Harmonia axyridis specific COI primer of harmonia axyridis, kit containing primer and detection method for harmonia axyridis

The technology of a heterochromia and detection method, applied in the field of molecular biology, can solve the problems that the predation of ladybugs in the ladybug group cannot be accurately evaluated, and the specific detection method of heterochromia has not been established, so as to achieve high practical value , High sensitivity, strong specificity

Inactive Publication Date: 2015-04-29
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

With the deepening of the research, it was found that traditional research methods such as routine intestinal anatomy and behavioral observation cannot accurately evaluate the predation among ladybugs in the ladybug group
At present, no specific detection method for Heterochromia has been established; polymerase chain reaction (PCR) technology has the characteristics of strong specificity, high sensitivity, simplicity and speed, and occupies an important position in the field of species identification

Method used

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  • Harmonia axyridis specific COI primer of harmonia axyridis, kit containing primer and detection method for harmonia axyridis
  • Harmonia axyridis specific COI primer of harmonia axyridis, kit containing primer and detection method for harmonia axyridis
  • Harmonia axyridis specific COI primer of harmonia axyridis, kit containing primer and detection method for harmonia axyridis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 The amplification effect of primer HaF3 / HaR3 on the heterochromia

[0024] 1. Preparation of Heterochromia genome

[0025] Put a single Heterochromia in a 1.5mL centrifuge tube and grind it, and use TIANamp Genomic DNA Kit blood / cell / tissue genomic DNA extraction kit (spin column type) (Tiangen Biological Co., Ltd., Beijing) to extract single The head ladybug genome. The DNA solution was stored at -20°C for later use, and 1 μL of the solution was taken as a DNA template during PCR amplification.

[0026] 2. Synthesis of specific COI primers for the detection of Heterochromia

[0027] The specific COI primer sequences are as follows:

[0028] HaF3: 5'-AATTGTTACAGCTCATGCT-3' (SEQ ID No. 1)

[0029] HaR3: 5'-CCCCTATTTCTACGATTG-3' (SEQ ID No. 2)

[0030] 3. PCR amplification

[0031] The reaction system is 20 μL, including: 10×EasyTaq buffer 2.0 μL, dNTP (2.5 mM) 0.4 μL, EasyTaq DNA polymerase (5U / μL) 0.2 μL, forward and reverse primers (10 μM) each 0.4 μL, ...

Embodiment 2

[0040] Example 2 Primer HaF3 / HaR3 Determination of the Minimum Detection Amount of Heterochromia

[0041] 1. Preparation of Heterochromia template DNA

[0042] According to Example 1, the genomic DNA of a single Heterochromia was extracted, and then the original template solution (the concentration of the DNA solution was 3ng / μL) was diluted by 2 times until no bands were detected, and 1 μL was used as a template for PCR amplification. Add directly to the PCR reaction system, and the reaction system is the same as that described in Example 1.

[0043] Using primers HaF3 / HaR3 to determine the minimum detection amount, PCR amplification was carried out with different dilutions of the heterochromia genome DNA as a template, and the detection limit of the DNA concentration was 0.01171875ng / μL ( figure 2 ).

[0044] 2. Preparation of Heterochromia plasmid

[0045] According to the implementation example 1, the genomic DNA of the ladybug heterochromia was amplified, and after th...

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Abstract

The invention relates to a harmonia axyridis specific COI primer and a detection kit containing the primer. According to a peculiar mtDAN sequence of harmonia axyridis, a pair of harmonia axyridis specific COI primers HaF3 and HaR3 (SEQ ID No.1 and 2) is designed and has specific amplification capability only for harmonia axyridis, the size of an application product is 197bp, the limit of detection of plasmid concentration is 1.193285577E+04, and the limit of detection of DNA concentration is 0.01171875ng / microliter. According to the method, a PCR (polymerase chain reaction) technology is adopted, so that the detection accuracy is improved, and the detection time is saved; a kit product is suitable for being primarily popularized and applied.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a heterochromia-specific COI primer, a kit containing the primer and a detection method thereof. Background technique [0002] Harmonia axyridis (Pallas) is an insect belonging to the class Insecta, order Coleoptera, and family Coccinoptera. It is widely distributed and preys on eggs and small larvae of various aphids, scale insects, psyllids, and moths. Heterochromia has a strong flight and dispersal ability, can search for hosts in a wide range, and quickly control the population of small individual pests such as aphids. It is an effective biological control species. Due to the long lifespan of adults and large amount of food, they can control the target pests immediately after release, and the control effect becomes stronger and stronger as the adults lay eggs and the larvae hatch. Our country has used Heterochromia ladybug to control the main pest of pine trees, Matsuma jap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2531/113
Inventor 杨帆王倩陆宴辉徐建祥
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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