• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.989-992
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• 2002

Sweet persimmon (Diospyros kaki Thunb.) is widely cultivated in the southern part of Korea and its cultivation is increasing. However, anthracnose disease caused by Colletotricuhum species is one of the major hinderances to the cultivation and production of sweet persimmon. Therefore, in the current study, PCR was used to specifically detect Colletotrichum spp., based on the sequences of the ITS II regions in the rDNA. Using the sequence data, CO-1 was designated to detect Colletotrichum together the with ITS 4 primer. The result showed that a single segment of ca. 500 bp was observed only in Colletotrichum, but not in any other fungal and bacterial isolates. The annealing temperatures and template DNA quantites were also investigated to identify optimal conditions for detection. Using these species-specific primers, a unique band was obtained at annealing temperatures ranging from $55^{\circ}C\;and\;61^{\circ}C$ and template DNA levels from 10 pg- $10{\mu}g$.

• Horticultural Science & Technology
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• v.33 no.2
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• pp.242-250
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• 2015

Insertional mutagenesis induced by T-DNA or transposon tagging offers possibilities for analysis of gene function. However, its potential remains limited unless good methods for detecting the target locus are developed. We describe a PCR technique for efficient identification of DNA sequences adjacent to the inserted T-DNA in a higher plant, Chinese cabbage (Brassica rapa ssp. pekinensis). This strategy, which we named variable argument thermal asymmetric interlaced PCR (VA-TAIL PCR), was designed by modifying a single-step annealing-extension PCR by including a touch-up PCR protocol and using long gene-specific primers. Amplification efficiency of this PCR program was significantly increased by employing an autosegment extension method and linked sequence strategy in nested long gene-specific primers. For this technique, arbitrary degenerate (AD) primers specific to B. rapa were designed by analyzing the Integr8 proteome database. These primers showed higher accuracy and utility in the identification of flanking DNA sequences from individual transgenic Chinese cabbages in a large T-DNA inserted population. The VA-TAIL PCR method described in this study allows the identification of DNA regions flanking known DNA fragments. This method has potential biotechnological applications, being highly suitable for identification of target genomic loci in insertional mutagenesis screens.

• Korean journal of applied entomology
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• v.56 no.4
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• pp.377-385
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• 2017

Estimates of evolutionary sequence divergence and inference of a phylogenetic tree for eight delphacid planthopper species were based on the full-length nucleotide sequence of the internal transcribed spacer 2 (ITS2) region. Size of the ITS2 DNA sequence varied from 550 bp in Sogatella furcifera to 699 bp in Nilaparvata muiri. Nucleotide sequence distance ($d{\pm}S.E.$) was lowest between N. muiri and N. bakeri ($0.001{\pm}0.001$), and highest between Ecdelphax cervina and Stenocranus matsumurai ($0.579{\pm}0.021$). Sequence distance between N. lugens and other planthoppers ranged from $0.056{\pm}0.008$ (N. muiri) to $0.548{\pm}0.021$ (S. matsumurai). In the neighbor-joining phylogenetic tree, all planthoppers were clustered separately into a species group, except N. muiri and N. bakeri. The ITS2 nucleotide sequence of N. lugens was used to design four loop-mediated isothermal amplification (LAMP) primer sets (BPH-38, BPH-38-1, BPH-207, and BPH-92) for N. lugens species-specific detection. After the LAMP reaction of three rice planthoppers, N. lugens, S. furcifera, and Laodelphax striatellus, with the four LAMP primer sets for 60 min at $65^{\circ}C$, LAMP products were observed in the genomic DNA of N. lugens only. In the BPH-92 LAMP primer set, the fluorescence relative to that of the negative control differed according to the amount of DNA (0.1 ng, 10 ng, and 100 ng) and incubation duration (20 min, 30 min, 40 min, and 60 min). At $65^{\circ}C$ incubation, the difference was clearly observed after 40 min with 10 ng and100 ng, but with a 60-min incubation period, the minimum DNA needed was 0.1 ng. However, there was little difference in fluorescence among all DNA amounts tested with 20 or 30 min incubations.

• Korean Journal of Poultry Science
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• v.35 no.1
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• pp.9-13
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• 2008

Salmonella enterica serotype gallinarum biovar Gallinarum or Pullorum and Salmonella enterica serotype Enteritidis are the most important diseases in poultry industry. Transitional diagnosis methods of these diseases such as direct isolation and identification by a biochemical test are time consuming with low specificity. In this study, we have focused on the suitable procedure for the rapid and accurate diagnosis of diseases derived from the three Salmonella strains. We initially confirmed Salmonella species by PCR using a specific ITSF/ITSR primer pair instead of biochemical test, and then the PCR-amplified phase 1 flagellin (fliC) using a specific fliCF/fliCR primer pair was digested with a restriction endonuclease, Bpm I and/or Bfa I, to discriminate among S. Pullorum, S. Gallinarum, and S. Enteritidis. We found that these methods could be applied to field isolates of the three Salmonella strains to detect and to discriminate rapidly for convenient diagnosis.

• The Korean Journal of Mycology
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• v.26 no.4 s.87
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• pp.466-477
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• 1998

Twenty-two Phellinus strains were characterized using colony morphologies and polymerase chain reaction (PCR) to divide into Phellinus linteus. There were some differences in mycelial growth and colony shapes among the strains when they were grown on various media such as PDA, MCM, MEA and YM. Phellinus linteus was slowly growing, formed golden-yellow colony, and produced blue pigment on PDA media. When the regions of internal transcribed spacer (ITS) were amplified from ribosomal RNA (rRNA) coding genes of P. igniarius and P. linteus strains by means of PCR, two types of band (700 bp and 800 bp) were appeared, respectively. For the amplified intergenic region I (IGRI), P. igniarius strains showed a different band among 500, 600, 700 and 800 bp according to the strains, whereas P. linteus strains did one specific band of 700 bp. By polymorphism analysis after digesting the amplified products with 6 different restriction enzymes, a band specific to P. linteus was generated when the products for ITS region were digested with HaeIII, suggesting that the enzyme digestion could provide effective method to distinguish between P. igniarius and P. linteus. And also, the analysis of genetic relationship showed that the genetic similarities were 89% and 95% in P. igniarius and P. linteus strains, respectively. Random amplification polymorphic DNA (RAPD) analysis using multiple primer sets and arbitrarily primed PCR (AP-PCR) with ITS3 primer could also result in a reproducible way to identify P. linteus strains.

• Journal of Ecology and Environment
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• v.29 no.2
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• pp.143-149
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• 2006

This study was conducted to identify native ectomycorrhizal (ECM) fungi colonizing Pinus densiflora for revegetation of abandoned coal mines in Korea. Seedlings of P. densiflora growing on coal mining spoils of a study site in Samcheok were collected. ECM roots were observed under stereomicroscope and their DNA were extracted from each root tip for a seedling for molecular identification. A PCR primer pair specific to fungi, ITS1F and ITS4, was used to amplify fungal DNA. Restriction enzymes, Alul and Hinfl were used for restriction fragment length polymorphism (RFLP). Combined with RFLP profiles and sequence analysis, total twenty one taxa were identified from the ECM root tips. Basidiomycetous fungi including Thelephoraceae, Pezizales, Laccaria, Pisolithus and Ascomycetous fungi including ericoid mycorrhizal fungi were identified from this study. Results showed that the most frequently found in the study sites was a species in Thelephoraceae. A possible use of ECM fungi identified in this study for the revegetation of abandoned coal mines with P. densiflora was discussed.

• BMB Reports
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• v.36 no.6
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• pp.529-532
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• 2003

The potential physiological role and technological application of the premature termination of DNA polymerization through the off-switch of exo+ polymerases were studied using 3' phosphorothioate-modified or unmodified primers with single base mismatch distal to the 3' terminus. With exonuclease-digestible unmodified primers, a gradient premature termination of DNA polymerization was observed when amplified with exo+ polymerases. With 3' allele specific phosphorothioate-modified primers, an efficient off-switch effect occurred in the discrimination of a single nucleotide polymorphism when directly using genomic DNA. Clearly, the off-switch of exo+ polymerases is useful in biomedical research.

• Mycobiology
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• v.51 no.6
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• pp.463-467
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• 2023

Soybean is one of the world's most widely cultivated food crops, and soybean seeds are supplied from national seed resources in Korea. However, the transmission of seed-borne diseases through infected soybean seeds is problematic. Among these diseases, soybean seed decay is caused by Diaporthe spp. Infecting the pods, and the infected seeds show rotting symptoms. Most diseased seeds are removed during the selection process; however, it is difficult to distinguish infected seeds that do not display symptoms. Hence, a sequencebased method was devised to screen Diaporthe-infected seeds. Based on the nuclear ribosomal internal transcribe spacer (ITS) region of the pathogen, a primer was designed to distinguish the infection from other soybean seed pathogens. As a result of the comparison between healthy and Diaporthe-diseased seeds by using the primers, Diaporthe was detected only in the diseased seeds. Therefore, it is possible to distribute healthy soybean seeds by detecting Diaporthe-diseased seeds at the genetic level using the Diaporthe-specific primers.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.114.2-115
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• 2003

Rice stripe virus causes severe damage to rice in Korea, Japan and China. RSV is a type member of the tenuivirus group and transmitted by the small brown planthopper, Laodeiphax striatellus, in a persistant manner. Until now, occurrence of RSV is limited in of southern part of Korea. But recently occurrence of RSV is increasing and spreading in central part of Korea including Chungcheong and Kyonggi province. So we analyzed recent occurrence trend of RSV which is increased and cloned and sequenced coat protein gene for isolating of RSV strain. Infected rice of each species(Ilpumbyeo, Sindongjinbyeo, Keumobyeo-2, Dongjinbyeo, Jongnambyeo, Samcheonbyeo, etc.)is collected, we extracted total RNA from infected leaves and detected RSV viral RNA by reverse transcription(RT)-PCR using specific primer of coat protein gene. The result of RT-PCR, we observed specific band. We already cloned cDNA from the band, is analyzing sequence variety and homology of each species.

• The Korean Journal of Mycology
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• v.26 no.4 s.87
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• pp.574-582
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• 1998

Five basidiocarps of Pholiota species have been collected from the areas of BubJu Temple for last two years, and identified to those of P. adiposa or Pholiota species. The taxonomy of these basidiocarps with the morphological aspects was compared with the analysis obtained from the polymorphisms of PCR-RAPD bands made after reacted with the random primers. The polymorphic variations were observed within the species of the basidiocarps, but not between genomic DNA's of the mycelia obtained and the basidiocarps. Several different bands made from the primers (28 and 36) in PCR-RAPD reactions were identified within the genus of Pholiota and speculated to be specific for the individual basidiocarp of P. adiposa collected. The primers employed here were considered to be very useful for distinguishing the individual isolates or basidiocarps collected from the fields. Also, the basidiospores were obtained from the sporeprints of the above basidiocarps as a simple agar and confirmed with observations of clamp connection under microscopes. The matings of them indicated the 'tetrapolar' type, being different from the 'bipolar' type reported by Japanese basidiocarps of P. adiposa or P. nameko. Based on our work, the edible fungi collected were speculated to be a new breeding resource for those of Pholiota commercialized in Japan.