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Fluorescence detection primer of culex wolbachia, detection method thereof and detection kit

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, strong specificity, and simple operation.

Inactive Publication Date: 2015-09-02
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 culex wolbachia, detection method thereof and detection kit
  • Fluorescence detection primer of culex wolbachia, detection method thereof and detection kit
  • Fluorescence detection primer of culex wolbachia, detection method thereof and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Sensitivity

[0028] 1. After a Culex mosquito (carrying Culex Wolbachia) was stunned by carbon dioxide, its abdomen was dissected and placed in a 0.2ul EP tube;

[0029] 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, the same below, shake before use, and add the white flake together);

[0030] 3. Fully mix;

[0031] 4. After centrifugation, incubate at 99°C for 10 minutes;

[0032] 5. Store the DNA extract at -20°C to obtain the genomic DNA of Culex mosquito Wolbachia;

[0033] 6. Perform 10, 100, and 1000-fold gradient dilution of the above-mentioned DNA extract containing the genomic DNA of Culex Wolbachia as the template DNA, and each gradient has two parallels, a total of 8 samples;

[0034] 7. PCR amplification system:

[0035] Template DNA 2μl, 10×Taqman buffer 5μl, 5mmol / L MgCl2 4μl, 2.5mol / L dNTPs 2μl, 20μmol / L probe (5'-CTTTCAATTGAAAAGATTCGATCAAC-3'...

Embodiment 2

[0045] Example 2: Repeatability

[0046]1. After two Culex mosquitoes (carrying Culex Wolbachia) were stunned by carbon dioxide, their abdomens were dissected and placed in 0.2ul EP tubes;

[0047] 2. Add 20ul DNA extraction solution respectively (shake before use, and add the white flakes together);

[0048] 3. Fully mix;

[0049] 4. After centrifugation, incubate at 99°C for 10 minutes;

[0050] 5. The DNA extraction solution was stored at -20°C, and thus the genomic DNA of Culex Wolbachia was obtained from two mosquitoes;

[0051] 6. The genomic DNA of each of the above Culex mosquito Wolbachia was used as template DNA for 10 repetitions, a total of 20 samples;

[0052] 7. PCR amplification system:

[0053] Template DNA 2μl, 10×Taqman buffer 5μl, 5mmol / L MgCl2 4μl, 2.5mol / L dNTPs 2μl, 20μmol / L probe 1μl, 20μmol / L fluorescence detection primer wPipF (5'-GTTTGTGCAGCTAATAG-3') 1μl, 20μmol / L fluorescence detection primer wPipR (5'-GTCTGCAAGGCCTATTTCTACTG-3') 1μl, 0.55U UN...

Embodiment 3

[0063] Example 3: Specificity

[0064] 1. Two Aedes albopictus (carrying type A and B Wolbachia), two single Aedes albopictus and two single B type Aedes albopictus, a total of 6 Aedes mosquitoes were fumigated by carbon dioxide After dizziness, the dissected abdomen was placed in 0.2ul EP tubes;

[0065] 2. Add 20ul DNA extraction solution respectively (shake before use, and add the white flakes together);

[0066] 3. Fully mix;

[0067] 4. After centrifugation, incubate at 99°C for 10 minutes;

[0068] 5. The DNA extraction solution was stored at -20°C, and thus the genomic DNA of Wolbachia was obtained from each of the six mosquitoes;

[0069] 6. Genomic DNA of each of the above Wolbachia as template DNA fluorescence quantitative PCR reaction;

[0070] 7. PCR amplification system:

[0071] Template DNA 2μl, 10×Taqman buffer 5μl, 5mmol / L MgCl2 4μl, 2.5mol / L dNTPs 2μl, 20μmol / L probe 1μl, 20μmol / L fluorescence detection primer wPipF (5'-GTTTGTGCAGCTAATAG-3') 1μl, 20μmol ...

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Abstract

The invention discloses a fluorescence detection primer of culex wolbachia, a detection method thereof and a detection kit. The fluorescence detection primer is characterized by comprising wPipF: 5minute-GTTTGTGCAGCTAATAG-3minute; wPipR: 5minute-GTCTGCAAGGCCT ATTTCTACTG-3minute and a probe: 5minute-CTTTCAATTGAAAAGATTCGATCAAC-3minute, wherein the two ends of the probe are respectively combined with a fluorescence generation group and a fluorescence quenching group. The culex wolbachia can be detected out in a fast-efficient-single manner by utilizing the fluorescence detection primer and the probe according to the detection method, and the fluorescence detection primer has the characteristics that the singleness is strong, the specificity is high (the culex wolbachia can be detected out, but no amplification of the aedes wolbachia occurs), the sensitivity is high and the lowest detection limit is 100copies per ml. Therefore, the fluorescence detection primer disclosed by the invention has the advantages of fastness, high efficiency, simple operation, high specificity, high sensitivity and simple identification and the like.

Description

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

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12N15/11C12Q1/04C12Q1/68C12Q1/6851C12Q1/6888C12Q2563/107C12Q2565/1015C12Q2545/114
Inventor 杨翠高秀洁奚志勇朱俭罗永平
Owner 奚志勇
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