• 한국유기농업학회지
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• 제27권3호
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• pp.353-363
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• 2019

To identify four cyst nematodes (Heterodera schachtii, H. trifolii, H. glycines, H. sojae) that are economically important plant-parasitic nematodes in Korea, restriction fragment length polymorphism (RFLP) by 8 endonucleases (PstI, VspI, AlwI, RsaI, MvaI, EcoRI, Eco72I, Hinf I) was performed based on sequence difference of mitochondrial DNA cytochrome c oxidase subunit I (COI) gene. As a result, species-specific DNA band patterns by RsaI endonuclease were observed in H. schachtii. The specific patterns was in H. trifolii by 3 endonucleases (VspI, AlwI, Hinf I), and was in H. glycines by Hinf I. While, H. sojae was not digested by 4 endonuclease (VspI, AlwI, RsaI, Hinf I). This study showed that four cyst nematodes could be distinguished using RFLP by 4 endonucleases (RsaI, VspI, AlwI, Hinf I) based on the sequence difference of COI gene.

• BMB Reports
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• 제38권4호
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• pp.491-499
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• 2005

DNA-based molecular markers for differentiation of five penaeid shrimps (Penaeus monodon, P. semisulcatus, Feneropenaeus merguiensis, Litopenaeus vannamei and Marsupenaeus japonicus) were developed based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single-stranded conformation polymorphism (SSCP) of 16S ribosomal (r) DNA. Differentiation of P. monodon, P. semisulcatus and L. vannamei can be unambiguously carried out by PCR-RFLP of 16S $rDNA_{560}$ whereas P. semisulcatus and M. japonicus shared a BABB mitotype. These shrimps were successfully discriminated by SSCP analysis of 16S $rDNA_{560}$. Nevertheless, the amplification success for L. vannamei and F. merguiensis was not consistent when tested against larger sample sizes. As a result, 16S $rDNA_{560}$ of an individual representing the most common mitotype of each species was cloned and sequenced. The new primer pair was designed and tested against the large sample sizes (312 bp product, N = 185). The amplification success was consistent across all species. PCR-RFLP of 16S $rDNA_{312}$ was as effective as that of 16S $rDNA_{560}$. Differentiation of all shrimp species were successfully carried out by SSCP analysis.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.284-289
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• 2004

A PCR assay for the rapid detection of Vibrio vulnificus strains was developed using a virulence gene for elastase found in various Vibrio species. The DNA sequences in the elastase gene facilitated the identification of a species-specific probe for pathogenic V. vulnificus strains from both clinical and environmental sources. Using an elastase gene-based PCR reaction, a species-specific 507-bp PCR product was visualized by agarose gel electrophoresis. Three different DNA extraction methods were then compared to improve the simplicity and rapidity of detection. A PCR assay using the conventional DNA extraction or boiling method was able to detect as few as 25 V. vulnificus cells, making the detection limits at least 1-log-scale lower than that for the EDT A-treated DNA extraction method. In particular, the boiling method, which does not require purification of the chromosomal DNA, was very effective in terms of simple and rapid detection. Meanwhile, the detection limit in a mixed bacterial culture that included other bacteria, such as Escherichia coli or Bacillus subtilis, was two V. vulnificus cells, which was 1-log-scale lower than that for the control. Accordingly, when coupled with a new DNA extraction method, the elastase gene-based PCR can provide a rapid, specific, and sensitive method for identifying V. vulnificus in clinical and environmental samples.

• BMB Reports
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• 제39권6호
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• pp.788-793
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• 2006

Bacterial DNA containing immunostimulatory CpG motifs can stimulate antigen-presenting cells to express co-stimulatory molecules and to produce various cytokines in vivo and in vitro. In this study, we fragmented macromolecular E.coli genomic DNA with DNase I, and analyzed the ability of the resulting DNA fragments to induce the NF-${\kappa}B$ activation and humoral immune response. Furthermore, using computational analysis and luciferase assay for synthetic ODNs based on the sequence of the immunostimulatory DNA fragments (DF-ODNs), an active component of DF-ODNs sequences was investigated. Experimental results demonstrated that DF-ODN is optimal for the NF-${\kappa}B$-responsive promoter activation in the mouse macrophage cell line and the humoral immune response in vivo. In agreement with the activity of the DF-ODNs processed by DNase I, a synthetic ODN based on the DF-ODN sequences is potent at inducing IL-12 mRNA expression in primary dendritic cells. These results suggest that the discovery and characterization of a highly active natural CpG-ODN may be achieved by the analyses of bacterial DNA fragments generated by a nuclease activity.

• 대한임상검사과학회지
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• 제38권3호
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• pp.158-165
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• 2006

Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. However, Mycobacterium species has a long period of incubation, and requires serious biochemical tests such as niacin, catalase, and nitrate test that are often tedious. The development of rapid and accurate diagnostics can aid in the early diagnosis of disease caused by Mycobacterium. The current DNA amplification and hybridization methods that have been developed target several genes for the detection of mycobacterial species such as hps65, 16S rDNA, rpoB, and dnaj. These methods produce rapid and accurate results. In this study, PCR-restriction fragment length polymorphism analysis(PCR-RFLP) based on the region of the rpoB gene was used to verify the identification of non-tuburculosis Mycobacterium species. A total of 8 mycobacterial reference strains and 13 clinical isolates were digested with restriction enzymes such as Msp I in this study. The results of using this process clearly demonstrated that all 13 specimens were identified by rpoB gene PRA method. The PCR-RFLP method based on the rpoB gene is a simple, rapid, and accurate test for the identification of Mycobacterium.

• 식물병연구
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• 제29권2호
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• pp.130-136
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• 2023

Citrus melanoses caused by Diaporthe citri, has been one of the serious diseases in many citrus orchards of Jeju Island. To protect melanose in citrus farms, a fast and exact diagnosis method is necessary. In this study, diseased leaves and dieback twigs were collected from a total of 49 farms within March to April in 2022. A total of 465 fungal isolates were obtained from a total of 358 isolated plant samples. Among these fungal isolates, 40 representatives of D. citri isolates which were isolated from 22 twigs and 18 leaves on 23 farms were found based on cultural characteristics on potato dextrose agar and conidial morphology. Additionally, the molecular assay was carried out and compared with those by morphological diagnosis. All isolates were identified as D. citri by analyzing the sequences at the internal transcribed spacer (ITS) rDNA region using primers of ITS1/ITS4 or at β-tubulin using primer Btdcitri-F/R. Therefore, based on the present study, where the results of morphological identification of conidial type were consistent with DNA sequence analysis of certain gene, choosing a suitable method for a fast diagnosis of citrus melanose was suggested.

• Journal of Microbiology and Biotechnology
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• 제32권8호
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• pp.1026-1033
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• 2022

This study presents a novel DNA part characterization technique that increases throughput by combinatorial DNA part assembly, solid plate-based quantitative fluorescence assay for phenotyping, and barcode tagging-based long-read sequencing for genotyping. We confirmed that the fluorescence intensities of colonies on plates were comparable to fluorescence at the single-cell level from a high-end, flow-cytometry device and developed a high-throughput image analysis pipeline. The barcode tagging-based long-read sequencing technique enabled rapid identification of all DNA parts and their combinations with a single sequencing experiment. Using our techniques, forty-four DNA parts (21 promoters and 23 RBSs) were successfully characterized in 72 h without any automated equipment. We anticipate that this high-throughput and easy-to-use part characterization technique will contribute to increasing part diversity and be useful for building genetic circuits and metabolic pathways in synthetic biology.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.82-85
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• 2005

Doxorubicin is a highly-valuable anthracycline-family polyketide drug with a very potent anticancer activity, typically produced by a Gram-positive soil bacterium called Streptomyces peucetius. Thanks to the recent development of Streptomyces genomics-based technologies, the random mutagenesis approach for Streptomyces strain improvement has been switched toward the genomics-based technologies including the application of DNA microarray systems. In order to identify and characterize the genomics-driven potential target genes critical for doxorubincin overproduction, three different types of doxorubicin overproducing strains, a dnrI(doxorubicin-specific positive regulatory gene)-overexpressor, a doxA (gene involved in the conversion from daunorubicin to doxorubicin)-overexpressor, and a recursively-mutated industrial strain, were generated and examined their genomic transcription profiles using Streptomyces DNA microarray systems. The DNA microarray results revealed several potential target genes in S. peucetius genome, whose expressions were significantly either up- or down-regulated comparing with the wild-type strain. A systematic understanding of doxorubicin overproduction at the genomic level presented in this research should lead us a rational design of molecular genetic strain improvement strategy.

• Mycobiology
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• 제28권1호
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• pp.17-26
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• 2000

The orchid symbiotic fungi were isolated from the roots of Korean native orchid (Cymbidium goeringii) collected and Chinese orchid (C. sinense) obtained from greenhouses. They were identified as a species of Rhizoctonia, based on the sequences of 18r rDNA, the microscopic observations of mycelia, and the symbiotic relationships with commercial orchids. The isolate collected from Chinese orchids was revealed to be a species of Ceratobasidium endophytica, and to be different from the other isolates at the thickness of the mycelia stained in the root cells of Korean native orchids. The other isolates collected from the Korean native orchids were considered to be a species of Tulsanella repens (anamorphic: Epulorhiza repens) or its related one. The physiologic or microscopic variations were oftenly observed among them, but the tendency of grouping these in the 18s rDNA sequences were observed to be consistent with those of the localities collected. The further taxonomical segregating for Korean symbiotic fungi was not made because the information concerned were limited in this moment, but was recognized as based on the sequences of 18s DNA.

• Parasites, Hosts and Diseases
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• 제49권4호
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• pp.381-384
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• 2011

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial largesubunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.