Test strip, preparation method and application thereof in transgenic protein AM79 EPSPS detection

A technology of test strips and detection areas, applied in the field of biochemical detection, can solve the problems of complicated operation, high cost, and long time consumption, and achieve the effect of simple operation, good sensitivity, and short detection time

Inactive Publication Date: 2021-03-19
ZHEJIANG XINAN CHEM INDAL GROUP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Previously, the conventional RT-PCR detection method was mostly used for the detection of AM79 EPSPS, which was complicated, time-consuming, costly, and not capable of high-throughput detection

Method used

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  • Test strip, preparation method and application thereof in transgenic protein AM79 EPSPS detection
  • Test strip, preparation method and application thereof in transgenic protein AM79 EPSPS detection
  • Test strip, preparation method and application thereof in transgenic protein AM79 EPSPS detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The making of embodiment 1 transgenic protein AM79 EPSPS immunoassay card

[0043] In each test card, the size of the sample pad is 4mm×15mm, the size of the marker pad is 3mm×4mm, the size of the nitrocellulose membrane is 4mm×28mm, and the size of the absorbent pad is 4mm×19mm.

[0044] Immobilize 40ng colloidal gold-labeled mouse anti-AM79 EPSPS monoclonal antibody on the gold standard pad of each detection card, immobilize 0.8μg mouse anti-AM79 EPSPS monoclonal antibody on the detection area on the nitrocellulose membrane and immobilize 1.0μg anti-AM79 EPSPS monoclonal antibody on the control area. Mouse IgG secondary antibody.

[0045] Paste sample pads, gold standard pads immobilized with colloidal gold-labeled specific monoclonal antibodies, nitrocellulose membranes, and absorbent pads in sequence on the bottom plate. Then it was dried under vacuum at 37 °C for 2 h.

Embodiment 2

[0046] Example 2 Effect Evaluation of Transgenic Protein AM79 EPSPS Immunoassay Card Detecting Protein Standards

[0047] ① Add 200 μL blank sample solution EB004 buffer solution (pH7.4, 0.2mol / LPBS: 8g sodium chloride, 3.35g disodium hydrogen phosphate dodecahydrate, 0.2g potassium dihydrogen phosphate, 0.2g potassium chloride, Dissolve in double-distilled water to 1 L), insert the quick test paper into the sample cup, react at room temperature for 8 minutes, and observe the color development result in the result observation area. The results show that the T line does not show color in the observation area, but the C line shows color.

[0048] ② Add 100 μL of 0.1 μg / ml AM79 EPSPS protein standard 1 (diluted with the same PBS solution as above) to the sample cup, and follow the same operation steps as the blank sample. The results show that the C line (control area) and T line (detection area) in the observation area develop color simultaneously.

[0049] ③ Add 100 μL of 0.1...

Embodiment 3

[0051] Example 3 Effect Evaluation of Transgenic Protein AM79 EPSPS Immunological Detection Card for Detecting Actual Transgenic Soybean Leaf Samples

[0052] 1. Plant leaf tissue extraction method:

[0053] 1. Place the plant leaf tissue between the lid and the body of the disposable tissue extraction tube, quickly cover the lid, and repeat 2 times to obtain 2 circular leaf tissues. Place the leaf blade at the bottom of the extraction tube with a pestle. Mark the tube wall with a marker pen.

[0054] 2. Insert the pestle into the tube, rotate the pestle to crush the leaves, and press continuously for 20-30 seconds.

[0055] 3. Add 0.2 mL (about 8 drops) of extraction buffer EB004.

[0056] 4. Repeat the crushing step to fully contact and mix the sample with the buffer. Remove the pestle (note that the pestle should be used only once to avoid cross-contamination between different samples), and obtain the sample to be tested.

[0057] 2. Sample testing

[0058] 1) Add 200...

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Abstract

The invention relates to the technical field of biochemical detection, in particular to a test strip, a preparation method of the test strip and application of the test strip in transgenic protein AM79 EPSPS detection. The handheld colloidal gold rapid test paper for the transgenic protein AM79 EPSPS provided by the invention can be widely applied to rapid detection of the transgenic protein AM79EPSPS, and has the following specific advantages that: (1) the test paper is simple to operate, does not need special instruments and equipment, and can be conveniently and directly used for detectionin fields and other fields; (2) the detection time is short (5-8 minutes); the result judgment standard is unified, i.e. for a negative result (-), only one C line appears; and for a positive result(+), T line and C line appear at the same time; and (3) the test paper is verified to have good sensitivity, and the lowest detection limit can reach 0.1microg / mL.

Description

technical field [0001] The invention relates to the technical field of biochemical detection, in particular to a test strip, its preparation method and its application in the detection of transgenic protein AM79 EPSPS. Background technique [0002] The herbicide-resistant EPSP synthase (5-enolpyruvyl-shikimate-3-phosphatesynthase, EPSPS) isolated from glyphosate-contaminated soil by metagenomics method is an aromatic amino acid in organisms—tryptophan, tyrosin, Glyphosate is a key enzyme in the biosynthesis of amino acid and phenylalanine; glyphosate blocks the synthesis of aromatic amino acids by inhibiting the activity of EPSP synthetase, and finally leads to the death of the tested plants. However, the activity of EPSP synthase of some bacteria is not inhibited by glyphosate, so the expression of bacterial EPSPS gene can protect plants from herbicide damage. The herbicide resistance gene EPSPS is transferred into soybeans, aiming at cultivating transgenic soybeans with e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573G01N33/558G01N33/543G01N33/577G01N33/58
CPCG01N33/543G01N33/558G01N33/573G01N33/577G01N33/585G01N2333/91182
Inventor 楼亿圆何军光翁嘉慧蒋红叶苗昱婷王云铃赵振宁周骏费月新陆建明
Owner ZHEJIANG XINAN CHEM INDAL GROUP
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