Fluorescence labeled cell agglutinin probe red tide biological detection reagent kit and detecting method thereof

A technology for fluorescent labeling and biological detection, applied in biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc., to achieve high accuracy and reliability, low detection cost, and convenient use

Inactive Publication Date: 2008-11-26
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because algal species usually do not have obvious morphological characteristics, but have genetic diversity and obvious regional genetic characteristics, the probes developed according to a specific species in a region are not suitable for the same species in other regions, so it is necessary to develop Applicable probes for geographical strains in China sea area

Method used

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  • Fluorescence labeled cell agglutinin probe red tide biological detection reagent kit and detecting method thereof
  • Fluorescence labeled cell agglutinin probe red tide biological detection reagent kit and detecting method thereof
  • Fluorescence labeled cell agglutinin probe red tide biological detection reagent kit and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Fluorescence-labeled lectin probes are used to identify and detect different cell lines and morphologically similar algal cells of the same genus or species

[0037] 1. Test materials: See Table 2 for the names and sources of 14 kinds of 23 strains of cells used in the experiment.

[0038] Table 2 Algae used in the test kit and test method verification experiment and their source and separation time

[0039]

[0040] 2. Detection method:

[0041] The cultivation method of the algae used in the experiment: the algae used in the experiment is separated from the red tide water sample in the sea area of ​​China, and the f / 2 medium is prepared after filtering the seawater with a filter membrane of 0.2 μm, under the light intensity of 5000LX, L:D=12 : The photoperiod of 12 and the temperature of 22°C were cultivated in the algae germplasm bank culture room of the State Key Laboratory of Offshore Marine Environmental Science, Xiamen University.

[0042] Preparation of lec...

Embodiment 2

[0061] Fluorescence-labeled lectin probe combined with fluorescence spectrophotometer for quantitative detection

[0062] One, material: with embodiment 1.

[0063] 2. Method:

[0064] 1. See Example 1 for methods such as cell staining and fixation and lectin probe binding.

[0065] 2. Quantitative determination of the binding of the lectin probe to the sample is detected by a spectrofluorometer (490nm excitation light and 515nm scattered light), and each sample is read twice to calculate the average value. In each case, the cell density after three centrifugation washes was controlled at 10 5 ±10 3 . No need to counterstain with DAPI. In both quantitative and qualitative assays, the cell density in f / 2 medium was 10 5 ±10 3 The cells without lectin were used as control. Each sample was measured 4 to 6 times in parallel, and then statistically analyzed.

[0066] 3. Results

[0067] Fluorescence spectrophotometer can be used to quantitatively measure the binding activ...

Embodiment 3

[0079] Quantitative and qualitative detection of fluorescently labeled lectin probes combined with flow cytometry

[0080] One, material: with embodiment 1.

[0081] 2. Method:

[0082] The algae liquid grown for 7 days was taken at a concentration of 100,000 cells / L, and fixed with 1% paraformaldehyde at 4°C for 1 hour.

[0083] Take about 10ml of algae liquid and centrifuge at 3000g / 10min, and transfer the precipitated algae cells to a 1.5ml centrifuge tube.

[0084] Decolorization: wash and centrifuge twice with salt-ethanol solution (70:30, v / v), 7500g / 2min, and filter once with a 30μm sieve.

[0085] The precipitate was treated with lectin at 100 μg / ml for 1 h at 20°C in the dark. After the reaction was finished, it was centrifuged and washed twice with filtered sterile artificial seawater (salinity 28, pH 8.0), 7500g / 2min.

[0086] Cells were resuspended in artificial seawater and analyzed by flow cytometry (Beckman Coulter Epics Altra2 model).

[0087] 3. Results ...

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Abstract

The biological red tide detecting kit with fluorescently-labeled cell agglutinin probe includes three components, component I, component II and component III. The component I is cell agglutinin probe system with n kinds of fluorescently-labeled cell agglutinin probes compounded with phosphate buffer; the component II is compounded with sterilized and filtered artificial sea water or phosphate buffer and Peck solution with silicon gel grain of vinyl pyrrolidone for cell separation; and the component III is sample fixing system selected from Lugol's iodine solution, polyformaldehyde and glutaraldehyde. The test method with the kit includes the following steps: culturing algae for experiment; fixing cell agglutinin configuration and algae cell; adding Peck solution to the cell cultured matter, adding cell agglutinin probe, adding phosphate buffer and Peck solution, suspending and depositing, re-dyeing with double-strand DNA fluorescent dye and observing; and estimating, counting and calculating fluorescent dying combination rate.

Description

technical field [0001] The present invention relates to a cell agglutinin (Lectin) probe, in particular to a fluorescent-labeled cell agglutinin probe and a low-abundance single target red tide algae in the detection and identification of purely cultured red tide algae cells and natural water bodies applications in cells. Background technique [0002] Red tide is an ecological phenomenon in which certain plankton reproduce explosively due to changes in the environmental conditions of the sea area, causing abnormal water color. Since the 20th century, red tides, especially toxic red tides, have occurred more and more frequently, and the area has continued to expand. It has become one of the major global marine disasters. It not only seriously damages the marine ecological environment and marine fishery resources, but also affects mariculture. Damage to the coastal tourism industry, and red tide biotoxins are transmitted and accumulated through the biological chain, and ultim...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/00G01N21/64
Inventor 侯建军黄邦钦胡俊林丽贞
Owner XIAMEN UNIV
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