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Fluorescence detection primer of A type and B type wolbachia as well as detection method and detection kit thereof

A detection kit and fluorescence detection technology, 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 strong specificity

Inactive Publication Date: 2015-08-26
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 of A type and B type wolbachia as well as detection method and detection kit thereof
  • Fluorescence detection primer of A type and B type wolbachia as well as detection method and detection kit thereof
  • Fluorescence detection primer of A type and B type wolbachia as well as detection method and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Sensitivity

[0041] 1. Sensitivity of Type A Wolbachia

[0042] 1. After a Guangzhou Aedes albopictus (carrying Aedes Wolbachia type A) was stunned by carbon dioxide, its abdomen was dissected and placed in a 0.2ul EP tube;

[0043] 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);

[0044] 3. Fully mix;

[0045] 4. After a brief centrifugation, incubate at 99°C for 10 minutes;

[0046] 5. Obtain the DNA extract and store it at -20°C to obtain the genomic DNA of Wolbachia type A in Aedes mosquitoes

[0047] 6. Perform 10, 100, and 1000-fold gradient dilution of the above-mentioned DNA extract containing the genomic DNA of Aedes Wolbachia type A as the template DNA, and each gradient has two parallels, a total of 8 samples;

[0048] 7. PCR amplification system:

[0049] Template DNA 2μl, PCR A solution 18μl and...

Embodiment 2

[0065] Example 2: Repeatability

[0066] 1. Two Aedes albopictus mosquitoes (carrying type A and type B Wolbachia) were stunned by carbon dioxide, and their abdomens were dissected and placed in 0.2ul EP tubes;

[0067] 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);

[0068] 3. Fully mix;

[0069] 4. After a brief centrifugation, incubate at 99°C for 10 minutes;

[0070] 5. Obtain the DNA extract and store it at -20°C to obtain two copies of genomic DNA containing Wolbachia type A and B of Aedes mosquitoes respectively;

[0071] 6. The genomic DNA of each of the above-mentioned Aedes mosquitoes type A and B Wolbachia was used as template DNA for 10 repetitions, a total of 20 samples;

[0072] 7. PCR amplification system:

[0073] Template DNA 2μl, PCR A solution 18μl and PCR B solution 2μl.

[0074] The PCR A solution conta...

Embodiment 3

[0085] Example 3: Specificity

[0086] 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 abdomens were dissected and placed in 0.2ul EP tubes;

[0087] 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);

[0088] 3. Fully mix;

[0089] 4. After a brief centrifugation, incubate at 99°C for 10 minutes;

[0090] 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.

[0091] 6....

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Abstract

The invention discloses a fluorescence detection primer of A type and B type wolbachia, as well as a detection method and a detection kit thereof. By adopting the fluorescence detection primer provided by the invention, the A-type and the B-type wolbachia can be rapidly, effectively and specifically detected according to the detection method, the fluorescence detection primer is strong in selectivity, high in specificity (the aedes wolbachia is detected, the A type, B type or A and B mixed type wolbachia can be distinguished, and the aedes wolbachia is not amplified), high in sensitivity, and the lowest detection limit is 100 copies / ml.

Description

Technical field: [0001] The invention belongs to the field of molecular biology, and in particular relates to a fluorescent detection primer for Aedes Wolbachia, 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 common ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12N15/11C12Q1/04C12Q1/68C12Q1/689C12Q1/6851C12Q2531/113C12Q2563/107
Inventor 杨翠高秀洁奚志勇朱俭罗永平
Owner 奚志勇
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