• 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.

• The Korean Journal of Mycology
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• v.44 no.2
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• pp.103-107
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• 2016

To establish a rapid and accurate detection of Phytophthora pinifolia, which is a quarantine pathogenic fungus in Korea, a species-specific primer was developed based on the ras-related protein (Ypt1) gene. Species-specific primer based on the DNA sequences of Ypt1 gene amplified 193 bp polymerase chain reaction (PCR) product for P. pinifolia. The primer pair yielded the predicted PCR product size exactly in testing with target pathogen DNAs, but not from the other 10 species of Phytophthora and 14 species of other phytopathogenic fungi. The primer pair also showed only the species-specific amplification curve on realtime PCR on target pathogen DNA. The detection sensitivity of real time PCR using species-specific primer pair was 10 to 100 times higher than conventional PCR, with 1 to $10pg/{\mu}L$.

• The Plant Pathology Journal
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• v.26 no.4
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• pp.409-416
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• 2010

Fusarium species, a large group of plant pathogens, potentially pose quarantine concerns worldwide. Here, we focus on the development of a method for detecting four Fusarium species in quarantined plants in Korea: F. solani f. sp. cucurbitae, F. stilboides, F. redolens, and F. semitectum var. majus. Species-specific primers were designed from the nucleotide sequences of either the translation elongation factor-1 alpha (TEF1) gene or RNA polymerase II subunit (RPB2) gene. Two different primer sets derived from TEF1, all specific to F. solani f. sp. cucurbitae, were able to differentiate the two races (1 and 2) of this species. A set of nested primers for each race was designed to confirm the PCR results. Similarly, two primer sets derived from RPB2 successfully amplified specific fragments from five F. stilboides isolates grouped within a single phylogenetic clade. A specific TEF1 primer set amplified a DNA fragment from only four of the 12 F. redolens strains examined, which were grouped within a single phylogenetic clade. All of the F. semitectum var. majus isolates could be specifically detected with a single RPB2 primer set. The specificity of the primer sets developed here was confirmed using a total of 130 Fusarium isolates.

• Preventive Nutrition and Food Science
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• v.15 no.2
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• pp.159-166
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• 2010

The aim of this study was to develop species-specific primer sets for kimchi Lactobacillus. Known gene sequences of Lactobacillus 16S rRNA were collected from the NCBI Gene bank, and 69 primer sets were designed using the homologous gene sequence. Six species of kimchi Lactobacilli were used as reference strains: Lactobacillus brevis KCTC3102, Lactobacillus farciminis KCTC3681, Lactobacillus fermentum KCTC3112, Lactobacillus hilgardii KCTC3500, Lactobacillus plantarum KCTC3099, and Lactobacillus sanfranciscensis KCTC3205. PCR amplification and gel electrophoresis were performed to identify the accuracy and specificity of the developed primer set. The results show that the primer set of 5'-aagcctgcgaaggcaag-3' & 5'-aggccaccggctttg-3', 5'-acatactatgcaaatctaagagattagacg-3' & 5'-actgagaatggctttaagagattagcttac-3' resulted in a specific PCR band on L. hilgardii, and primer set of 5'-ctaataccgcataacaactactttcacat-3' & 5'-aacttaataaaccgcctacattctctttac-3' on L. farciminis. The results indicate that the developed primer sets can provide a useful tool for the identification and differentiation of L. hilgardii and L. farciminis from other Lactobacillus species of kimchi.

• Research in Plant Disease
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• v.20 no.1
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• pp.21-24
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• 2014

Clubroot caused by the obligate biotrophic protist Plasmodiophora brassicae Woronin is one of the most damaging diseases of Brassicaceae family. In this study, we developed species-specific primer sets for rapid and accurate detection of P. brassicae. The primer sets developed amplified a specific fragment only from P. brassicae DNA while they did not amplify a band from 10 other soilborne pathogens or from Kimchi cabbage. In sensitivity test, the species-specific primer set ITS1-1/ITS1-2 could work for approximately 10 spores/ml of genomic DNA showing more sensitivity and accuracy than previous methods. With quantitative real-time PCR test, the primer set detected less spores of P. brassicae than before, confirming that the species-specific primer set could be useful for rapid and accurate detection of P. brassicae.

• Development and Reproduction
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• v.18 no.1
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• pp.43-49
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• 2014

Three species of Nortamea concinua (NC) and Haliotis discus hannai (HDH) from Tongyeong and Sulculus diversicolor supertexta (SDS) are widely distributed on the coast of the Yellow Sea, southern sea and Jeju Island in the Korean Peninsula under the innate ecosystem. There is a need to understand the genetic traits and composition of three mollusk species in order to evaluate exactly the patent genetic effect. PCR analysis was performed on DNA samples extracted from a total of 21 individuals using seven decamer oligonucleotides primers. Seven primers were shown to generate the unique shared loci to each species and shared loci by the three species which could be clearly scored. A hierarchical clustering tree was constructed using similarity matrices to generate a dendrogram, which was facilitated by the Systat version 10. 236 specific loci, with an average of 56.3 per primer, were identified in the NC species. 142 specific loci, with an average of 44.7 per primer, were identified in the HDH species. Especially, 126 numbers of shared loci by the three species, with an average of 18 per primer, were observed among the three species. Especially, the decamer primer BION-75 generated 7 unique loci to each species, which were identifying each species, in 700 bp NC species. Interestingly, the primer BION-50detected 42 shared loci by the three species, major and/or minor fragments of sizes 100 bp and 150 bp, respectively, which were identical in all samples. As regards average bandsharing value (BS) results, individuals from HDH species (0.772) exhibited higher bandsharing values than did individuals from NC species (0.655). In this study, the dendrogram obtained by the seven decamer primers indicates three genetic clusters: cluster 1 (CONCINNA 01~CONCINNA 07), cluster 2 (HANNAI 08~HANNAI 14), cluster 3 (SUPERTEXTA 15~SUPERTEXTA 21). Comparatively, individuals of HDH species were fairly closely related to that of SDS species, as shown in the hierarchical dendrogram of genetic distances.

• The Plant Pathology Journal
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• v.31 no.3
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• pp.212-218
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• 2015

In this study, we developed a species-specific PCR assay for rapid and accurate detection of three Xanthomonas species, X. axonopodis pv. poinsettiicola (XAP), X. hyacinthi (XH) and X. campestris pv. zantedeschiae (XCZ), based on their draft genome sequences. XAP, XH and XCZ genomes consist of single chromosomes that contain 5,221, 4,395 and 7,986 protein coding genes, respectively. Species-specific primers were designed from variable regions of the draft genome sequence data and assessed by a PCR-based detection method. These primers were also tested for specificity against 17 allied Xanthomonas species as well as against the host DNA and the microbial community of the host surface. Three primer sets were found to be very specific and no amplification product was obtained with the host DNA and the microbial community of the host surface. In addition, a detection limit of $1pg/{\mu}l$ per PCR reaction was detected when these primer sets were used to amplify corresponding bacterial DNAs. Therefore, these primer sets and the developed species-specific PCR assay represent a valuable, sensitive, and rapid diagnostic tool that can be used to detect three specific pathogens at early stages of infection and may help control diseases.

• Journal of Life Science
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• v.28 no.9
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• pp.1062-1067
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• 2018

Reliable labeling of fish products can reassure consumers regarding the identity and quality of seafoods. Therefore, techniques that can identify adulteration or mislabeling are valuable. To rapidly identify two Trachurus species, Trachurus japonicus and Trachurus novaezelandiae, a highly efficient, rapid, duplex polymerase chain reaction (PCR) having two species-specific primers simultaneously was identified. This species-specific primer focused on a single nucleotide mismatch in the 3'-terminal base of a primer designed in the mitochondrial cytochrome c oxidase (COI) subunit I DNA. To optimize the duplex PCR condition, gradient PCR reactions were conducted to determine the primer annealing temperature and the primer concentration. The PCR's product was observed on the gel, suggesting that DNA molecules may be useful in differentiating the two species. The length of the amplification fragments were 103 bp for Trachurus japonicus and 214 bp for Trachurus novaezelandiae, which, along with the species-specific primer visualized by agarose gel electrophoresis, enabled accurate distinction of the species of the Trachurus genus. These results indicate that the duplex PCR, which has a species-specific primer based on single nucleotide polymorphism (SNP), can be useful for rapidly differentiating the two species of Trachurus. This duplex PCR analysis is simple, rapid, and reliable, and could be beneficial to protecting consumers' rights.

• Korean Journal of Medicinal Crop Science
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• v.13 no.2
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• pp.121-125
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• 2005

An analysis of RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) was performed with three Angelica species (A. gigas Nakai, A. sinensis (Olive.) Diels and A. acutiloba Kitag) in an effort to distinguish between members of these three species. Two arbitrary primers (OPC02, OPD11) out of80 primers tested, produced 17 species-specific fragments among the three species. Eight fragments were specific for A. sinensis, four fragments specific for A. gigas, five specific for A. acutiloba. When primers OPC02 and OPD11 were used in the polymerase chain reaction, RAPD-PCR fragments that were specific for each of the three species were generated simultaneously. Primer OPC02 produced eight species-specific fragments: four were specific for A. sinensis, one for A. gigas, and three for A. acutiloba. Primer OPD11 produced nine speciesspecific fragments: four for A. sinensis, three for A. gigas, and two for A. acutiloba. The RAPD-PCR markers that were generated with these two primers should rapidly identify members of the three Angelica species. The consistency of the identifications made with these species-specific RAPD-PCR markers was demonstrated by the observation that each respective marker was generated from three accessions of each species, all with different origins. We also performed the RAPD-PCR analysis with the dried Angelica root samples that randomly collected from marketed and from the OPC02 primer, obtained a A. gigasspecific band and the band were cloned and sequenced.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.12
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• pp.1887-1894
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• 2007

In this paper, a rapid and reliable gene-targeted species-specific polymerase chain reaction (PCR) technique based on a two-step process was established to identify bifidobacteria in dairy products. The first step was the PCR assay for genus Bifidobacterium with genus specific primers followed by the second step, which identified the species level with species-specific primer mixtures. Ten specific primer pairs, designed from nucleotide sequences of the 16-23S rRNA region, were developed for the Bifidobacterium species including B. angulatum, B. animalis, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum, B. minimum, B. subtile, and B. thermophilum. This technique was applied to the identification of Bifidobacterium species isolated from 6 probiotic products, and four different Bifidobacterium spp. (B. bifidum, B. longum, B. infantis, and B. breve) were identified. The findings indicated that the 16S-23S rDNA gene-targeted species-specific PCR technique is a simple and reliable method for identification of bifidobacteria in probiotic products. PCR combined with Denaturing Gradient Gel Electrophoresis (DGGE) for identification of the bifidobacteria was also evaluated and compared with the gene-targeted species-specific technique. Results indicated that for fermented milk products consistency was found for both species-specific PCR and PCR-DGGE in detecting species. However, in some lyophilized products, the bands corresponding to these species were not visualized in the DGGE profile but the specific PCR gave a positive result.