• Restorative Dentistry and Endodontics
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• v.23 no.1
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• pp.328-338
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• 1998

Porphyromonas endodontalis, an anaerobic Gram negative cocobacillus which was known to be associated with the infected root canals and periapical lesions, is very difficult to culture and to detect by the traditional method in that it requires much time to induce the specific black pigmentation, and it is very sensitive to oxygen and the antibiotics added in the culture medium. In this study, the nucleotide sequences of the 'probe h' (0.73kb), one of the specific DNA probes top. endodontalis (ATCC 35406) which had been developed by our department, was determined and then a pair of primers for PCR amplification was fabricated to identify P. endodontalis. The plasmids containing 'probe h' were purified by $Wizard^{TM}$ Midipreps DNA Purification System (Promega Corp.), and the nucleotide sequences of the 'probe h' were determined by the dideoxy chain termination method using TaqTrack Sequencing System (Promega Corp.) and detected by fluorescent labelling method. The sense/antisense PCR primers were designed with computer software (Lasergene, DNASTAR Ind. PCR was done with a programmable GeneAmp PCR System 2400 (Perkin Elmer-Cetus Co.). Each sample containing the whole genomic DNA of P. endodontalis and other black-pigmented Bacteroides was itailly denatured at $94^{\circ}C$ for 5 min and then subjected to 30 cycles, each of them consisting of 60s at $94^{\circ}C$, 60s at $60^{\circ}C$, and 90s. at $72^{\circ}C$. The amplified DNA was resolved electrophoretically in a 1.0 % agarose gel in 1X TAE buffer, stained with EtBr, and photographed on a UV transilluminator. The results were as follows : 1. The nucleotide sequences of 'probe h' (743 base pairs) were obtained by dideoxy chain termination method, and from that results the specific primers to P. endodontalis (ATCC 35406), 'Primer H1/ Primer H2', were designed. 2. It has been found that 'Primer H1/H2' could detect P. endodontalis (ATCC 35406) using PCR. 3. The PCR system with this primers may be a powerful technique to amplify the specific sequences of 'probe h' of P. endodontalis (ATCC 35406) that produce distinct identification of it from other black-pigmented Bacteroides, and this could help us to determine the nature of periapical disease.

• The Plant Pathology Journal
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• v.36 no.5
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• pp.468-475
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• 2020

Malva vein clearing virus (MVCV) is a member of the Potyvirus species, and has a negative impact on the aesthetic development of Alcea rosea. It was first reported in Germany in 1957, but its complete genome sequence data are still scarce. In the present work, A. rosea leaves with vein-clearing and mosaic symptoms were sampled and analyzed with small RNA deep sequencing. By denovo assembly the raw sequences of virus-derived small interfering RNAs (vsiRs) and whole genome amplification of malva vein cleaning virus SX strain (MVCV-SX) by specific primers targeting identified contig gaps, the full-length genome sequences (9,645 nucleotides) of MVCV-SX were characterized, constituting of an open reading frame that is long enough to encode 3,096 amino acids. Phylogenetic analysis showed that MVCV-SX was clustered with euphorbia ringspot virus and yam mosaic virus. Further analyses of the vsiR profiles revealed that the most abundant MVCV-vsiRs were between 21 and 22 nucleotides in length and a strong bias was found for "A" and "U" at the 5′-terminal residue. The results of polarity assessment indicated that the amount of sense strand was almost equal to that of the antisense strand in MVCV-vsiRs, and the main hot-spot region in MVCV-SX genome was found at cylindrical inclusion. In conclusion, our findings could provide new insights into the RNA silencing-mediated host defence mechanism in A. rosea infected with MVCV-SX, and offer a basis for the prevention and treatment of this virus disease.

• Korean Journal of Clinical Laboratory Science
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• v.39 no.1
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• pp.49-55
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• 2007

HCV is a single-stranded RNA virus and more than 1 million new cases are reported annually worldwide. The six major HCV genotypes and numerous subtypes vary in their geographic distribution. It is thought that genetic heterogeneity of HCV may account for some of the differences in disease outcome and response to treatment observed in HCV infected persons. In this study, we determined HCV genotypes among chronic Korean HCV patients and evaluated direct sequence PCR protocols developed. For the study, 232 chronic HCV patient sera were used. HCV RNA was extracted and two pairs of consensus PCR primers were selected in 5'UTR region for amplification of HCV RNA. Amplification products obtained from the HCV positive cases were subjected to automatic sequencing. Sequences were compared with those in GenBank by using the BLAST program. From this study, five HCV genotypes, 1b, 2a, 2b, 2c and 3a were found. HCV genotypes 4, 5 and 6 were not determined. HCV genotype 1b (53.9%, 125/232) and 2a (35.8%, 83/232) were most frequently found. This group was followed by 2b (3.9%, 9/232), 3a (3.4%, 8/232) and 2c (3.0%, 7/232). The data presented here suggest a complex distribution of HCV types and they were well correlated with other reports on Koreans and will be helpful for type-specific follow-up of Korean HCV patients. This study showed that 5'UTR direct sequence analysis is a sensitive and rapid method to identify HCV genotypes.

• BMB Reports
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• v.29 no.3
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• pp.272-275
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• 1996

The expression of genes induced by Dichloroacetate (DCA) treatment was analyzed by mRNA differential display. Purified total RNAs from rat liver treated with saline or DCA (100 mg/100 g b.w.) were reverse transcribed by using a set of oligonucleotide primers. The PCR products were resolved on a denaturing sequencing gel. PCR band representing mRNA expressed specifically in DCA-treated liver was excised and reamplified by PCR. A 120-bp c-DNA clone named IC1 was isolated and the DNA sequence of IC1 was analyzed. IC1 revealed 50% homology with 3' end of a mouse fibroblast growth factor mRNA This result indicates that DCA induces the expression of a gene which has a 50% homology with a Mouse fibroblast growth factor, and expression of this gene might be involved in non genotoxic process caused by DCA.

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.881-884
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• 2000

A polymerase chain reaction (PCR) was performed to rapidly identify Lactobacillus plantarum from type strains and kimchi samples. The PCR experiments were carried out using specific oligonucleotide primer sets based on the 16S rRNA gene sequences of L. plantarum. The expected DNA amplificate of 419 bp was obtained when either purified DNA or whole cells of L. plantarum strains reacted with LP primers, yet not with any of the other strains. The PCR product was confirmed by DNA sequencing. Accordingly, since the PCR method used is simple, specific, and rapid, it will be useful for monitoring and evaluation L. plantarum in the mixed microbial population found in kimchi.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3065-3069
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• 2016

Single nucleotide polymorphism (SNP) detection has been used extensively for genetic association studies of diseases including cancer. For mass, yet accurate and more economic SNP detection we have optimized tetra primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) to detect three SNPs in the cytochrome P450 2E1 (CYP2E1) gene locus; i.e. rs3813865, rs2070672 and rs3813867. The optimization system strategies used were (1) designing inner and outer primers; (2) determining of their optimum primer concentration ratios; and (3) determining of the optimum PCR annealing temperature. The tetra primer ARMS PCR result could be directly observed using agarose gel electrophoresis. The method succesfully determined three SNPs in CYP2E1 locus, the results being consistent with validation using DNA sequencing and restriction fragment length polymorphisms (RFLP).

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.48-55
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• 2004

Six strains of an obligate predatory bdellovibrio isolate that preys on Burkholderia glumae in rice paddy field water and rhizosphere soil, were identified and characterized. The numbers of Bdellovibrio cells varied from $3.2{\times}10^3$ to $9.2{\times}10^3$ plaque-forming unit/g after enrichment in cells of B. glumae. Prey range tests with six Bdellovibrio strains and 17 prey strains of rice-pathogenic, antibiosis-related, or nitrogen-fixing bacteria resulted in unique predation patterns in related prey cells. Strain BG282 had the widest prey range on 7 plant pathogenic bacteria among the 17 prey strains tested. However, no predation occurred with strains of Azospirillum brasilense, Paenibacillus polymyxa, Pseudomonas fluorescens, P. putida, and Serratia marcescens that are associated with antibiosis or nitrogen fixation in the rice ecosystem. Identification was confirmed by the presence of typical bdelloplast in the prey cells of B. glumae and by a PCR assay using B. bacteriovorus-specific primers. Furthermore, 16S rDNA sequencing of the six bdellovibrio strains showed a homology range of 97.2% to 99.2% to the type strain of B. bacteriovorus.

• Parasites, Hosts and Diseases
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• v.52 no.1
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• pp.99-103
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• 2014

Mitochondrial genome sequence of malaria parasites has served as a potential marker for inferring evolutionary history of the Plasmodium genus. In Plasmodium falciparum, the mitochondrial genome sequences from around the globe have provided important evolutionary understanding, but no Indian sequence has yet been utilized. We have sequenced the whole mitochondrial genome of a single P. falciparum field isolate from India using novel primers and compared with the 3D7 reference sequence and 1 previously reported Indian sequence. While the 2 Indian sequences were highly divergent from each other, the presently sequenced isolate was highly similar to the reference 3D7 strain.

• BMB Reports
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• v.37 no.2
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• pp.144-147
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• 2004

Molecular amplification and sequencing of genomic DNA that encodes camel polyubiquitin (PUBC1) was performed by a polymerase chain reaction (PCR) using various sets of primers. The amplification generated a number of DNA fragments, which were sequenced and compared with the polyubiquitin coding sequences of various species. One DNA fragment that conformed to 325 bp was found to be 95 and 88% homologous to the sequences of human polyubiquitin B and C, respectively. The DNA translated into 108 amino acids that corresponded to two fused units of ubiquitin with no intervening sequence, which indicates that it is a polyubiquitin and contains at least two units of ubiquitin. Although, variations were found in the nucleotide sequence when compared to those of other species, the amino acid sequence was 100% homologous to the polyubiquitin sequences of humans, mice, and rats. This is the first report of the polyubiquitin DNA coding sequence and its corresponding amino acid sequence from camels, amplified using direct genomic DNA preparations.

• Journal of Microbiology
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• v.40 no.2
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• pp.151-155
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• 2002

A Superoxide Dismutase (SOD) gene from the obligate intracellular bacterium Orientia tsutsugamushi has been cloned by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Nucleotide sequencing revealed that the predicted amino acid sequence was significantly more homologous to known iron-containing SODs (FeSOD) than to manganese-containing SODs (MnSOD). Conserved regions in bacterial FeSOD could also be seen. Isolation of the oriential SOD gene may provide an opportunity to examine its role in the intracellular survival of this bacterium.