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Early stage molecular detection and identification method and application for eight kinds of oophagous trichogrammae in trichogrammatidae family

A technology of molecular detection and identification method, which is applied in the field of species identification, can solve problems such as time-consuming, and achieve the effects of fast detection, wide application range, and simple operation

Inactive Publication Date: 2018-09-28
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It takes a long time to rear Trichogramma eggs and larvae to adult stage identification, so a new method is urgently needed to make up for the shortcomings of traditional investigation methods

Method used

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  • Early stage molecular detection and identification method and application for eight kinds of oophagous trichogrammae in trichogrammatidae family
  • Early stage molecular detection and identification method and application for eight kinds of oophagous trichogrammae in trichogrammatidae family
  • Early stage molecular detection and identification method and application for eight kinds of oophagous trichogrammae in trichogrammatidae family

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Determination of the SspI standard band spectrum and the VspI enzyme digestion standard band spectrum of 8 species of Trichogramma

[0033] 1 Trichogramma sample

[0034] 8 species of Trichogramma, including Trichogramma brassicae, T.cacoeciae, T.chilonis, T.evanescens, T. japonicum, T. ostriniae, T. bilingensis and T. dendrolimi.

[0035] 2 test hosts

[0036] Rice moth eggs.

[0037] 3 Production of egg cards

[0038] Take fresh rice moth eggs with similar size and shape and good plumpness, use glue to paste single rice moth eggs on coordinate paper to make egg cards (3cm×2cm), equidistant (0.4cm) on one egg card Paste 20 eggs, the degree of paste is easy to peel and not easy to fall off. The prepared egg card is placed in a glass test tube (the specification is 10cm×3cm). At the same time, put a 1cm x 1cm piece of filter paper in the test tube and drip two drops of distilled water to keep it moist, and seal the glass test tube mouth with a absorbent c...

Embodiment 2

[0081] The detection verification of embodiment 2 corn borer Trichogramma

[0082] The host eggs to be tested are the eggs of the Asian corn borer, and the parasitic Trichogramma is T. ostriniae.

[0083] Take the Trichogramma DNA in the eggs of the Asian corn borer to be detected. DNA was extracted using ProMEGA's SVGenomic DNA Purification System Kit. The extraction method is as follows:

[0084] (1) Configure Mix as a buffer for extracting DNA. The buffer system is 275 μl, including Nuclei Lysis Solution (200 μl), 0.5M EDTA (pH8.0) (50 μl), Proteinase K (20 mg / ml) (20 μl), Rnase ASolution (4 mg / ml) (5 μl).

[0085] (2) Dip the mix with a dissecting needle and transfer the host eggs containing unemerged Trichogramma / unparasitized host eggs to a 1.5ml centrifuge tube, add 50μl Mix with a pipette (Gilson, Eppendorf) and use a plastic grinding rod Grind.

[0086](3) After grinding sufficiently, draw 225 μl of Mix to rinse the grinding rod.

[0087] (4) Vortex and mix we...

Embodiment 3

[0099] The detection verification of embodiment 3 rice borer Trichogramma

[0100] The host eggs to be detected are eggs of cotton bollworm, and the parasitic Trichogramma is T. japonicum.

[0101] Other operations are the same as in Example 2.

[0102] The resulting SspI digested products and VspI digested products were detected by agarose gel electrophoresis, and DNAMAN 7.0 software was used to analyze the electrophoresis results. The estimated values ​​of the three bands cut by SspI were 812bp, 28bp, and 88bp, respectively, and the VspI enzyme The estimated value of the excised band is 928bp, and the number and size of the found bands match the standard band of Trichogramma rice borer T. japonicum, confirming that the detection is correct.

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Abstract

The invention provides an early stage molecular detection and identification method for eight kinds of oophagous trichogrammae in a trichogrammatidae family. The COI gene (cytochrome oxidase I of mtDNA) of the oophagous trichogrammae is obtained by a molecular biology method; a PCR technology is used as the basis; an oophagous trichogrammae COI gene specific primer is designed to amplify the oophagous trichogrammae COI gene in a host egg to be detected; the amplified PCR product of the oophagous trichogrammae COI gene fragment is used as a template; specific incision enzymes SspI and VspI arerespectively used for enzyme digestion; according to different enzyme digestion product electrophoretic bands, the comparison is performed with the standard band spectrum of the 8 kinds of oophagous trichogrammae; the identification on the types of the oophagous trichogrammae parasitized in the host eggs to be detected can be realized; in addition, the application to biological prevention and control of agricultural pest study is provided. The detection and identification method provided by the invention has the advantages that the requirement on instrument equipment is low; the identificationtime is shortened; the defect that the form cannot be used for identifying the oophagous trichogrammae in the non-adult period can be overcome; accuracy and high speed are realized; the cost is reduced.

Description

technical field [0001] The invention relates to the field of species identification, in particular to an early molecular detection and identification method for eight Trichogramma species of Trichogramma, which can be applied to investigate Trichogramma species and parasitic conditions, and evaluate biocontrol effects. Background technique [0002] Trichogramma Trichogramma belongs to Hymenoptera Hymenoptera Trichogrammatidae, is a parasitic insect widely used in biological control, can parasitize Lepidoptera, Diptera, Hymenoptera, Coleoptera, In the eggs of more than 400 kinds of harmful insects from 6 orders including Hemiptera and Megaloptera, the eradication of pests at the egg stage has been valued by the world. [0003] At present, morphological characteristics are mostly used in the identification of Trichogramma adult stage, and some molecular biological characteristics are used; however, there is no literature report on the identification of non-adult stage Trichogr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888
CPCC12Q1/6888C12Q2600/158
Inventor 李元喜华海青王伟
Owner NANJING AGRICULTURAL UNIVERSITY
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