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Rapid diagnosis method for diagnosing exotic insect western flower thrips and diagnosis primer thereof

A western flower thrips, rapid diagnosis technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. impact, etc.

Inactive Publication Date: 2008-07-30
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantages of this method are: the materials required by this method are easy to obtain and the cost is low; the disadvantages are: high quality requirements for glass slides, a large number of specimens are required, larvae and eggs cannot be identified, and the professional knowledge level of appraisers is high. Accuracy is affected by subjective factors (identification operator's experience)
The advantage of this method is: it breaks through the defects of traditional morphological identification methods, and uses the means of molecular biotechnology to identify thrips, and the identification results are accurate and reliable; the disadvantages are: sequencing requires high quality of PCR products, and the cost of sequencing is high. At least one sequencing reaction is required to identify a thrips, and the current market price of each reaction is 35-40 yuan. Sequence comparison and analysis are required. The result analysis is difficult and requires high experimental conditions (sequencer is required). Operators are required to have proficient operating skills, such as handing over to a specialized sequencing company for sequencing is time-consuming (3-5 working days), etc.
The advantage of this method is: Compared with the method of Brunner et al, this method can identify Thrips occidentalis without sequencing, which is more economical and faster; but the disadvantage is: poor repeatability and low accuracy

Method used

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  • Rapid diagnosis method for diagnosing exotic insect western flower thrips and diagnosis primer thereof
  • Rapid diagnosis method for diagnosing exotic insect western flower thrips and diagnosis primer thereof
  • Rapid diagnosis method for diagnosing exotic insect western flower thrips and diagnosis primer thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: extract the total DNA of thrips to be checked

[0029]a. Immerse the thrips to be tested in distilled water for 1 hour to remove surface impurities and avoid DNA contamination. b. Transfer the worms to a 1.5ml Eppendorf tube filled with 200μL of extract (10mmol / Ltris-cl, 1mmol / LEDTA, 1% SDS, pH=8.0), use a round glass rod or a burnt blue gun The head is thoroughly ground to a homogeneous state. c. Add 2.5 μL of 20 mg / mL Proteinase K (Proteinase K) to the homogenate after grinding, mix well, and then put the Eppendorf tube in a 56°C water bath for 2 hours. d. First extraction: Take the Eppendorf tube out of the water bath, add 1 volume of phenol / chloroform (1:1), shake and mix for 2-3 minutes, and then centrifuge for 10 minutes (4°C, 10000r / min) , Aspirate the supernatant into another new Eppendorf tube (the volume of the aspirated supernatant should preferably not exceed 100 μL). e Second extraction: Use an equal volume of chloroform to extract the supe...

Embodiment 2

[0030] Embodiment 2: PCR reaction

[0031] The primers used in the PCR reaction are the developed Western flower thrips specific forward primer 5'-CAGGGTGGTCGCTTCACCGCTTC-3'reverse primer: 5'-GCGAGAAAATAATGCAAACTGCG-3', PCR reaction uses 25 μL standard reaction system, which contains 2 μL DNA solution, 2.5 μL 10× Taq buffer, 0.5 μL of 10 mM dNTP mixture, 0.5 μL of each primer (10 μm / L), 0.5 μL of Taq DNA polymerase (2.5 U / μL). The reaction conditions were: denaturation at 94°C for 5 minutes, 30s at 94°C, 45s at 50°C, 45s at 72°C, and 10 minutes at 72°C after 30 cycles.

Embodiment 3

[0032] Embodiment 3 agarose gel electrophoresis

[0033] The concentration of agarose gel used for agarose gel electrophoresis was 2%, and EB was added to the gel. The total loading volume is 7 μL, including 5 μL of PCR product and 2 μL of loading buffer. The electrophoresis voltage was a constant voltage of 120v, and the electrophoresis time was 30 minutes.

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Abstract

The invention relates to a diagnostic method in which a specific PCR primer can be used to amplify a to-be-tested sample, thereby quickly identifying Frankliniella occidentalis. In addition, the invention also relates to a specific primer which can be used for quickly diagnosing Frankliniella occidentalis, thereby quickly diagnosing Frankliniella occidentalis.

Description

technical field [0001] The present invention relates to a kind of rapid diagnosis method of the invasive pest Thrips occidentalis, more specifically, the present invention relates to the diagnostic method for quickly identifying and identifying Thrips occidentalis by amplifying the sample to be tested with specific PCR primers; in addition, the present invention The invention also relates to a specific primer for rapid diagnosis of Western flower thrips, so as to achieve the purpose of rapid diagnosis of Western flower thrips. Background technique [0002] Western flower thrips Frankliniella occidentalis belongs to Thysanoptera, Thripidae, is a world-renowned dangerous pest. It hosts more than 60 families and more than 500 species of plants, especially for greenhouse flowers and solanaceous plants. According to data, since Western flower thrips was discovered in the western mountains of the United States and Canada in 1895, the distribution of western flower thrips has spre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 路虹游中华石宝才宫亚军
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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