• Korean Journal of Food Science and Technology
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• v.35 no.3
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• pp.470-474
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• 2003

DNA sequence information on small-subunit rRNA gene (16S rDNA) obtained from food-poisoning bacterial culture was used to investigate the presence of bacterial pathogens in food. By reverse dot blot detection method, presence of food-poisoning bacteria could be confirmed on hybridization of digoxigenin-labeled 16S rDNA Polymerase Chain Reaction (PCR) primer product and biotin-labeled specific oligonucleotide probe. Escherichia coli, Bacillus cereus. and Salmonella sp. were used as the representative food-poisoning bacterial microorganisms. An oligonucleotide probe, based on the variable region of 16S rRNA gene, was used as the specific probe. These tools may be more useful than classic biochemical method for rapid identification of contaminated food.

• Microbiology and Biotechnology Letters
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• v.20 no.4
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• pp.483-490
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• 1992

In order to understand the transfer behavior of a particular gene in water environments, kanamycin resistance ($Km^r$) gene was tracked by Southern hybridization with DNA probe in its conjugal transfer. A $Km^r$ natural bacterial isolate and genetically modified microorganisms (GMMs) constructed from the isolate were used as donor for conjugal transfer of the $Km^r$ gene. The transfer frequencies of the $Km^r$ gene from GMM strains were generally 10 to 100 times higher than those from the natural isolate. The conjugants obtained from GMM strains in LB broth had more plasmids newly appeared, and particularly the conjugants in A Wand FW waters revealed more rearrangement in their plasmids as a function of conjugation time. When plasmids of the conjugants obtained in LB broth were Southern hybridized with DNA probe of the $Km^r$ gene, the $Km^r$ plasmids in the conjugants were detected at the same position of the plasmids in donor cells, in spite of the fact that the plasmids were highly rearranged in conjugant cells. But the $Km^r$ plasmids in the donor of DKI and DKC601, and DKC600 were not identified in the conjugants obtained after 50 h conjugation in AW and after 30 h in AW, respectively. The size of the $Km^r$ plasmids showing hybridization signal were a little changed in the conjugants obtained in A Wand FW waters. Therefore, the method of Southern hybridization with DNA probe was proved to be very specific and useful for tracking of particular genes in water environments.

• Molecules and Cells
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• v.27 no.2
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• pp.205-209
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• 2009

The loci of the 5S and 45S rRNA genes were localized on chromosomes in five species of Capsicum, namely, annuum, chacoense, frutescens, baccatum, and chinense by FISH. The 5S rDNA was localized to the distal region of one chromosome in all species observed. The number of 45S rDNA loci varied among species; one in annuum, two in chacoense and frutescens, and chinense, and four in baccatum, with the exceptions that 'CM334' of annuum had three loci and 'tabasco' of frutescens gad one locus. 'CM334'-derived BAC clones, 384B09 and 365P05, were screened with 5S rDNA as a probe, and BACs 278M03 and 262A23 were screened with 25S rDNA as a probe. Both ends of these BAC clones were sequenced. FISH with these BAC probes on pachytenes from 'CM334' plant showed one 5S rDNA locus and three 45S rDNA loci, consistent with the patterns on the somatic chromosomes. The 5S rDNA probe was also applied on extended DNA fibers to reveal that its coverage measured as long as 0.439 Mb in the pepper genome. FISH techniques applied on somatic and meiotic chromosomes and fibers have been established for chili to provide valuable information about the copy number variation of 45S rDNA and the actual physical size of the 5S rDNA in chili.

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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• pp.132-133
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• 2003

The marine dinoflagellate genus Alexandrium comprise PSP producing A. acatenella, A. angustitabuzatum, A. catenella, A. fundyense, A. minutum, A. ostenfezdii, A. tamiyavanichii and A. tamarense. In monitoring toxic Alexandrium, rapid and exact species identification is one of the significant prerequisite work, however we have suffered confusion of species definition in Alexandrium. To surmount this problem, we chose DNA probing, which has long been used as an alternative for conventional identification methods, primarily relying on morphological approaches using microscope in microbial field. Oligonucleotide DNA probes targeting rRNA or rDNA have been commonly used in diverse studies to detect and enumerate cells concerned as a culture-indetendent powerful tool. Despite of the massive literature on the HAB species containing Alexandrium, application of DNA probing for species identification and detection has been limited to a few documents. DNA probes of toxic A. tamarense, A. catenella and A. tamiyavanichii, and non-toxic A. affine, A. fraterculus, A. insuetum and A. pseudogonyaulax were designed from LSU rDNA D1-D2, and applied to whole cell-FISH. Each DNA probes reacted only the targeted Alexandrium cells with very high species-specificity within Alexandrium. The probes could detect each targeted cells obtained from the natural sea water samples without cross-reactivity. Labeling intensity varied in the growth stage, this showed that the contents of probe-targeted cellular rRNA decreased with reduced growth rate. Double probe TAMID2S1 achieved approximately two times higher fluorescent intensity than that with single probe TAMID2. This double probe did not cross-react with any kinds of microorganisms in the natural sea waters. Therefore we can say that in whole-cell FISH procedure this double DNA probe successfully labeled targeted A. tamiyavanichii without cross-reaction with congeners and diverse natural bio-communities.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.133-137
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• 1986

17S-ribosomal RNA gene from the hygromycin resistant protozoan Tetrahymena thermophila hmr 3 was cloned on E. coli vector pBR 322 as part of study to work the 17S-rRNA structure and the mechanism of hygromycin resistance. The 17S-rDNA was inserted into the Hind 111 site of pBR 322. The clones having recombinant plasmid were selected by the method of colony hybridization with a 17S-rDNA probe of wild type B1868. The orientation of 17S-rDNA insert was located near the tetracycline resistant gene of pBR 322 in a clone 5-19 with the recombinant plasmid.

• Korean Journal of Veterinary Service
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• v.35 no.3
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• pp.215-222
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• 2012

Species-specific $TaqMan^{(R)}$ probe-based real-time PCR assays were developed for detection of beef, pork, chicken, duck, goose and turkey. The primer and probe sets used in this study were designed to be complementary to fibroblast growth factor (FGF) for cattle and pig, mitochondrial NADH dehydrogenase (ND) subunit 3 and ND2 for chicken and duck, 12S rRNA for goose and turkey, respectively. As internal positive control we used conserved region in the ribosomal 18S RNA gene to ensure the accuracy of the detection of target DNA by real-time PCR. We confirmed that real-time PCR assays with the primer and probe sets were positive for cattle, pig and chicken intended target animal species with no cross-reactivity with other non-target animal species. Only >50 ng DNA of beef show cross-reactivity in the determination of duck. Using species-specific primer and probe sets, it was possible to detect amounts of 0.1 ng DNA of cattle and pig, 1.0 pg DNA of chicken, duck and turkey, and 0.1 pg DNA of goose for raw samples, respectively. The detection limits were 0.1 ng DNA of cattle, 1.0 ng DNA of pig and 1.0 pg DNA of chicken for DNA mixtures (beef, pork and chicken) extracted from heat-treated ($121^{\circ}C$/5 min) meat samples. In conclusion, it can be suggested that the $TaqMan^{(R)}$ probe-based assay developed in this study might be a rapid and specific method for the identification of meat species in raw or cooked meat products.

• Fisheries and Aquatic Sciences
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• v.7 no.4
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• pp.175-183
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• 2004

The dinoflagellate Alexandrium tamiyavanichii Balech, a producer of toxins causing paralytic shellfish poisoning (PSP), has recently been considered as one of main organisms responsible for toxication of shellfish in Japan. In this study, A. tamiyavanichii was subjected to a molecular phylogenetic analysis inferred from 28S rDNA D1-D2 sequences and a species-specific LSU rRNA-targeted oligonucleotide DNA probe was designed to identify A. tamiyavanichii using the whole cell-FISH (fluorescence in situ hybridization). The sequences of the 28S rDNA D1-D2 region of A. tamiyavanichii showed no difference from A. cohorticular AF1746l4 (present name A. tamiyavanichii) and formed a distinct clade from the 'tamarensis species complex'. The probe, TAMID2, reacted specifically with A. tamiyavanichii cultured cells, without any cross-reaction with other species belonging to the same genus, including A. tamarense, A. catenella, A. affine, A. fraterculus, A. insuetum and A. pseudogonyaulax. In a test of cross-reactivity with a field sample, TAMID2 reacted consistently with only A. tamiyavanichii, indicating that the present protocol involving the TAMID2 probe might be useful for detecting toxic A. tamiyavanichii in a simple and rapid manner.

• Korean Journal of Microbiology
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• v.42 no.2
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• pp.89-94
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• 2006

The purpose of this study is to develop the strain-specific PCR primers for the identification of prevotella inter-media ATCC 49046 which is frequently used in the pathogenesis studies of periodontitis. The Hind III-digested genomic DNA of P. intermedia ATCC 49046 were cloned by random cloning method. The specificity of cloned DNA fragments were determined by Southern blot analysis. The nucleotide sequence of cloned DNA probes was determined by chain termination method. The PCR primers were designed based on the nucleotide sequence of cloned DNA fragment. The data showed that Pig6 DNA probe were hybridized with the genomic DNA from P. intermedia strains (ATCC $25611^T$ and 49046) isolated from the Westerns, not the strains isolated from Koreans. The Pig6 DNA probe were consisted of 813 bp. Pig6-F3 and Pig6-R3 primers, designed base on the nucleotide Sequences Of Pig6 DNA Probe, were 3150 specific to the only both P. intermedia ATCC $25611^T$ and P. intermedia ATCC 49046. In the other hand, Pig6-60F and Pig6-770R primers were specific to the only P. intermedia ATCC 49046. The two PCR primer sets could detect as little as 4 pg of chromosomal DNA of P. intermedia. These results indicate that Pig6-60F and Pig6-770R primers have proven useful for the identification of P. intermedia ATCC 49046, especially with regard to the maintenance of the strain.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.212-220
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• 2008

A Pn10 DNA probe was introduced as a Prevotella nigrescens ATCC $33563^T$-specific DNA probe. In that study, the specificity of the Pn10 was tested with only type or reference strains of 5 oral bacterial species. The purpose of this study is to evaluate the specificity of the Pn10 using the wild type strains of P. nigrescens and is to develop the P. nigrescens ATCC $33563^T$-specific PCR primers based on the nucleotide sequence of the Pn10. The specificity of the Pn10 DNA probe was determined by Southern blot analysis. The nucleotide sequence of Pn10 DNA probes was determined by chain termination method. The PCR primers were designed based on the nucleotide sequence of cloned DNA fragment. The data showed that Pn10 DNA probe were hybridized with the genomic DNAs from P. nigrescens ATCC $33563^T$ and KB6. The Pn10 homologous region, KB6-Pn10, of P. nigrescens KB6 was cloned by PCR and sequenced. The Pn10 and KB6-Pn10 DNA fragments were consisted of 1,875 bp and 1,873 bp, respectively. The percent identity of the two was 98.8% and the divergence of them was 0.6%. The two primer sets (Pn10-F-AC/ Pn10-R-AC and Pn10-F-A/ Pn10-R-A), designed base on the nucleotide sequences of Pn10 DNA probe, were specific to the P. nigrescens ATCC $33563^T$. The two PCR primer sets could detect as little as 4 pg of genomic DNA of P. nigrescens ATCC $33563^T$. These results indicate that the two PCR primer sets have proven useful for the identification of P. nigrescens ATCC $33563^T$, especially with regard to the maintenance of the strain.

• Journal of fish pathology
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• v.23 no.3
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• pp.313-322
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• 2010

Edible tunicate Halocynthia roretzi, one of the most commercially important aquatic organisms in Korea, has been killed by tunic softness syndrome since last decade. The intracellular protistan parasite observed by the transmission electron microscope in hemocytes of the tunicate was considered to be the causative agent of the mass mortality. The goal of the present work is to examine the characteristic features of the parasite by identifying the 18S rDNA sequences of the parasite. The experiments conducted include amplification of presumptive 18S rDNA from diseased tunicate tissues with UNonMet-PCR and sequencing the product. A preliminary phylogenetic analysis was performed on the presumptive parasite rDNA. A digoxigenin labeled DNA probe was designed on the basis of the sequences of rDNA. Dig-ISH assay was conducted to diagnose the protistan parasite. A PCR using UNonMet-PCR primer generated 595 bp SSU rDNA fragment. Subsequently, PCRs with primer pair expended this sequence to 1542 bp. This is the first partial sequences of SSU rDNA gene to be published on the protistan parasite that has presumed causing the mass mortality of tunicate. Since the Dig-ISH technique demonstrated the presence of infection in hemocytes on the all host tissues, the fragment was confirmed to be the intracellular protistan parasite SSU rDNA. A phylogenetic analysis suggested that the protistan parasite may be a unique eukaryote that is closely related to Apicomplexa.