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Measuring method of immune PCR of abscisic acid

An assay method and abscisic acid technology, applied in the field of immuno-PCR assay of abscisic acid, can solve the problems of complicated operation, low cost, expensive equipment, etc., and achieve the effects of simple and convenient operation, improved binding ability, and improved repeatability

Pending Publication Date: 2018-05-29
HUNAN AGRICULTURAL UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

CN103592291A provides a method for measuring abscisic acid based on nano-gold labeling and tyramide signal amplification technology. The method belongs to chemiluminescent technology, using biotinylated abscisic acid antibody-modified nano-gold as a marker, using magnetic beads as a carrier, and combining tyramide Amine signal amplification technique and PIP-HRP-H 2 o 2 The chemiluminescent system is highly sensitive for the determination of abscisic acid. This method is low in cost and high in sensitivity, but the operation is more complicated.
CN104297410A provides a kind of analytical method of detecting abscisic acid and jasmonic acid in fresh tobacco leaves by liquid mass spectrometry. high price
[0006] The existing methods for the determination of abscisic acid have problems such as complicated sample preparation and operation steps, expensive equipment, high cost of consumables, and strict technical requirements, which limit the application of the method.

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  • Measuring method of immune PCR of abscisic acid
  • Measuring method of immune PCR of abscisic acid
  • Measuring method of immune PCR of abscisic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] In order to facilitate the high-throughput quantitative analysis of immuno-PCR, this example compares the uniformity of different types of commonly used polypropylene (PP) PCR tubes.

[0043] The specific method is to add a certain amount of water to different types of polypropylene (PP) PCR tubes, and observe the height of the water line to verify the uniformity of the PCR tubes. The results are as follows: figure 2 shown.

[0044] In immuno-PCR, the consistency and stability of the solidified probe mixture (ie DNA template) on the surface of the PCR tube is the decisive factor for the accurate quantification of the analyte before the PCR program runs. Therefore, before coating with ABA monoclonal antibody, it is necessary to select high-quality PCR tubes / plates, high-performance cross-linking agents, and suitable washing buffers. From figure 2 It can be seen that ordinary PCR tubes and eight-tube tubes have high deviations on the inner surface, which are not suita...

Embodiment 2

[0046] This example compares the effect of glutaraldehyde pretreatment on the binding ability between PCR tubes / plates and ABA antibodies.

[0047] The specific test steps include:

[0048] (1) Pretreat the PCR tube / plate with glutaraldehyde: drop 100 μL of 0.8% glutaraldehyde into the PCR tube / plate, soak at room temperature for 2 hours, then remove the glutaraldehyde and rinse the PCR with PBS Tube / plate 3 times;

[0049] (2) ABA monoclonal antibody coating: Then add 10 μL of ABA monoclonal antibody with a concentration of 20 μg to the bottom of the PCR tube / plate and incubate at 4°C for 2 hours, then remove the monoclonal antibody solution and wash it 3 times with buffer washing solution, The buffer washing solutions used were TBS (Tris buffer, pH 7.5), PBS (phosphate-buffered saline, pH 7.4), PBST (phosphate-buffered saline and Tween, pH 7.5) and ultrapure water. Add 100 μL of 1% bovine serum albumin to each tube / well, incubate at 4°C for 1 hour, wash the PCR tube / plate ...

Embodiment 3

[0058] Based on the immuno-PCR method of the present invention, the ABA content in eight different tissues of Arabidopsis thaliana and rice was measured using real-time immuno-PCR method (RT-Im-PCR), and each tissue was repeated 12 times in a 96-well plate test, the process is as figure 1 shown.

[0059] The specific test steps are:

[0060] (1) Plant material and sampling:

[0061] Disinfect the surfaces of Arabidopsis and rice seeds with 70% alcohol and 5% sodium hypochlorite, wash them three times, and incubate in the dark for 48 hours, then start to germinate simultaneously at 4°C. Arabidopsis and rice seeds were cultured in 0.4% (w / v) phytocondensed solidified MS medium. Arabidopsis and rice seedlings were placed vertically in a growth chamber at 22°C and 32°C, respectively, with 16 hours of light followed by 8 hours of darkness. To collect stems and flowers, ten-day-old Arabidopsis seedlings were moved into pots for maturity. About 100mg of fresh plant tissue was co...

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Abstract

The invention belongs to the field of measurement of plant hormones, and provides a measuring method of immune PCR of abscisic acid. The method comprises the steps of usingglutaraldehyde to pretreat the inner surface of a PCR tube / plate, using a single anti-abscisic-acid antibody to cover the surface of the treated PCR tube / plate to synthesize a probe mixture, adding the probe mixture into the treated PCR tube / plate, then adding an abscisic acid sample to be measured into the PCR tube / plate to be reacted, conducting amplification in real-time PCR equipment, and according to the amplification result, determining the content ofabscisic acid. A comparison experiment shows that by utilizing glutaraldehyde to pretreat the inner surface of the PCR tube / plate, the antibody covering capability can be improved by 20-30%; the three substances of a biotinylation ABA polyclonal antibody, biotinylation DNA and avidin are further utilized to synthesize the probe mixture, the repeatability of ABA measurement is improved, the detection lower limit of the immune PCR method can reach 10 ng / L, close to the detection result of liquidchromatography mass, and the problem that organism in-vitro plant hormones aredifficult to directly react with biomacromolecules is solved.

Description

technical field [0001] The invention belongs to the field of plant hormone determination, in particular to an immuno-PCR assay method for abscisic acid. Background technique [0002] Phytohormones are usually small molecular organic compounds that play an important role in plant growth and development. For decades, advances in research on plant hormones have been the most important driving force behind agricultural scientific and technological progress and the "Green Revolution". In order to better study the molecular mechanism of plant hormones, it is necessary to study the structure, distribution and polar distribution of plant hormones in different organs, tissues and cells. Therefore, high-throughput quantitative detection of plant hormones with high sensitivity is A powerful tool for studying the effects of plant hormones. Determination of plant hormones includes biological assays, immunoassays and biosensors, electrochemical analysis, chromatography, mass spectrometr...

Claims

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Application Information

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IPC IPC(8): C12Q1/6804
CPCC12Q1/6804C12Q2531/113C12Q2563/131
Inventor 肖浪涛苏益李维黄志刚王若仲
Owner HUNAN AGRICULTURAL UNIV
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