New method for rapidly detecting ochratoxin A through molecular beacon probe

A technology of molecular beacons and probes, which can be used in analytical materials, biological testing, measuring devices, etc., can solve problems such as expensive, time-consuming and labor-intensive, and achieve the effects of simple operation, high sensitivity, and good accuracy

Inactive Publication Date: 2017-03-29
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, OTA monitoring mainly relies on liquid chromatography-fluorescence detection (HPLC-FLD) and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Although the detection accuracy is high, it is expensive and requires multi-step pretreatment ,Time-consuming

Method used

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  • New method for rapidly detecting ochratoxin A through molecular beacon probe
  • New method for rapidly detecting ochratoxin A through molecular beacon probe
  • New method for rapidly detecting ochratoxin A through molecular beacon probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the selection of detection condition

[0042] (1) Preparation process:

[0043] a. Preparation of aptamer Apt solution: Dissolve 2.7 nmol of aptamer in 270 μL of aptamer solution (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0), and set aside;

[0044] b. Preparation of Molecular Beacon MB Solution: Add 5.4 nmol of aptamer to 540 μL of annealing solution (50 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), refold at 95°C for 3 min, and place at room temperature for 30 min to make it Slowly cool down to room temperature.

[0045] (2) Selection of the best detection conditions:

[0046] a. Competition method:

[0047] ①Test group: Precisely pipette 350 μL of detection solution, add 50 μL of 10 μM Apt solution and 10 μM MB solution respectively, react for 2 minutes, add 1 μg·mL -1 50 μL of OTA solution, record the fluorescence intensity: control group: accurately pipette 350 μL of detection solution, add 50 μL of 10 μM Apt solution and 50 μL of 10 μM MB solution r...

Embodiment 2

[0052] Example 2: Detection of OTA in malt and beer by MB probes

[0053] 1. Sample extraction:

[0054] Malt sample: Accurately weigh 2.0 g (accurate to 0.1 g) of malt powder (passed through a No. 2 sieve), add 5 mL of acetonitrile-water (80:20, v / v) solution, vortex-assisted extraction for 1 min, and after ultrasonic extraction for 5 min, Centrifuge at 4000rpm for 10min. Precision supernatant 1mL, add detection solution to dilute to 10mL, mix well, pass through 022μm filter membrane, and take the subsequent filtrate.

[0055] Beer sample: take commercially available canned beer, open the cap, degass it by ultrasonic, accurately measure 10mL and place it in a 100mL volumetric flask, add the test solution to make up to the mark line, mix well, pass through a 0.22μm filter membrane, and take the subsequent filtrate. have to.

[0056] 2. Fluorescence detection conditions: F7000 fluorescence spectrophotometer (Hitachi, Japan). Instrument parameter settings include excitation ...

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Abstract

The present invention relates to a new method for rapidly detecting ochratoxin A through a molecular beacon probe. According to the present invention, based on the DNA key site for specifically recognizing OTA, a molecular beacon having the stem comprising sixes complementary bases and the ring having 8 bases is designed, wherein the sequence of the molecular beacon is 5'-FAM-CCGGGTCCACCCACACCCGG-DABCYL-3'; and the probe is stable and is easy to synthesize, is used for sample analysis, has the simple operation, can complete the detection within 20 min, further has advantages of good accuracy and high sensitivity, and can be used for the rapid screening of the ochratoxin A in malt, beer, and other samples.

Description

technical field [0001] The invention relates to a new method for detecting ochratoxin A (OTA) with a molecular beacon probe, in particular to artificially designing the molecular beacon probe MB for the nucleotide sequence Apt that specifically recognizes the OTA molecule, and using competitive Combined with Apt, it is suitable for the quantitative detection of OTA in malt, beer and other samples, and belongs to the field of rapid detection of mycotoxins. Background technique [0002] Malt is made from the mature fruit of the gramineous plant barley Hordeum vulgare L. after germination and drying, and is widely distributed in various parts of our country. Due to its high nutritional value, it is not only an excellent raw material for brewing beer, but also plays an extremely important role in clinical medical care as a traditional Chinese medicine. The growth of malt needs to be wetted with water and dried at low temperature. The storage requires dry and low temperature con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N21/64
CPCG01N21/6428G01N33/5308
Inventor 杨美华豆小文褚先锋孔维军
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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