10}CFU/g$, 0.60, 0.72 log10 MPN/100g respectively. Total aerobic counts of Chungmukimbab ingredients increased $0.83{\sim}l.33 log_{10}CFU/g$ at different time

• Environmental Analysis Health and Toxicology
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• v.15 no.4
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• pp.131-137
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• 2000

The release of hazardous waste materials into the environment poses serious risks in humans and ecosystems. The risk assessment of environmental pollutants including hazardous chemicals requires a comprehensive measurement of hazard and exposure of the chemicals that can be achieved by toxicity evaluation using a biological system such as biomarkers. In this report we have tried to develop a biomarker used to elucidate a molecular basis of, and to monitor abnormal behaviors caused by diazinon in Japanese medaka (Oryzias latipes) as a model organism. First, an attempt was made to clone tyrosine hydroxylase gene from Japanese medaka that would be a candidate for a biomarker for neuronal modulations and behaviors. For monitoring experiments at behavioral and molecular biological levels, the fish were treated under different sublethal conditions of diazinon and their behavioral responses were observed . In this study we have successfully cloned a partial TH gene from the medaka fish through PCR screening of an ovary cDNA library. DNA sequencing analysis revealed that the amplified fragment was 327 bp encoding 109 amino acids. Comparing the DNA sequence of medaka TH with other species, TH gene revealed the DNA sequence was completely identical to that of rat TH. In the RT-PCR, 330 Up of mRNA was consistently amplified in all the treated samples including control There were no significant differences in the TH expression level regardless of treating concentrations (1∼5,000 ppb) and time (0∼48 hr) The reason appeared to be that RT-PCR was not performed using through a quantitative analysis normalized against an actin gene expression. Organ or tissue - specific detection of TH activity and mRNA as biomarkers will be a useful monitoring tool for neurobehavioral changes in fish influenced by toxic chemicals. Furthermore, quantitative analysis of locomotive patterns and its correlation with the neurochemical and molecular data would be highly useful in measuring toxicity and hazard ofvarious environmental pollutants.

• Journal of fish pathology
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• v.11 no.1
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• pp.77-81
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• 1998

By standardized method, Bacillus subtilis BGA, Bacillus cereus var. mycoides ATCC 11778, and Micrococcus luteus ATCC 9341 were seeded on the muller hinton agar (Difco) plate, and pH was adjusted to 6.0, 7.2, and 8.0. Five agar plates, B. subtilis (pH 6.0), B. cereus (pH 6.0), B. subtilis (pH 7.2), B. subtilis (pH 8.0), and M. luteus (pH 8.0), were employed as test plates of modified EEC 4-plate method. Oxytetracycline (OTC) with a diet was orally administered to flounder, Paralichthys olivaceus, at 100 mg/kg once a day. After oral administration, modified EEC 4-plate method by the three screening test using muscle-direct, extraction-disk and direct-disk methods was conducted for 3 fish at 1, 3, 5, 10, 15, 20, 25 and 30 days. Muscle-direct treatment of B. subtilis (pH 6.0) was found to be dubious positive (${\pm}$) at the 1st day after the administration; thereafter, it was found to be negative to the last day of the experiment. Extraction-disk and direct-disk treatment of B. subtilis (pH 6.0) were found to be negative from the 1st day to the last day after the administration. B. subtilis (pH 7.2), B. subtilis (pH 8.0), and M. luteus (pH 8.0) by the three screening tests, were found to be negative all the way after the administration. On the other hand, B. cereus (pH 6.0) by the three screening tests was clearly found to be positive for the first 15 days after the administration, and then muscle-direct and direct-disk treatment of B. cereus (pH 6.0) were found to be dubious positive at 20th days after the administration. However extraction-disk treatment of B. cereus (pH 6.0) was clearly found to be negative at the same stage; thereafter, the three screening tests of B. cereus (pH 6.0) were found negative to the last of the experiment. These findings showed that to have equal sensitivity to those determination for the residual detection of OTC, and also confirmed that B. cereus was effective test organism for the monitoring of OTC.

• Korean Journal of Environmental Agriculture
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• v.30 no.3
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• pp.288-294
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• 2011

BACKGROUND: Methylmercury is analyzed by HPLC-ICP/MS because of the simplicity for sample preparation and interference. However, most of the pre-treatment methods for methylmercury need a further pH adjustment of the extracted solution and removal of organic matter for HPLC. The purpose of this study was to establish a rapid and accurate analytical method for determination of methylmercury in fish by using HPLC-ICP/MS. METHOD AND RESULTS: We conducted an experiment for pre-treatment and instrument conditions and analytical method verification. Pre-treatment condition was established with aqueous 1% L-cysteine HCl and heated at $60^{\circ}C$ in microwave for 20 min. Methylmercury in $50{\mu}L$ of filtered extract was separated by a C18 column and aqueous 0.1% L-cysteine HCl + 0.1% L-cysteine mobile phase at $25^{\circ}C$. The presence of cysteine in mobile phase and sample solution was essential to eliminate adsorption, peak tailing and memory effect problems. Correlation coefficient($r^2$) for the linearity was 0.9998. The limits of detection and quantitation for this method were 0.15 and $0.45{\mu}g/kg$ respectively. CONCLUSION: Result for analytical method verification, accuracy and repeatability of the analytes were in good agreement with the certified reference materials values of methylmercury at a 95% confidence level. The advantage of the established method is that the extracted solution can be directly injected into the HPLC column without additional processes and the memory effect of mercury in the ICP-MS can be eliminated.