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Method for detecting non-specific esterase activity of aphids and detection reagent box

A non-specific, aphid-based technology, applied in the field of detection of aphid non-specific esterase activity and detection kits, can solve the problems of high operating conditions, complicated operation, and large number of insects

Inactive Publication Date: 2014-01-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In insect resistance detection, bioassay and enzyme activity analysis are mainly used at present. The former uses a large number of insects, the operation is complicated, and it is difficult to master; the latter requires high operating conditions and requires certain equipment and other conditions, which is not suitable. Promote use at the grassroots level and in the field

Method used

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  • Method for detecting non-specific esterase activity of aphids and detection reagent box
  • Method for detecting non-specific esterase activity of aphids and detection reagent box
  • Method for detecting non-specific esterase activity of aphids and detection reagent box

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Experimental program
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Effect test

Embodiment 1

[0030] 1. Research object

[0031] The research objects of the present invention are field samples collected all over the country, and the aphid populations are reared indoors.

[0032] 2. Method

[0033] Use the mixed substrate filter paper color method to detect the non-specific esterase activity of aphid field populations. According to the color depth, compare the statistical results with the standard color plate. For the specific process, see the attached figure 1 .

[0034] 2.1 Collection and processing of aphid samples

[0035] The wingless adult aphids were collected in centrifuge tubes, quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0036] 2.2 Preparation of filter paper substrate

[0037] Prepare 1% (w / v) α-NA and β-NA with acetone, weigh 1g of α-NA and β-NA each and dilute to 100ml with acetone, store in a refrigerator at 4°C, take an equal volume before use Mix the two, soak the filter paper in it for 2 minutes, take it out and dry it i...

Embodiment 2

[0046] 1. Research object

[0047] The research objects were field samples collected from all over the country, and the aphid populations were reared indoors.

[0048] 2. Method

[0049] Use a single substrate, α-NA or β-NA, and use ordinary phosphate buffer as the homogenate.

[0050] 2.1 Collection and processing of aphid samples

[0051] The wingless adult aphids were collected in centrifuge tubes, quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0052] 2.2 Preparation of substrate

[0053] Prepare 1% (w / v) α-NA or β-NA with acetone and store in a 4°C refrigerator.

[0054] 2.3 Preparation of developer

[0055] Prepare 0.15% Fast Blue B salt with distilled water as a color developer. Note that this homogenate should be prepared and used immediately. Take 1 / 20 of the volume of the homogenate and drop it on the filter paper prepared above, and place it at room temperature in the dark for 15 minutes. More than 100 heads of the tested population must ...

Embodiment 3

[0060] Preparation and application of embodiment 3 kit

[0061] 1. Filter paper adsorbed with mixed substrates:

[0062] Mix equal volumes of α-naphthyl acetate with a mass concentration of 1% and β-naphthyl acetate with a mass concentration of 1% prepared in acetone, absorb on qualitative filter paper, dry in the shade, wrap in tin foil and store at 4°C;

[0063] 2. Chromogenic agent buffer solution: prepare 0.15% chromogenic agent Fast Blue B with 0.04M, pH7.0 phosphate buffer solution, and this chromogenic agent must be prepared and used immediately.

[0064] The specific application is the same as in Example 1: collect the field population of aphids, after homogenizing a single aphid according to the above method, drop the homogenate onto the filter paper for color development, compare with the standard color plate, count the color development of resistant individuals, repeat for each population Detect more than 100 heads, calculate the frequency of resistant individuals ...

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Abstract

The invention provides a method for detecting non-specific esterase activity of aphids. The method includes isometrically mixing a substrate alpha-naphthyl acetate and a substrate beta-naphthyl acetate according to a ratio of 1:1 to obtain a mixed substrate, and preparing filter paper adsorbing the mixed substrate; collecting field population of the aphids, after homogenizing single-head aphids, dropping homogenate on the filter paper for color development, and comparing with a standard color palette, wherein the darker the filter paper develops, the higher the individual enzymatic activity is. By the method, resistance level of the field population can be judged by detecting individual non-specific esterase (NSE) activity. By comparing the individual color developed on the filer paper adsorbing the mixed substrate with the standard color palette, resistance individual frequency is acquired, and then population resistance level is known.

Description

technical field [0001] The invention relates to a rapid biochemical detection method and a mixed substrate detection kit for the resistance of agricultural pests to organophosphorus Background technique [0002] The formation and development of insect resistance to chemical control has caused great difficulties, how to quickly and accurately detect the level of aphid resistance is very important. In the aspect of insect resistance detection, bioassay and enzyme activity analysis are mainly used at present. The former uses a large number of insects, the operation is complicated, and it is difficult to master; the latter requires high operating conditions and requires certain equipment and other conditions, which is not suitable. Promote use at the grassroots level and in the field. [0003] At present, the resistance detection of aphids to organophosphorus is mainly carried out through the traditional bioassay-leaf coating method: take fresh and non-polluted cabbage leaves a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/44
Inventor 高希武刘晓岚汤秋玲李永丹梁沛
Owner CHINA AGRI UNIV
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