• Journal of Soil and Groundwater Environment
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• v.11 no.5
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• pp.43-50
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• 2006

Bioreporter bacteria, such as recombinant bioluminescent bacteria, have been used for the detection of specific compounds in complex environmental media. In this study, optimum conditions for the preparation and application of deep-freezed and Iyophilized recombinant bioluminescent strain KG1206 were investigated for the future application on contaminated environmental sites. Genetically engineered microorganism, Pseudomonas putida mt-2 KG1206, contains TOL plasmid and the plasmid inserted $P_{m}$, promoter on the upper part of lux gone in vector pUCD615, and m-toluate and benzoate are considered direct inducers for bioluminescence. Optimum conditions determined for the preparation and application of the deep-freezed and lyophilized strain were followings: cryoprotective agent (24% sucrose), lyophilization time (12 hrs), strain concentration ($OD_{600}=0.6$), reconstitution for freezed strain (quick reconstitution at $35^{\circ}C$), reconstitution for lyophilized strain ($3{\sim}6$ hrs exposure on LB medium), carrying conditions (keep at $20^{\circ}C$ after reconstitution). These results demonstrate the feasibility of deep-freezed or lyophilized state of genetically engineered bioluminescent strain for environmental usage.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.5
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• pp.731-738
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• 2015

Nematode infection in the epithelial tissue of cultured rockfish Sebastes schlegeli was first reported in 2012. Since then, nematode infections have caused serious economic losses in rockfish aquaculture on the west coast of Korea. Taxonomic and life cycle information for this parasite are currently unknown. In this study, 18S rRNA and cytochrome c oxidase subunit I (COI) genes were used for molecular identification and polymerase chain reaction (PCR) to detect the invisible stages of this parasite. Nucleotide sequences of the 18S rRNA of the rockfish nematode showed 98% identity with that of Philometra morii. Therefore, this rockfish nematode was classified to the Philometridae family. However, we could not identify it to genus level using 18S rRNA. Its COI nucleotide sequences shared 85% and 82% identities with those of Bursaphelenchus sinensis and Philometra overstreeti, respectively. In addition, two gene-specific primer sets were designed based on the 18S rRNA gene to detect the intermediate host and nematode larvae. These primers were specific to this rockfish nematode without cross-reacting to other pathogens. The detection limit of the PCR assay using these primers was 1,000 copies of nematoda plasmid DNA. Therefore, the PCR assay described here is suitable for the detection of nematode DNA within rockfish. In addition, this PCR assay could be used to detect nematode larvae and the intermediate host.

• Journal of Periodontal and Implant Science
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• v.38 no.sup2
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• pp.335-342
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• 2008

Purpose: Cytolethal distending toxin (CDT) considered as a key factor of localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis is composed of five open reading frames (ORFs). Among of them, the individual role of CdtA and CdtC is not clear; several reports presents that CDT is an AB2 toxin and they enters the host cell via clathrin-coated pits or through the interaction with GM3 ganglioside. So, CdtA, CdtC, or both seem to be required for the delivery of the CdtB protein into the host cell. Moreover, recombinant CDT was suggested as good vaccine material and antibody against CDT can be used for neutralization or for a detection kit. Materials and Methods: We constructed the pET28a-cdtC plasmid from Aggregatibacter actinomycetemcomitans Y4 by genomic DNA PCR and expressed in BL21 (DE3) Escherichia coli system. We obtained the antibody against the recombinant CdtC in mice system. Using the anti-CdtC antibody, we test the native CdtC detection by ELISA and Western Blotting and confirm the expression time of native CdtC protein during the growth phase of A. actinomycetemcomitans. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and generated the anti CdtC antibody against recombinant CdtC subunit expressed in E. coli system. Our anti CdtC antibody can be interacting with recombinant CdtC and native CDT in ELISA and Western system. Also, CDT holotoxin existed at 24h but not at 48h meaning that CDT holotoxin was assembled at specific time during the bacterial growth. Conclusion: In conclusion, we thought that our anti CdtC antibody could be used mucosal adjuvant or detection kit development, because it could interact with native CDT holotoxin.

• Korean Journal of Agricultural Science
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• v.42 no.2
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• pp.99-103
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• 2015

Potato virus T (PVT) is a plant pathogen in the family Betaflexiviridae, group IV single-stranded positive sense RNA viruses. The major host of PVT is potato, and it has been reported in Ullucus tuberosus, Oxalis tuberosa and Tropaeolum tuberosum. This study aimed at developing reverse transcription (RT)-polymerase chain reaction (PCR) and nested PCR techniques for specific detection of PVT. Finally, Two RT-PCR primer sets were developed and verified. The RT-PCR products were amplified to 734 (PVT RT-PCR primer set 6) and 828 bp (PVT RT-PCR primer set 29) long to detect PVT. The nested PCR primer sets [PVT-N70/C20 ($734{\rightarrow}315bp$) and PVT-N75/C30 ($828{\rightarrow}529bp$)] were developed which are high sensitivity and verification for detection of PVT. Furthermore, a modified-positive control plasmid is use to verify contamination of laboratory in PVT detection. This study supported the diagnose PVT in potato or PVT related hosts.

• KSBB Journal
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• v.26 no.2
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• pp.172-176
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• 2011

This study was aimed to verify suppressive effect of pine-needle extract on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression. In order to evaluate suppressive effect on iNOS expression, RAW 264.7 cells were stably transfected using an iNOS promoterluciferase reporter plasmid yielding RAW 264.7/pGL2-NeomiNOS_ pro11 cells. Established in vitro detection system revealed to diminish LPS-induced iNOS expression by 0.1~500 ${\mu}g/mL$ of saponin at the concentration-dependant manner. Pine needle extract also diminished LPS-induced iNOS expression to 92 and 88% at 500 and 50 ${\mu}g/mL$, respectively. These results suggest that the in vitro detection system developed here could be useful for the verification of suppressive materials on iNOS expression and pine needle extract could be used for the development of functional foods.

• Journal of Food Hygiene and Safety
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• v.15 no.3
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• pp.262-266
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• 2000

The polymerase chain reaction was used to selectively detect sequences within the fimbrial antigen of Salmonella enteritidis. Sterile milk was artificially inoculated with known amount of S. enteritidis and then DNA was extracted with guanidine thiocyanate/phenol/chloroform, followed by PCR. A detection limit of as few as 100 colony forming unit (cfu) per 0.5 ml milk was obtained with this method. For the whole procedure, it took only 5 h. A semi-quantitative polymerase chain reaction assay which allows an estimation of colony forming unit of S. enteritidis was developed. Known amount of standard plasmid pGem-4Z-Sef B(-) containing cloned S. enteritidis fimbrial antigen gene was co-amplified with Salmonella genomic DNA isolated from artificially inoculated milk. The same set of primers were used for the amplification and the products were cleaved with Bam HI. The concentration of the target DNA could be estimated by comparing the intensity of the two bands after electrophoresis. The PCR-based protocol described in this paper provides a rapid, simple, and sensitive method for detecting S. enteritidis in milk.

• Journal of Life Science
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• v.9 no.4
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• pp.358-367
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• 1999

Thirty strains of methicillin resistant Staphylococcus aureus were obtained from the clinical isolates. In order to investigate the pursuit of the pathogens of nosocomial infection, these strains were studied for antibiotic sensitivity as well as its resistant pattern. Among the methods of hybridization which directly confirm the specific antibiotic resistant genes by means of the recently developed specific probe DNA, dot blot hybridization and southern blot hybridization were performed and these two methods were compared in their sensitivity and specificity. Strains that is sensitive to cephalothin to the subject of methicillin resistant Staphylococcus aureus were in 43%. Those that are sensitive to cefoperazone and cefuroxime were 26% and 23%, respectively. In case of MIC, MIC50 of cefoperazone was 8 $\mu\textrm{g}$/$m\ell$, and MIC90 was 128 $\mu\textrm{g}$/$m\ell$ to be the lowest. As the results of plasmid DNA electrophoresis, most of methicillin resistant Staphylococcus aureus strains had more than 4 plasmids. These plasmids digested by BamHI, methicillin resistant Staphylococcus aureus is distributed as 10 fragments with the size of 65 kb to 1.5 kb. Dot blot hybridization were performed to examine the existence of mecA gene to show the detection rate of 50%. Southern blot hybridization were done to see if DNA bands which amplify the activity of digoxigenium-labeled probe by PCR were actually PCR products of mecA gene and it showed the detection rate of 53%. It can be concluded that the southern blot hybridization seemed to be better in sensitivity and specificity when it is compared with the results of dot blot hybridization.

• Korean Journal of Agricultural Science
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• v.42 no.2
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• pp.105-109
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• 2015

Andean potato latent virus (APLV) is a phytopathogenic virus that belongs to the Group IV (+) sense ssRNA viruses of the genus Tymovirus. It mainly infects potatoes and is specified as a controlled quarantine virus in Korea. In this study, two primer sets of RT-PCR and nested PCR [set 2 ($404{\rightarrow}259bp$) and set 23 ($501{\rightarrow}349bp$)], were selected, which can rapidly and accurately diagnose APLV in quarantine sites. In addition, a modified-positive control plasmid is development, can possible verification of laboratory contamination in diagnosis of APLV detection. The PCR-base system developed in this study is expected to diagnose APLV and contribute to the plant quarantine in Korea.

• Journal of Life Science
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• v.13 no.6
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• pp.799-804
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• 2003

In order to verify the occurrence of an estrogenic compound in natural products, the estrogenic activity was measured using an in vitro detection system. For this system, human breast cancer cell line MCF7 was transfected using an estrogen responsive CAT reporter plasmid. Estrogenic activities of photosynthetic algae spirulina and sea lettuce were evaluated using this system. Estrogenic activities of a $500\mug/ml\; and\; 50 \mug/ml$ ethanol extracts of spirulina were as much as that of $10^{-8}$M standard solution (17$\beta$-estradiol) and activity of $5\mug/ml$ ethanol extract of spirulina was as much as that of $10^{-10}$ M standard solution. However, no significant estrogenic activity was observed using sea lettuce extract. Estrogenic activities of marine animals, such as star fish and shrimp, were also evaluated using this system, however, no significant estrogenic activity was observed in these extracts. In this result, it is confirmed that spirulina extract possesses estrogenic compound.

• Fisheries and Aquatic Sciences
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• v.24 no.11
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• pp.351-359
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• 2021

The red sea bream iridovirus (RSIV) belonging to genus Megalocytivirus is responsible for red sea bream iridoviral disease (RSIVD) in marine and freshwater fishes. Although several diagnostic assays for RSIV have been developed, diagnostic sensitivity (DSe) and specificity (DSp) of real-time polymerase chain reaction (PCR) assays are not yet evaluated. In this study, we developed a TaqMan probe-based real-time PCR method and evaluated its DSe and DSp. To detect RSIV, the probe and primers were designed based on consensus sequences of the major capsid protein (MCP) genes from megalocytiviruses including RSIV, infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primers were shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A 95% limit of detection (LOD_95%) was determined to be 5.3 viral genome copies/µL of plasmid DNA containing the MCP gene from RSIV. The DSe and DSp of the developed real-time PCR assay for field samples (n = 112) were compared with those of conventional PCR assays and found to be 100% and 95.2%, respectively. The quantitative results for SYBR Green and TaqMan probe-based real-time PCR were not significantly different. The TaqMan probe-based real-time PCR assay for RSIV may be used as an appropriate diagnostic tool for qualitative and quantitative analysis.