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PNA probe for detecting red tide organism Takayama pulchellum and its uses

A technology of Gymnodinium and peptide nucleic acid, which is applied to the application field of low-abundance single target red tide algae cells, and can solve the problems such as the use of PNA probes that are not seen.

Inactive Publication Date: 2009-09-09
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, people have devoted themselves to the development of PNA probe technology with broad application prospects to monitor red tide groups, so that these cells can be detected by optical or chemical means, but there is no report on the use of PNA probes in red tide detection.

Method used

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  • PNA probe for detecting red tide organism Takayama pulchellum and its uses
  • PNA probe for detecting red tide organism Takayama pulchellum and its uses
  • PNA probe for detecting red tide organism Takayama pulchellum and its uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Whole cell fluorescence in situ hybridization detection of T. pulchellum pure algal cells and other algae for cross experiments.

[0048] 1. Sources and culture methods of all testing materials:

[0049] On-site sample collection method: collect 5-10L seawater with a water collector, filter and concentrate with 10μm sieve, reduce the water volume to 100-200mL, measure the volume, and add 1 / 10 volume of paraformaldehyde ( 10%), the final concentration was 1%, and counted by microscopy. Use a capillary glass tube or a 10 μl micropipette to separate live cells from freshly collected unfixed samples to pick out multiple single cells of the target algae under a stereo microscope (according to the situation, pre-filter with sieve or add f / 2 medium pre-culture), placed in a multi-well cell culture plate, observed under microscope and added to f / 2 medium for culture. cultured in culture flasks. The algae that have been separated and can grow normally are filtered...

Embodiment 2

[0058] Example 2: Comparison of hybridization effects between PNA probes and DNA probes.

[0059] 1. Testing materials:

[0060] The names and sources of the algal cell strains used in the experiment are shown in Table 3. For the cultivation method, see item 1 of Example 1 for details.

[0061] 2. Detection method:

[0062] See item 2 of Example 1 for details. The quantitative analysis of the fluorescence signal intensity of in situ hybridization of DNA and PNA probes was performed using the Simple PCI professional image analysis software system of C-image Company.

[0063] 3. Results:

[0064] Under the same probe concentration, comparing the hybridization efficiency and fluorescence intensity after hybridization of the DNA probe DNATP18S02 with the highest hybridization rate and the DNA probe DNATP18S02 with a similar sequence to the PNA probe, it was found that under the same hybridization conditions, the PNA The hybridization rate (95%) of the probe was significantly h...

Embodiment 3

[0068] Example 3: Whole-cell fluorescent hybridization detection of mixed algal species samples with fluorescently labeled PNA probes.

[0069] 1. Testing materials:

[0070] The names and sources of the algal cell strains used in the experiment are shown in Table 3. For the cultivation method of algae, refer to the first clause of embodiment 1.

[0071] 2. Detection method:

[0072] For details, see Article 2 of Example 1.

[0073] 3. Results:

[0074] Mixed samples I (Mixed samples I) are composed of Heterophyta akashiwo, Stigneria conicerii, Dunaliella salina, Chrysopsis zhanjiangensis, Sclerostenella mesospergiae and Thalassiosira wischii. Mixed samples II (Mixed samples II) consisted of Prorodinium minutiae, Alexandrium minutis, Prorocentrum donghaiense, Karenia mikii and Gymnodinium brevis. In the two mixed samples, about 1 / 5 of the mixed volume of the target algae cell culture liquid was added and mixed with natural seawater to simulate the sample characteristics o...

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Abstract

Peptide nucleic acid probes for detection of red tide organism Pyrodinanophyllum and their applications, involving a molecular probe, providing a fluorescently labeled peptide nucleic acid probe capable of rapidly detecting and identifying harmful red tide organisms Pyrodinanophyllum and its design method and application. The molecular name is PNATP28S01, and the molecular structure composition sequence is [Flu]-OO ATG CCA TCT CAA GA, which is electrically neutral. The four monomers carry ATGC4 bases and are connected end to end to form a chain structure. When designing probes, the rDNA target sequence is sorted by nucleotide sequence software and the sequences of closely related organisms obtained from major nucleic acid data; the conserved sequence of the target species is selected, and the specific sequence is searched for matching, and the The specific sequence of the conserved genus and species in the known sequence; detection of probe sequence parameters; design and calculation of PNA probes to obtain negative and positive control probes.

Description

technical field [0001] The present invention relates to a molecular probe, in particular to the design of a fluorescently labeled peptide nucleic acid probe and the rapid detection and identification of red tide algae Takayama pulchellum cells and low-abundance single targets in natural water bodies Application in red tide algal cells. Background technique [0002] Peptide nucleic acid (PNA) is a kind of uncharged new artificial information molecule similar to the backbone structure of peptide chain and carrying bases. The backbone of the molecule is composed of monomer N-(2-aminoethyl )-glycine is a long chain repeatedly connected by an amide bond, and the side chain is formed by connecting the base to the backbone of the main chain by a methylene carbonyl bond. Since the discovery of PNA 10 years ago, it has been widely used in molecular biology research and related fields as an excellent oligonucleotide substitute. It is considered to be a very promising probe tool. The ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 黄邦钦侯建军
Owner XIAMEN UNIV
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