• Title/Summary/Keyword: fluorescent detection

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Development of Liposome Immunoassay for Salmonella spp. using Immunomagnetic Separation and Immunoliposome

  • Shin, Jung-Hee;Kim, Myung-Hee
    • Journal of Microbiology and Biotechnology
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    • v.18 no.10
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    • pp.1689-1694
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    • 2008
  • The ability to detect Salmonella spp. is essential in the prevention of foodborne illness. This study examined a Salmonella spp. detection method involving the application of immunomagnetic separation and immunoliposomes (IMS/IL) encapsulating sulforhodamine B (SRB), a fluorescent dye. A quantitative assay was conducted by measuring the fluorescence intensity of SRB that was produced from an immunomagnetic bead-Salmonella spp.-immunoliposome complex. The results indicated detection limits of $2.7{\times}10^{5}$ and $5.2{\times}10^{3}$ CFU/ml for Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterka subsp. enterka serovar Typhimurium (S. Typhimurium), respectivley. The signal/noise ratio was improved by using 4% skim milk as a wash solution rather than 2% BSA. In addition, higher fluorescence intensity was obtained by increasing the liposome size. Compared with the conventional plating method, which takes 3-4 days for the isolation and identification of Salmonella spp., the total assay time of to h only including 6 h of culture enrichment was necessary for the Salmonella detection by IMS/IL. These results indicate that the IMS/ IL has great potential as an alternative rapid method for Salmonella detection.

Colloidal Textile Dye-Based Dipstick Immunoassay for the Detection of Infectious Flacherie of Silkworm, Bombyx mori L.

  • Sivaprasad, V.;Nataraju, B.;Renu, S.;Datta, R.K.
    • International Journal of Industrial Entomology and Biomaterials
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    • v.6 no.1
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    • pp.27-31
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    • 2003
  • Infectious flacherie of silkworm Bombyx mori is caused by B. mori infectious flacherie virus (BmIFV) and causes severe crop loss to sericulturists. In the present study, a colloidal textile dye-based dipstick immunoassay is developed for the detection of infectious flacherie in silkworms. Colloidal textile dye (blue D2R) with Aλ$_{max}$ at 620 nm was sensitised with 500 $\mu\textrm{g}$/ml of purified anti-BmIFV IgG. The dye-antibody reagent detects purified antigen up to 10 ng/ml and BmIFV infection in diseased larval extracts $(up to a dilution of {10^-5})$ and faecal matter extracts $(up to a dilution of {10^-2})$ by forming clear blue dot within 30 min. It was observed to be stable for three months period at $4^{\circ}C$. The efficacy of textile dye-based dipstick immunoassay was on pay with HRP-based dipstick immunoassay and fluorescent antibody test, and better than latex agglutination and ouchterlony tests in the detection of BmIFV The dye-based dipstick immunoassay method provides a simple, sensitive and less expensive test for the detection of BmIFV infection in silkworms.s.

Toxic detection in mine water based on proteomic analysis of lysosomal enzymes in Saccharomyces cerevisiae

  • Nguyen, Ngoc-Tu;Kim, Yang-Hoon;Bang, Seung Hyuck;Hong, Ji Hye;Kwon, Soon Dong;Min, Jiho
    • Environmental Analysis Health and Toxicology
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    • v.29
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    • pp.19.1-19.10
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    • 2014
  • Objectives Lysosome is the cell-organelle which is commonly used as biomonitoring tool in environmental pollution. In this study, the lysosomal proteomic of the yeast Saccharomyces cerevisiae was analyzed for utilization in the detection of toxic substances in mine water samples. Methods This work informs the expression of lysosomal proteomic in yeast in response with toxic chemicals, such as sodium meta-arsenite and tetracycline, for screening specific biomarkers. After that, a recombinant yeast contained this biomarker were constructed for toxic detection in pure toxic chemicals and mine water samples. Results Each chemical had an optimal dose at which the fluorescent protein intensity reached the peak. In the case of water samples, the yeast showed the response with sample 1, 3, 4, and 5; whereas there is no response with sample 2, 6, and 7. Conclusions The recombinant yeast showed a high ability of toxic detection in response with several chemicals such as heavy metals and pharmaceuticals. In the case of mine water samples, the response varied depending on the sample content.

Analysis of Saikosaponins by HPLC with Photoreduction Fluorescence Detection (광반응 HPLC를 이용한 시호 사포닌의 분석)

  • Shin, Young-Geun;Cho, Kyung-Hee;Kwon, Soo-Jin;Do, Young-Mi;Hwang, Gwi-Seo;Park, Jeong-Hill;Park, Man-Ki
    • YAKHAK HOEJI
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    • v.40 no.1
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    • pp.41-45
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    • 1996
  • A high performance liquid chromatography using photoreduction fluorescence detection was described for the analysis of saikosaponins. Saikosaponins were separated on an $NH_2$ column using acetonitrile and aqueous 2-tert-butylanthraquinone(t-BAQ) as mobile phase. Column effluent was passed through a 40cm PTFE capillary tube coiled around a 10W UV lamp to reduce t-BAQ to a highly fluorescent dihydroxyanthracene derivative which was detected by a fluorescence detector. The optimal concentration of t-BAQ was found to be $6{\times}10^{-5}M$ and the optimal reaction time to be 2 seconds. The detection limit for saikosaponin a and d by this method was found to be about 280ng and 80ng. The dynamic linear range was over two orders and the correlation coefficient of the calibration curve of them was 0.998.

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Fluorimetric Determination of Dichloroacetamide by RPLC with Postcolumn Detection

  • Choi, Yong-Wook;Reckhow, David A.
    • Bulletin of the Korean Chemical Society
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    • v.25 no.6
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    • pp.900-906
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    • 2004
  • An RPLC-postcolumn detection method has been developed for the fluorimetric determination of dichloroacetamide (DCAD) in water. After ammonia and DCAD were separated on a $C_{18}$ nonpolar stationary phase with 2.5% methanol-0.02 M phosphate buffer at pH 3, the column eluant was reacted with post column reagents, o-phthaldialdehyde (OPA) and sulfite ion at pH 11.5, to produce a highly fluorescent isoindole fluorophore, which was measured with a fluorescence detector ( ${\lambda}_{ex}$ = 363 nm, ${\lambda}_{em}$ = 425 nm). With the optimized conditions for RPLC and the postcolumn derivatization, the calibration curve was found to be linear in the concentration ranges of 0.5 and 20 ${\mu}$M for DCAD, and the detection limit for DCAD was 0.18 ${\mu}$M (23${\mu}$g/L). This corresponded to 18 pmol per 100 ${\mu}$L injection volume for a signal-to-noise ratio of 3, and the repeatability and reproducibility of this method were 1.0% and 2.5% for five replicate analyzes of 2 ${\mu}$M DCAD, respectively. The degradation yields DCAD to ammonia were 94 and 99%, and the percent recoveries of DCAD from 4 and 6 ${\mu}$M DCAD-spiked tap water were shown mean more than 97%.

Simple and Rapid Detection of Potato leafroll virus by Reverse Transcription Loop-mediated Isothermal Amplification

  • Ju, Ho-Jong
    • The Plant Pathology Journal
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    • v.27 no.4
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    • pp.385-389
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    • 2011
  • A new reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the Potato leafroll virus (PLRV) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to address its advantages over RTPCR. RT-LAMP primers were designed from the open reading frame 3 (ORF3) sequence of PLRV. The RT-LAMP reactions were conducted without or with a set of loop primers. By real-time monitoring using Turbimeter, the RT-LAMP (with loop primers) detects PLRV in less than 30 min, compared to 120 min of RT-PCR. By adding fluorescent reagent during the reaction, final products of the RT-LAMP were fluorescently visualized under UV light or could be differentiated by naked-eye inspection under normal light. The RT-LAMP was extremely sensitive, about 2000-fold more sensitive than RT-PCR. This study presents great potential of the RT-LAMP for diagnosis and PLRV epidemiology because RT-LAMP method is speedy, sensitive, inexpensive, and convenient.

In Situ Identification of Cyanobacteria

  • Ahn Tae-Seok;Hong Sun-Hee;Chung Hyun-Mi;Belkova Natalia L.
    • Proceedings of the Microbiological Society of Korea Conference
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    • 2001.11a
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    • pp.121-127
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    • 2001
  • Seven cyanobacteria strains (Anabaena macrospora NIERl0016, Oscillatoria sp. NIER10042, Microcystis aeruginosa NIER10015, M. ichtyoblabe BIER10025, BIER10040, M. novacekii NIER10029, M. wesenbergii NIER10068) were tested with four rRNA - targeted oligonucleotide probes labelled with horseradish peroxidase (HRP) and specific for cyanobacteria. Non- fluorescent detection of hybridization signal was used. The hybridization with artificial mixture of cyanobacteria have shown the possibility to use 2 species-specific probes in duplicate hybridization and detection with different colored substrates.

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Detection and Quantification Method of Beta-amyloid for Alzheimer Disease Diagnosis (알츠하이머 질병의 조기진단을 위한 베타 아밀로이드의 검출 및 정량화 방법)

  • Kim, Kwan-Su;Kang, Jae-Min;Chae, Cheol-Joo;Song, Ki-Bong
    • Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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    • 2010.06a
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    • pp.220-220
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    • 2010
  • The beta-amyloid protein ($A_{\beta}$) is well known for main cause of Alzheimer disease (AD). Generally, detection of $A_{\beta}$ is carried out by using fluorescent material or DNA test, but these process is long time and expensive process. Therefore, in this research, we investigated the simple diagnosis method to detect the $A_{\beta}$ by using photo-transistor.

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Design and Synthesis of Metallopeptide Sensors: Tuning Selectivity with Ligand Variation

  • Kim, Joung-Min;Joshi, Bishnu Prasad;Lee, Keun-Hyeung
    • Bulletin of the Korean Chemical Society
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    • v.31 no.9
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    • pp.2537-2541
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    • 2010
  • We chose a fluorescent pentapeptide sensor (-CPGHE) containing a dansyl fluorophore as a model peptide and investigated whether the selectivity and sensitivity of the peptides for heavy and transition metal ions could be tuned by changing amino acid sequence. In this process, we developed a selective peptide sensor, Cp1-d (-HHPGE, $K_d\;=\;670\;nM$) for detection of $Zn^{2+}$ in 100% aqueous solution and a selective and sensitive peptide sensor, Cp1-e (-CCHPGE, $K_d\;=\;24\;nM$) for detection of $Cd^{2+}$ in 100% aqueous solution. Overall results indicate that the selectivity and sensitivity of the metallopeptide sensors to specific heavy and transition metal ions can be tuned by changing amino acid sequence.

Analysis of Ginseng Saponins by HPLC with Photoreduction Fluorescence Detection

  • Kim, Bae-Yuan;Lee, Mi-Young;Cho, Kyung-Hee;Park, Jeong-Hill;Park, Man-Ki
    • Archives of Pharmacal Research
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    • v.15 no.4
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    • pp.328-332
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    • 1992
  • A new high performance liquid chromatographic procedure is described for the analysis of ginseng saponins. Ginseng saponins were separated on Lichrosorb $NH_2$ column and anthraquinone-2, 6-disulfonate (AQDS) solution was added to the column effluent. The effluent was passed through 1.5m-PTFE capillary coiled around 10 W-UV lamp to reduce AQDS to highly fluorescent 9. 10-dihydroxyanthracene-2, 6-disulfonate which was detected by fluorescence detector. The detection limit for the ginsenoside $Rg_1$ by this method was found to be about 350 ng, the dynamic linear range was $10^2$ and the correlation coefficient of the calibration curve was 0.9999.

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