Detection primer of culex wolbachia, detection method thereof and detection kit
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A technology for detection kits and detection primers, 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 strong specificity
Active Publication Date: 2015-09-02
GUANGZHOU WOLBAKI BIOTECH CO LTD
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The current method for PCR detection of different Wolbachia is not perfect
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Embodiment 1
[0019] Example 1: Specificity
[0020] 1. Take six 0.2ml EP tubes containing 20μl DNA extraction solution (the formula of DNA extraction solution is: 30mM NaOH, 0.25mM EDTA, 15mM Tris-HCl, the solvent is water, the same below);
[0021] 2. After three Culex mosquitoes (containing Culex Wolbachia) and three Aedes albopictus (containing Wolbachia) were stunned by carbon dioxide, their abdomens were dissected, and the above 6 in an EP tube;
[0022] 3. After a brief centrifugation, incubate at 95°C for 10 minutes;
[0023] 4. After instantaneous centrifugation, obtain the DNA extract, store at -20°C, and obtain the genomic DNA of Wolbachia from six mosquitoes respectively (specifically: three genomic DNAs of Culex Wolbachia, three Genomic DNA of Aedes Wolbachia) was used to carry out PCR reactions using the Wolbachia genomic DNA of six mosquitoes as templates.
[0024] 5. PCR amplification system:
[0025]
[0026] 6. Perform PCR amplification after mixing and centrifugati...
Embodiment 2
[0031] Example 2: Sensitivity
[0032] Get the genomic DNA of a portion of Culex Wolbachia in Example 1, then carry out 10, 100, 1000, 10000, 100000 times of gradient dilution, and then use the diluted dilution as template DNA, according to the PCR of Example 1 method to amplify, the specific results are as follows figure 2 As shown, the dilution solution diluted 100000 times can amplify the PCR product, and then calculate the concentration, thus the final detection limit is 855×10 -5 ng / μl (that is, the concentration of genomic DNA of Culex mosquito Wolbachia in the 100000-fold dilution).
[0033]
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Abstract
The invention discloses a detection primer of culex wolbachia, a detection method thereof and a detection kit. The detection primer is characterized by comprising wPipF: 5 minute-GTGCAGCTAATAGCAAGGAACA-3minute;and wPipR: 5 minute-CCTATCTTTTCTGGCATCTTCG-3 minute. The culex wolbachia can be detected out in a fast-efficient-single manner by utilizing the detection primer according to the detection method, and the 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 855 times 10<-5> ng per mul. Therefore, the 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 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 most common typ...
Claims
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