• Journal of Microbiology and Biotechnology
• /
• v.15 no.5
• /
• pp.1022-1027
• /
• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• Korean Journal of Clinical Laboratory Science
• /
• v.45 no.1
• /
• pp.1-4
• /
• 2013

Vancomycin-resistant enterococci (VRE) have emerged as important healthcare-associated infection since last two decades. ChromID VRE agar (cIDVA) is useful for VRE rectal swab screening. We investigated all VRE were isolated on the cIDVA. A total of 363 rectal swabs of 85 patients to test VRE screening were inoculated into bile-esculin (B-E) broth with $6{\mu}g/mL$ vancomycin. After 24 hours incubation, we subcultured B-E broths were changed to black onto cIDVA. All isolates were identified by the MICROSCAN and VITEK2. The vanA gene and vancomycin minimal inhibition concentration (MIC) were detected by PCR and E-test respectively. 277 E. faecium (84.7%), 16 E. faecalis (4.9%), 25 E. avium (7.6%), 8 E. gallinarum (2.4%) and 1 E. raffinosus (0.3%) were isolated. 10.3% of VRE detected on cIDVA were other than E. faecium and E. faecalis that presented various color from colorless to pale violet. All isolates contained vanA and vancomycin MIC were > $256{\mu}g/mL$. VRE isolates other than E. faecium and E. faecalis should be objective to the contact precautions for healthcare-associated infection control if they possess vanA gene. Due to emerging enterococci carrying vanA such as E. avium, E. gallinarum, and E. raffinosus, VRE surveillance should be expanded to all isolates on chromogenic agar.

• BMB Reports
• /
• v.37 no.5
• /
• pp.527-532
• /
• 2004

A subtracted cDNA library specific to osmotic stress of Haloxylon ammodendron (Mey.) Bge seedlings was constructed by suppression subtractive hybridization (SSH) and T/A cloning. SSH was performed between two groups of H. ammodendron seedlings, one was cultivated in Hoagland (H) solution as a driver and the other group was treated with osmotic stress of the Hoagland solution by the addition of 400 mM mannitol (M), as a tester. The library consisted of about 400 recombinant clones, with the average size being of 500 bp, ranging from 300 bp to 1500 bp. Using a PCR-select differential screening kit, 100 recombinant clones were randomly chosen from the subtracted cDNA library and hybridized with forward,reverse subtracted and unsubtracted probes for two rounds. As a result, 21 positive clones specific to osmotic stress were obtained and some of them were verified by Northern blot analysis. The sequencing analysis of 6 positive clones and the following homology comparison to GenBank [blastx] non-redundant databases characterized that two sequences obtained in this experiment may contribute to novel drought-related genes.

• Journal of Microbiology
• /
• v.38 no.4
• /
• pp.230-238
• /
• 2000

Class III chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class III chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5'flanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at -716 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for past-transnational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P chrysogenum and genus Aspergillus.

• Food Science of Animal Resources
• /
• v.33 no.5
• /
• pp.655-663
• /
• 2013

In this study, a rapid and sensitive detection tool for screening Salmonella spp. by using a loop-mediated isothermal amplification (LAMP) assay targeting the genomic sequence of the invA gene was developed. The inclusivity and exclusivity were both at 100% in the analysis, and the limit of detection (LOD) in a pure S. Enteritidis culture suspended in saline was $3.2{\times}10^3$ CFU/mL at 18.17 min ($R^2$ = 0.9446). The LODs of the LAMP assay in buffered peptone water with Salmonella (BPW) and duck carcass swab sample enriched in BPW with Salmonella (BPWS) after 0 and 12 h incubation were $3.2{\times}10^3$ CFU/mL and $3.2{\times}10^0$ CFU/mL, respectively. Comparing the LODs in BPW with those in BPWS, the LAMP assay was less influenced by compounds of duck carcass swab sample than the PCR assay. Sensitivity and specificity of the LAMP assay in 50 duck carcass swab samples enriched in BPW for 6 h were 96% and 84%, respectively, indicating that the LAMP assay is a rapid, simple and sensitive assay, which can be utilized as a potential screening tool of Salmonella spp. in duck carcass sample.

• Journal of Microbiology and Biotechnology
• /
• v.30 no.3
• /
• pp.459-468
• /
• 2020

This study established a new polymerase spiral reaction (PSR) that combines with reverse transcription reactions for HCV detection targeting 5'UTR gene. To avoid cross-contamination of aerosols, an isothermal amplification tube (IAT), as a separate containment control, was used to judge the result. After optimizing the RT-PSR reaction system, its effectiveness and specificity were tested against 15 different virus strains which included 8 that were HCV positive and 7 as non-HCV controls. The results showed that the RT-PSR assay effectively detected all 8 HCV strains, and no false positives were found among the 7 non-HCV strains. The detection limit of our RT-PSR assay is comparable to the real-time RT-PCR, but is more sensitive than the RT-LAMP. The established RT-PSR assay was further evaluated for detection of HCV in clinical blood samples, and the resulting 80.25% detection rate demonstrated better or similar effectiveness compared to the RT-LAMP (79.63%) and real-time RT-PCR (80.25%). Overall, the results showed that the RT-PSR assay offers high specificity and sensitivity for HCV detection with great potential for screening HCV in clinical blood samples.

• Korean Journal of Veterinary Service
• /
• v.30 no.4
• /
• pp.557-562
• /
• 2007

This study was carried out to discriminate Hanwoo from the milking and hybrid cattle by detection of MC1R gene related to bovine hair color. One hundred sixty six samples were collected from the abattoir (n = 106) and local market (n = 60). The beef from abattoir were originated from Hanwoo (n=27), Holstein (n=29), Hybrid (n=45) and imported cattle (n=5), respectively. The beef from market consisted of Hanwoo (n=36), Holstein (n=7) and imported ones (n=17). Commercialized screening kit (Kogenebiotec, Korea) was used for MC1R gene analysis. As a result, Hanwoo was discriminated from Holstein. However, 9 of 45 hybrid and 11 of 22 imported beef samples were indistinguishable from Hanwoo. It could be explained by second generation of crossing of Hanwoo with Holstein or the cattle with silver or yellow hair. This results suggest that additional tests as well as MC1R gene detection be needed to confirm Hanwoo beef among cattle beef.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 2003.10a
• /
• pp.113.2-114
• /
• 2003

Five pepper-infecting potyviruses, Pepper mottle virus (PepMoV), Chilli veinal mottle virus (CVMV), Pepper veinal mottle virus (PVMV), Pepper severe mosaic virus (PSMV) and Tobacco each virus (TEV), are known filamentous virus and can be infected pepper crops systemically. To understand pathology and genome information of the five viruses on pepper plants, host reactions and sequences were compared to the 5 viruses. Five potyviruses were inoculated onto some typical cultivars of hot peppers and compared their symptoms, and virus accumulations. A set of degenerate primers for potyviruses were applied to 5 viruses and RT-PCR was performed. RT-PCR products containing partial nuclear inclusion b and coat protein (CP) genes were cloned. Then, oligo dT primer and species-specific primer were redesigned to amplify the C-terminal part of CP and 3' noncoding regions of each viruses. Sequences of the viruses were analyzed and compared to serological relationships among the viruses. The data can be useful for screening of potyviruses in pepper plants and pathogen-derived transgenic pepper plant development.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2001.11a
• /
• pp.146-156
• /
• 2001

The biosynthetic gene clusters for bluensomycin and spectinomycin were isolated and characterized from the bluensomycin producer, Streptomyces bluensis ATCC27420 and the spectinomycin producer, Streptomyces spectabilis ATCC27741, respectively. PCR primers were designed specifically to amplify a segment of dTDP-glucose synthase gene based on its conserved sequences of several actinomycete strains. By screening cosmid libraries using amplified PCR fragments, 30-kb and 45-kb DNA fragments were isolated from Streptomyces bluensis and Streptomyces spectabilis, respectively. Sequencing analysis of them revealed that each contains 15 open reading frames (ORFs). Some of these ORFs were turned out to be antibiotic resistance genes (blmA and speN), dTDP-glucose synthase genes (blmD and spcD), and dTDP-D-glucose 4,6-dehydratase genes (blmE and spcE), suggesting that the blm and spec gene clusters are likely involved in the biosynthesis of bluensomycin and spectinomycin, respectively.

• Journal of Veterinary Clinics
• /
• v.25 no.4
• /
• pp.289-291
• /
• 2008

3-year-old female Pitbull terrier dog that had been moved to Republic of Korea was diagnosed with Ehrlichia canis infection. Abnormal findings on a complete blood count (CBC) and serum chemistry profile were moderate anemia, mild thrombocytopenia, hyperproteinemia and hyperglobulinemia. Serologic screening test by a commercial ELISA kit for Ehrlichia canis was positive, and serum antibody titer was markedly high (> 1 : 10240). The morula of Ehrlichia organisms was not detected in buffy coat blood smears. Polymerase chain reaction (PCR) was done using the peripheral blood and the result was negative. Based on the serologic test results and the clinical signs, the dog was diagnosed as ehrlichiosis. The dog responded well to doxycycline and was uneventfully recovered.