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Arma chinensis virus specific CP detection primer and PCR detection method

A technology for detection of primers and detection methods, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc. The effect of high accuracy, strong specificity and simple operation process

Pending Publication Date: 2022-02-01
TOBACCO RES INST CHIN AGRI SCI ACAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Chinese researchers have carried out detailed research on the control effect and artificial breeding technology of gnat stink bugs, including population dynamics, artificial feed, pest control species, etc. This problem seriously threatens the large-scale production and popularization of the stinkbug, and it is urgent to carry out relevant research on the pathogenic species and their functions in the stinkbug.

Method used

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  • Arma chinensis virus specific CP detection primer and PCR detection method
  • Arma chinensis virus specific CP detection primer and PCR detection method
  • Arma chinensis virus specific CP detection primer and PCR detection method

Examples

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Effect test

preparation example Construction

[0031] Step 2, preparation of the cDNA of the stinkbug;

[0032] Step 3, checking the synthesis of the specific primer of AcV-1;

[0033] Step 4, PCR amplification reaction, PCR amplification program and PCR product identification;

[0034]Step 5: Recheck the test results.

[0035] Specifically, it includes the following steps: according to the operation steps in step 1, after grinding the gnat stink bug sample, take 0.1 g into a 1.5 mL sterile enzyme-free centrifuge tube, add TRIzol™ Reagent and mix well, let it stand at room temperature for 5 min, add 200 µL chloroform, shake vigorously up and down for 25-30 s, let stand at room temperature for 3 minutes, then centrifuge at 12,000 rpm at 4°C for 15 minutes. Add 500 µL of isopropanol, mix well, let stand at room temperature for 10 minutes, centrifuge at 4°C at 12,000 rpm for 10 minutes, discard the supernatant, add 1 mL of 75% ethanol, blow the precipitate to suspend it, after washing, spin at 4°C at 7,500 rpm , centrifuge...

Embodiment 1

[0047] Embodiment 1: The present invention provides a kind of stinkbug virus-specific CP detection primer and PCR detection method, comprising the following steps: step 1, extraction of stinkbug total RNA, grinding, extraction and purification in turn, after the stinkbug sample is ground , take 0.1 g in a 1.5 mL sterile enzyme-free centrifuge tube, add TRIzol™ Reagent and mix well, let stand at room temperature for 5 minutes, add 200 µL chloroform, shake vigorously up and down for 25-30 s, stand at room temperature for 3 minutes, 4 Centrifuge at 12,000 rpm for 15 min. After centrifugation, pipette 400 µL of the supernatant into a new 1.5 mL sterile, enzyme-free centrifuge tube, add 500 µL of isopropanol, mix well, and let stand at room temperature for 10 min. rpm, centrifuge for 10 min, discard the supernatant, add 1 mL of 75% ethanol, pipette the precipitate to suspend it, after washing, centrifuge at 7500 rpm at 4°C for 5 min, discard the supernatant, centrifuge at 7500 rpm a...

Embodiment 2

[0048]Example 2: Extraction of total RNA of stink bugs, preparation of cDNA, synthesis of AcV-1F / R, PCR amplification and identification of PCR products are all the same as Example 1, and the target fragment in Example 1 is referred to EasyPure® Quick Gel Extraction Kit (TransGen, Beijing, China) Instructions for gel cutting recovery, recovery of target fragments and vector pEASY-T1 25 ℃ 30 min ligation, heat shock method to transform Escherichia coli DH5α competent cells, spread in the medium containing ampicillin (Amp) overnight Cultivate, pick the clones on the plate for colony PCR, and send the bacterial liquid samples corresponding to the correct size bands in the PCR results to Shanghai Passino Biotechnology Co., Ltd. for sequence determination. The sequencing results are compared with the correct bacterial liquid. Cultivate overnight in a shaker at 200 rpm, and extract plasmids according to the instructions of EasyPure® Plasmid MiniPrep Kit (TransGen, Beijing, China). Th...

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Abstract

The invention discloses an arma chinensis virus specific CP detection primer and a PCR detection method, and belongs to the technical field of gene detection. The arma chinensis virus specific CP detection primer and the PCR detection method comprise the following steps: 1, extraction of arma chinensis total RNA: sequentially grinding, extracting, and purifying; 2, preparation of arma chinensis cDNA; 3, synthesis of a specific primer for detecting AcV-1; 4, PCR amplification reaction, PCR amplification program and PCR product identification; and 5, reinspection of the detection result. According to the invention, the method can be used for detecting single imago, five-year nymphs and four-year nymphs of arma chinensis, can also be used for detecting single three-year nymphs, two-year nymphs, newly hatched nymphs and egg masses, is simple, convenient and rapid to operate, adopts a PCR technology, is simple, rapid and efficient in operation process, generally can be completed within 2 hours, and is strong in specificity; and the specific primer designed by the invention only has an amplification capability on AcV-1.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a primer for CP detection specific to stinkbug virus and a PCR detection method. Background technique [0002] Arma chinensis, also known as Arma chinensis, belongs to Hemiptera (Hemiptera), Pentatomidae (Pentatomidae), Asopinae (Asopinae), Arma (Arma), the nymphs and adults of Arma chinensis can prey on sticky There are more than 40 kinds of common pests, such as insects, moths, leaf beetles, scale insects, aphids, and Spodoptera frugiperda. They are a kind of predatory natural enemy insects. In nearly 20 provinces such as Guizhou, based on the characteristics of broad-spectrum feeding habits of stinkbugs, it has a significant inhibitory effect on the occurrence of serious agricultural pests such as Lepidoptera and Coleoptera. However, the number of stinkbugs is very difficult under natural conditions. To meet the needs of pest control, it is necessary to carry out indoo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686
Inventor 孙梅雪徐蓬军任广伟王秀芳王新伟
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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