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Proportional aptamer fluorescent probe and method for detecting ochratoxin A by same

A nucleic acid aptamer, ochratoxin technology, which is applied in fluorescence/phosphorescence, chemical instruments and methods, measuring devices, etc., can solve the problems of complex detection of ochratoxin A, etc., and achieves easy sample pretreatment, high sensitivity, and high sensitivity. Environmentally friendly effects

Active Publication Date: 2019-03-08
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the characteristics of structural responsiveness, high stability, high specificity, easy modification, and low cost, nucleic acid aptamers have gradually become a substitute for antibodies. At present, they have been initially used in the detection of food hazard factors, but some of them have been used The detection method of ochratoxin A by nucleic acid aptamer is complicated

Method used

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  • Proportional aptamer fluorescent probe and method for detecting ochratoxin A by same
  • Proportional aptamer fluorescent probe and method for detecting ochratoxin A by same
  • Proportional aptamer fluorescent probe and method for detecting ochratoxin A by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Preparation of fluorescent ratio probe solution

[0034] Add 35 μL of binding buffer solution (composed of 20-40 mM Tris, 8-10 mM magnesium salt ions, 50-70 mM potassium salt ions and 1 mM DTT), 35 μL of 10 μM OTA nucleic acid in a clean reaction tube Aptamer (obtained from prior art), 35 μL ThT solution with a concentration of 300 μM, 225 μL ultrapure water, a total of 330 μL, mixed well and stored at low temperature (4°C) for future use.

Embodiment 2

[0035] Example 2: Establishing a standard curve of ochratoxin A concentration and fluorescence ratio signal

[0036] The steps of this embodiment are as follows:

[0037](1) Take eight clean disposables, add ultrapure water and ochratoxin A respectively to prepare six different concentrations of ochratoxin A standard solution, 2 μL each, the concentration of ochratoxin A standard solution is 0 ng / mL, 5 ng / mL, 40 ng / mL, 70 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 700 ng / mL;

[0038] (2) Add 33 μL of the fluorescent proportional probe solution prepared in Example 1 to each of the eight test tubes, mix well, and then let stand at room temperature for 10 min;

[0039] (3) Use a full-featured microplate detector to perform two consecutive fluorescence detections. The excitation wavelengths for fluorescence detection are 425 nm and 485 nm, and the emission wavelength ranges are 470-650 nm and 515-650 nm. According to the two fluorescence detections The ratio result of the signal di...

Embodiment 3

[0040] Example 3: Detection of Ochratoxin A Contained in Coffee

[0041] The steps of this embodiment are as follows:

[0042] (1) Take three clean centrifuge tubes, add coffee and different concentrations of ochratoxin A respectively and dilute 20 times, the final concentration of ochratoxin A in the three test samples is 200 ng / mL and 300 ng respectively / mL, 400 ng / mL, the test sample with a final concentration of ochratoxin A of 200 ng / mL was named test sample No. 1, and the test sample with a final concentration of ochratoxin A of 300 ng / mL was named test No. 2 Sample, the test sample whose final concentration of ochratoxin A is 400 ng / mL is named as No. 3 test sample;

[0043] (2) Take three clean centrifuge tubes, add 2 μL of the test samples with final concentrations of ochratoxin A of 200 ng / mL, 300 ng / mL, and 400 ng / mL and 33 μL of Example 1 to the three clean centrifuge tubes respectively. Prepare the fluorescent ratio probe solution and mix it evenly, then let it...

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Abstract

The invention discloses a fluorescent proportional probe constructed by utilizing a labeled aptamer and nucleic acid quadruplex dye. The aptamer can specifically identify ochratoxin A, namely an OTA aptamer. The nucleotide sequence of the OTA aptamer is shown as SEQ ID NO:1 in a sequence table. A detecting method of the ochratoxin A comprises the following steps: (1), preparing an OTA aptamer-thioflavin T mixed reaction solution, namely a proportional aptamer fluorescent probe solution; (2), establishing a standard curve for quantitative detection of the ochratoxin A; (3), detecting the ochratoxin A in a real sample. By using the method disclosed by the invention to detect the ochratoxin A through the fluorescent proportional probe, the interference of other nucleic acids or impurities canbe resisted, and experimental errors caused by primary fluorescent signal detection in a traditional method are reduced; the method has the advantages of controllable process, simple operation, highsensitivity and only 0.38 ng / mL of detection limit; meanwhile, the method can carry out fluorescence detection in a homogeneous solution environment, does not need elution separation and has greater application potential and popularization value in on-spot rapid detection.

Description

technical field [0001] The invention belongs to the field of ochratoxin A detection, and relates to a proportional nucleic acid aptamer fluorescent probe, including a nucleic acid aptamer and a nucleic acid quadruplex that can specifically recognize ochratoxin A and are marked with a fluorescent chromogenic group A dye thioflavin T, and a method for rapid and homogeneous detection of ochratoxin A using the probe. Background technique [0002] Ochratoxin A (Ochratoxin A) is a metabolite produced by some Aspergillus or Penicillium. It is a mycotoxin widely distributed in nature and highly toxic. It often contaminates cereals, soybean products, coffee and wine, etc. Agricultural products or food. As early as 1993, the International Agency for Research on Cancer listed ochratoxin A as a human carcinogen. At present, the detection methods of ochratoxin A are mainly chromatography (such as high performance liquid chromatography, thin layer chromatography, etc.) and enzyme-linked...

Claims

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Application Information

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IPC IPC(8): G01N21/64C09K11/07
CPCC09K11/07C09K2211/1007C09K2211/1037G01N21/6428G01N21/6486
Inventor 邓锐杰夏许寒曾红棱任尧赵志峰何强
Owner SICHUAN UNIV
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