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Method for quantitatively detecting toxigenic microcystis and special standard substance thereof

A technology of microcystin and standard products, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, introduction of foreign genetic material using carriers, etc., can solve the problems that microcystins cannot meet early warning and water quality safety control, etc. Achieve the effects of wide linear detection range, good purity, and simple preparation method

Inactive Publication Date: 2011-02-16
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, direct detection of microcystin content in water cannot meet the needs of early warning of cyanobacterial blooms and water quality safety control. The isolation and functional studies of microcystin synthase genes not only revealed the genetic basis of microcystin synthesis, Provides a method for accurate detection of toxin-producing Microcystis

Method used

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  • Method for quantitatively detecting toxigenic microcystis and special standard substance thereof
  • Method for quantitatively detecting toxigenic microcystis and special standard substance thereof
  • Method for quantitatively detecting toxigenic microcystis and special standard substance thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, preparation of the standard product pMD-18T-mcyA plasmid standard product of quantitative detection Microcystis

[0037] 1. Culture of Microcystis aeruginosa

[0038] The toxin-producing strain Microcystis aeruginosa (Microcystis aeruginosa) numbered FACHB-905 was purchased from Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. Microcystis aeruginosa was cultured according to the culture method provided by the Institute of Hydrobiology, Chinese Academy of Sciences. Prepare BG11 medium according to the provided formula (Table 1), that is, in 1000mL deionized water, add the following substances respectively, and store at 4°C after sterilization.

[0039] Table 1BG11 medium formula

[0040]

[0041]

[0042] For the cultivation of algae, avoid inoculating a small amount of algae in a large amount of medium. The entire process of cultivating algae requires absolute aseptic operation, and the experimental tools required for cultivation and ...

Embodiment 2

[0125] Embodiment 2, establishment of Microcystis quantitative PCR detection method

[0126] 1. Optimization of quantitative PCR reaction conditions

[0127] The DNA of Microcystis aeruginosa (number FACHB-905) (toxin-producing strain) was extracted using the method in Example 1. according to Premix Ex Taq TM (Code: DRR041S) manual, the reaction system of qPCR is (20μL): Premix Ex Taq TM 10 μL, 0.1-0.5 μL each of the upstream and downstream primers (mcyA-F, mcyA-R) (final concentration 0.1-0.5 μmol / L), the Microcystis aeruginosa obtained above (number FACHB-905) (toxin-producing strain ) DNA 2 μL, double distilled water 7.6 μL to make up the volume to 20 μL.

[0128] The qPCR reaction procedure is as follows, 1 cycle: 95°C, 5min; 40 cycles: 95°C, 5s, 50-60°C, 30s, 72°C, 30s, fluorescence is collected during annealing; the process of the melting curve is: 95°C , 1min, 1min at 65°C, the temperature rises by 0.5°C every 30s starting from 65°C, and the junction temperatur...

Embodiment 3

[0152] Embodiment 3, utilize quantitative PCR method to detect Microcystis in actual water environment sample

[0153] 1. Actual water sample collection

[0154] The actual water samples were taken from Taihu Lake and its surrounding rivers, and the sampling time was August 10-11, 2010. The sampling method shall be taken at 0.5m below the water surface according to the standard sampling method. The samples were transported back to the laboratory in an ice box and stored at 4°C.

[0155] 2. Determination of chlorophyll a and algae density

[0156] August is the season for cyanobacteria blooms in the Taihu Lake Basin. The instrument YSI6600V2 was used to detect the algae density and chlorophyll a of the water samples. The results are shown in Table 5. The concentration of chlorophyll a is 1.7×10 -3 -1.35×10 -2 mg / L, the density of algae is 8.2×10 5 -2.4×10 7 a / L. (Chlorophyll-a and algae density are routine indicators of water eutrophication.)

[0157] Table 5 Chlorophyl...

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Abstract

The invention discloses a method for quantitatively detecting toxigenic microcystis and a special standard substance thereof. The standard substance for detecting the toxigenic microcystis in a sample to be detected is recombinant plasmid or recombinant bacterium or recombinant cell which contains mcyA gene; and the sequence of the mcyA gene is sequence 1 in the sequence list. The departure plasmid of the recombinant plasmid is pMD-18T.The standard substance has the characteristic of combination of broad spectrum identification and specificity, high sensitivity, simple preparation method, long retention period, good purity and wide linear detection range, thus being capable of being applied to fast quantitative detection of the toxigenic microcystis in a sample in the water environment.

Description

technical field [0001] The invention relates to a method for quantitatively detecting toxin-producing Microcystis and a special standard product thereof. Background technique [0002] Microcystins (MCs for short) are monocyclic heptapeptides produced by cyanobacteria (cyanobacteria), especially some Microcystis (Microcystis), and are commonly found in water bodies where cyanobacteria blooms occur. Microcystin is a liver toxin, long-term drinking of water containing microcystin can cause liver damage and even liver cancer. [0003] Microcystins are a class of toxins that are regulated by toxin-producing genes and metabolized in cells. Studies have shown that not all Microcystis can produce microcystins, and the toxicity of Microcystis strains (Toxic strains) and nontoxic strains (Nontoxic strains) is determined by genetics, and there is no obvious difference in morphology. difference. Microcystins are non-ribosomally synthesized by the peptide synthase complex gene cluster...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12Q1/68C12N1/19C12N1/21C12N5/10C12Q1/06C12N1/15G01N21/64
Inventor 李丹何苗刘丽施汉昌
Owner TSINGHUA UNIV
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