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Protein chip for rapid quantitative determination of bacterial fruit blotch of watermelon and preparation method thereof

A quantitative detection and protein chip technology, which is applied in measurement devices, instruments, scientific instruments, etc., to achieve the effects of simple operation, good promotion prospects, and good specificity

Inactive Publication Date: 2011-10-05
上海慧耘生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the beginning of its birth, the protein chip was mainly used for proteomics research, and then it was gradually extended to related fields of b

Method used

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  • Protein chip for rapid quantitative determination of bacterial fruit blotch of watermelon and preparation method thereof
  • Protein chip for rapid quantitative determination of bacterial fruit blotch of watermelon and preparation method thereof
  • Protein chip for rapid quantitative determination of bacterial fruit blotch of watermelon and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1: as figure 1 As shown, a protein chip for rapid quantitative detection of watermelon fruit spot bacteria is a biochip in which at least one watermelon fruit spot bacteria capture antibody D is immobilized on a chip substrate. An enzyme-labeled IgG antibody is also immobilized on the substrate as a positive control P, and a spotting buffer as a negative control N; the substrate is a nitrocellulose membrane (NC). The enzyme-labeled IgG is alkaline phosphatase-labeled IgG, which is fixed on the chip base as a positive control for protein chip quality control. See figure 1 , on the substrate from left to right in the order of positive control P, negative control N, and capture antibody D, and spot 0.2 μL of each point sequentially, including 4 replicates of positive control and 4 replicates of negative control, and 8 replicates of capture antibody.

Embodiment 2

[0029] Embodiment 2: A kind of preparation method of the protein chip of rapid quantitative detection watermelon fruit spot bacterium, comprises the following steps:

[0030] a. Substrate pretreatment: the substrate needs to be treated with ddH before use 2 O soaking, and drying constant; The soaking time is at least 15min, preferably 30min; The drying temperature is 15-37°C, preferably 25°C;

[0031] b. Chip spotting preparation: Dilute the capture antibody with spotting buffer, and take enzyme-labeled IgG and spotting buffer as positive control and negative control respectively, spot and fix according to the design; the spotting buffer Liquid is 0.001-0.5mol / L, the carbonate buffer solution that pH value is 7.0-10.0, preferably 0.05mol / L, the carbonate buffer solution that pH is 9.6, and carbonate buffer solution is Na 2 CO 3 1.59 g, NaHCO 3 2.93 g, NaN 3 0.2 g, ddH 2 O 1000 mL; the concentration of the capture antibody is 20-80mg / L, preferably 80 mg / L; the described...

Embodiment 3

[0032] Embodiment 3: the preparation of watermelon fruit blotch protein chip

[0033] Nitrocellulose membrane substrate with ddH 2 Soak in O for 30 min and dry at 25°C for 30 min. Dilute the capture antibody to 80mg / L with 0.05mol / L carbonate buffer, and use alkaline phosphatase-labeled IgG as a positive control, and carbonate buffer as a negative control. See figure 1 , on the substrate from left to right in the order of positive control P, negative control N, and capture antibody D, and spot 0.2 μL of each point sequentially, including 4 replicates of positive control and 4 replicates of negative control, and 8 replicates of capture antibody. After sample application, the substrates were placed in a wet box, fixed at 37°C for 20 min, and stored at 4°C for later use. The capture antibody and detection antibody used were purchased from Agdia Company, Cat. No. SRA 14800.

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Abstract

The invention discloses a protein chip for rapid quantitative determination of bacterial fruit blotch of watermelon and a preparation method thereof. The protein chip for rapid quantitative determination of bacterial fruit blotch of watermelon is prepared by fixing at least one capture antibody of bacterial fruit blotch of watermelon on a chip substrate. Cellulose nitrate membrane is used as the chip substrate, specific antibodies are used for capturing bacterial fruit blotch of watermelon, IgG labeled with alkaline phosphatase is used as a positive control, a point sample buffer is used as a negative control, and the detection result can be obtained by direct naked eye judgement or quantitative determination using grey analysis. Compared with traditional ELISA, the protein chip provided by the invention can attain the same detection capability with ELISA by using a twelfth of amount of the capture antibody and an eighth of detection time, with simple operation and long-term preservation of detection results, and the protein chip can be used for rapid self-detection of bacterial fruit blotch of watermelon on the planting line with a wide popularization prospect.

Description

technical field [0001] The invention relates to the field of plant protection, mainly for watermelon fruit spot fungus ( Acidovorax avenae subsp. citrulli (Schaad) Willems et al . 1992, Aac) Protein chip for rapid quantitative detection. Background technique [0002] Bacterial fruit blotch of watermelon is a disease that seriously endangers the production of Cucurbitaceae plants such as watermelon and muskmelon. The scientific name of watermelon fruit spot fungus is acidophage oats subsp. watermelon, and it is a specific pathogen that causes fruit spot disease. This fungus is an important quarantine disease in the field of plant protection. [0003] Watermelon fruit spot disease was first reported by Webb and Goth in 1965, but it has not been paid attention to until 1989. The disease generally occurred in Florida, Indiana and other areas of the United States, making up to 80% of the watermelons in the diseased areas unable to be marketed. The sale of watermelons cause...

Claims

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Application Information

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IPC IPC(8): G01N33/569
Inventor 刘箐熊亮斌孔君
Owner 上海慧耘生物科技有限公司
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