• Korean Journal of Microbiology
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• v.41 no.2
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• pp.117-124
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• 2005

Two bacterial isolates obtained from rotifer and diseased olive flounder larvae, Paralichthys olivaceus, were identified as Vibrio ichthyoenteri based on the results of phenotypic characterization. In an attempt to develop rapid PCR method for the detection of V. ichthyoenteri, we examined the 16S-23S rRNA intergenic spacer region(ISR) of V. ichthyoenteri and developed species-specific primer for V. ichthyoenteri. Analysis of the ISR sequences showed that V. ichthyoenteri contains one type of polymorphic ISRs. The size of ISRs was 348 bp length and did not contain tRNA genes. Mutiple alignment of representative sequences from different V. species revealed several domains of high sequence variability, and allowed to design species-specific primer for detection of V. ichthyoenteri. The specificity of the primer was examined using genomic DNA prepared from 19 different V. species, isolated 18group Vibrio species and most similar sequence of other known Vibrio species. The results showed that the PCR reaction using species-specific primer designed in this study can be used to detect V. ichthyoenteri.

• Mycobiology
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• v.31 no.3
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• pp.133-138
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• 2003

Analysis of phylogenetic relationship was performed among Phellinus species based on 18S ribosomal subunit sequence data. Twenty-five strains of 19 Phellinus species including P. linteus were examined in this study. Regions of 18S ribosomal subunit were very conserved, but some variable regions between Phellinus species were observed. The species-specific detection primers, modified by 2 or 3 nucleotides in sense primer were designed based on 18S ribosomal DNA(rDNA) sequence data. The 210 by PCR bands were detected with annealing temperature $48^{\circ}C$. The 18S 2F-18S 4R detection primer set distinguished P. linteus from various Phellinus species but some species like P. baumii, P. weirianius, P. rhabarberinus and P. pomaceus also had weak reactivity on this primer set. The 18S 3F-18S 4R primer set distinguished only P. linteus from various Phellinus species, although sensitivity with this primer set was lower than that of 18S 2F-18 4R primer set. These primer sets would be useful for the detection of only P. linteus among unknown Phellinus species rapidly.

• Journal of fish pathology
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• v.17 no.3
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• pp.159-169
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• 2004

Bacterial wihte enteritis ocurred by infection of V. ichthyoenteri is a devastating disease in olive flounder (Paralichthys olivaceus) hatcheries in Korea. Since white enteritis has been a problem in aquqtic industries, necessity of a rapid detection method is increased. In an attempt to develop rapid PCR method the detection of V. ichthyoenteri, we examined the 16S-23S rRNA intergenic spacer region(ISR) of V. ichthyoenteri and developed species-specific primer for V. ichthyoenteri. The intergenic spacers were amplified by primers complementary to conserved region of 16S and 23S rRNA genes. The intergenic spacer region between the 16S and 23S rRNA genes of V. ichthoenteri were investigated by PCR fragment length typing and DNA sequencing. Analysis of the ISR sequences showed that V. ichthyoenteri contains one types of polymorphic ISRs. The size of ISRs ranged 348bp length and not contains tRNA genes. Mutiple alignment of representative sequences from different Vibrio species revealed several domains of high sequence variability, and allowed to design species-specific primer for detection of Vibrio ichthyoenteri. PCR. The specific of the primer was examined using genomic DNA prepared from 19 different Vibrio species, isolated 18group Vibrio species. The results showed that the PCR reaction using species-specific primer designed in this study can be used to detect V. ichthyoenteri.

• Research in Plant Disease
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• v.29 no.1
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• pp.94-99
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• 2023

Blueberry red ringspot virus (BRRSV) and blueberry scorch virus (BlScV) are included in the quarantine virus list managed by the Korean Animal and Plant Quarantine Agency. A multiplex polymerase chain reaction (PCR) assay with an internal control was developed for the simultaneous detection of both viruses. The specific primers used here were designed based on the highly conserved regions of the genomic sequences of each virus, obtained from the National Center for Biotechnology Information nucleotide databases. The primers were designed to amplify a partial sequence within coat protein (CP) for detecting BRRSV and a partial sequence within the CP-16 kDa for detecting BlScV. 18S ribosomal RNA (rRNA) was used as internal control, and the primer set used in a previous study was modified in this study for detecting 18S rRNA. Each conventional PCR using the BRRSV, BlScV, and 18S rRNA primers exhibited a sensitivity of approximately 1 fg plasmid DNA. The multiplex PCR assay using the BRRSV, BlScV, and 18S rRNA primers was effective in simultaneously detecting the two viruses and 18S rRNA with a sensitivity of 1 fg plasmid DNA, similar to that of conventional PCR assays. The multiplex PCR assay developed in this study was performed using 14 blueberry cultivars grown in South Korea. BRRSV and BlScV were not detected, but 18S rRNA was all detected in all the plants tested. Therefore, our optimized multiplex PCR assay could simultaneously detect the two viruses and 18S rRNA in field samples collected from South Korea in a time-efficient manner. This approach could be valuable in crop protection and plant quarantine management.

• The Plant Pathology Journal
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• v.18 no.2
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• pp.98-101
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• 2002

Using phytoplasma universal primer pair Pl and P7, a fragment of about 1.8 kb nucleotide sequences of 16S rRNA gene and 16S-23S rRNA intergenic spacer region, and a portion of 23S rRNA gene of jujube witches'broom (JWB) and mulberry dwarf(MD) phytoplasmas were determined. The nucleotide sequences of JWB and MD were 1,850 bp and 1,831 bp long, respectively. The JWB phytoplasma sequence was aligned with the homologous sequence of MD phytoplasma. Twenty-eight base insertions and nine base deletions were found in the JWB phytoplasma sequence compared with that of MD phytoplasma. The similarity of the aligned sequences of JWB and MD was 84.8%. The near-complete 16S rRNA gene DNA sequences of JWB and MD were 1,529 bp and 1,530 bp in length, respectively, and revealed 89.0% homology. The 16S-23S rRNA intergenic spacer region DNA sequences were 263 bp and 243 bp in lengths respectively, while homology was only 70% and the conserved tRNA-lle gene of JWB and MD was located into the intergenic space region between 16S-23S rRNA gene. The nucleotide sequences were 77 bp long in both JWB and MD, and showed 97.4% sequence homology. Based on the phylogenetic analysis of the two phytoplasmas, the JWB phytoplasma belongs to the Elm yellow phytoplasma group (16S rV), whereas, the MD phytoplasma belongs to the Aster yellow group (16S rI).

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.194-198
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• 2008

A newly designed Cytophaga-Flavobacteria-specific 16S rRNA gene primer pair was employed to investigate the CF community structure in the Bering Sea, revealing a previously unknown and unexpected high CF diversity in this high latitude cold sea. In total, 56 clones were sequenced and 50 unique CF 16 rRNA gene fragments were obtained, clustering into 16 CF subgroups, including nine cosmopolitan subgroups, five psychrophilic subgroups, and two putatively autochthonous subgroups. The majority of sequences (82%) were closely related to uncultured CF species and could not be classified into known CF genera, indicating the presence of a large number of so-far uncultivated CF species in the Bering Sea.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.5
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• pp.731-738
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• 2015

Nematode infection in the epithelial tissue of cultured rockfish Sebastes schlegeli was first reported in 2012. Since then, nematode infections have caused serious economic losses in rockfish aquaculture on the west coast of Korea. Taxonomic and life cycle information for this parasite are currently unknown. In this study, 18S rRNA and cytochrome c oxidase subunit I (COI) genes were used for molecular identification and polymerase chain reaction (PCR) to detect the invisible stages of this parasite. Nucleotide sequences of the 18S rRNA of the rockfish nematode showed 98% identity with that of Philometra morii. Therefore, this rockfish nematode was classified to the Philometridae family. However, we could not identify it to genus level using 18S rRNA. Its COI nucleotide sequences shared 85% and 82% identities with those of Bursaphelenchus sinensis and Philometra overstreeti, respectively. In addition, two gene-specific primer sets were designed based on the 18S rRNA gene to detect the intermediate host and nematode larvae. These primers were specific to this rockfish nematode without cross-reacting to other pathogens. The detection limit of the PCR assay using these primers was 1,000 copies of nematoda plasmid DNA. Therefore, the PCR assay described here is suitable for the detection of nematode DNA within rockfish. In addition, this PCR assay could be used to detect nematode larvae and the intermediate host.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.36 no.3
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• pp.239-246
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• 2003

We have examined the 16S-23S rRNA intergenic spacer region (ISR) of Vibrio vulnificus KCTC 2959. ISRs were amplified by primers complementary to conserved regions of 16S and 23S rRNA genes. ISR amplicons were cloned and sequenced. Analysis of the ISR sequences showed that V. vulnificus KCTC 2959 contains five types of polymorphic ISRs. Size of ISRs ranged from 424 to 741 bp in length and the number of tRNA genes ranged from one to four. The ISRs were designated as ISR-E $(tRNA^{Glu}),\;ISR-IA\;(tRNA^{Ile}-tRNA^{Ala})$, ISR-EKV $(tRNA^{Glu}-tRNA^{Lys}-tRNA^{Val})$, ISR-IAV $(tRNA^{Ile}-tRNA^{Ala}-tRNA^{val})$ and ISR-EKAV $(tRNA^{Glu}-tRNA^{Lys}-tRNA^{Ala}-tRNA^{Val})$ based on their tRNA genes. Multiple alignment of representative sequences from different Vibrio species revealed several domains of high sequence variability. We used the sequences of variable domains to design species-specific primer for detection PCR. Specificity of the primers was examined using genomic DNA prepared from 18 different Vibrio species. The results showed that the PCR using primers designed in this study can be used to detect V. vulnificus from other Vibrio species.

• Fisheries and Aquatic Sciences
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• v.9 no.2
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• pp.70-74
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• 2006

DNA sequence-based typing is considered a robust tool for the discrimination of dinoflagellate species because of the availability of extensive rDNA sequences. Here, we present a rapid, cost-effective DNA-sequencing technique for various PCR products. This sequencing strategy relies on 'nested' or 'tailed' primer labeled with near-infrared dye, and uses a minimal volume of unpurified PCR product (ca. $5{\mu}L$) as the DNA template for sequencing reactions. Reliable and accurate base identification was obtained for several hundred PCR fragments of rRNA genes. This quick, inexpensive technique is widely applicable to sequence-based typing in clinical applications, as well as to large-scale DNA sequencing of the same genomic regions from related species for studies of molecular evolution.

• Korean Journal of Plant Resources
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• v.19 no.1
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• pp.161-168
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• 2006

To investigate the origin of backyeoncho (Opuntia ficus-indica var. saboten), we isolated 685 bp clone using ITS primer pairs. The rDNA consists of the genes coding for the partial 54 bp 185, 162 bp 5.8S, and partial 56 bp 26S. The coding regions are interrupted by two internal transcribed spacers, 193 bp ITS1 and 220 bp ITS2. The ITS2 of backnyeoncho in length was shorter than that previously registered in Cucurbitoideae plants. The GC contents was 66.8% in ITS1, and 67.7% in ITS2. The rDNA of backnyeoncho matched to the previously reported genes and showed a high similarity with the 95% identity with Pereskiopsis porteri (L708037). In the phylogenetic analysis, the backnyeoncho rDNA was clustered with Pereskiopsis porteri (L708037).