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Detection primer, kit and detection method for wolbachia in brown planthopper

A detection kit and detection primer technology are applied in the field of molecular biology to achieve the effects of high specificity, easy identification and high specificity

Inactive Publication Date: 2018-01-12
GUANGZHOU WOLBAKI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, only the common type of Wolbachia in planthoppers can be detected. Since different planthoppers carry different types of Wolbachia, it is particularly important to find specific detection primers and detection methods

Method used

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  • Detection primer, kit and detection method for wolbachia in brown planthopper
  • Detection primer, kit and detection method for wolbachia in brown planthopper
  • Detection primer, kit and detection method for wolbachia in brown planthopper

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The detection kit of a kind of brown planthopper Wolbachia described in this embodiment, comprises detection primer and PCR reagent, so

[0040] The detection primers described are as follows.

[0041] wLugF: 5'-GGCTGATAATCCAGCAATTGC-3'; SEQ ID NO.1

[0042] wLugR: 5'-AAAAATTAAACGCTACTCCA-3' SEQ ID NO.2.

[0043]DNA extraction reagent: the dilution buffer is: 30 mM NaOH, 0.25 mM EDTA, 15 mM Tris-HCl, and the solvent is water; 0.5 μl of DNARelease is added to every 20 μl of the dilution buffer.

Embodiment 2

[0044] Example 2: Specificity

[0045] 1. Take six 0.2ml EP tubes filled with 20μl dilution buffer and 0.5μl DNARelease (the formulation of the dilution buffer is: 30mM NaOH, 0.25mM EDTA, 15mM Tris-HCl, and the solvent is water);

[0046] 2. Three brown planthoppers of Guangzhou (confirmed to contain Wolbachia of brown planthopper) and three brown planthoppers (confirmed to contain Wolbachia of brown planthopper) were soaked in absolute ethanol for 2 minutes, and the whole planthoppers were taken out and respectively Put it into the above 6 EP tubes;

[0047] 3. After transient centrifugation, put it into a PCR instrument for DNA extraction;

[0048] 4. DNA extraction procedure: 55°C, 5min; 98°C, 5min; 30°C, 10s.

[0049] 5. After instantaneous centrifugation, the DNA extract was obtained, diluted 10 times, and stored at -20°C to obtain the genomic DNA of Wolbachia from six planthoppers (specifically: three copies of Wolbachia from Guangzhou planthopper). Genomic DNA of the...

Embodiment 3

[0060] Example 3: Sensitivity

[0061] Get the genomic DNA of three parts of brown planthopper Wolbachia in Example 2, measure its genomic DNA concentration as 50ng / μl, 42ng / μl and 30ng / μl, then perform 10, 100, 1000, 10000, 100000, 1,000,000-fold gradient dilution, and then use the diluted dilution as template DNA to amplify according to the PCR method in Example 2. The specific results are as follows: figure 2 As shown (only the test results of one DNA sample are given, and the test results of other samples are basically the same), the diluted solution after dilution of 10000 times can amplify the PCR product, and then calculate the concentration, thus the final detection limit is 30×10 -4 ng / μl (that is, the concentration of genomic DNA of N. lugens Wolbachia in the 10000-fold dilution).

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Abstract

The invention discloses a detection primer, kit and a detection method for wolbachia in brown planthopper. The detection primer for wolbachia in brown planthopper is shown as wLugF: 5'-GGCTGATAATCCAGCAATTGC-3' and wLugR: 5'-AAAAATTAAACGCTACTCCA-3'. By utilizing the detection primer disclosed by the invention, the wolbachia in brown planthopper can be rapidly, efficiently and specifically detectedaccording to the detection method in the invention, the detection primer has high specificity (only the wolbachia in brown planthopper is detected, but amplification of wolbachia in small brown rice planthopper is avoided) and high sensitivity, and the lowest detection limit is 30*10<-4>ng / ul.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a detection primer for brown planthopper Wolbachia, a detection method and a detection kit. Background technique [0002] As a maternally inherited obligate intracellular symbiont, Wolbachia exists widely in a large number of arthropods. Existing studies have shown that theoretically about 60% of insects are infected with Wolbachia worldwide. Wolbachia can not only induce the cytoplasmic incompatibility (Cytoplasmic Incompatibility, CI) phenotype in the host to achieve population suppression, but also block arboviruses through the population replacement it promotes. CI refers to the death of offspring embryos produced when males infected with one type of Wolbachia mate with females not infected with Wolbachia or females infected with a different strain of Wolbachia, thereby expressing For adult sterility. [0003] In the control target area, the continuous release ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6806C12Q1/6848C12N15/11
Inventor 不公告发明人
Owner GUANGZHOU WOLBAKI BIOTECH CO LTD
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