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Specific COI primers for peristenus spretus Chen&van Achterberg, kit containing same and application of primers and kit

The technology of the red-necked Braconidae and the kit is applied in the field of molecular biology, and can solve the problems of death of chlorophyll bugs, inability to study chlorophyll bugs, restrictions on identification work, etc., and achieves strong specificity, improved accuracy, and improved operation. simple effect

Active Publication Date: 2015-05-20
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the fact that parasitic wasps are difficult to observe in the field, and the parasitic wasps are easy to die in the field, it has been impossible to conduct better research on the parasitic relationship between them and the green Lygus, and the identification work is also restricted, and it is impossible to accurately evaluate the red-necked The Controlling Effect of Braconus persicum on Lygus japonica

Method used

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  • Specific COI primers for peristenus spretus Chen&van Achterberg, kit containing same and application of primers and kit
  • Specific COI primers for peristenus spretus Chen&van Achterberg, kit containing same and application of primers and kit
  • Specific COI primers for peristenus spretus Chen&van Achterberg, kit containing same and application of primers and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Amplification effect of the red-necked Braconid spp. specific primers PsF1 and PsR1 on the red-necked B.

[0026] 1. Preparation of the genome of Brassica spp.

[0027] Put the single-headed Braconus persicae into a 1.5mL centrifuge tube and grind it for extraction. And the obtained DNA solution was stored at -20°C for future use.

[0028] 2. Synthesis of specific COI primers for the detection of B.

[0029] The sequence of the specific COI primers of Braconus red-necked is as follows:

[0030] Forward primer PsF1: 5'-TATTAAGAAGAATAATTGGGATG-3'

[0031] Reverse primer PsR1: 5'-ATTTAAATTTCGATCTGTTAATAAC-3'

[0032] 3. PCR amplification

[0033] The reaction system is 20 μL, including: 10×EasyTaq buffer 2.0 μL, dNTPs (2.5 mM) 0.4 μL, EasyTaq DNA polymerase (5U / μL) 0.2 μL, forward and reverse primers (10 μM) each 0.4 μL, template DNA 1.0 μL, ddH 2 O 15.6 μL.

[0034] The PCR reaction conditions were: 94°C for 4 minutes; 94°C for 30 seconds, 58°C for 30 se...

Embodiment 2

[0043] Example 2 Determination of the minimum detectable amount of PsF1 / PsR1 primers PsF1 / PsR1

[0044] According to Example 1, the genomic DNA of the single-headed Braconus persicae was extracted, and amplified according to the reaction system and PCR reaction conditions described in Example 1. Then the original template solution (the concentration of the DNA solution is 20ng / μL) is gradually diluted by 2 times, and 1 μL of the diluted solution is taken as a template for PCR amplification, and directly added to the PCR reaction system. The reaction system is the same as that described in Example 1. . Dilute until no bands can be detected.

[0045] Use primers PsF1 / PsR1 to determine the minimum detection amount, and use different dilution multiples of Braconus persicae genomic DNA as a template for PCR amplification. The concentration of DNA template added in lane 1 is 20ng / μL, and the concentration is reduced by 2 times The diluted DNA template was amplified, and the result...

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Abstract

The invention relates to the field of molecular biology and in particular provides specific COI primers for peristenus spretus Chen&van Achterberg, a kit containing the same and an application of the primers and the kit. A pair of specific COI primers PsF1 and PsR1 (shown in SEQ ID No.1 and SEQ ID No.2) for peristenus spretus Chen&van Achterberg is designed according to a conserved sequence of mtDNA (mitochondrial deoxyribonucleic acid) of peristenus spretus Chen&van Achterberg. Through detection, the primers have a specific amplification capacity only toward peristenus spretus Chen&van Achterberg, the sizes of amplified products are 263bp, and the concentration detection limit of DNA with sensitivity is 0.625ng / mu L. A specific single band is obtained by amplifying the specific primers by utilizing a PCR (polymerase chain reaction) detection technique, is simple to operate, has the characteristics of high efficiency, low price and strong specificity and conduces to improving the detection accuracy.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular, to a specific COI primer for Brachydontia red-necked, a kit containing the primer and an application thereof. Background technique [0002] The green mirid bug Apolygus lucorum (Meyer-Dür) belongs to the family Miridae of the order Homoptera. It is the dominant species of mirid bug in various cotton areas in my country, and has now become a major pest in cotton fields. There have been no very effective measures for the prevention and control of the green Lygus for a long time, and chemical pesticides are generally used as the main method. However, with the use of pesticides, the drug resistance of the green ligus has been increasing, and the control of the green ligus has become more difficult. As one of the means of biological control, parasitic natural enemies rely on parasitic factors such as venom, polymorphic DNA viruses, virus-like particles, virus-like filaments, ovarian pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 李金花杨帆罗素萍袁海滨陆宴辉
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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