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Fluorescence detection primer for three kinds of Wolbachia as well as detection method and detection kit thereof

A detection kit and fluorescence detection technology, which is applied in the field of molecular biology, can solve problems such as imperfect detection methods, and achieve the effects of simple identification, high specificity, and high sensitivity

Inactive Publication Date: 2015-09-16
奚志勇
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current method for PCR detection of different Wolbachia is not perfect

Method used

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  • Fluorescence detection primer for three kinds of Wolbachia as well as detection method and detection kit thereof
  • Fluorescence detection primer for three kinds of Wolbachia as well as detection method and detection kit thereof
  • Fluorescence detection primer for three kinds of Wolbachia as well as detection method and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Sensitivity

[0054] 1. Sensitivity of Wolbachia type A in Aedes mosquitoes

[0055] 1. After a mosquito carrying Aedes Wolbachia type A is stunned by carbon dioxide, its abdomen is dissected and placed in a 0.2ul EP tube;

[0056] 2. Add 20ul of DNA extraction solution (the formula of DNA extraction solution is: 30mM NaOH, 0.25mM EDTA, 15mM Tris-HCl, the solvent is water, shake before use, and add the white flake together);

[0057] 3. Fully mix;

[0058] 4. After a brief centrifugation, incubate at 99°C for 10 minutes;

[0059] 5. Obtain the DNA extract and store it at -20°C to obtain the genomic DNA of Wolbachia Aedes mosquito;

[0060] 6. Dilute the DNA extract in gradients of 10, 100, and 1000. Each gradient has two parallels, a total of 8 samples, which are used as template DNA for PCR amplification;

[0061] 7. PCR amplification system:

[0062] Template DNA 2μl, PCR A solution 18μl and PCR B solution 2μl.

[0063] The PCR A solution contains 10 ...

Embodiment 2

[0082] Example 2: Repeatability

[0083] 1. Two triple-infected Aedes albopictus (carrying Aedes type A, Aedes type B and Culex Wolbachia) were stunned by carbon dioxide, and their dissected abdomens were placed in 0.2ul EP tubes;

[0084] 2. Add 20ul DNA extraction solution (the formula of the DNA extraction solution is: 30mM NaOH, 0.25mM EDTA, 15mM Tris-HCl, the solvent is water, shake before use, and add the white flake together);

[0085] 3. Fully mix;

[0086] 4. After a brief centrifugation, incubate at 99°C for 10 minutes;

[0087] 5. Obtain the DNA extract and store it at -20°C to obtain the genomic DNA of Aedes type A, Aedes type B and Culex Wolbachia;

[0088] 6. The above-mentioned genomic DNAs of Aedes type A, Aedes type B and Culex Wolbachia were repeated 10 times as template DNA, with a total of 20 samples;

[0089] 7. PCR amplification system:

[0090] Template DNA 2μl, PCR A solution 18μl and PCR B solution 2μl.

[0091]The PCR A solution contains 10 pmol ...

Embodiment 3

[0102] Example 3: Specificity

[0103] 1. Two Aedes albopictus containing a single type A Wolbachia, two Aedes albopictus containing a single B type Wolbachia and two Culex mosquitoes (containing Culex Wolbachia) , a total of 6 mosquitoes were stunned by carbon dioxide, and their dissected abdomens were placed in 0.2ul EP tubes;

[0104] 2. Add 20ul of DNA extraction solution (the formula of DNA extraction solution is: 30mM NaOH, 0.25mM EDTA, 15mM Tris-HCl, the solvent is water, shake before use, and add the white flake together);

[0105] 3. Fully mix;

[0106] 4. After a brief centrifugation, incubate at 99°C for 10 minutes;

[0107] 5. Obtain the DNA extract and store it at -20°C, that is, two copies of genomic DNA of Aedes mosquito type Wolbachia, two copies of genomic DNA of Aedes mosquito type B Wolbachia and two copies of Culex There are six genome DNAs of Wolbachia, and the six genome DNAs are respectively used as template DNA for PCR amplification.

[0108] 6.

...

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Abstract

The invention discloses a fluorescence detection primer for three kinds of Wolbachia as well as a detection method and a detection kit thereof. By utilizing the fluorescence detection primer disclosed by the invention, the aedes type A, the aedes type B and culex Wolbachia can be rapidly, efficiently and specifically detected according to the detection method disclosed by the invention, the primer has the advantages of high singleness and high specificity, only the aedes Wolbachia is detected in aedes, only the culex Wolbachia is detected in the culex, the sensitivity is high, and the lowest detection limit is 100copies / ml.

Description

Technical field: [0001] The invention belongs to the field of molecular biology, and in particular relates to fluorescent detection primers for three Wolbachia species, a detection method and a detection kit. Background technique: [0002] Wolbachia is a symbiotic microorganism widely distributed in arthropods, and it may be the most abundant group of insect symbiotic microorganisms. It is distributed in Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Lepidoptera and other insect species. It uses vertical transmission as its basic mode of transmission among host generations. It exists stably in the germ cells of the host, is transmitted to the offspring of the host through egg cells, and can regulate the reproductive activities of the host through various methods such as cytoplasmic incompatibility, feminization and andricide. These regulatory functions promote its widespread spread within the host population. [0003] Cytoplasmic incompatibility (CI) is the most ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12N15/11C12Q1/04C12Q1/68C12Q1/689C12Q1/6851C12Q2531/113C12Q2563/107
Inventor 高秀洁杨翠周其伟李丽梅奚志勇朱俭
Owner 奚志勇
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