• Title/Summary/Keyword: RFLP.

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Strain differentiation of canine distemper virus by reverse transcriptase polymerase chain reaction and restriction fragment length polymorphism analysis

  • An, Dong-jun;Song, Jae-young;Lee, Joung-bok;Park, Jong-hyeon;Shin, Jin-ho;Kim, Yong-hwan;An, Soo-hwan
    • Korean Journal of Veterinary Research
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    • v.39 no.4
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    • pp.778-785
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    • 1999
  • To detect CDV RNA in clinical samples and differentiate prevailing CDV virulent strains affecting susceptible animals from attenuated vaccine strains, we performed RT-PCR, RFLP, and sequencing. CDV specific primers were generated from the middle part of nucleocapsid gene. The expected size of PCR products, 519 bp, was observed in tissues of Jindo dog, poodle dog, badger, fourteen of nineteen blood samples as well as 5 vaccine strains including domestic and imported products. The PCR products obtained from tissues and PBMCs of infected animals were digested to 317- and 202-bp fragments by Bam HI, but the products obtained from four of five vaccine strains and Lederle strain were not digesed by Bam HI. Only one vaccine strain of which the PCR products were digested by Bam HI was confirmed as imported vaccine, modified Synider Hill strain. Based on seqencing data obtained from the 519-bp products, it was confirmed that Bam HI restriction site tends to be conserved in field isolates compared to the commercially available attenuated vaccine strains. Partial nucleotide sequences of CDV NP gene obtained from tissues of Jindo dog, poodle and badger shared 100% homology each other, whereas the nucleotide sequences showed 96.3, 96.5, 93.6 and 93.4% homology with Yanaka (virulent), Han95 (virulent), Lederle (attenuated) and Onderstepoort (attenuated) strain, respectively.

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Phytophthora citricola, a Causal Agent of Jujube (Zizyphus jujuba) Fruit Rot (대추 역병균의 Phytophthora citricola의 동정)

  • 지형진;임양숙;정기채;조원대
    • Korean Journal Plant Pathology
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    • v.14 no.5
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    • pp.402-407
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    • 1998
  • Phytophthora rot on jujube fruit has occurred at several cultivation areas in Kyung-buk and Kyung-nam provinces. Symptoms consisted of brownish to reddish rot on fruits resulting in early drop or mummification. The causal fungus isolated from infected fruits and adjacent leaf stalks was identified as Phytophthora citricola, which has never been reported in Korea. Sporangia were semi-papillate, noncaducous and highly variable in shapes. Plerotic oospores with paragynous antheridia were abundant is single cultures. Sporangia of two isolates were measured as 38-76$\times$20-40 ${\mu}{\textrm}{m}$ and averaged 51.4$\times$27.0 and 55.6$\times$36.0 ${\mu}{\textrm}{m}$. Oogonia were ranged from 26 to 36 ${\mu}{\textrm}{m}$ and averaged 31.3 and 32.0 ${\mu}{\textrm}{m}$. Colony pattern was slightly radiated with sparse aerial mycelia on common media. Minium, optimum and maximum temperatures for mycelial growth were recorded at 7, 25, and 32$^{\circ}C$, respectively. Among tested media, 10% V8A was the best and $25^{\circ}C$ was better than 15$^{\circ}C$ for oospore formation of the fungus. The jujube isolates of P. citricola were readily differentiated from other closely related species in the genus, namely; P. nicotianae, P. citrophthora, P. cactorum, P. capsici, and P. plalmivora on the basis of PCR-RFLP of r-DNA. The fungus showed strong pathogenicty to jujube, apple, pear, orange, persimmon and eggplant, and relatively weak to citron, tomato, pepper and cucumber. In this study, P. citrocola is firstly identified and jujube fruit rot caused by the fungus is recorded as a new disease in Korea.

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Unrecorded Phytophthora Diseases of Flowering Plants Caused by Phytophthora nicotianae in Korea (Phytophthora nicotianae에 의한 국내 미기록 화훼류 역병)

  • 지형진;김완규;김재영;임성언
    • Korean Journal Plant Pathology
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    • v.14 no.5
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    • pp.452-457
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    • 1998
  • Thirty-eight isolates of Phytophthora sp. caused rots on roots and basal stems were collected from five flowering plants from 1992 to 1997 at eight cultivation areas in Korea. All the isolates were identified as P. nicotianae based on following characteristics. The fungus produced markedly papillate, not caducous and ovoid to spherical sporangia, abundant chlamydospores, and small oospores with amphigynous antheridia only when paired with either A1 or A2 mating type. All isolates grew well at 35$^{\circ}C$ and showed distinct arachnoid colony patterns on CMA and PDA. Sizes of sporangia and chlamydospores of five representative isolates from each plant averaged 43-52$\times$30-38 ${\mu}{\textrm}{m}$ and 28 ~34 ${\mu}{\textrm}{m}$. Mating type of the isolates was either A1 or A2, and oogonia and oospores were measured as 28~31 ${\mu}{\textrm}{m}$ and 21~25 ${\mu}{\textrm}{m}$. PCR-RFLP analysis of rDNA of the five isolates resulted that restriction band patterns of the small subunit and ITS regions were identical to a perilla isolate of P. nicotianae, but distinct from P. cactorum and P. capsici. Cross inoculation tests showed that the five isolates had pathogenicity to lily, christmas cactus, anthurium, baby's breath and carnation with different degrees. However, each isolate showed stronger pathogenicity to its corresponding original host than others. Among five lily cultivars Georgia and Quririna were more susceptible than Napoli and others. This is first report of Phytophthora root and stem rot of lily, Christmas cactus, anthurium, baby's breath and monochoria in Korea.

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Stem Rot on Adzuki Bean (Vigna angularis) Caused by Rhizoctonia solani AG 4 HGI in China

  • Sun, Suli;Xia, Changjian;Zhang, Jiqing;Duan, Canxing;Wang, Xiaoming;Wu, Xiaofei;Lee, Suk-Ha;Zhu, Zhendong
    • The Plant Pathology Journal
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    • v.31 no.1
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    • pp.67-71
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    • 2015
  • During late August and early September 2011, stem rot symptoms were observed on adzuki bean plants (Vigna angularis) growing in fields located in Beijing and Hebei Province, China, respectively. In this study, four isolates were obtained from infected stems of adzuki bean plants. Based on their morphology, and sequence and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses of the ribosomal DNA internal transcribed spacers (rDNA-ITS) region, the four isolates were identified as Rhizoctonia solani in anastomosis group (AG) 4 HGI. Pathogenicity tests showed that all isolates were strongly pathogenic to adzuki bean and resulted in serious wilt symptoms which was similar to observations in the fields. Additionally, the isolates infected several other crops and induced related rot on the roots and basal stems. To our knowledge, this is the first report of Rhizoctonia solani AG 4 HGI causing stem rot on adzuki bean.

Characterization of an Isolate of Cucumber mosaic virus Isolated from Chinese aster (Callistephus chinensis) (과꽃에서 분리한 Cucumber mosaic virus의 성질)

  • Oh, Sun-Mi;Kim, Sung-Ryul;Hong, Jin-Sung;Ryu, Ki-Hyun;Lee, Gung-Pyo;Choi, Jang-Kyung
    • Research in Plant Disease
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    • v.14 no.3
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    • pp.229-232
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    • 2008
  • An isolate of Cucumber mosaic virus (CMV), designated as Cas-CMV, was isolated from Chinese aster (Callistephus chinensis) showing severe mosaic symptom, and its properties was compared to the well-characterized Fny-CMV (subgroup IA) and As-CMV (subgroup IB) by host reaction in several indicator plants, dsRNA analysis, RT-PCR analysis, and restriction enzyme profile of the PCR products. Cas-CMV differed markedly in their host reaction to Fny-CMV or As-CMV in Cucurbita pepo cv. Black beauty. In the zucchini squash, all strains induced chlorotic spot on inoculated leaves and mosaic symptoms on upper leaves. However, symptoms induced by Cas-CMV were developed lethal necrosis on the young plants 15 to 20 days after inoculation. In experiments of dsRNA analysis and RT-PCR analysis, properties of Cas-CMV was come within subgroup I CMV. Moreover, restriction enzyme analysis using HindIII of the RT-PCR products showed that Cas-CMV belong to a member of CMV subgroup IA.

Polychlorobiphenyl (PCB) 토양오염복원: PCB 제거 토양미생물들의 군집과 기능을 효과적으로 분석하는 신 genomics 방법개발에 관한 연구

  • Park Jun-Hong
    • Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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    • 2005.04a
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    • pp.28-30
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    • 2005
  • Because of high population diversity in soil microbial communities, it is difficult to accurately assess the capability of biodegradation of toxicant by microbes in soil and sediment. Identifying biodegradative microorganisms is an important step in designing and analyzing soil bioremediation. To remove non-important noise information, it is necessary to selectively enrich genomes of biodegradative microorganisms fromnon-biodegradative populations. For this purpose, a stable isotope probing (SIP) technique was applied in selectively harvesting the genomes of biphenyl-utilizing bacteria from soil microbial communities. Since many biphenyl-using microorganisms are responsible for aerobic PCB degradation In soil and sediments, biphenyl-utilizing bacteria were chosen as the target organisms. In soil microcosms, 13C-biphenyl was added as a selective carbon source for biphenyl users, According to $13C-CO_2$ analysis by GC-MS, 13C-biphenyl mineralization was detected after a 7-day of incubation. The heavy portion of DNA(13C-DNA) was separated from the light portion of DNA (12C-DNA) using equilibrium density gradient ultracentrifuge. Bacterial community structure in the 13C-DNAsample was analyzed by t-RFLP (terminal restriction fragment length polymorphism) method. The t-RFLP result demonstates that the use of SIP efficiently and selectively enriched the genomes of biphenyl degrading bacteria from non-degradative microbes. Furthermore, the bacterial diversity of biphenyl degrading populations was small enough for environmental genomes tools (metagenomics and DNA microarrays) to be used to detect functional (biphenyl degradation) genes from soil microbial communities, which may provide a significant progress in assessing microbial capability of PCB bioremediation in soil and groundwater.

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Discrimination of Korean Cattle (Hanwoo) with Imported Beef from USA Based on the SNP Markers

  • Shim, Jung-Mi;Seo, Dong-Won;Seo, Seong-Won;Kim, Jong-Joo;Min, Dong-Myung;Kim, Ik-Chul;Jeon, Jin-Tae;Lee, Jun-Heon
    • Food Science of Animal Resources
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    • v.30 no.6
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    • pp.918-922
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    • 2010
  • Due to the large amount of beef imported from the USA to Korea, Korean consumers have become increasingly interested in the country of origin since it can affect market prices. Previously, Bos indicus and Bos taurus-specific markers were developed for the purpose of cattle breed identification, specifically discrimination of Australian beef. In this study, six SNP markers derived from Illumina 50K bovine SNP chip data were used for the discrimination between Korean cattle (Hanwoo) and imported beef from USA. PCR-RFLP genotyping methods were also developed, which indicates that these markers can be applied relatively easily compared to other markers. Taking into account a discrimination rate of 55% based on MC1R marker between Hanwoo and imported beef from USA, two additional markers, SNPs 23803 and 34776, were ideal and resulted in probability of identification of 0.942 and probability of misjudgment of 0.03. Therefore, the markers developed in this study can greatly contribute to the correct discrimination between beef from USA and Hanwoo beef.

Bacterial Diversity in the Mud Flat of Sunchon Bay, Chunnam Provice, by 16S rRNA Gene Analysis (16S rRNA 유전자 분석에 의한 전남 순천만 갯벌의 세균 다양성)

  • 이명숙;홍순규;이동훈;배경숙
    • Korean Journal of Microbiology
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    • v.37 no.2
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    • pp.137-144
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    • 2001
  • In order to investigate the diversity of bacterial community in the mud flat of Sunchon Bay, Chunnam province, diversity of amplified 16S rDNA was examined. Total DNA was extracted from sediment soils and 16S rDNAs were amplified using PCR primers based on the universally conserved sequences in bacteria. Clonal libraries were constructed and 111 clones were examined by amplified rDNA restriction analysis (ARDRA) using HaeIII. Clones were clustered based on restriction patterns using computer program, GelCompar II. One hundred different RFLP types were detected from 111 clones. The 20 clones were selected and sequenced according to dendrograms derived from ARDRA, to cover most of the bacterial diversity in the clone libraries. None of the clones were identical to any representatives in the Ribosomal Database Project small subunit RNA databases and GenBank. All sequences showed between 77 and 96.8% similarity to the known 16s rRNA sequence from cultured organisms. The 20 clones sequenced fell into seven major lineages of the domain Bacteria: alpha-, delta-, gamma-Proteobacteria, low G+C Gram positive bacteria, high G+C Gram positive bacteria, Sphingobacteria (Cytophaga) and Cyanobacteria (chloroplast). Among the clones, the Proteobacteria were dominant.

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Molecular Phylogenetic Analysis of Botrytis cinerea Occurring in Korea (우리나라에 발생하는 잿빛곰팡이병균 Botrytis cinerea의 분자계통학적 유연관계)

  • Back, Chang-Gi;Lee, Seung-Yeol;Jung, Hee-Young
    • The Korean Journal of Mycology
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    • v.42 no.2
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    • pp.138-143
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    • 2014
  • Several isolates were collected from apple, pepper, strawberry, cucumber and tomato having typical gray mold symptoms. All the isolates were identified as Botrytis cinerea by using morphological characteristics and PCR-RFLP method. It was difficult to analyze the phylogenetic relationship of these isolates by using ITS region, HSP60 and G3PDH because these genes were highly homologous in their nucleotide in inter-species of B. cinerea and intra-species of genus Botrytis. However, phylogenetic analysis using combined sequences (RPB2, HSP60 and G3PDH genes) clearly showed that all isolate of B. cinerea were different from Botrytis spp. Furthermore, it was also confirmed that strawberry isolate was distantly related to apple, pepper, cucumber and tomato isolates that were closely related to each other in nucleotide level.

Gene Polymorphism of TNF-α in Korean Generalized Aggressive Periodontitis (한국인 전반적 급진성 치주염 환자에서 발견된 TNF-α 유전자의 다변성)

  • Kim, Il-Shin
    • Journal of Digital Convergence
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    • v.14 no.1
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    • pp.321-326
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    • 2016
  • The aim of this study was to evaluate the association between TNF polymorphism and generalized aggressive periodontitis (GAP) in Korean subjects. The study population consisted of 60 subjects with GAP and 81 reference group. Genomic DNA was extracted from the buccal swabs and the polymorphisms of $TNF-{\alpha}-308$, -238 promoter genes, $TNF-{\beta}+252$ and TNFR 2+587 were determined by PCR-RFLP using restriction enzymes. The genotype distribution in the GAP were 3.2%, 38.7%, and 82.35% for A/A, A/G and G/G genotypes of $TNF-{\alpha}-308$. At the position of $TNF-{\alpha}-238$, the genotype distribution in the GAP were 25.5% and 74.5% for A/G and G/G genotypes. Allele A frequency of $TNF-{\alpha}-238$ were 67.6% in GAP and 72.2% in reference group. According to these findings, the polymorphism at $TNF-{\alpha}-308$ and -238 may be associated with GAP in Korean.