• Korean Journal of Food Science and Technology
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• v.35 no.3
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• pp.470-474
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• 2003

DNA sequence information on small-subunit rRNA gene (16S rDNA) obtained from food-poisoning bacterial culture was used to investigate the presence of bacterial pathogens in food. By reverse dot blot detection method, presence of food-poisoning bacteria could be confirmed on hybridization of digoxigenin-labeled 16S rDNA Polymerase Chain Reaction (PCR) primer product and biotin-labeled specific oligonucleotide probe. Escherichia coli, Bacillus cereus. and Salmonella sp. were used as the representative food-poisoning bacterial microorganisms. An oligonucleotide probe, based on the variable region of 16S rRNA gene, was used as the specific probe. These tools may be more useful than classic biochemical method for rapid identification of contaminated food.

• Proceedings of the KIEE Conference
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• 2002.07c
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• pp.1569-1571
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• 2002

The determination of DNA hybridization can apply the molecular biology research, clinic diagnostics, bioengineering, environment monitoring, food science and other application area. So, The determination of hybridization is very important for the improvement of DNA detection system. In this study, we report the characterization of the DNA hybridization by the electricalchemical methods. A new electrochemical biosensor is described for voltammetric detection of gene sequence related to probe oligonucleotide of bacterium Escherichia coli O157:H7. The biosensor involves the immobilization of a 18-mer probe oligonucleotide, which is complemetary to a specific gene sequence related to Escherichia coli O157:H7 on a gold electrode through specific adsorption. The probe oligonucleotide was used to determine the amount of target oligonucleotide in solution using mitoxantrone(MTX) as the electrochemical indicators. The cathodic peak currents $(I_{peak})$ of MTX were linearly related to the concentration of the target oligonucleotide sequence in the range $1[{\mu}M]{\sim}0.1[nM]$. The detection limit of this approach was 0.01[nM]. In addition, these indicators were capable of selectivity discriminating against various mismatching condition.

• Tuberculosis and Respiratory Diseases
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• v.49 no.5
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• pp.546-557
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• 2000

Background : Oligonucleotide chip technology has proven to be a very useful tool in the rapid diagnosis of infectious disease. Rifampin resistance is considered as a useful marker of multidrug-resistance in tuberculosis. Mutations in the rpoB gene coding $\beta$ subunit of RNA polymerase represent the main mechanism of rifampin resistance. The purpose of this study was to develop a diagnosis kit using oligonucleotide chip for the rapid and accurate detection of rifampin-resistance in Mycobacterium tuberculosis. Method : The sequence specific probes for mutations in the rpoB gene were designed and spotted onto the glass slide, oligonucleotide chip. 38 clinical isolates of Mycobacterium were tested. A part of rpoB was amplified, labelled, and hybridized on the oligonucleotide chip with probes. Results were analyzed with a laser scanner. Direct sequencing was done to verify the results. Result : The low-density oligonucleotide chip design어 to determine the specific mutations in the rpoB gene of M. tuberculosis accurately detected rifampin resistance associated with mutations in 28 clinical isolates. Mutations at codons 531, 526, and 513 were confirmed by direct sequencing analysis. Conclusion : Mutant detection using oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of multidrug-resistance in M. tuberculosis.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.658-665
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• 2009

Quality control QC for spot-uniformity is a critical point in fabricating an oligonucleotide array, and quantification of targets is very important in array analysis. We developed two new types of QC probes as a means of confirming the quality of the uniformity of attached probes and the quantification of targets. We compared the signal intensities and fluorescent images of the QC and target-specific probes of arrays containing only target-specific probes and those containing both QC and target-specific probes. In a comparison of quality control methods, it was found that the arrays containing QC probes could check spot-uniformity or spot defects during all processes of array fabrication, including after spotting, after washing, and after hybridization. In a comparison of quantification results, the array fabricated by the method using QC probes showed linear and regular results because it was possible to normalize variations in spot size and morphology and amount of attached probe. This method could avoid errors originating in probe concentration and spot morphology because it could be normalized by QC probes. There were significant differences in the signal intensities of all mixtures (P<0.05). This result indicates that the method using QC probes is more useful than the ordinary method for quantification of mixed target. In the quantification of mixed targets, this method could determine a range for mixed targets of various amounts. Our results suggest that methods using QC probes for array fabrication are very useful to the quality control of spots in the fabrication processes of quantitative oligonucleotide arrays.

• Journal of Soil and Groundwater Environment
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• v.11 no.1
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• pp.1-6
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• 2006

The analysis of functional population and its dynamics on the environment is essential for understanding bioremediation in environment. Here, we report a method for oligonucleotide microarray for the monitoring of aliphatic and aromatic degradative genes. This microarray contained 15 unique and group-specific probes which were based on 100 known genes involved pathways in biodegradation. Hybridization specificity tests with pure cultures, strain Pseudomonas aeruginosa KCTC 1636 indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. It was found that the presence of 8 genes encoding alkane, naphthalene, biphenyl, pyrene (PAH ring-hydroxylating) degradation pathway could be detected in oil contaminated soil sample. Therefore, the findings of this study strongly suggest that oligonucleotide microarray is an effective diagnostic tool for evaluating biodegradation capability in oil contaminated subsurface environment.

• Biomedical Science Letters
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• v.2 no.2
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• pp.249-255
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• 1996

In this paper, a in situ hybridization(ISH) has been used to investigate the yield of viral RNA expression from each organ tissues. It is studied to establish a rapidly, specific diagnostic method detecting rabbit haemorrhagic disease virus(RHDV) RNA in 10% formalin-fixed, paraffin-em-bedded tissues of naturally RHDV-infected rabbits using oligonucleotide probe to be made by RHDV total sequences. Biotin was used as the oligonucleotide probe marker. in situ hybridization is detected the virus genome in the cells and tissue as specifically compared with others nucleic acid hybridization method. All ISH procedure of RHDV were completed to Mi-croProbe$^{TM}$ capillary action system within 1-2 hours. In this report, RHDV was distributed widely in the cytoplasm of liver cell and the cortex of kidney but lung tissue and medulla of kidney were showed to positive reaction at locally. Although not entirely free of technical limitations, nucleic acid identification holds advantages over other diagnostic tests, including exquisite sensitivity, specificity, interchangeability and speed. It is expected that, in the immediate future viral nucleic acid detection will be a prominent part of the methods used in histopathology.

• Fisheries and Aquatic Sciences
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• v.3 no.1
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• pp.64-70
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• 2000

DNA probes for the l6S rRNA have been designed for the detection of Edwardsiella tarda. In order to accomplish this purpose, the l6S rRNA gene from E. tarda has been cloned and sequenced. Two highly feasible oligonucleotide probe sites have been determined by the database analysis programs presented by PCGENE and BLAST. These two probes have been evaluated by slot blot hybridization analysis. Hetero- and homo-trimeric templates have been synthesized using these two probe sites. The templates have been further multimerized by PCR to generate between 150 and 300 bp long DIG-11-dUTP labeled probes. Unlike 3' end labeled oligonucleotide probes or templates, multimerized probes showed no cross­hybridization in the given experimental condition. Furthermore, a significant increase in sensitivity has been observed with these probes. This method, we presented here, may be useful for the designing of probes for the detection of other fish pathogenic microorganisms also.

• Journal of Microbiology and Biotechnology
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• v.16 no.5
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• pp.740-747
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• 2006

Amplification by universal consensus sequences in pathogenic bacterial DNA would allow rapid identification of pathogenic bacteria, and amplification of genus-specific and species-specific sequences of pathogenic bacterial DNA might be used for genotyping at the genus and species levels. For design of probes for molecular diagnostics, several tools are available as stand-alone programs or as Web application. However, since most programs can design only a few probe sets at one time, they are not suitable for large-scale and automatic probes design. Therefore, for high-throughput design of specific probes in diagnostic array development, an automated design tool is necessary. Thus, we developed a Web-based automatic system for design of genus-specific and species-specific probes for pathogen detection. The system is available at http://www.miprobe.com.

• Microbiology and Biotechnology Letters
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• v.45 no.4
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• pp.358-364
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• 2017

Asymmetric PCR preferentially amplifies one DNA strand for use in DNA hybridization studies. Linear-After-The-Exponential-PCR (LATE-PCR) is an advanced asymmetric PCR method which uses innovatively designed primers at different concentrations. This study aimed to optimise LATE-PCR parameters to produce single-stranded DNA of Candida spp. and Aspergillus spp. for detection via probe hybridisation. The internal transcribed spacer (ITS) region was used to design limiting primer and excess primer for LATE-PCR. Primer annealing and melting temperature, difference of melting temperature between limiting and excess primer and concentration of primers were optimized. In order to confirm the presence of single-stranded DNA, the LATE-PCR product was hybridised with digoxigenin labeled complementary oligonucleotide probe specific for each fungal genus and detected using anti-digoxigenin antibody by dot blotting. Important parameters that determine the production of single-stranded DNA in a LATE-PCR reaction are difference of melting temperature between the limiting and excess primer of at least $5^{\circ}C$ and primer concentration ratio of excess primer to limiting primer at 20:1. LATE-PCR products of Candida albicans, Candida parapsilosis, Candida tropicalis and Aspergillus terreus at up to 1:100 dilution and after 1 h hybridization time, successfully hybridised to respective oligonucleotide probes with no cross reactivity observed between each fungal genus probe and non-target products. For Aspergillus fumigatus, LATE-PCR products were detected at 1:10 dilution and after overnight hybridisation. These results indicate high detection sensitivity for single-stranded DNA produced by LATE-PCR. In conclusion, this advancement of PCR may be utilised to detect fungal pathogens which can aid the diagnosis of invasive fungal disease.

• Fisheries and Aquatic Sciences
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• v.7 no.4
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• pp.175-183
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• 2004

The dinoflagellate Alexandrium tamiyavanichii Balech, a producer of toxins causing paralytic shellfish poisoning (PSP), has recently been considered as one of main organisms responsible for toxication of shellfish in Japan. In this study, A. tamiyavanichii was subjected to a molecular phylogenetic analysis inferred from 28S rDNA D1-D2 sequences and a species-specific LSU rRNA-targeted oligonucleotide DNA probe was designed to identify A. tamiyavanichii using the whole cell-FISH (fluorescence in situ hybridization). The sequences of the 28S rDNA D1-D2 region of A. tamiyavanichii showed no difference from A. cohorticular AF1746l4 (present name A. tamiyavanichii) and formed a distinct clade from the 'tamarensis species complex'. The probe, TAMID2, reacted specifically with A. tamiyavanichii cultured cells, without any cross-reaction with other species belonging to the same genus, including A. tamarense, A. catenella, A. affine, A. fraterculus, A. insuetum and A. pseudogonyaulax. In a test of cross-reactivity with a field sample, TAMID2 reacted consistently with only A. tamiyavanichii, indicating that the present protocol involving the TAMID2 probe might be useful for detecting toxic A. tamiyavanichii in a simple and rapid manner.