Detection primer for wolbachia in small brown rice planthopper as well as detection method and detection kit thereof

A detection kit and detection primer technology, which is applied in the field of molecular biology, can solve the problems of non-specific primers and methods for the detection of SBPH, and achieve the effect of simple identification, high sensitivity and high sensitivity

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

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Problems solved by technology

[0006] There are mainly three kinds of rice planthoppers that harm rice in my country: brown planthopper, brown planthopper and white-backed planthopper. The white-backed planthopper naturally carries Wolbachia in nature, but at present, the Wolbachia in the planthopper There are no specific primers and methods for detection, so it is particularly important to find specific detection primers and detection methods

Method used

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  • Detection primer for wolbachia in small brown rice planthopper as well as detection method and detection kit thereof
  • Detection primer for wolbachia in small brown rice planthopper as well as detection method and detection kit thereof
  • Detection primer for wolbachia in small brown rice planthopper as well as detection method and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] A detection kit for Wolbachia striatellus described in this embodiment includes detection primers and PCR reagents, and the detection primers are as follows.

[0039] wStriF: 5'-GTAAATACTTCTGAAACAAATG-3'; SEQ ID NO.1

[0040] wStriR: 5'-AAAAATTAAACGCTACTCCA-3'; SEQ ID NO.2.

Embodiment 2

[0041] Example 2: Specificity

[0042] 1. Take six 0.2ml EP tubes filled with 20μl dilution buffer and 0.5μl DNARelease (the formula of dilution buffer is: 30mM NaOH, 0.25mM EDTA, 15mM Tris-HCl, the solvent is water);

[0043] 2. Soak three Guangzhou striatellus (containing Wolbachia striatellus) and three brown planthoppers (containing Wolbachia brown planthopper) in absolute ethanol for 2 minutes, then take out the whole planthopper and put them in In the above 6 EP tubes;

[0044] 3. After transient centrifugation, put it into a PCR instrument for DNA extraction;

[0045] 4. DNA extraction procedure: 55°C, 5min; 98°C, 5min; 30°C, 10s.

[0046]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 Guangzhou striatellus Wolbachia). The genomic DNA of Baklava, the genomic DNA of three parts of brown planthopper Wolbachia), respectiv...

Embodiment 3

[0058] Example 3: Sensitivity

[0059] Get three parts of Example 2 diluted 10-fold Wolbachia genomic DNA, then carry out 100, 1000, 10000, 100000, 1000000 times of gradient dilution, and then use the diluted dilution as template DNA, according to the implementation The kit described in Example 1 and the PCR method described in Example 2 carry out amplification, and the specific results are as follows figure 2 , image 3 and Figure 4 As shown, the PCR product can be amplified in the diluted solution diluted 10,000 times, and then the concentration is calculated, so that the final detection limit is 55×10 -4 ng / μl (that is, the concentration of the genomic DNA of Wolbachia striatellus in the 10000-fold dilution).

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Abstract

The invention discloses a detection primer for wolbachia in small brown rice planthopper as well as a detection method and a detection kit thereof. The detection primer for wolbachia in small brown rice planthopper is shown as SEQ ID NO.1 and SEQ ID NO.2. By utilizing the detection primer disclosed by the invention, the wolbachia in small brown rice planthopper can be rapidly, efficiently and specifically detected according to the detection method in the invention, the detection primer has high specificity (only the wolbachia in small brown rice planthopper is detected, but amplification of wolbachia in brown planthopper is avoided) and high sensitivity, and the lowest detection limit is 55*10<-4>ng / ul. Therefore, the detection primer disclosed by the invention has the advantages of beingrapid, efficient, high in specificity, high in sensitivity, simple and convenient in 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 Wolbachia striatellus striatellus, 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 the population suppression effect, but also block the arbovirus through the population replacement promoted by it. 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, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/6806C12Q1/04C12N15/11C12R1/01
Inventor 不公告发明人
Owner GUANGZHOU WOLBAKI BIOTECH CO LTD
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