• The Plant Pathology Journal
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• 제25권3호
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• pp.247-255
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• 2009

Root-knot nematode species, such as Meloidogyne hapla, M. incognita, M. arenaria, and M. javanica are the most economically notorious nematode pests, causing serious damage to a variety of crops throughout the world. In this study, DNA sequence analyses were performed on the D3 expansion segment of the 28S gene in the ribosomal DNA in an effort to characterize genetic variations in the three Meloidogyne species obtained from Korea and four species from the United States. Further, PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism), SCAR (Sequence Characterized Amplified Region) PCR and RAPD (Randomly Amplified Polymorphic DNA) were also utilized to develop methods for the accurate and rapid species identification of the root-knot nematode species. In the sequence analysis of the D3 expansion segment, only a few nucleotide sequence variations were detected among M. incognita, M. arenaria, and M, javanica, but not M. hapla. As a result of our haplotype analysis, haplotype 5 was shown to be common in M. arenaria, M. incognita, M. javanica, but not in the facultatively parthenogenetic species, M. hapla. PCR-RFLP analysis involving the amplification of the mitochondrial COII and large ribosomal RNA (lrRNA) regions yielded one distinct amplicon for M. hapla at 500 bp, thereby enabling us to distinguish M. hapla from M. incognita, M. arenaria, and M. javanica reproduced via obligate mitotic parthenogenesis. SCAR markers were used to successfully identify the four tested root-knot nematode species. Furthermore, newly attempted RAPD primers for some available root-knot nematodes also provided some species-specific amplification patterns that could also be used to distinguish among root-knot nematode species for quarantine purposes.

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.176-181
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• 2002

In order to isolate and identify Bifidobacterium spp. that originated in Korea, feces were sampled from healthy Korean adults and children living in three villages, the first having a history of longevity and the other two where the diet did not include fermented milk or any pharmaceutical preparations. Through the use of Gram staining and microscopic examination for cell morphology, 23 bacterial strains presumed to be the Bifidobacterium genus were isolated from the feces of 13 out of a total of 59 Korean people. To identify the Bifidobacterium strains at the genus level, these bacteria were then analyzed by TLC and the fructose-6-phosphate phosphoketolase (F6PPK) test. The result showed that 22 of the isolated strains were confirmed to be members of the genus Bifidobacterium. All of these bifidobacteria were also identified as Bifidobacterium spp. by the fermentation test. Using a RFLP analysis, an attempt was made to identify the Bifidobacterium spp. that had been isolated from both Korean adults and children. In a genomic Southern blot analysis after digestion with two restriction enzymes (EcoRI, HindIII), all of the 14 randomly selected Korean isolates showed patterns identical to those of three different B. longum species. Another restriction enzyme, CfoI (4-bp recognition enzyme), was then used to identify the strain. Interestingly, all the Korean isolates were identified as B. longum ATCC 15708, indicating that a RFLP analysis was effective for identifying Bifidobacterium spp. at both the strain and species levels.

• 대한수의학회지
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• 제38권2호
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• pp.304-313
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• 1998

Sixteen Korean field transmissible gastroenteritis viruses (TGEVs) were isolated using swine testicular cell (STC) and the genomic diversity of them was analyzed. All TGEV isolates produced a typical cytopathic effect in STC and were confirmed as TGEV by immunofluorescence assay using monoclonal antibody against TGEV and PCR using TGEV specific primers. RNAs from TGEV field isolates and vaccine TGEV were extracted and amplified by RT and PCR. The RT-PCR products were digested with selected restriction enzymes and analyzed RFLP patterns. The N-terminal end region of S gene and ORF 3 and 3-1 genes of TGEV amplified by TGEV specific primer pairs seemed to be conserved. Most specific variations were detected in S gene amplified by TGEV 4/6 primer pairs which includes antigenic sites A and D. When the PCR products were treated with Sau3AI and Ssp I, Bvac(vaccine strain), field isolates 133 and 347 were differentiated from Miller and Purdue types. In the case of D5 field isolates, it was classified into Purdue type by Sau 3AI but classified into independent TGEV by Ssp I. Two different TGEV strains from D2 sample were confirmed by plaque purification and RT-PCR-RFLP analysis. To investigate the change occurring in TGEV genome after serial passage, the TGEV P44 strain was passaged through STC. There were specific changes in S gene and a large deletion was observed in ORF 3 and 3-1 genes. These studies showed that a distinct difference in genome exists among TGEV field isolates.

• Journal of Ecology and Environment
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• 제31권4호
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• pp.309-316
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• 2008

To develop a monitoring method for soil microbial communities in rice paddy fields, we used terminal restriction fragment length polymorphism (T-RFLP) to compare soil bacterial community structure in rice paddy fields experiencing different management practices: organic practices, conventional practices without a winter barley rotation, and conventional practices with a winter barley rotation. Restriction fragment length profiles from soils farmed using organic practices showed very different patterns from those from conventional practices with and without barley rotation. In principal component analyses, restriction fragment profiles in organic practice samples were clearly separated from those in conventional practice samples, while principal component analysis did not show a clear separation for soils farmed using conventional practices with and without barley rotation. The cluster analysis showed that the bacterial species compositions of soils under organic practices were significantly different from those under conventional practices at the 95% level, but soils under conventional practice with and without barley rotation did not significantly differ. Although the loadings from principal component analyses and the Ribosomal DNA Project II databases suggested candidate species important for soils under organic farming practices, it was very difficult to get detailed bacterial species information from terminal restriction fragment length polymorphism. Rank-abundance diagrams and diversity indices showed that restriction fragment peaks under organic farming showed high Pielou's Evenness Index and the reciprocal of Simpson Index suggesting high bacterial diversity in organically farmed soils.

• 대한바이러스학회지
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• 제27권2호
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• pp.169-176
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• 1997

In this study, the feasibility of identification and genotypic differentiation of enteroviruses was investigated by using nested reverse transcription-polymerase chain reaction (nested RT-PCR), single-stranded conformation polymorphism (SSCP), and restriction fragment length polymorphism (RFLP) techniques. Two hundred seventy-four clinical samples were assayed by both nested RT-PCR and tube culture method using MRC-5 and MK cells; 58 (86.6%) out of 67 enterovirus culture-positive samples contained enteroviral RNA. In addition, 114 (55.1%) of 207 samples from patients with suspected enteroviral CNS disease with negative viral cultures were positive by the nested RT-PCR. The nested RT-PCR products were genotyped by the SSCP method and the results were compared with serotypes. We could differentiate 6 subtypes, 3 of which are similar to coxsackievirus B3, B5, echovirus 11, plus 3 other subtypes. RFLP cleaved with Sty I, Bgl I, and Xmn I yielded characteristic patterns for each laboratory strains. This study demonstrates the usefulness of the RT-PCR for the rapid diagnosis of enterovirus infection and the potentials of the SSCP method for differentiation of enterovirus strains.

• Parasites, Hosts and Diseases
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• 제52권1호
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• pp.79-83
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• 2014

Human taeniases had been not uncommon in the Republic of Korea (=Korea) until the 1980s. The prevalence decreased and a national survey in 2004 revealed no Taenia egg positive cases. However, a subsequent national survey in 2012 showed 0.04% (10 cases) prevalence of Taenia spp. eggs suggesting its resurgence in Korea. We recently encountered 4 cases of Taenia saginata infection who had symptoms of taeniasis that included discharge of proglottids. We obtained several proglottids from each case. Because the morphological features of T. saginata are almost indistinguishable from those of Taenia asiatica, molecular analyses using the PCR-RFLP and DNA sequencing of the cytochrome c oxidase subunit 1 (cox1) were performed to identify the species. The PCR-RFLP patterns of all of the 4 specimens were consistent with T. saginata, and the cox1 gene sequence showed 99.8-100% identity with that of T. saginata reported previously from Korea, Japan, China, and Cambodia. All of the 4 patients had the history of travel abroad but its relation with contracting taeniasis was unclear. Our findings may suggest resurgence of T. saginata infection among people in Korea.

• Parasites, Hosts and Diseases
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• 제44권1호
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• pp.27-33
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• 2006

Two species of Cryptosporidium are known to infect man; C. hominis which shows anthroponotic transmission between humans, and C. parvum which shows zoonotic transmission between animals or between animals and man. In this study, we focused on identifying genotypes of Cryptosporidium prevalent among inhabitants and domestic animals (cattle and goats), to elucidate transmittal routes in a known endemic area in Hwasun-gun, Jeollanam-do, Republic of Korea. The existence of Cryptosporidium oocysts was confirmed using a modified ZiehlNeelsen stain. Human infections were found in 7 $(25.9\%)$ of 27 people examined. Cattle cryptosporidiosis cases constituted 7 $(41.2\%)$ of 17 examined, and goat cases 3 $(42.9\%)$ of 7 examined. Species characterizations were performed on the small subunit of the rRNA gene using both PCR-RFLP and sequence analysis. Most of the human isolates were mixtures of C. hominis and C. parvum genotypes and similar PCR-RFLP patterns were observed in cattle and goat isolates. However, sequence analyses identified only C. hominis in all isolates examined. The natural infection of cattle and goats with C. hominis is a new and unique finding in the present study. It is suggested that human cryptosporidiosis in the studied area is caused by mixtures of C. hominis and C. parvum oocysts originating from both inhabitants and domestic animals.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 1993년도 학술대회지
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• pp.138-142
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• 1993

Korean ginseng has been widely used as medicine from ancient times in Asia. Current breeding efforts in Korea include the individual plant selection and the subsequent pure - line isolation, and considerable number of lines with desirable traits have thus been isolated. However, there were rare data on genetic maker and its analysis for selection of superior varieties. For taxonomic characterization and development of genetic markers for ginseng breeding, molecular biological methods including the RFLP and RAPD methods were applied. Cytoplasmic DNA of ginseng was analyzed for RFLP analysis. However. there is no different pattern among the chloroplast DNA or mitochondrial DNA of variants. In the case of RAPD analysis, the band patterns using 4 of 10 RAPD primers show the distinctive polymorphism among 9 ginseng variants, and lines, and Similarity Index(SI) on polymorphism was calculated for the extent and nature of these variabilities in ginseng. The sequences of 4 selected primers were TGCCGAGCTG, AATCGGGCTG. GAAACGGGTG, and GTGACGTAGG. By SI based on the polymorphic band patterns, Chungkyung - Chong and Hwangskoog - Chong, and JakyungChong 81783 and Jinjakyung of Russia showed the most close SI. The data of KG10l coincided with the fact that it was released from Hwangskoog - Chong. and Jakyung - Chong 81783 and Jinjakyung of Russia showed the most close SI. The data of KG101 coincided with the fact that it was released from Hwangskoog - Chong by breeding process. The data of Jakyung strains indicated the significant variation among the strains. From these results, RAPD analysis method could be succesively applied to the classification and genetic analysis for breeding of Korean ginseng.

• 한국식물병리학회지
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• 제14권5호
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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.

• Journal of Microbiology
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• 제44권2호
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• pp.155-161
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• 2006

Comparative analysis of microbial communities in a sequencing batch reactor which performed enhanced biological phosphorus removal (EBPR) was carried out using a cultivation-based technique and 16S rRNA gene clone libraries. A standard PCR protocol and a modified PCR protocol with low PCR cycle was applied to the two clone libraries of the 16S rRNA gene sequences obtained from EBPR sludge, respectively, and the resulting 424 clones were analyzed using restriction fragment length polymorphisms (RFLPs) on 16S rRNA gene inserts. Comparison of two clone libraries showed that the modified PCR protocol decreased the incidence of distinct fragment patterns from about 63 % (137 of 217) in the standard PCR method to about 34 % (70 of 207) under the modified protocol, suggesting that just a low level of PCR cycling (5 cycles after 15 cycles) can significantly reduce the formation of chimeric DNA in the final PCR products. Phylogenetic analysis of 81 groups with distinct RFLP patterns that were obtained using the modified PCR method revealed that the clones were affiliated with at least 11 phyla or classes of the domain Bacteria. However, the analyses of 327 colonies, which were grouped into just 41 distinct types by RFLP analysis, showed that they could be classified into five major bacterial lineages: ${\alpha},\;{\beta},\;{\gamma}-$ Proteobacteria, Actinobacteria, and the phylum Bacteroidetes, which indicated that the microbial community yielded from the cultivation-based method was still much simpler than that yielded from the PCR-based molecular method. In this study, the discrepancy observed between the communities obtained from PCR-based and cultivation-based methods seems to result from low culturabilities of bacteria or PCR bias even though modified culture and PCR methods were used. Therefore, continuous development of PCR protocol and cultivation techniques is needed to reduce this discrepancy.