• 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.

• International Journal of Oral Biology
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• v.38 no.1
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• pp.21-27
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• 2013

Anginosus group streptococci (AGS) were classified based on the nucleotide sequences of the 16S rRNA gene (16S rDNA) and comprised Streptococcus anginosus, Streptococcus intermedius, and Streptococcus constellatus. It is known that AGS is a causative factor of oral and systematic diseases. The purpose of this study was to discriminate the 56 clinical strains of AGS isolated from Korean oral cavities using phylogenetic analysis of 16S rDNA and species-specific PCR at the species-level. The 16S rDNA of clinical strains of AGS was sequenced using the dideoxy chain termination method and analyzed using MEGA version 5 software. PCR was performed to identify the clinical strains using species-specific primers described in previous studies and S. intermedius-specific PCR primers developed in our laboratory. The resulting phylogenetic data showed that the 16S rDNA sequences can delineate the S. anginosus, S. intermedius, and S. constellatus strains even though the 16S rDNA sequence similarity between S. intermedius and S. constellatus is above 98%. The PCR data showed that each species-specific PCR primer pair could discriminate between clinical strains at the species-level through phylogenetic analysis of 16S rDNA nucleotide sequences. These results suggest that phylogenetic analysis of 16S rDNA and PCR are useful tools for discriminating between AGS strains at the species-level.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1401-1409
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• 2015

The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 10⁰ fg/µl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.

• Korean Journal of Microbiology
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• v.34 no.1_2
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• pp.51-57
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• 1998

A study was carried out to understand some parameters affecting on RAPD analysis with Lactobacillus species. From the results, we found that appearance of specific DNA bands were very influenced by the concentration of $MgCl_2$ but it was overcome by applying enough amount of Taq DNA polymerase. Other parameters such as concentrations of template DNA, random primers and Taq DNA polymerase have enhanced the production of specific DNA bands by increasing their concentration applied. However, we noticed that G/C contents of random primers did not show any correlations with number of specific RAPD bands generated but the RAPD results were heavily influenced by the characteristics of the random primers, that is, the sequences of the oli.

• The Korean Journal of Mycology
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• v.26 no.4 s.87
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• pp.574-582
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• 1998

Five basidiocarps of Pholiota species have been collected from the areas of BubJu Temple for last two years, and identified to those of P. adiposa or Pholiota species. The taxonomy of these basidiocarps with the morphological aspects was compared with the analysis obtained from the polymorphisms of PCR-RAPD bands made after reacted with the random primers. The polymorphic variations were observed within the species of the basidiocarps, but not between genomic DNA's of the mycelia obtained and the basidiocarps. Several different bands made from the primers (28 and 36) in PCR-RAPD reactions were identified within the genus of Pholiota and speculated to be specific for the individual basidiocarp of P. adiposa collected. The primers employed here were considered to be very useful for distinguishing the individual isolates or basidiocarps collected from the fields. Also, the basidiospores were obtained from the sporeprints of the above basidiocarps as a simple agar and confirmed with observations of clamp connection under microscopes. The matings of them indicated the 'tetrapolar' type, being different from the 'bipolar' type reported by Japanese basidiocarps of P. adiposa or P. nameko. Based on our work, the edible fungi collected were speculated to be a new breeding resource for those of Pholiota commercialized in Japan.

• Journal of Food Hygiene and Safety
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• v.30 no.3
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• pp.289-294
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• 2015

The aim of this study was to develop rapid screening method for the identification of Chinese herbal medicine species with similar appearance, Polygonum multiflorum, Cynanchum wilfordii and C. auriculatum, by using genetic markers. As a genetic marker, psbA-trnH gene in chloroplast was selected due to differences in sequence among the three species. Species-specific primers were designed based on the sequences of the marker gene of P. multiflorum, C. wilfordii, and C. auriculatum, and the expected size of PCR products was 160, 147, and 119 bp, respectively. Under the developed conditions, cross-reaction was not detected among these three plant species. To confirm the efficiency of our species-specific primers, the optimized method was applied to a variety of processed products composed of mostly P. multiflorum and C. wilfordii, demonstrating that our method was a rapid and easy screening assay. Our findings suggest this screening method can be utilized to prevent the distribution of economically motivated adulteration food and to improve consumer's right.

• Proceedings of the National Institute of Ecology of the Republic of Korea
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• v.3 no.4
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• pp.212-220
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• 2022

DNA markers have been studied and used intensively to identify plant species based on molecular approaches. The genus Medicago belongs to the family Fabaceae and contains 87 species distributed from the Mediterranean to central Asia. Five species of Medicago are known to be distributed in South Korea; however, their morphological characteristics alone cannot distinguish the species. In this study, we analyzed the phylogenetic relationships using collected five species of Medicago from South Korea and 44 taxa nucleotide information from NCBI. The constructed phylogenetic tree using gibberellin 3-oxidase 1 and tRNA^Lys (UUU) to maturase K gene sequences showed the monophyly of the genus Medicago, with five species each forming a single clade. These results suggest that there are five species of Medicago distributed in South Korea. In addition, we designed polymerase chain reaction primers for species-specific detection of Medicago by comparing the plastid sequences. The accuracy of the designed primer pairs was confirmed for each Medicago species. The findings of this study provide efficient and novel species identification methods for Medicago, which will assist in the identification of wild plants for the management of alien species and living modified organisms.

• Korean Journal of Microbiology
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• v.42 no.1
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• pp.19-25
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• 2006

This study was undertaken to develop PCR primers for the identification and detection of Streptococcus mutans (by)using species-specific forward and universal reverse primers. These primers targeted the variable regions of the 16S ribosomal RNA coding gene (rDNA). The primer specificity was tested against 11S. mutans strains and 10 different species (22 strains) of oral bacteria. The primer sensitivity was determined by testing serial dilutions of the purified genomic DNA of S. mutans ATCC $25175^T$. The data showed that species-specific amplicons were obtained from all the S. mutans strains tested, which was not observed in the other species. The direct and nested PCR could detect as little as 2 pg and 2 fg of the chromosomal DNA from S. mutans ATCC $25175^T$, respectively. This shows that the PCR primers are highly sensitive and applicable to the detection and identification of S. mutans.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.42 no.2
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• pp.146-150
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• 2009

Genetic variation and relationship of two wild (Pampus argenteus and P. echinogaster) and one cultured (P. chinesis) pomfret fish belonging to the genus Pampus were assessed. Specimens were collected from Korea and China and subjected to amplified fragment length polymorphism (AFLP) DNA fingerprinting. Four primer combinations generated a total of 304 DNA fragments ranging from 153 to 251 bands. Polymorphism and genetic diversity of cultured P. chinensis (22.9% and 0.038) were significantly lower than the two wild species of P. argenteus (93.6% and 0.311) and P. echinogaster (94.0% and 0.290). Genetic distance ranged from 0.335 (P. argenteus and P. echinogaster) to 0.646 (P. argenteus and P. chinensis) and showed a congeneric relationship within this genus. Twenty one of specific AFLP markers from four primer combinations bands were produced. These results suggest that AFLP polymorphism may be a useful marker for genetic identification among the three species studies here.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.113.2-114
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• 2003

Five pepper-infecting potyviruses, Pepper mottle virus (PepMoV), Chilli veinal mottle virus (CVMV), Pepper veinal mottle virus (PVMV), Pepper severe mosaic virus (PSMV) and Tobacco each virus (TEV), are known filamentous virus and can be infected pepper crops systemically. To understand pathology and genome information of the five viruses on pepper plants, host reactions and sequences were compared to the 5 viruses. Five potyviruses were inoculated onto some typical cultivars of hot peppers and compared their symptoms, and virus accumulations. A set of degenerate primers for potyviruses were applied to 5 viruses and RT-PCR was performed. RT-PCR products containing partial nuclear inclusion b and coat protein (CP) genes were cloned. Then, oligo dT primer and species-specific primer were redesigned to amplify the C-terminal part of CP and 3' noncoding regions of each viruses. Sequences of the viruses were analyzed and compared to serological relationships among the viruses. The data can be useful for screening of potyviruses in pepper plants and pathogen-derived transgenic pepper plant development.