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Kit for detecting existence and content of insecticide in water body

A technology of pesticides and kits, which is applied in the field of kits for detecting whether there are pesticides in the water body and the range of pesticide content, can solve the problems of time-consuming and laborious, and achieve the effect of improving sensitivity, low cost and rapid detection

Inactive Publication Date: 2012-01-04
INST OF ZOOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection of pesticide residues in most sewage requires the use of large instruments, which is time-consuming and laborious

Method used

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  • Kit for detecting existence and content of insecticide in water body
  • Kit for detecting existence and content of insecticide in water body
  • Kit for detecting existence and content of insecticide in water body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Expression of xb1 fusion gene

[0041] 1. Cloning of xb1 gene

[0042] 1. Extract the total RNA of Spodoptera litura and reverse transcribed into cDNA.

[0043] 2. Using the cDNA extracted in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product (about 1611 bp).

[0044] F1 (upstream primer): 5'-ATGGTGCAAGTGAGAGTGAATGA-3';

[0045] R1 (downstream primer): 5'-TCACAATACATATTTTTCATACAATTT-3'.

[0046] PCR reaction system (50μl): 10×buffer 5μl, dNTP (10mM) 1μl, F1 (10uM) 1.25μl, R1 (10uM) 1.25μl, Taq (Expand High Fidelity) 0.75μl, water 39.75μl, cDNA template 1μl.

[0047] PCR reaction conditions: pre-denaturation at 94°C for 2min; then 30 cycles: 94°C for 15s, 55°C for 30s, 68°C for 2min; finally 68°C extension for 7min, storage at 4°C.

[0048] 3. Use agarose gel DNA recovery kit (Tiangen, product number DP208) to recover and purify the PCR amplified product of step 2, and ligate with pGEM-T easy vecto...

Embodiment 2

[0067] Example 2. Assembly of the kit

[0068] The kit (100 times) consists of 22 enzyme tubes, 22 substrate tubes, 22 color reagent tubes, 1 bottle of buffer (40ml) and 250 0.2ml centrifuge tubes.

[0069] The buffer is made of 50mM phosphate buffer (pH7.0); it is the disodium hydrogen phosphate Na 2 HPO 4 ·12H 2 O (Chemical Reagent Company of Sinopharm Group; Product No. 10020318) and sodium dihydrogen phosphate NaH 2 PO 4 ·2H 2 O (Sinopharm Chemical Reagent Company; product number 20040718) is obtained by dissolving in water.

[0070] Chromogenic reagent tube: 1.5ml centrifuge tube containing 5mg Gulan B salt (China National Pharmaceutical Group Chemical Reagent Co., Ltd., catalog number 70117082).

[0071] Substrate tube: 1.5ml centrifuge tube containing 1mg β-naphthyl acetate (China National Pharmaceutical Group Chemical Reagent Co., Ltd., product catalog number 30127624).

[0072] Enzyme tube: the enzyme tube prepared in step 3 of Example 1.

[0073] The storage conditions of the k...

Embodiment 3

[0074] Example 3. Use of the kit

[0075] 1. Pretreatment of each component in the kit

[0076] The components in the kit prepared in Example 2 were processed as follows:

[0077] Add 1ml of the buffer provided by the pre-cooled kit to each enzyme tube to obtain an enzyme solution (the concentration of XB1 fusion protein in the enzyme solution is about 20μg / ml).

[0078] Add 1ml of distilled water to each developer tube to obtain a developer solution (the mass percentage of solid blue B salt is 0.5%).

[0079] Add 0.4ml of absolute ethanol and 0.6ml of the buffer provided by the kit to each substrate tube to obtain a substrate solution (the mass percentage of β-naphthyl acetate is 0.1%).

[0080] Enzyme solution, substrate tube solution and color developer solution should all be used and prepared; in consideration of cost saving, it is best to make at least 5 samples to be tested each time.

[0081] Second, the application of the kit (detection of propoxur)

[0082] 1. Sample preparation

...

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Abstract

The invention discloses a kit for detecting the existence and content of an insecticide in a water body. The kit comprises the following components: XB1 protein, fast blue B salt, beta-naphthyl acetate and 50mM phosphate buffer with the pH of 7.0. The XB1 protein is protein (a) or protein (b), wherein the protein (a) is shown by a sequence 1 in a sequence table, and the protein (b) is shown by amino acid residues from a position-39 to a position-574 at the N terminal of the sequence 1 in the sequence table. Experiments prove that the XB1 protein from spodoptera litura has carboxylesterase activity and can react with the beta-naphthyl acetate serving as a natural substrate to form beta-naphthol, the fast blue B salt performs color development, and red is obtained; and carboxylesterase can completely or partially lose enzymatic reaction capacity to decrease the red after being inhibited by organic phosphorus, carbamates or pyrethroid insecticides, and the decrease degree of the red is in direct proportion to the type and concentration of the insecticide. The kit can quickly detect the insecticide in the water body.

Description

Technical field [0001] The invention relates to a kit for detecting whether there are pesticides in water bodies and the content range of the pesticides. Background technique [0002] The application of pesticides to control crop diseases, pests, weeds, and rodents is one of the important measures to ensure the sustainable and stable development of my country's "two highs and one excellent" agriculture. However, the use of pesticides is a double-edged sword. Due to the massive abuse of pesticides, the water quality in the application areas is contaminated and the ecological balance is destroyed. At the same time, the unreasonable use of chemical pesticides causes the rapid development and enhancement of pest resistance, forcing pesticides Corresponding increase in the amount and frequency, the use and dependence of pesticides presents a vicious circle, causing the phenomenon of pesticide application and residue exceeding the standard to increase. In the use of pesticides, only a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78C12N15/55C12N15/70C12N9/18C12Q1/44
Inventor 江红崔峰兰文升乔传令
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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