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Dual-label rapid response nucleic acid adapter probe and method for detection of aflatoxin B1

A nucleic acid aptamer and aflatoxin technology, which is applied in the direction of measuring devices, instruments, fluorescence/phosphorescence, etc., can solve problems such as unfavorable rapid detection, difficult storage, complicated and cumbersome procedures, and achieve great application potential and promotion value. Effects of detection sensitivity and detection efficiency improvement

Pending Publication Date: 2019-03-01
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Detection methods that rely on large-scale instruments are very limited in terms of timeliness and application scenarios, and require professional instrument operators, while methods based on antibody recognition also have many limiting factors, such as cumbersome preparation of antibodies, difficulty in storage, and high cost. Not conducive to rapid on-site detection
[0004] Nucleic acid aptamer (aptamer) is a kind of single-stranded nucleic acid (DNA or RNA) chain with molecular recognition, which has the advantages of easy synthesis and modification, stability and low cost, and is widely used in basic research, molecular diagnosis and agricultural products and farmland environmental pollutants. It has shown broad application prospects in many fields such as monitoring. At present, most of the methods for using nucleic acid aptamers to detect aflatoxin B1 use a single-label signal. One toxin target molecule can only trigger a response of one fluorescent signal, so the detection sensitivity low, or need to combine with other nanomaterials or enzymes to catalyze the amplification process, the procedure is more complex and cumbersome, it takes a long time, and it is impossible to quickly complete the entire detection process in the same reaction system

Method used

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  • Dual-label rapid response nucleic acid adapter probe and method for detection of aflatoxin B1
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  • Dual-label rapid response nucleic acid adapter probe and method for detection of aflatoxin B1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Design and synthesis of double-labeled rapid response nucleic acid aptamer probes

[0039] This example uses https: / / sg.idtdna.com / calc / analyzer and http: / / www.nupack.org websites to design complementary probes complementary to both ends of the AFB1 nucleic acid aptamer through nucleic acid hybridization thermodynamic assisted calculation . The AFB1 nucleic acid aptamer is obtained from the prior art (see Patent: PCT / CA2010 / 001292), its nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing, the nucleotide sequence of the complementary probe It is shown as SEQ ID NO: 2 in the sequence listing.

[0040] The above-mentioned nucleotide sequence was handed over to a DNA synthesis company—Sangon Bioengineering (Shanghai) Co., Ltd., which was commissioned to synthesize and label the fluorescent chromogenic group (FITC) at both ends of the AFB1 nucleic acid aptamer; The needles are double-ended labeled with a fluorescent quencher (BHQ1).

Embodiment 2

[0041] Example 2: Preparation of double-labeled rapid response nucleic acid aptamer probes and establishment of a standard curve of aflatoxin B1 concentration and fluorescence signal

[0042] (1) Add binding buffer solution (20-40 mM Tris, 8-10 mM magnesium salt ions, 50-70 mM potassium salt ions, and 1 mM DTT) into a clean transparent container, double-terminal labeled fluorescent chromogenic group The AFB1 nucleic acid aptamer (concentration: 10 μM), the complementary probe (concentration: 12 μM) of double-terminal labeled fluorescent quencher group and deionized water (volume ratio: 2:1:1:14 respectively), the total The volume was 1 mL, and after mixing, it was allowed to stand still at room temperature for 30 min, so that the preparation of the double-labeled fast-response nucleic acid aptamer probe was completed;

[0043] (2) Add 18 μL of the solution prepared in the example to seven clean 200 μL centrifuge tubes, and add 2 μL to 20 μL of the solution containing different...

Embodiment 3

[0045] Example 3: Detection and recovery calculation of aflatoxin B1 contained in bean paste

[0046] The steps of this embodiment are as follows:

[0047] (1) Take three clean 50 mL centrifuge tubes, add 5 g of bean paste without aflatoxin B1, 12 mL of methanol, 8 mL of deionized water and different amounts of aflatoxin B1 and mix well, then shake for 30 min. After shaking, centrifuge at 7000 rpm for 5 min, and take the supernatant as the sample to be tested. The concentrations of aflatoxin B1 in the three centrifuge tubes are 50 ng / mL, 100 ng / mL, and 200 ng / mL, respectively;

[0048] (2) Take another three clean 200 μL centrifuge tubes, and add 18 μL of the solution containing the double-labeled rapid response nucleic acid aptamer probe prepared in Example 2 and 2 μL of Aspergillus flavus to the three 200 μL centrifuge tubes Toxin B1 concentration of 50 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL samples to be tested, then mix well and let stand at room temperature for 1 min;

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Abstract

The invention relates to a dual-label rapid response nucleic acid adapter probe, which consists of a nucleic acid adaptor, the 5' end and 3' end of which are labeled by a fluorescent chromogenic grouprespectively, and a complementary probe, the 5' end and 3' end of which are labeled by a fluorescent quenching group respectively. The nucleic acid adaptor is a nucleic acid adaptor which can especially identify aflatoxin B1, namely, AFB1 nucleic acid adaptor. The nucleotide sequence of the AFB1 nucleic acid adaptor is shown as SEQ ID NO: 1 in the sequence list. The complementary probe is complementary to two ends of the AFB1 nucleic acid adaptor. The nucleotide sequence of the complementary probe is shown as SEQ ID NO: 2 in the sequence list. The method can be used for rapidly detecting aflatoxin B1. The identification process, after the aflatoxin B1 is added, can be completed in one minute, and one aflatoxin B1 molecule can trigger the recovery of two fluorescent group signals with highsensitivity. The whole reaction is carried out in a homogeneous and constant temperature solution environment, and the operation is simple.

Description

technical field [0001] The invention belongs to the field of detection of aflatoxin B1, and relates to a double-labeled rapid-response nucleic acid aptamer probe, including a double-terminal labeled fluorescent chromogenic group, which can specifically recognize aflatoxin B1 nucleic acid aptamer, that is, AFB1 A nucleic acid aptamer, a complementary probe complementary to the aptamer and double-terminal labeled with a fluorescence quenching group, and a method for rapidly and homogeneously detecting aflatoxin B1 using the probe for non-enzyme-dependent amplification. Background technique [0002] Aflatoxin is a secondary metabolite produced by Aspergillus flavus. In hot and humid areas, such as our Sichuan Province, the probability of aflatoxin appearing in agricultural products is very high. It is the most toxic mycotoxin and is extremely harmful to human health. Among them, aflatoxin B1 (Aflatoxin B1, referred to as AFB1) is the most toxic aflatoxin, which has been confirm...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6486G01N2021/6432
Inventor 邓锐杰夏许寒何强杨淏赵志峰
Owner SICHUAN UNIV
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