• The Korean Journal of Mycology
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• v.46 no.4
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• pp.467-477
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• 2018

Colletotrichum circinans, an anthracnose pathogen, causes serious damage to onions worldwide. In this study, specific molecular markers were developed to detect C. circinans accurately and quickly with both conventional and real-time PCR methods. The cirTef-F/cirTef-R and cirTu-F/cirTu-R primer sets, which are specific for C. circinans, were constructed by analyzing $tef-1{\alpha}$ and ${\beta}-tubulin$ genes in the fungus. Using the conventional PCR method, 100 pg and 1 ng of fungal DNA could be detected using the cirTef-F/cirTef-R and cirTu-F/cirTu-R sets, respectively. Using the real-time PCR method, 10 pg and 100 pg of fungal DNA could be detected more sensitively with the cirTef-F/cirTef-R and cirTu-F/cirTu-R sets, respectively. Detection of C. circinans from the artificially infected onion seeds was possible by using both conventional and real-time PCR methods and the developed cirTef-F/cirTef-R primer set. The PCR markers specific for C. circinans developed in this study may enhance the efficiency of fungal pathogen detection in imported vegetables and seeds.

• Research in Plant Disease
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• v.14 no.3
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• pp.219-222
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• 2008

Real time PCR was used to discriminate Colletotrichum acutatum and C. gloeosporioides for analysis of population density. Two primers, caInt2 and cgint, used for conventional PCR to discriminate two species were modified with fluorescent dye to make probe for real time PCR. Fluorescence signals were successfully detected by fCaInt2 and vCgint probe coupled with primer pair Unicon and Unicor1 resulting in discrimination of C. acutatum and C. gloeosporioides by comparison of delta Rn value.

• Korean Journal of Clinical Laboratory Science
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• v.36 no.1
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• pp.33-37
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• 2004

In order to study the effect of temperature of denaturation, annealing and extension and cycles on amplification of DNA by PCR method, We isolated the hepatitis B virus DNA from hepatitis B patient blood and compared the density of DNA amplified by Reference PCR Program (denaturation at $94^{\circ}C$ for 30 sec., annealing at $60^{\circ}C$ for 1 min., extension at $72^{\circ}C$ for 1 min., holding at $72^{\circ}C$ for 5min., 30 cycles) that is usually used in laboratory to the density of DNA amplified by PCR program changed only the denaturation temperature or annealing temperature or extension temperature. We amplified about 341bp of hepatitis B virus DNA by Reference PCR Program from hepatitis patient blood, but the DNAs denatured at $72^{\circ}C$ or $60^{\circ}C$ were not detectable on photoradiography film. The DNA amplified at $37^{\circ}C$ of annealing temperature was not detectable, but the DNA annealed at $72^{\circ}C$ was detectable the lower density of DNA than the DNA amplified by Reference PCR Program. Each DNA amplified by PCR program changed only the extension temperature to $37^{\circ}C$ or $60^{\circ}C$ was almost same density as DNA amplified by Reference PCR Program. We compared the density of hepatitis B virus DNA amplified by Reference PCR Program for 30 cycles, 20 cycles, 10 cycles, and 5 cycles. The DNA cycled for 20 cycles was not amplified well as cycled for 30 cycles, but the DNA was detectable on the photoradiography film. The DNAs amplified for 10 cycles or 5 cycles were not detectable on photoradiorgaphy film. The concentration of hepatitis B virus DNA amplified in Reference PCR condition for 30 cycles, 20 cycles, 10 cycles, and 5 cycles were $72{\mu}g/m{\ell}$, $83{\times}10^{-3}{\mu}g/m{\ell}$, $27{\times}10^{-6}{\mu}g/m{\ell}$, and nondetectable, respectively.

• Korean Journal of Plant Resources
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• v.16 no.1
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• pp.40-48
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• 2003

Cylindrocarpon destructans is the major pathogen inducing the root rot disease in ginseng. Up to now, there is no reliable and convenient method to analyze the spore density or population of this pathogen in ginseng-growing soil or any contaminated farmlands. Therefore, it will be very valuable to develop a new and reliable method in detecting the spore of this pathogen. In this study, a molecular biological technique using two step nested PCR method, was developed. Two universal ITS primers, ITS5F and ITS4R were used in the first round of PCR to amplify a fragment of ITS region from the genomic DNA of C. destructans. The specific prmers Nest 1 and Nest 2 were designed and used in the second round of PCR to amplify a inner fragment from the first round PCR product of C. destructans. C. destructans spore, only soil samples from the diseased ginseng farm produced the positive bands, suggesting its usefulness in detecting the C. destructans spores in soil samples. Thus it is recommended to first extract the whole genomic DNA from soil samples and use it for the PCR reaction, thereby eliminating the inhibitory activity of soil components.

• The Korean Journal of Mycology
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• v.46 no.2
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• pp.186-192
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• 2018

Rapid and accurate detection methods for Colletotrichum coccodes, an anthracnose pathogen of pepper and tomato, were developed using PCR. A specific primer set, coccoTef-F/coccoTef-R, which was constructed by analyzing tef-$1{\alpha}$ genes from 13 species and 22 strains of Colletotrichum, could specifically detect C. coccodes at a level of 10 ng by conventional PCR method and at 10 pg by real-time PCR. The PCR-based methods were also capable of detecting C. coccodes in pepper and tomato seeds artificially infected with the pathogen. The developed PCR methods can be applied for rapid and accurate inspection of C. coccodes in the seeds intended for export or import.

• 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.

• Research in Plant Disease
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• v.11 no.1
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• pp.48-55
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• 2005

Cylindrocarpon destructans is a soil-borne plant pathogenic fungus causing root rot on ginseng and trees. Rapid and exact detection of this pathogen was practiced on ginseng seedlings by nested PCR using speciesspecific primer set. The second round of PCR amplification by Dest 1 and Dest 4 primer set formed 400 bp of species-specific fragment of C. destructans from the product of first round of amplification by ITS 1 and ITS 4 primer set. In the PCR sensitivity test based on DNA density, nested PCR detected to the limit of one fg and it meant the nested PCR could detect up to a few spores of C. destructans. Also, nested PCR made it possible to detect the pathogen from ginseng seedlings infected by replantation on artificial infested soil. Our nested PCR results using species-specific primer set could be utilized for diagnosis of root rot disease in ginseng cultivation.

• Journal of Life Science
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• v.26 no.9
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• pp.1007-1014
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• 2016

A highly efficient, rapid, and reliable multiplex polymerase chain reaction based method for distinguishing ten species of genus Cynoglossus (C. senegalensis, C. abbreviates, C. macrolepidotus, C. arel, C. semilaevis, C. interruptus, C. joyneri, C. lingua, C. robustus, and C. monodi) is described. The species-specific primer sets were designed base on the cytochrome oxidase subunit I gene (1,500 bp). The optimal PCR conditions and primers were selected for ten of Cynoglossus species to determine target base sequences using single PCR. Multiplex PCR using the ten pairs of primers either specifically amplified a DNA fragment of a unique size or failed, depending on each species DNA. The length of amplification fragment of 208 bp for C. senegalensis, 322 bp for C. abbreviates, 493 bp for C. macrolepidotus, 754 bp for C. arel, 874 bp for C. semilaevis, 952 bp for C. interruptus, 1,084 bp for C. joyneri, 1,198 bp for C. lingua, 1,307 bp for C. robustus, and 1,483 bp for C. monodi with the species-specific primers, visualized by agarose gel electrophoresis, allowed perfectly distinction of the Cynoglossus species. The multiplex PCR assay can be easily performed on multiple samples and attain final results in less than 6 hours. This technique should be a useful addition to the molecular typing tools for the tentative identification of Cynoglossus species.

• Journal of fish pathology
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• v.5 no.2
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• pp.143-152
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• 1992

Metallothionein is an essential and common protein to regulate the intracellular concentration of heavy metals, which exist in most organisms from bacteria to vertebrates. Although the detailed function of metallothianein has not been fully identified until yet, it may be involoved in the cellular protection against the heavy metal toxicity and in the global regulation of several other genes and the expression of metalloproteins. We have cloned the full cDNA clone of metallothionein gene in Channel Catfish by Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR) starting from poly(A)-containing mRNAs. All PCR fragments have been subcloned into EcoRV site of pBluescript SK+ and dT-tailed at Smal site of pUC19, then PCR products are recovered by the double digestion of recombinant plasmids wiht EcoRI and HindIII, which are adjacent to EcoRV site in multicloning sites or by rapid PCR screening. The nucleotide sequence analysis of pMT150(one of the PCR clones) showed high homology with several other piscine metallothionein cDNA genes.

• Journal of IKEEE
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• v.23 no.1
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• pp.295-301
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• 2019

A heater in a portable real-time polymerase chain reaction(PCR) system is one of the important factors for controlling the PCR thermocycle precisely. Since heaters are integrated on a small-sized PCR chip for rapid heating and fabricated by semiconductor processes, the cost of producing PCR chips is high. Here, we propose to use chip resistors as an inexpensive and accurate temperature control method. The temperature distribution was simulated using one or two chip resistors on a real-time PCR chip and the PCR chip with uniform temperature distribution was fabricated. The temperature rise and fall rates were $18^{\circ}C/s$ and $3^{\circ}C/s$, respectively.