Competitive PCR법을 이용한 Marine birnavirus(MABV)의 정량적 검출

김영진,김석렬,정성주,오명주 한국어병학회 2001 한국어병학회지 Vol.14 No.3

기존의 어류 병원성 바이러스 연구에 있어 주화세포를 이용한 TCID_50과 plaque assay는 미량의 바이러스에 대해, 그리고 현재 많이 사용되고 있는 polymerase chain reaction(PCR)은 정확한 정량이 힘들다는 것이 각각 단점으로 지적된 바 있다. 이에 어류 병원 바이러스를 효율적으로 조사하기 위한 방법을 개발하고자 연안 양식장에 만연해 있는 marine birnavirus(MABV)를 대상으로 competitive PCR을 이용하여 그 감도 및 정량성 여부를 시험하였다. 본 연구에서 1.44×10^7 PFU/㎖의 바이러스 액에 대해 10^4배 희석한 바이러스 시료까지 검출이 가능하였고, 이 바이러스 액 200㎕에는 약 100 copy의 MABV cDNA가 존재함을 확인할 수 있어 이와 같은 바이러스 유전자의 양을 토대로 간접적인 추정이 가능할 것으로 판단되었다. 감염된 개체에 있는 viral genome의 수는 질병의 진행을 monitor하는 데 중요한 parameter의 하나로서 이 방법을 이용하여 얻어진 정보는 infection stage의 구분에 대한 기초자료로 이용할 수 있을 것으로 판단된다. It has been shown that TCID_50 and plaque assay might be hard to apply for the analysis of small amount of virus. Additionally polymerase chain reaction(PCR) does reply the quantity of virus correctly. Thus we developed the competitve PCR, and effective mean to investigate fish pathogenic virus, and analyzed the sensitivity against marine birnavirus (MABV). In this study, the virus sample of 1.44×10^7PFU/㎖ was detected after up to 10^4 dilution. And the existence of 100 copies MABV cDNA was identified with the competitive PCR in this virus sample. These results indicated that the indirect presumption was available on the basis of the amounts the viral genome. Viral genome's number in the infected individual can be used as the fundamental data to divide the infection stage and to monitor the progress of disease.

Is Koi Herpesvirus (KHV) Related to the Mass Mortality Occurring among Cultured Carp, Cyprinus carpio, in Korea?

Kim, Wi-Sik,Jung, Sung-Ju,Kim, Du-Woon,Kim, Seok-Ryel,Kim, Jeong-Ho,Oh, Myung-Joo The Korean Society of Fisheries and Aquatic Scienc 2010 Fisheries and Aquatic Sciences Vol.13 No.1

Since 1998, a new viral disease with high mortality has been consistently recorded in Korea in cultured carp, Cyprinus carpio. In this study, we investigated an epizootic of the disease that caused high mortality rates in carp obtained from 11 farms in Korea between 1999 and 2007. Assessment of koi herpesvirus (KHV) levels in diseased carp was carried out to determine if this virus was the etiologic agent of disease in this instance. High mortality rates in carp were recorded mainly in the spring and autumn at water temperatures between $19^{\circ}C$ and $24^{\circ}C$. Diseased fish typically showed surface discoloration, with a thick opaque mucus covering the body and gills. Protozoan parasites and bacteria were recovered from 7/29 (24%) and 2/26 (8%) of fish, respectively. Evidence of viral infection was marked; cytopathic effects (CPEs), characterized by cell rounding and an extended cytoplasm in fathead minnow (FHM) cells, were detected in 40/41 fish (98%). A high mortality rate (80%) resulted when supernatants of cell cultures showing CPEs were applied to previously healthy fish. KHV was detected by polymerase chain reaction in 6/41 fish (15%), but was not detected in supernatants obtained from cell cultures showing CPEs. These results suggest that KHV may not be the etiologic agent of the high mortality occurring among cultured carp in Korea; therefore, some other-as yet unidentified-infective agent must be responsible.

Detection of Norovirus in Contaminated Ham by Reverse Transcriptase-PCR and Nested PCR

Seok Ryel Kim,Duwoon Kim,Ki-Sung Kwon,In-Gyun Hwang,Myung-Joo Oh 한국식품과학회 2008 Food Science and Biotechnology Vol.17 No.3

In order to enhance the efficacy of norovirus detection by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR, this study developed a norovirus mRNA concentration method using poly oligo dT-conjugated magnetic beads. An efficient norovirus detection protocol was performed on commercial ham using 2 viral elution buffers (glycine buffer and Tris beef extract buffer) and 2 concentration solutions [polyethylene glycol (PEG) and zirconium hydroxide]. The different approaches were verified by RT-PCR and nested PCR. This method was performed on ham in less than 8 hr by artificial inoculation of serial dilutions of the virus ranging from 1,000 to 1 RT-PCR unit/mL. The viral extraction and concentration method had 10-fold higher sensitivity using the combination of Tris beef extract buffer and PEG as compared to glycine buffer and zirconium hydroxide. This method proved that RT-PCR and nested PCR have the sensitive ability to detect norovirus in commercial ham, in that norovirus was successfully detected in artificially contaminated samples at a detection level as low as 1-10 RT-PCR unit/mL. Overall, such a detection limit suggests this protocol is both quick and efficient in terms of its potential use for detecting norovirus in meat products.

Comparison of WSSV (white spot syndrome virus) DNA using Different oligonucleotide primers

Seok Ryel Kim,Yeong Jin Kim,Sung Ju Jung,Myung Joo Oh 전남대학교 수산과학연구소 2006 수산과학연구소논문집 Vol.15 No.2

White spot syndrome (WSS) is a viral disease that affects on most of the commercially cultivated shrimp species in worldwide. WSSV causing white spot syndrome in Korea were genetically similar to PRDV in Japan. In this study, we compared PCR products of WSSV with those of PRDV, using their specific oligonucleotide primers. As a result of sequence analysis, amplified PCR products were almost identical to the described sequences used in this experiment. Therefore, the results obtained in this study suggest that WSSV causing white spot syndrome in Korea were genetically similar to PRDV in Japan. Naturally, more detailed studies will be needed to identify the taxonomic correlation between these viruses.

Overexpression of Recombinant Arylsulfatase Cloned from Pseudoalteromonas carrageenovora

Kim Jong-Oh,Kim Seok-Ryel,Lim Jae-Myung,Nam Soo-Wan,Kim Hyeung-Rak The Korean Society of Fisheries and Aquatic Scienc 2005 Fisheries and Aquatic Sciences Vol.8 No.3

Arylsulfatase cloned from a marine bacterium, Pseudoalteromonas carrageenovora, was over-expressed in Escherichia coli. Most of the recombinant arylsulfatase was found in the cell lysate with induction up to $10{\mu}M$ IPTG. However, enzyme activity was observed both in the culture supernatant and cell lysate by induction with IPTG concentration of $50-5,000{\mu}M$. Most of the recombinant enzyme was localized in the periplasmic space with $10{\mu}M$ IPTG induction, while half of the enzyme was distributed in the periplasmic space with $50{\mu}M$ IPTG induction. Cell growth and arylsulfatase activity did not change with the induction time, and the level of recombinant arylsulfatase expression was maintained at 4-5 U/mL after 6 to 14 hr of culture.

Recovery of Pseudomonas anguilliseptica from Diseased Striped Beakperch (Oplegnathus fasciatus) in Korea

( Seok Ryel Kim ),( Myoung Ae Park ),( Shin Ichi Kitamura ),( Sung Ju Jung ),( Myung Joo Oh ) 한국수산과학회(구 한국수산학회) 2010 Fisheries and Aquatic Sciences Vol.13 No.2

Antimicrobial Effects of Chemical Disinfectants on Fish Pathogenic Bacteria

Seok-Ryel Kim,Kyung-Hee Park,Duwoon Kim,Sung-Ju Jung,So-Yong Kang,Myung-Joo Oh 한국식품과학회 2008 Food Science and Biotechnology Vol.17 No.5

This study was to examine the potential disinfection efficiencies of 10 compounds by determining their antimicrobial capacity and ichthyotoxicity. Antimicrobial effects against Vibrio sp., Edwadsiella tarda, Streptococcus sp., and Staphylococcus sp. were tested using 10 different disinfectants; hydrogen peroxide, sodium hypochlorite, chlorine dioxide, povidon iodine, formaldehyde, glutaraldehyde, quaternary ammonium compounds (QACs), didecyl dimethyl ammonium chloride (DDAC), ortho-dichlorobenzen, and copper sulfate. Chlorine dioxide (ClO₂) containing 5% ClO₂ and copper sulfate had no effects on bactericidal activity, while the other disinfectants resulted in 99.99% bactericidal activity against 4 strains of fish pathogenic bacteria. The ichthyotoxicity of the 10 disinfectants was investigated using 3 kinds of fish species; flounder (Paralichthys olivaceus), rockfish (Sebastes pachycephalus), and black sea bream (Acanthopagrus schlegelii). Median lethal concentration (LC₅₀) values of the 10 disinfectants were estimated to determine toxicity ranges of the doses within 24 hr. Among test disinfectant solutions, hydrogen peroxide showed the highest LC₅₀ in flounder (201.3), rockfish (269.7), and black sea bream (139.3 ppm). DDAC revealed the lowest LC₅₀ in flounder (2.1), rockfish (1.0), and black sea bream (1.5 ppm). These results suggest that DDAC, quaternary ammonium compounds, glutaraldehyde, and sodium hypochlorite are effective disinfectants for fish and bacterial species examined in this study.

Relationship between White Spot Symptom and Physiological Status of Two Penaeid Shrimps

Kim, Su Kyoung,Kim, Myung Seok,Park, Myoung Ae,Kim, Su mi,Jang, In Kwon,Kim, Seok Ryel,Cho, Miyoung Korean Society of Environmental Biology 2017 환경생물 : 환경생물학회지 Vol.35 No.4

Shrimps infected with WSSV(White Spot Syndrome Virus) generally exhibit white spots in their inner space of carapaces as an acute clinical sign. In an effort to identify the correlation between this acute clinical sign and the condition, the index factors (RNA/DNA concentration and ratio, trypsin activity) were analyzed. A total 580 farmed Fenneropenaeus chinensis and 130 Lithopenaeus vannamei were collected from western and southern fifteen outdoor ponds in Korea. The status of the white spot pathology was divided into four stages (stage 0, stage I, stage II, and stage III), in accordance with the clinical signs as to the size and area of white spots. A significant decrease in RNA concentration and RNA/DNA ratio for multi-infected fleshy prawn (WSSV and vibrio sp.) occurred during the stage III (the whole carapace is covered with a white spot). In particular, RNA/DNA ratio was significantly lower as $1.47{\pm}0.04$ than other groups. A similar trend was also found in the single infection (WSSV), but the decrease was less than the multi-infection. In the species comparison, both species were vulnerable to the multi-infection, but L. vannamei was more sensitive than F. chinensis(ANOVA, p<0.05): A significant decrease in RNA concentration and RNA/DNA ratio was first found in stage II for the former species, while it was found in stage III for the latter species. Trypsin activity was also showed a similar tendency with nucleic acid variation. Multi-infected shrimp showed drastically decrease of trypsin activity. According to the results, clinical signs of the white spot under carapace have an only physiological effect on shrimp if they covered entirely with white spots.

Experimental Transfer of Tetracycline Resistance Genes from Fish-derived Bacteria to Escherichia coli

Kim Seok-Ryel,Kim Hyeung-Rak,Suzuki Satoru The Korean Society of Fisheries and Aquatic Scienc 2006 Fisheries and Aquatic Sciences Vol.9 No.2

To determine whether the tetracycline resistance genes tet (34), tet (M), and tet (S) can be transferred among bacteria, we used a filter mating experiment allowing intimate cell-cell contact between donor and recipient. The tet(34) gene, conveyed on a chromosome of Vibrio species (No. 6 and SW-42) was not transferred to Escherichia coli JM109, suggesting that it is not transferred among bacterial species. The tet (M) gene was transferred from three Vibrio strains (4-E, SW-18, and SW-38) to E. coli at frequencies of $8.5{\times}10^{-5}\;to\;2.1{\times}10^{-6}$. The tet(S) gene was transferred from Lactococcus garvieae KHS98032 to E. coli at a frequency of $1.8{\times}10^{-6}$. Transconjugated recipients showed increased minimum inhibitory concentrations against oxytetracycline. Although the donors possess the Tn916-Tn1545 transposons, they were not detected in transformed recipients, suggesting that the transfer of tet(M) and tet(S) is mediated by elements or mechanisms. Two ribosomal protect protein genes were also transmissible from marine bacteria to E. coli, suggesting gene hopping among marine, terrestrial, and human environments.

Notes : Experimental Transfer of Tetracycline Resistance Genes from Fish-derived Bacteria to Escherichia coli

( Seok Ryel Kim ),( Hyeung Rak Kim ),( Satoru Suzuki ) 한국수산학회 2006 Fisheries and Aquatic Sciences Vol.9 No.2