Apolygus lucorum specific COI primer, kit containing apolygus lucorum specific COI primer and application of kit

A kit, the technology of Lygus viridans, applied in the field of molecular biology, can solve the problems of low specificity, high cost, and time-consuming, etc., and achieve the effects of strong specificity, improved accuracy, and simple operation

Active Publication Date: 2015-05-13
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 traditional research methods such as routine intestinal anatomy, behavioral observation, serological analysis or isotope labeling, not only need to spend a lot of time, but also expensive, specificity is not strong, subject to certain restrictions in use
In order to accurately assess the control effect of the predatory natural enemies of the green ligus on the green ligus, it is necessary to establish a set of rapid and accurate molecular detection methods, but there is no standard detection method for the green ligus

Method used

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  • Apolygus lucorum specific COI primer, kit containing apolygus lucorum specific COI primer and application of kit
  • Apolygus lucorum specific COI primer, kit containing apolygus lucorum specific COI primer and application of kit
  • Apolygus lucorum specific COI primer, kit containing apolygus lucorum specific COI primer and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Amplification effect of Lygus aeruginosa specific primers AluF1 and AluR1 on Lygus aeruginosa

[0025] 1. Preparation of the Lygus green bug genome

[0026] Put a single Lygus viridans into a 1.5mL centrifuge tube and grind it for extraction. The obtained DNA solution was stored at -20°C for later use, and 1 μL of the solution was taken as a DNA template when performing PCR amplification.

[0027] 2. Synthesis of specific COI primers for the detection of Lygus chlorophylla

[0028] The sequence of the specific COI primer for Lygus green bug is as follows:

[0029] Forward primer AluF1:5'-TTTGGAAATTGACTAGTACCA-3'

[0030] Reverse primer AluR1:5'-GGAAGTGATAATAATAACAGTAGG-3'

[0031] 3. PCR amplification

[0032] 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.

[0033] The PCR reaction cond...

Embodiment 2

[0042] Example 2 Determination of Primer AluF1 / AluR1 to the Minimum Detection Amount of Lygus chlorophylla

[0043] According to Example 1, the genomic DNA of a single Lygus lucidum 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 160ng / 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.

[0044] Utilize the primer AluF1 / AluR1 to do the determination of the minimum detection amount, use different dilution multiples of Lygus chlorophylla genomic DNA as the template for PCR amplification, such as figure 2 As shown in , the DNA concentration of swimming lane 1 is 160ng / μL, and the DNA template diluted by 2-fold method is use...

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Abstract

The invention relates to the molecular biological field and particularly provides an apolygus lucorum specific COI primer, a kit containing the apolygus lucorum specific COI primer and application of the kit. According to a conserved sequence of apolygus lucorum mtDNA, a pair of apolygus lucorum specific COI primers AluF1 and AluR1 (as shown in SEQ ID No.1 and SEQ ID NO.2) are designed; according to the detection, the pair of primers have specific amplification ability, the size of amplified products is 323bp, and the sensitivity DNA concentration detection limit is 0.15625ng / mu L. By utilizing a PCR (polymerase chain reaction) detecting technology, a single specific stripe is obtained by amplifying the specific primers. The apolygus lucorum specific COI primer is simple to operate, and has the characteristics of being efficient, low in price and strong in specificity, so that the detecting accuracy is improved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the specific COI primer of Lygus viridans, a kit containing it and application thereof. Background technique [0002] The green bug Apolygus lucorum (Meyer-Dür) belongs to the family Miridae of the order Homoptera. It has a green body, is good at hiding, and has a quick response. Its nymphs and adults suck plant buds, leaves, flower buds, and young fruits with piercing-sucking mouthparts. . The green Lygus almost spreads all over the country's cotton areas, harming cotton and affecting cotton production. Especially in recent years, with the large-scale planting of Bt cotton, its population has increased sharply, and its damage has increased. It has risen from a minor pest to a major pest in cotton fields, and even affected jujube trees, grapes, pears, tea trees, etc. grow crops. At present, the green Lygus has become an important pest on many crops in my country, seriously aff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888
Inventor 李金花杨帆袁海滨陆宴辉
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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