• Korean Journal of Veterinary Research
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• v.42 no.3
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• pp.363-370
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• 2002

For the development of diagnostic polymerase chain reaction (PCR) to fungal infection by dermatophytes Trichophyton and Microsporum, detection of the fungal DNA by PCR and analysis of the DNA pattern were undertaken in the present study. A total of 15 strains were tested and those consisted of 3 reference strains and 12 isolates such as: reference strains of T mentagrophytes (downy type, ATCC 9533), T rubrum (IFO 6204) and M gypseum (ATCC 9083), and each isolate of T mentogrophytes (powdery type), T mentagrophytes (granular type), T mentogrophytes (purple-red type), T rubrum, T raubitschekii, T tonsurans, T equinum, T ajelloi, T verrucosum, M cookei, M nanum and M gypseum. The DNA were purely isolated from all strains of Trichophyton spp. and Microsporum spp. by a simple method partly consisted of disruption of fungal cells by lyophilization and grinding and extraction of fungal DNA without phenol treatment which is a routine procedure in DNA isolation. For the detection of fungal DNAs, optimal condition of PCR was determined as preheating once at $94^{\circ}C$ for 5 min, 35 cycles of denaturation at $94^{\circ}C$ for 1 min, annealing at $38^{\circ}C$ for 1 min and polymerization at $72^{\circ}C$ for 2 min, and 1 cycle of final extension at $72^{\circ}C$ for 5 min. In PCR using arbitrary primers AP-1 (5' ACCCGACCTG3') and AP-2 (5' ACGGGCCAGT3'), DNAs in various numbers and sizes were detected from different species of Trichophyton and Microsporum, while DNAs in similar size were also detected in all strains of Trichophyton spp. and Microsporum spp. There were unique DNAs observed from certain dermatophytes by AP-1 such as 1,900 bases in T rubrum, 950 and 1,100 bases in T raubitscheldi, 2,100 bases in T equinum, 400 bases in T verrucosum and 1,150 bases in M gypseum. The unique DNAs were also observed by AP-2 such as 1,200 bases in T ajelloi, 250 bases in T verrucosum, 1,150 bases in M cookei and 2,000 bases in M nanum. The results indicated that PCR can detect a specific DNA from certain Trychophyton and Microsporum spp, which can be the information for further development of diagoomc PCR to dennatophytes.

• Korean Journal of Microbiology
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• v.43 no.4
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• pp.264-272
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• 2007

For the detection of human immunodeficiency virus (HIV), ultra-rapid real-time PCR methods were developed. The target DNA sequences were used 495 bp HIV-1-specific env gene (gi_1184090) and 294 bp HIV-2-specific env gene (gi_1332355). Ultra-rapid real-time PCR was peformed by $Genspector^{TM}$ (Samsung, Korea) using microchip-based, $6\;{\mu}l$ of reaction volume with extremely short running time in only 2 steps (denaturation, annealing/extension) in each cycle of PCR. Total reaction for 30 cycled ultra-rapid PCR detection including melting temperature analysis was completed in 7 min and 30 sec. The HIV-1-specific 117 bp-long or HIV-2-spe-cific 119 bp-long PCR products were successfully amplified from the minimum of template, $2.3{\times}10^3$ copies of each euv gene using 30 cycled two-steps ultra-rapid PCR. This kind of ultra-rapid real-time PCR method would be useful not only for the rapid-detection of HIV, but also rapid-detection of other pathogens.

• Korean Journal of Plant Tissue Culture
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• v.25 no.6
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• pp.519-524
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• 1998

A simple and reliable method to diagnose cucumber green mottle mosaic virus of watermelon in Korea (CGMMV-WK) was determined by RT-PCR, and coat protein gene for CGMMV-WK was cloned. Comparing to a method reported by Lee et al. (1996), the method developed here showed a better RT-PCR reaction. RT-PCR was possible by one step in the PCR reaction mixture that contains 20 pmol of primer, reverse transcriptase (30 unit), RNasin (5 unit) using the crude RNA solution. RT-PCR condition for specifically diagnosing CGMMV-WK was that cDNA was synthesized at 42$^{\circ}C$ for 45 min followed by pre-denaturation at 95$^{\circ}C$ for 2 min, and then PCR reaction was carried out with a programmed condition that consisted of 36 sequential cycles at 96$^{\circ}C$ for 30 sec, 6$0^{\circ}C$ for 30 sec, and 72$^{\circ}C$ for 1 min. A gene encoding the coat protein of CGMMV-WK was cloned and characterized. Nucleotide sequence of coat protein gene of CGMMV-WK shared 98.77% and 99.38% of sequence identity with those of CGMMV-W and CGMMV-SH, respecitvely, however, all of amino acid sequences were same.

• Applied Biological Chemistry
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• v.38 no.2
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• pp.118-122
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• 1995

Escherichia Coli ornithine transcarbamylase is the enzyme which catalyzes the L-citrulline biosynthesis from L-ornithine and carbamyl phosphate. To facilitate the purification of enzyme which will be used for many biochemical studies such as structure and function relationships and catalytic mechanisms, the cloning and expression of E. coli argI gene for ornithine transcarbamylase was conducted. argI was amplified from genomic DNA of E. coli strain of $DH5{\alpha}$, by polymerization chain reaction (PCR) method. The amplified argI gene was ligated to the prokaryotic expression vector pKK223-3 and used for transformation of E. coli TB2 which was deficient of ornithine transcarbamylase. The over-produced enzyme by the tnansformant was purified by ammonium sulfate fractionation, heat denaturation and affinity chromatography. The result of SDS denaturation gel electrophoresis for the purified enzyme showed a single band of about 38 kDa of ornithine transcarbamylase. Kinetic data for the expressed enzyme gave almost the s?????? values as those of the wild type enzyme. The $k_{cat}$, of the enzyme was $1.0{\times}10^5min^{-1}$, and $K_ms$ for ornithine and carbamyl phosphate were 0.35 mM and 0.06 mM, respectively.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.474-481
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• 1996

Since the commercially available rabbit anti-cyclin D3, generated from c-terminal 16 amino acid residues which are common to human and murine cyclin D3, is highly cross-reactive with many other cellular proteins of mouse, a new rabbit polyclonal anti-cyclin D3 has been raised by using murine cyclin D3 protein expressed at a high level in Escherichia coli as the immunogen. To express murine cyclin D3 protein in E. coli, the cyclin D3 cDNA fragment encoding c-terminal 236 amino acid residues obtained by polymerase chain reaction (PCR) was inserted into the NcoI/BamHI site of protein expression vector, pET 3d. Molecular mass of the cyclin D3 overexpressed in the presence of IPTG (Isopropyl $\beta$-D-thiogalactopyranoside) was approximately 26 kDa as calculated from the reading frame on the DNA sequence, and the protein was insoluble and mainly localized in the inclusion bodies that could be easily purified from the other cellular soluble proteins. When renaturation was performed following denaturation of the insoluble cyclin D3 protein in the inclusion bodies using guanidine hydrochloride, 4.4 mg of soluble form of cyclin D3 protein was produced from the transformant cultured in 100ml of LB media under the optimum conditions. Four-hundred micrograms of the soluble form of cyclin D3 protein was used for each immunization of a rabbit. When the antiserum obtained 2 weeks after tertiary immunization was applied to Western blot analysis, it was able to detect 33 kDa cyclin D3 protein in both murine lymphoma cell line BW5147.G.1.4 and human Jurkat T cells at 3,000-fold dilution with higher specificity to murine cyclin D3, demonstrating that the new rabbit polyclonal anti-murine cyclin D3 generated against c-terminal 236 amino acid residues more specifically recognizes murine cyclin D3 protein than does the commercially available rabbit polyclonal antibody raised against c-terminal 16 amino acids residues.

• Applied Chemistry for Engineering
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• v.34 no.5
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• pp.479-487
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• 2023

Copper is cost-effective and abundantly available as a biocidal coating agent for a wide range of material surfaces. Natural oxidation does not compromise the efficacy of copper, allowing it to maintain antimicrobial activity under prolonged exposure conditions. Furthermore, copper compounds exhibit a broad spectrum of antimicrobial activity against pathogenic yeast, both enveloped and non-enveloped types of viruses, as well as gram-negative and gram-positive bacteria. Contact killing of copper-coated surfaces causes the denaturation of proteins and damage to the cell membrane, leading to the release of essential components such as nucleotides and cytoplasm. Additionally, redox-active copper generates reactive oxygen species (ROS), which cause permanent cell damage through enzyme deactivation and DNA destruction. Owing to its robust stability, copper has been utilized in diverse forms, such as nanoparticles, ions, composites, and alloys, resulting in the creation of various coating methods. This mini-review describes representative coating processes involving copper ions and copper oxides on various material surfaces, highlighting the antibacterial and antiviral properties associated with different oxidation states of copper.

• Horticultural Science & Technology
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• v.18 no.4
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• pp.513-517
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• 2000

This experiment was carried out to detect the cymbidium mosaic virus (CymMV) and the odontoglossum ringspot virus (ORSV) in the seed-derived plantlets of Phalaenopsis imported from Taiwan by one-step reverse transcription-polymerase chain reaction (RT-PCR). Simple and rapid crude plant extracts for RT-PCR were prepared. The reverse transcription step was performed at $42^{\circ}C$ for 45 min and the following thermal cycling scheme was used for 36 reaction cycles: template predenaturation at $96^{\circ}C$ for 2 min, template denaturation at $96^{\circ}C$ for 30 s, primer annealing at $60^{\circ}C$ for 30 s, and DNA synthesis at $72^{\circ}C$ for 1 min. Of the 40 seed-derived plantlets of Phalaenopsis imported from Taiwan, all of them were infected with CymMV, but ORSV was not detected.

• Journal of Life Science
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• v.14 no.2
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• pp.309-314
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• 2004

Cyclic AMP receptor protein (CRP) is involved in the transcriptional regulation of more than 100 genes in E. coli. CRP dimer is converted into active form via the sequential conformation change of cAMP binding pocket, hinge region and HTH DNA binding motif by binding of cAMP. The temperature gradient gel electrophoresis (TGGE) was applied to CRP protein to know whether it was an efficient technique to study the conformational transitions and the thermal stability. TGGE showed the unfolding process of wild-type and S83G CRP proteins with the temperature gradient set from 29 to 71$^{\circ}C$ on nondenaturing polyacrylamide gel. Melting temperature (Tm) was 57$\pm$1 and 55$\pm$1$^{\circ}C$ for wild-type and S83G CRP, respectively in acidic buffer[89.8 mM Glycine and 24 mM Boric acid (pH 5.8)].

• Journal of Plant Biotechnology
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• v.5 no.2
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• pp.83-86
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• 2003

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two specific 22-mer primers located in coat protein gene of SPFMV. A 411 bp PCR-product was detected in virus infected plants as well as tissue culture raised sweet potato but not in healthy plants. For optimization of RT-PCR protocol, the optimum crude nucleic acid concentration, annealing temperature, primer concentration and numbers of PCR-cycle for maximum sensitivity and specificity were determined. The optimum condition for RT-PCR was as follows: RT-PCR reaction mixture was one-step mixture, containing 50 pmol of primer, 30 units of reverse transcriptase, 5 units of RNasin, and the crude nucleic acid extracts (200 ng). In RT-PCR, cDNA was synthesized at $42^{\circ}C$ for 45 min before a quick incubation on ice after pre-denaturation at $95^{\circ}C$ for 5 min. The PCR reaction was carried out for 40 cycles at $96^{\circ}C$ for 30 see, $63^{\circ}C$ for 30 sec, $72^{\circ}C$ for 1 min, and finally at $72^{\circ}C$ for 10 min. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having $95-98\%$ homology with published sequence data for SPFMV. The benefits of this RT-PCR based detection of SPFMV would be simple, rapid and specific.

• Korean Journal of Materials Research
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• v.11 no.2
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• pp.120-125
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• 2001

Generally, hydroxyapatite(HAp), zeolite, carbon molecular sieve , activated carbon and alumina are used as heavy metal ions adsorption materials. Among those adsorption materials, HAp which has good positive ion-exchange ability with metal ion, and zeolite are utilized in wastewater treatment. Most of water pollutions are caused by hazardous heavy metals ions as well as bacteria in waste water. In this study, a adsorption materials (HAP and zeolite) are ion-exchanged with a well known antimicrobial metal ions, such as $Ag^+,\;Cu^{2+},\;and\;Zn^{2+}$, in order to give a adsorption of heavy metal ions and a killing effects of bacteria. The antimicrobial effects of adsorption materials are observed using by E. Coli. The results show that there is a complete antimicrobial effect in the adsorption materials with $Ag^+$ at the concentration of $1{\times}10^{-4}$cell/$m\ell$ of E. Coli until 24 hours. However, there is not good antimicrobial effects in the adsorption materials with $Cu^{2+},\;and\;Zn^{2+}$ substitution. Feng et. al. showed the denaturation effects of silver ions which induces the condensed DNA molecules and losing their replication abilities.