• Title/Summary/Keyword: specific probe

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Detection of Biodegradative Genes in Oil Contaminated Soil Microbial Community by Oligonucleotide Microarray (Oligonucleotide Microarray를 이용한 유류 오염 토양 미생물 군집내 난분해성 화합물 분해 유전자의 검출)

  • Lee Jong-Kwang;Kim Hee;Lee Doo-Myoung;Lee Seok-Jae;Kim Moo-Hoon
    • 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.

Comparison between Dot Blot Hybridization and Southern Blot Hybridization in Detecting Methicillin Resistant Staphylococcus aureus (Methicillin 내성 Staphylococcus aureus의 검출을 위한 분자유전학적 기법에 관한 연구)

  • 조태흠;김민정;오양효
    • Journal of Life Science
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    • v.9 no.4
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    • pp.358-367
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    • 1999
  • Thirty strains of methicillin resistant Staphylococcus aureus were obtained from the clinical isolates. In order to investigate the pursuit of the pathogens of nosocomial infection, these strains were studied for antibiotic sensitivity as well as its resistant pattern. Among the methods of hybridization which directly confirm the specific antibiotic resistant genes by means of the recently developed specific probe DNA, dot blot hybridization and southern blot hybridization were performed and these two methods were compared in their sensitivity and specificity. Strains that is sensitive to cephalothin to the subject of methicillin resistant Staphylococcus aureus were in 43%. Those that are sensitive to cefoperazone and cefuroxime were 26% and 23%, respectively. In case of MIC, MIC50 of cefoperazone was 8 $\mu\textrm{g}$/$m\ell$, and MIC90 was 128 $\mu\textrm{g}$/$m\ell$ to be the lowest. As the results of plasmid DNA electrophoresis, most of methicillin resistant Staphylococcus aureus strains had more than 4 plasmids. These plasmids digested by BamHI, methicillin resistant Staphylococcus aureus is distributed as 10 fragments with the size of 65 kb to 1.5 kb. Dot blot hybridization were performed to examine the existence of mecA gene to show the detection rate of 50%. Southern blot hybridization were done to see if DNA bands which amplify the activity of digoxigenium-labeled probe by PCR were actually PCR products of mecA gene and it showed the detection rate of 53%. It can be concluded that the southern blot hybridization seemed to be better in sensitivity and specificity when it is compared with the results of dot blot hybridization.

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Detection of Ralstonia solanacearum with Nested PCR and DNA Enzyme-Linked Immunosorbent Assay (Nested PCR과 DNA Enzyme-Linked Immunosorbent Assays를 이용한 Ralstonia solanacearum의 검출)

  • Ko, Young-Jin;Cho, Hong-Bum
    • Korean Journal of Microbiology
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    • v.43 no.3
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    • pp.179-185
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    • 2007
  • In this study, we used the method of guanidin isothiocyanate and boiling with Chelex-100 resin to extract genomic DNA of Ralstonia solanacearum from soil. It is more efficient than general protocols to remove inhibitory compounds in soil and R. solanacearum on. Then, we applied polymerase chain reaction and DNA enzyme-linked immunosorbent assay (ELISA) to identify and detect pathogen. The fliC gene of R. solanacearum was selected for specific detection of pathogen and primer sets were designed. Among the primer sets, two specific and sensitive primer sets, RsolfliC(forward: 5-GAACGCCAACGGTGCGAACT-3 and reverse; 5-GGCGGCCTTCAGGGAGGTC-3, designed by J. $Sch\ddot{o}nfeld$ et al.) and RS_247 (forward: 5-GGCGGTCTGTCGGCRG-3 and reverse; 5-CGGTCGCGTTGGCAAC-3 designed by this study), were designed to perform nested PCR. Nested PCR primer was labeled with biotin for hybridization between nested PCR product and probe to analyze with DNA ELISA.

Application of Temperature-compensated Resistivity Probe in the Field (온도보상형 전기저항 프로브의 현장 적용성 평가)

  • Jung, Soon-Hyuck;Yoon, Hyung-Koo;Lee, Jong-Sub
    • KSCE Journal of Civil and Environmental Engineering Research
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    • v.31 no.4C
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    • pp.117-125
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    • 2011
  • The practical use of the electrical resistivity, which can makes the acquirement of the high resolution data in specific area, is increased in order to obtain a reasonable data for a ground investigation. The objective of this study is development of TRPF(Temperature-compensated Resistivity Probe for Field test), and an application in the field test for obtaining a reliable electrical resistivity value about considering the temperature effects. Temperature sensor is attached at 15mm, 30mm, 90mm below from the cone tip in consideration with the results of temperature transient process of cone probe and safety, and the angle of cone tip is $60^{\circ}$ for geometrical reason and minimizing the disturbance during the penetration test. Diameter of the cone probe is equally 35.7mm and penetration rate is 2 cm/sec for a comparison with standard cones such as CPT and SPT, and others. The temperature change is instantly observed around $4^{\circ}C$ when touching the ground, and the comparing results among the other cones indicates that the temperature compensation should be conducted in the ground survey using the electrical resistivity. This study shows that the necessity of temperature effects compensation during penetration test through the development and field verification of TRPF (Temperature-compensated Resistivity Probe for Field test).

Application of LATE-PCR to Detect Candida and Aspergillus Fungal Pathogens by a DNA Hybridization Assay

  • Gopal, Dhayaalini Bala;Lim, Chua Ang;Khaithir, Tzar Mohd Nizam;Santhanam, Jacinta
    • 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.

Rapid Sex Identification of Chicken by Fluorescence In Situ Hybridization Using a W Chromosome-specific DNA Probe

  • Sohn, S.H.;Lee, C.Y.;Ryu, E.K.;Han, J.Y.;Multani, A.S.;Pathak, S.
    • Asian-Australasian Journal of Animal Sciences
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    • v.15 no.11
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    • pp.1531-1535
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    • 2002
  • It has been known that the sex of chicken cells can be most accurately identified by fluorescence in situ hybridization (FISH). However, the presently available FISH has not been widely used for sex identification, because the procedures for cell preparation and FISH itself are complicated and time-consuming. The present study was undertaken to test a rapid FISH procedure for sexing chicken. A FISH probe was simultaneously synthesized and labeled with digoxigenin by polymerase chain reaction (PCR) targeting a 416 bp segment of the 717 bp XhoI family fragment which is repeated over 10 thousand times exclusively in the W chromosome. Sexing by FISH was performed on cytological preparations of early embryos, adult lymphocytes and feather pulps of newly hatched chicks. The DNA probe hybridized to all types of uncultured interphase as well as metaphase female but not male cells that had been examined. Moreover, consistent with the known site of the XhoI family, the hybridization signal was localized to the pericentromeric region of the W chromosome. We, therefore, conclude that the present PCR-based FISH can be used as a rapid and reliable sex identification procedure for chicken.

RELIABILITY and VALIDITY of DUAL PROBE-FIXING FRAME for REHABILITATIVE ULTRASOUND IMAGING for EXERCISES with VISUAL FEEDBACK

  • Na-eun Byeon;Jang-hoon Shin;Wan-hee Lee
    • Physical Therapy Rehabilitation Science
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    • v.12 no.3
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    • pp.259-267
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    • 2023
  • Objective: Rehabilitative ultrasound imaging is a safe and noninvasive technique for evaluating muscle thickness. A dual probe-fixing frame (DPF) can provide visual feedback during exercises targeting specific muscles. The purpose of this research was to verify the reliability and validity of the DPF for dual-probe ultrasound (DPU)-based visual feedback exercises, allowing users to use both hands freely. Design: This cross-sectional study used repeated measures to compare muscle thickness measurements obtained using the handheld device and DPF with DPU. Methods: Twenty healthy adults participated in the study. Measurements were taken over two sessions, with a two-day interval between the sessions. The thicknesses of the rectus abdominis (RA) and transverse abdominis (TrA) muscles were measured using DPU. The DPF with DPU developed by the research team, was used along with a laptop-based muscle viewer. Bland-Altman analysis and intraclass correlation coefficients (ICCs) calculations were used in statistical analyses to evaluate agreement and reliability, respectively. Results: The results of the Bland-Altman analysis showed small average differences between the handheld and DPF methods for both RA and TrA muscle thicknesses. Inter-rater reliability analysis showed high ICC values for DPF measurements of both RA (0.908-0.912) and TrA (0.892-741) muscle thicknesses. Intra-rater reliability analysis also showed good ICC values for measurements taken by a single examiner over two days. Conclusion: The findings of this study demonstrate that the DPF provides reliable and valid measurements of muscle thickness during visual feedback exercises using the DPU.

Microplate hybridization assay for detection of isoniazid resistance in Mycobacterium tuberculosis

  • Han, Hye-Eun;Lee, In-Soo;Hwang, Joo-Hwan;Bang, Hye-Eun;Kim, Yeun;Cho, Sang-Nae;Kim, Tae-Ue;Lee, Hye-Young
    • BMB Reports
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    • v.42 no.2
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    • pp.81-85
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    • 2009
  • Early and accurate detection of drug resistant Mycobacterium tuberculosis can improve both the treatment outcome and public health control of tuberculosis. A number of molecular-based techniques have been developed including ones using probe molecules that target drug resistance-related mutations. Although these techniques are highly specific and sensitive, mixed signals can be obtained when the drug resistant isolates are mixed with drug susceptible isolates. In order to overcome this problem, we developed a new drug susceptibility test (DST) for one of the most effective anti-tuberculosis drug, isoniazid. This technique employed a microplate hybridization assay that quantified signals from each probe molecule, and was evaluated using clinical isolates. The evaluation analysis clearly showed that the microplate hybridization assay was an accurate and rapid method that overcame the limitations of DST based on conventional molecular techniques.

Genome Detection Using an Integrated type DNA Chip Microelectrode-array and Non-labeling Target DNA (집적형 DNA칩 미소 전극 어레이 및 비수식화 표적 DNA를 이용한 유전자 검출)

  • Choi, Yong-Sung;Lee, Hea-Yeon;Tanaka, Hiroyuki;Tanaka, Hidekafu;Kwon, Young-Soo;Kawai, Tomoii
    • Proceedings of the KIEE Conference
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    • 2001.11a
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    • pp.274-276
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    • 2001
  • This research aims to develop the multiple channel electrochemical DNA chip using microfabrication technology. At first, we fabricated a high integration type DNA chip array by lithography technology. Several probe DNAs consisting of thiol group at their 5-end were immobilized on the sold electrodes. Then target DNAs were hybridized and reacted. Cyclic voltammetry showed a difference between target DNA and control DNA in the anodic peak current values. Therefore, it is able to detect a plural genes electrochemically after immobilization of a plural probe DNA and hybridization of non-labeling target DNA on the electrodes simultaneously. It suggested that this DNA chip could recognize the sequence specific genes.

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Optimal Fuzzy Sliding-Mode Control for Microcontroller-based Microfluidic Manipulation in Biochip System

  • Chung, Yung-Chiang;Wen, Bor-Jiunn
    • 제어로봇시스템학회:학술대회논문집
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    • 2004.08a
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    • pp.196-201
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    • 2004
  • In biometric and biomedical applications, a special transporting mechanism must be designed for the ${\mu}$TAS (micro total analysis system) to move samples and reagents through the microchannels that connect the unit procedure components in the system. An important issue for this miniaturization and integration is microfluid management technique, i.e., microfluid transportation, metering, and mixing. In view of this, this study presents an optimal fuzzy sliding-mode control (OFSMC) design based on the 8051 microprocessor and implementation of a complete microfluidic manipulated system implementation of biochip system with a pneumatic pumping actuator, a feedback-signal photodiodes and flowmeter. The new microfluid management technique successfully improved the efficiency of molecular biology reaction by increasing the velocity of the target nucleic acid molecules, which increases the effective collision into the probe molecules as the target molecules flow back and forth. Therefore, this hybridization chip was able to increase hybridization signal 6-fold and reduce non-specific target-probe binding and background noises within 30 minutes, as compared to conventional hybridization methods, which may take from 4 hours to overnight. In addition, the new technique was also used in DNA extraction. When serum existed in the fluid, the extraction efficiency of immobilized beads with solution flowing back and forth was 88-fold higher than that of free-beads.

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