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Specific rapid PCR (Polymerase Chain Reaction) detecting method for cotton aphid as well as SCAR (Sequence Characterized Amplified Region) primer and kit thereby

A specific and kit-based technology, applied in the field of molecular biology, can solve the problem of no detection method for cotton aphids, and achieve the effects of saving detection time, simple operation process and high application value

Inactive Publication Date: 2011-08-10
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

However, there is currently no standard detection method for cotton aphids in China.

Method used

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  • Specific rapid PCR (Polymerase Chain Reaction) detecting method for cotton aphid as well as SCAR (Sequence Characterized Amplified Region) primer and kit thereby
  • Specific rapid PCR (Polymerase Chain Reaction) detecting method for cotton aphid as well as SCAR (Sequence Characterized Amplified Region) primer and kit thereby
  • Specific rapid PCR (Polymerase Chain Reaction) detecting method for cotton aphid as well as SCAR (Sequence Characterized Amplified Region) primer and kit thereby

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Detection of the amplification effect of primers AGZWSE / AGZWSF on cotton aphids

[0018] (1) Preparation of aphid template DNA

[0019] Place a single aphid on a parafilm dripped with 20 μL of extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 20 mM NaCl, pH 8.0), and use the bottom of a 0.2 mL PCR tube as a homogenizer to thoroughly grind , the homogenate was transferred into a 1.5mL centrifuge tube with a micropipette; then the homogenizer was washed four times with 200μL buffer, transferred into the same centrifuge tube, mixed evenly, and 5μL proteinase K (20mg / mL) was added, mixed well and then placed in Water bath at 65°C for 1 hour (mix once in the middle); then bath in boiling water for 5 minutes, add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, mix gently dozens of times, and place on ice for 30 minutes; Centrifuge at 4°C and 12000r / min for 20min, take the supernatant, add 440μL of pre-cooled absolute ethanol, mix gently, and wait for ...

Embodiment 2

[0034] Example 2: Detection of the amplification effect of primers AGZWSE / AGZWSF on different stages and sexes of cotton aphids

[0035] (1) Preparation of cotton aphid template DNA

[0036] Place the single head / single aphid of different insect states and sexes on the parafilm membrane dripped with 20uL extraction buffer (50mM Tris-HCl, 1mM EDTA, 1% SDS, 20mM NaCl, pH 8.0), the preparation process is the same as the implementation example 1.

[0037] (2) Synthesize SCAR-specific primers for testing cotton aphids, and the nucleotide sequences of the primers are the same as in Example 1.

[0038] (3) Carry out PCR amplification reaction, reaction system and PCR amplification procedure are the same as embodiment 1.

[0039] (4) To identify the PCR product, the identification method is the same as in Example 1.

[0040] (5) Implementation results

[0041] Using primers AGZWSE / AGZWSF, the genomic DNA of cotton aphids of different stages and sexes was used as template for PCR a...

Embodiment 3

[0042] Example 3: Determination of the minimum detection amount of cotton aphid with primer AGZWSE / AGZWSF

[0043] (1) Preparation of cotton aphid template DNA

[0044] Single-headed female adults of cotton aphid were placed on a parafilm dripped with 20 μL of extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 20 mM NaCl, pH 8.0), and the preparation process was the same as in Example 1. Then the original template solution was serially diluted to 1 / 2560 head by 2 times, and 2 μL was used as the template for PCR amplification, which was directly added to the PCR reaction system.

[0045] (2) Synthesize specific primers for testing cotton aphids, and the nucleotide sequences of the primers are the same as in Example 1.

[0046] (3) Carry out PCR amplification reaction, reaction system and PCR amplification procedure are the same as embodiment 1.

[0047] (4) To identify the PCR product, the identification method is the same as in Example 1.

[0048] (5) Implementation resu...

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Abstract

The invention discloses a specific rapid PCR (Polymerase Chain Reaction) detecting method for cotton aphid as well as an SCAR (Sequence Characterized Amplified Region) primer and a kit thereby, belonging to the field of molecular biology. In the method, a pair of specific primers is designed according to a specific genome DNA sequence of cotton aphid; and the primer pair only has amplification capability on the cotton aphid, the size of an amplification product is 358bp, and the detecting sensitivity reaches 1 / 320 female imagoes. The method is supplementation and improvement on an RAPD (Random Amplified Polymorphism DNA) technology and an SSR (Simple Sequence Repeats) technology detecting method of cotton aphid. Meanwhile, by adopting an SCAR-PCR technology, the method improves detecting accuracy, saves the detecting time and can be popularized in China south breeding bases (Hainan), fruit, vegetable and flower seed production bases (such as Gansu), organic fruit and vegetable production bases and flower production bases in a kit way.

Description

technical field [0001] The invention relates to a specific rapid PCR detection method for cotton aphids, used SCAR primers and a kit, and belongs to the field of molecular biology. Background technique [0002] Cotton aphid Aphis gossypii Glover is the melon aphid, commonly known as greasy insects, honey insects, oil insects, belonging to Homoptera, Aphididae, and Aphids. It is a worldwide pest. It occurs all over the country except Tibet. Among them, the Yellow River Basin, Liaohe River Basin and Northwest Cotton Region were severely affected. Cotton aphid is a polyphagous pest with a wide range of hosts. There are 285 species of known host plants in 74 families in the world, and 113 species have been recorded in my country. Plants that live for a period of time after overwintering and hatching in the next spring, including hibiscus, prickly ash, pomegranate, buckthorn, plantain, summer solstice, purple lily, bitter pickle, etc.; resident hosts (also known as second hosts, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 万方浩吴霞张桂芬郭建英刘万学
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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