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A kind of detection method and detection kit of ochratoxin A

A detection kit and technology for ochratoxin, which are applied in the directions of biochemical equipment and methods, microbial determination/inspection, measurement devices, etc., can solve the problems of long sample pretreatment time, difficulty in realizing rapid detection, and expensive instruments, etc. Achieving the effect of economical cheapness, rapid response and low cost

Active Publication Date: 2019-10-18
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional detection methods mainly include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry, etc., but the instruments used in these methods are expensive, time-consuming, and pretreatment of samples It takes a long time and it is difficult to achieve rapid detection

Method used

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  • A kind of detection method and detection kit of ochratoxin A
  • A kind of detection method and detection kit of ochratoxin A
  • A kind of detection method and detection kit of ochratoxin A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] A detection method for ochratoxin A is carried out according to the following steps:

[0076] (1) Use buffer (10 mM Tris-HCl, pH 8.5, 120 mM NaCl, 5 mM KCl, 20 mM CaCl 2 , 15 mM MgCl 2 ) to fully dissolve H1 (500 nM), add different concentrations of ochratoxin A, mix thoroughly, and react at room temperature for 30 minutes.

[0077] (2) Add H2, mix thoroughly, and react at room temperature for 90 minutes.

[0078] (3) Add AuNPs1 and AuNPs2 at the same time, mix well, leave at room temperature for 15 minutes, and observe the color change. The presence or absence of ochratoxin A in the detection system can be judged by the color change.

Embodiment 2

[0080] A detection kit for ochratoxin A comprises the following components:

[0081] (1) Stem-loop structure sequence H1, the sequence is as follows:

[0082] H1: 3'-ACAGGCTACGAGGGAAATGCGGTGGGTGTGGGCTAG(A)-TCTCATTGTACCCACTTGGACCAATTAGCGACTATAG(B)-CTAGCCCAC(C)-5' (SEQ ID NO: 1);

[0083] (2) Substrate sequence H2, the sequence is as follows:

[0084] H2: 5'-GCTGGACTATTCAGAGTA(D)-TrAG-GATATCTAACTGAGTCCAGC(E)-3' (SEQ ID NO: 2);

[0085] (3) F sequence modified colloidal gold 1 (AuNPs1):

[0086] AuPNs1: 3'-SH-AAAAAA-CTATAGATTG(F)-5' (SEQ ID NO: 3);

[0087] (4) G-sequence modified colloidal gold 2 (AuNPs2):

[0088] AuPNs2: 5'-SH-AAAAAA-GCTGGACTCA(G)-3' (SEQ ID NO: 4);

[0089] (5) The buffer is 10 mM Tris-HCl, pH 8.5, 120 mM NaCl, 5 mM KCl, 20 mM CaCl 2 , 15 mM MgCl 2 .

Embodiment 3

[0091] Detection of different concentrations of ochratoxin A:

[0092] Prepare ochratoxin A standard solutions with concentrations of 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL and 100 ng / mL, and store them in the dark at 4°C.

[0093] Ochratoxin A solutions of different concentrations were added to the reaction system described in Example 1 respectively, and the experimental results were observed after sufficient reaction, such as figure 2 As shown, 10 pg / mL of ochratoxin A can produce a clear blue change, indicating that its detection limit is 10 pg / mL. As the concentration of ochratoxin A increased, the blue intensity increased and gradually became saturated.

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Abstract

The invention discloses a method and a kit for detecting ochratoxin A. According to the invention, a nucleic acid aptamer for the ochratoxin A is taken as a molecular recognition element. When a system to be detected contains the ochratoxin A, the molecular recognition element can be activated by the ochratoxin A to form a nucleic acid having a catalytic function that is capable of continuously and cyclically incising a substrate sequence to achieve the purpose of signal amplification. In combination with color change of colloidal gold, detection of the ochratoxin A through direct observation with bare eyes can be achieved. The kit for detecting ochratoxin A have relatively high sensitivity with a detection limit of 10 pg / mL to the ochratoxin A, and is highly specific in detection with the detection being not affected by common disruptors. The method for detecting ochratoin A employs the colloidal gold as a signal reporter molecule and can obtain a detection result that is directly visible to bare eyes with no need of use any detection instrument; moreover, the method is simple to operate, low in cost, and extensively applicable to primary detection of ochratoxin A contamination condition.

Description

technical field [0001] The invention belongs to the field of biological toxin detection, and relates to a detection method and a detection kit for ochratoxin A. Background technique [0002] Ochratoxin A (OTA) is a kind of mycotoxin, which is a toxic metabolite produced by some strains of aspergillus and penicillin. It is highly teratogenic, carcinogenic and mutagenic, and widely exists in Various foods, including cereals, beans and soy products, dried fruits, coffee and coffee beans, and alcohol. [0003] In view of the harmfulness and wide distribution of ochratoxin A, the development of a rapid and sensitive method for the detection of ochratoxin A is of great significance in terms of food safety and environmental health. Traditional detection methods mainly include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry, etc., but the instruments used in these methods are expensive, time-consuming, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/682G01N21/78G01N33/558
Inventor 陈俊华李定强陈曼佳
Owner GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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