• Journal of Microbiology
• /
• v.43 no.2
• /
• pp.209-212
• /
• 2005

The purpose of this study was to develop species-specific PCR primers for use in the identification and detection of Actinobacillus actinomycetemcomitans. These primers target variable regions of the 168 ribosomal RNA coding gene (rDNA). We assessed the specificity of the primers against 9 A. actinomycetemcomitans strains and 11 strains (3 species) of the Haemophilus genus. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC$ 33384^$T Our obtained data revealed that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 4 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these PCR primers are incredibly sensitive, and should prove suitable for application in epidemiological studies, as well as the diagnosis and monitoring of periodontal pathogens after treatment for periodontitis.

• Restorative Dentistry and Endodontics
• /
• v.24 no.1
• /
• pp.13-25
• /
• 1999

P. endodontalis which was known to be associated with the infected root canals and periapical lesions is very difficult to detect by culture methods or traditional methods. Detection of bacteria using polymerase chain reaction(PCR) for 16S ribosomal DNA(rDNA) is fast, simple, and accurate with relatively small amount of target cells. 16S rDNA consist of conserved regions those are same to all species, and variable regions which represent species specificity. The 16S rDNA sequences of P. endodontalis and P. gingivalis were aligned and two highly variable regions were selected as a pair of species specific oligonucleotide primers for P. endodontalis. And then the pair of primers for PCR amplification was synthesized to identify P. endodontalis. The sequences of the species specific primers for the 16S rDNA of P. endodontalis were as follows ; sense primer[endo1]: 5'-CTATATTCTTCTTTCTCCGCATGGAGGAGG-3' antisense primer[endo2]: 5'-GCATACCTTCGGTCTCCTCTAGCATAT-3' In this study, for the identification of P. endodontalis without culture from the mixed clinical samples, PCR was done with species specific primers for the 16S rDNA sequences of P. endodontalis. The results were as follows : 1. The species specificity of the primers for the 16S rDNA of P. endodntalis was determined by the PCR methods. About 490bp amplicon which was specific only for P. endodntalis was produced with P. endodontalis. No amplicon was produced by PCR with other strains similar to P. endodontalis. 2. The synthesized species specific primers reacted with conventionally identified P. endodontalis which we have in conservative dentistry laboratory. 3. The identification of P. endodontalis using PCR technique with samples collected from infected root canals or periapical lesions was more sensitive than that of culture methods. 4. Seven samples revealed including P. endodontalis by PCR technique. Five of them were related with pains, two of them with sinus tract, three of them with foul odor, and three of them with purulent drainage. P. endodontalis was shown to have great relation with pains.

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

• Korean Journal of Medicinal Crop Science
• /
• v.11 no.4
• /
• pp.268-273
• /
• 2003

To identify the variation of the RAPD patterns between two Atractylodes species, 52 kinds of random primers were applied to each eight of A japonica and A. macrocephala genomic DNA. Ten primers of 52 primers could be used to discriminate between the species and 18 polymorphisms among 67 scored DNA fragments (18 fragments are specific for A. japonica and A. macrocephala) were generated using these primers, 26.9% of which were polymorphic. RAPD data from the 10 primers was used for cluster analysis. The cluster analysis of RAPD markers showed that the two groups are genetically distinct. On the other hand, to identify the variation of the AFLP patterns and select the species specific AFLP markers, eight combinations of EcoRI/MseI primers were applied to the bulked A. japonica and A. macrocephala genomic DNA. Consequently, three combinations of EcoRI/MseI primers (EcoRI /Mse I ; AAC/CTA, AAC/CAA, AAG/CTA) used in this study revealed 176 reliable AFLP markers, 42.0% of which were polymorphic. 74 polymorphisms out of 176 scored DNA fragments were enough to clearly discriminate between two Atractylodes species.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 2003.10a
• /
• pp.120.2-120
• /
• 2003

Agrobacterium vitis is a causal agent of crown-gall disease on grapevine. In Korea, grapevine variety (GeoBong) have severely been infected by the bacteria since stems of the variety were buried in soil for overwintering. Infection ratio over 70-80％ was observed on 7 years old GeoBong grapevine in Ansung and Cheonan. PCR specific primers for A. vitis strains were designed using nucleotide sequences of vir A gene in Ti-Plasmid, pheA gene in chromosomal DNA and a URP-PCR polymorphic band. Three hundred bacterial strains were isolated from the different 80 galls formed on GeoBong grapevine in Cheonan and Ansung of Korea and were screened to identify A. vitis using the three specific PCR primers for Agrobacterium vitis. Twenty-four bacterial strains that are detected by the primers were further confirmed by pathogenicity and biochemical methods. To investigate the genomic diversity of the bacterial strains, twenty primers of 20 mer referred to universal rice primers (URP) were applied for PCR fingerprinting, Of them, URP2R and URP2F primers could effectively be used to detect polymorphism within the bacterial strains.

• Korean Journal of Plant Resources
• /
• v.17 no.2
• /
• pp.107-115
• /
• 2004

To develop universal primers for phylogenetic analysis and species-specific marker for breeding program of kiwifruit, eighteens primers were designed from kiwifruit genome-specific repeat sequences. Seven species including twenty two varieties collected from native eastern Asia were examined using 18 to 22 mer kiwifruit target(KT) primers. among eighteen primers, we selected seven primers for phylogenetic relationship. The genus Actinidia was divided into two large groups; group I,A. arguta, A. melanandra, A. kolomikta, and A. marcrosperma, characterized by the non-hair in fruits and loaves or a few pubescences only in young stage, which belongs to the section Leiocarpae, and group II, A. chinensis, A. deliciosa, and A. eriantha, characterized by a lot of hairs only in young fruit stage and with a lot of hairs or fuzzes in leaves and branches, which belongs to the section Stellatae. Group II especially belongs to the series Perfectae of the section Stellatae and was divided into two subgroups; subgroup I containing A. chinensis and A. deliciosa, and subgroup II containing A. eriantha. In contrast, the two species, A. chinensis and A. deliciosa, which are known to have common parents, were divided into two independent subgroups with 80％ of a similarity value. On the other hand, we selected KT6F for variety specific bands, KT12E primers for 'Hayward' and 'Tomuri'. KT7F or KT12F primers were useful for analysis of inheritance pattern in kiwifruit cross-breeding. We suggest that these primers will be a powerful tool for elucidating phylogenetic relationship and selection of novelty kiwifruit in a breeding program.

• Development and Reproduction
• /
• v.23 no.4
• /
• pp.367-375
• /
• 2019

Pufferfish (Takifugu spp.) are economically important edible marine fish. Mistakes in pufferfish classification can lead to poisoning; therefore, accurate species identification is critical. In this study, we used the mtDNA cytochrome c oxidase subunit I gene (COI) to design specific primers for six Takifugu species among the 21 domestic or imported pufferfish species legally sold for consumption in Korea. We rapidly and simultaneously identified these pufferfish species using a highly efficient, multiplex polymerase chain reaction (PCR) system with the six species-specific primers. The results showed that species-specific multiplex PCR (multiplex species-specific polymerase chain reaction; MSS-PCR) either specifically amplified PCR products of a unique size or failed. MSS-PCR yielded amplification fragment lengths of 897 bp for Takifugu pardalis, 822 bp for T. porphyreus, 667 bp for T. niphobles, 454 bp for T. poecilonotus, 366 bp for T. rubripes, and 230 bp for T. xanthpterus using the species-specific primers and a control primer (ca. 1,200 bp). We visualized the results using agarose gel electrophoresis to obtain accurate contrasts of the six Takifugu species. MSS-PCR analysis is easily performed and provides identification results within 6 h. This technique is a powerful tool for the discrimination of Takifugu species and will help prevent falsified labeling, protect consumer rights, and reduce the risk of pufferfish poisoning..

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

• Development and Reproduction
• /
• v.20 no.2
• /
• pp.135-140
• /
• 2016

Genomic DNAs isolated from crucian carp of four rivers, belonging to the family Cyprinidae was amplified by seven oligonucleotides primers. In the present study, we employed hierarchical clustering method in order to reveal genetic distances and variations. Crucian carp was acquired from Hangang river (CAH), Geumgang river (CAG), Nakdonggang river (CAN) and Yeongsangang river (CAY). The primer BION-12 generated the most loci (a total of 50) with an average of 10 in the CAY population. The primer BION-10 generated the least loci (a total of 19), with an average of 3.8 in the CAG population, in comparison to the other primers used. Seven oligonucleotides primers made 16.7 average no. per primer of specific loci in the CAH population, 7.4 in the CAG population, 8.6 in the CAN population and 0.9 in the CAY population, respectively. The specific loci generated by oligonucleotides primers revealed inter-individual-specific characteristics, thus disclosing DNA polymorphisms. The dendrogram obtained by the seven oligonucleotides primers indicates four genetic clusters. The genetic distance that displayed significant molecular differences was between individuals no.06 and no.08 from the CAG population (genetic distance = 0.036), while the genetic distance among the five individuals that displayed significant molecular differences was between individuals no.08 and no.09 from the CAG population (genetic distance = 0.088). With regard to average bandsharing value (BS) results, individuals from CAY population ($0.985{\pm}0.009$) exhibited higher bandsharing values than did individuals from CAH population ($0.779{\pm}0.049$) (P<0.05). Relatively, individuals of CAY population were fairly closely related to that of CAN location (genetic distance between two populations<0.016).

• Journal of Microbiology
• /
• v.43 no.4
• /
• pp.331-336
• /
• 2005

The objective of this study was to assess the strain-specificity of a DNA probe, Fu12, for Fusobacterium nucleatum subsp. nucleatum ATCC $25586^T$ (F. nucleatum ATCC $25586^T$), and to develop sets of strain-specific polymerase chain reaction (PCR) primers. Strain-specificity was tested against 16 strains of F. nucleatum and 3 strains of distinct Fusobacterium species. Southern blot hybridization revealed that the Fu12 reacted exclusively with the HindIII-digested genomic DNA of F. nucleatum ATCC $25586^T$. The results of PCR revealed that three pairs of PCR primers, based on the nucleotide sequence of Fu12, generated the strain-specific amplicons from F. nucleatum ATCC $25586^T$. These results suggest that the DNA probe Fu12 and the three pairs of PCR primers could be useful in the identification of F. nucleatum ATCC $25586^T$, especially with regard to the determination of the authenticity of the strain.