Marine phaeocystis globosa single-cell detection method based on a TaqMan probe technology

A technology of Phaeocystis globosa and detection method, applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as detection of single-celled Phaeocystis globosa, and achieve easy analysis of experimental results and high detection throughput , the effect of improving sensitivity

Pending Publication Date: 2020-11-27
GUANGXI ACAD OF SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the detection of single-cell Phaeocystis sphaeroides based on TaqMan probes at home and abroad.

Method used

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  • Marine phaeocystis globosa single-cell detection method based on a TaqMan probe technology
  • Marine phaeocystis globosa single-cell detection method based on a TaqMan probe technology
  • Marine phaeocystis globosa single-cell detection method based on a TaqMan probe technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Detection of marine unicellular Phaeocystis sphaeroides by TaqMan probe method based on real-time fluorescent quantitative PCR:

[0063] (1) Sample collection:

[0064] There are a total of 5 samples in this embodiment, numbered respectively as sample 1, sample 2, sample 3, sample 4, and sample 5, which were collected by the Ocean Center of Guangxi Academy of Sciences during the red tide voyage investigation;

[0065] (2) Genomic DNA extraction:

[0066] Use the Takara lysate extraction protocol (product number: 9770), and refer to the method in the manual of the reagent for detailed steps;

[0067] (3) Configuration of the reaction system:

[0068] The reaction system of fluorescent quantitative PCR detection is 20uL, and the probe method reagent of Kangwei Century Company is used. The components and volumes of each reaction system are shown in Table 1; the negative control reaction system uses 1uL sterile water as a DNA template, and the addition of othe...

Embodiment 2

[0075] Embodiment 2: the present invention is to the repetitive experiment of same sample:

[0076] In order to detect the repeatability and stability of the detection of the present invention, the same number (10,000) of indoor purified Phaeocystis globosa cells were detected, and a total of 6 samples were detected, respectively numbered as repeated sample 1, repeated sample 2, repeated Sample 3, replicate 4, replicate 5, replicate 6; 3 replicates each. The test results are attached Figure 5 and Table 3; the results of the repeatability amplification curve show that the method has good repeatability. According to the statistical results, the standard deviation of the Ct value of 6 copies of the same number of Phaeocystis sphaeroides cell DNA templates is 0.19, indicating that the method has good repeatability and stability;

[0077]

[0078] Table 3: The detection results of the present invention on 6 indoor purified cultured cells of Phaeocystis globosa with the same nu...

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Abstract

The invention belongs to the technical field of detection of environmental microorganisms by a biological method, and discloses a marine phaeocystis globosa single-cell detection method based on a TaqMan probe technology. The method comprises the following steps: S1, designing and synthesizing a primer and a probe; S2, preparing a positive control sample of the phaeocystis globosa; S3, performingnegative control quality control; S4, extracting genomic DNA of a marine single-cell phaeocystis globosa sample and a positive control sample; S5, performing fluorescent quantitative PCR detection byadopting the primer and the probe designed in the S1; S6, judging a real-time fluorescent quantitative PCR amplification signal; S7, carrying out agarose gel electrophoresis analysis on a fluorescentquantitative PCR product; S8, carrying out sequencing analysis on a fluorescent quantitative PCR product; and S9, detecting and analyzing marine single-cell phaeocystis globosa. According to the method, low-density harmful red tide algae in the early stage of harmful red tide outbreak can be detected, so that an effective early warning tool is provided for early warning and early prevention and control of red tide outbreak, and the economic loss of mass culture and tourism nuclear power industries is avoided.

Description

technical field [0001] The invention relates to the technical field of detection of environmental microorganisms by molecular biology methods, in particular to a method for single-cell detection of Phaeocystis globosa based on TaqMan probe technology. Background technique [0002] Phaeocystis belongs to Chrysophyta Dinophyceae, has a wide range of temperature and salinity adaptability, and is a kind of harmful red tide cause species that commonly exists in the world, including my country's coastal waters, among which Phaeocystis globosa is the most common. Phaeocystis globosa has unique physiological and ecological characteristics. It is a marine microalgae with a complex and heterogeneous life cycle. It has two morphological stages that can be converted from free single cells (3-8 μm) and groups. It mainly forms red tides in groups. The cysts of Phaeocystis strains in my country's sea areas are huge, which is rare in the world, with a diameter of up to 3cm, which is signifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6895C12Q1/04
CPCC12Q1/6851C12Q1/6895C12Q2600/166C12Q2531/113C12Q2561/101C12Q2545/113C12Q2563/107
Inventor 王佳乐王英辉赖俊翔李杰许铭本陆家昌
Owner GUANGXI ACAD OF SCI
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