Specific primer pair for identifying lissorhoptrus oryzophilus and application of specific primer pair

A rice water weevil and primer pair technology, applied in the biological field, can solve the problems of unstable results, poor repeatability, non-specific PCR products and the like

Pending Publication Date: 2021-07-06
山西省植物保护植物检疫总站 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RAPD has advantages such as no special design of primers for PCR amplification reaction, no need to know the information of the genome of the organism being studied, and easy to obtain PCR products. certain difficulty

Method used

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  • Specific primer pair for identifying lissorhoptrus oryzophilus and application of specific primer pair
  • Specific primer pair for identifying lissorhoptrus oryzophilus and application of specific primer pair

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Example 1 Sample DNA extraction

[0022] The genomic DNA extraction of rice water weevil and rice weevil adopts the kit method, and the kit used is Ezup column animal genomic DNA extraction kit; purchased from Sangon Bioengineering Co., Ltd. The specific operation steps are as follows:

[0023] (1) Insect samples preserved by immersion in absolute ethanol, the single-headed worms were separated, washed twice with sterile water, and placed in sterile water for 24 hours to completely remove ethanol residues. Then the worms were placed on sterilized filter paper to dry;

[0024] (2) Put the dried sample into a new 1.5mL sterilized centrifuge tube, add the digestive solution and fully grind the sample with an electric grinder, and perform DNA extraction according to the DNAEzup Columnar Animal Genome Extraction Kit Operation Manual;

[0025] (3) When the sample DNA is on the adsorption column in the last step, add 50 μL of sterilized deionized water and let it stand for 3...

Embodiment 2

[0027] Example 2: RAPD amplification of sample DNA

[0028] According to the principle of RAPD, 20 primers of RAPD 10-mer Set F of BGI random primer set were selected for PCR screening of rice water weevil and rice weevil DNA samples.

[0029] Tiangen 2×Taq PCR MasterMix II reagent was used for PCR amplification reaction, and the total reaction system was 25 μL, including: 1 μL of DNA to be tested; 1 μL of random primers; 12.5 μL of 2×Taq PCR MasterMix II; 10.5 μL of sterile water. The concentration of the DNA of the sample to be tested is not less than 400pg / μL; the molar concentration of the random primer is 10μM. The PCR program was: 95°C for 3 min; 95°C for 30s, 40°C for 45s; 72°C for 1 min, a total of 35 cycles; 72°C for 2 min, and the PCR product was stored at 4°C. 3 μL of PCR products were used for agarose gel electrophoresis to detect amplification results. The agarose gel was 1.0%, the voltage used was 180V, and the gel imaging system was used to take pictures. The...

Embodiment 3

[0030] Example 3: Design and identification of rice water weevil-specific primers

[0031] The obtained base sequences were analyzed, and specific primers were designed using Primer Premier 5 software. Upstream primer sequence: SEQ ID NO: 1: ATGTGCCCACCTCTGTTCTT, downstream primer sequence: SEQ ID NO: 2: ACTGCATCGTCAGTAACTAT. The primers were sent to Sangon Bioengineering Co., Ltd. for synthesis.

[0032] The DNA of existing samples of rice water weevil at different developmental stages (egg, early larvae, mature larva, pupa, female adult and male adult) and rice weevil DNA were amplified and identified. Tiangen 2×Taq PCR MasterMixⅡ reagent was used for PCR amplification reaction, and the total reaction system was 25 μL, including: 1 μL of DNA to be tested; 0.5 μL of upstream primers; 0.5 μL of downstream primers; 12.5 μL of 2×Taq PCR MasterMixⅡ; 10.5 μL of sterile water μL. The concentration of the DNA of the sample to be tested is not less than 400pg / μL; the molar concent...

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Abstract

The invention provides a method for detecting lissorhoptrus oryzophilus by using a specific PCR primer. According to the method, on the basis that the sequence of the lissorhoptrus oryzophilus is obtained through the RAPD technology, specific primers which are paired and can be used for rapidly identifying the species are designed. A PCR detection system is established by utilizing the primer pair, and a single DNA band which is about 370bp and specific to the lissorhoptrus oryzophilus is amplified. The method has the advantages of being accurate in identification, convenient to operate, good in specificity and the like, rapid molecular identification of the lissorhoptrus oryzophilus can be achieved, and the efficiency of detection and quarantine work is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a rice water weevil specific primer and a rapid identification method thereof. Background technique [0002] Rice water weevil (Lissorhoptrus oryzophilus Kuschel), belonging to Coleoptera (Coleoptera), Curculionidae (Curculionidae), is native to the southern Texas region of the United States. The pest has spread rapidly around the world in just a few decades, and has now become an important rice pest worldwide. Phytosanitary pests. The rice water weevil was first discovered in Tanghai County, Tangshan City, Hebei Province, my country in 1988, and there are currently 23 species in Hebei, Tianjin, Beijing, Liaoning, Shandong, Zhejiang, Jilin, Anhui, Hunan, Shanxi, Shaanxi, Guizhou, Xinjiang, etc. Provinces (autonomous regions, municipalities directly under the Central Government) reported the occurrence of this pest. Rice water weevil mainly damages rice. Adults feed on leaves and la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6888C12Q2600/156C12Q2531/113C12Q2535/139C12Q2565/125
Inventor 张萌刘一景令狐芮平董瑞李晋
Owner 山西省植物保护植物检疫总站
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