Detection kit of ochratoxin and method thereof for detecting ochratoxin

A technology of ochratoxin and detection kit, which is applied in the direction of measuring devices, instruments, fluorescence/phosphorescence, etc., can solve the problems of low detection sensitivity and inconvenient operation of ochratoxin, achieve high sensitivity and specificity, rapid detection and easy operation simple effect

Active Publication Date: 2019-07-19
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a kit for detecting ochratoxin and a method

Method used

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  • Detection kit of ochratoxin and method thereof for detecting ochratoxin
  • Detection kit of ochratoxin and method thereof for detecting ochratoxin
  • Detection kit of ochratoxin and method thereof for detecting ochratoxin

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0055] Example 1 Kit for detecting ochratoxin

[0056] This embodiment provides a kit for detecting ochratoxin, the kit includes the following substances:

[0057] The nucleic acid aptamer of ochratoxin has the sequence shown in SEQ ID NO. 1:

[0058] 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3' (SEQ ID NO. 1);

[0059] The cDNA that is not completely complementary to the ochratoxin nucleic acid aptamer has the sequence shown in SEQ ID NO.2.

[0060] 5'-TGTCCGTAGCTCCCAATTCACCTCCGTCACCCGATC-3' (SEQ ID NO. 2);

[0061] The card issuing sequence H1 has the sequence shown in SEQ ID NO.3:

[0062] 5'-AGGGCGGGGGTGTGGGGGCGTCTAACGGAGGTGAATTGGGTTT-3' (SEQ ID NO.3);

[0063] The card issuing sequence H2 has the sequence shown in SEQ ID NO.4:

[0064] 5’-TGGGTAGACGCCCCCACACCAAGAAGAAGAATGCCTCCACTTAACCCTA-3’ (SEQ ID NO. 4)

[0065] Methemoglobin;

[0066] Tetramethylbenzidine (TMB).

[0067] Among them, the ochratoxin aptamer and cDNA are not completely complementary to each other, and H1 and H2 each form a ...

Example Embodiment

[0068] Example 2 Method for detecting ochratoxin

[0069] This example provides a method for using the kit provided in Example 1, including the following steps:

[0070] (1) After mixing the ochratoxin nucleic acid aptamer and the cDNA that is not completely complementary to the nucleic acid aptamer, place it in a water bath at 95°C for 5 minutes, and then cool it naturally at room temperature, so that the two will form a double strand through complementary nucleic acid pairing;

[0071] (2) Add 2μL of PBS to the system to dilute the ochratoxin sample to be tested, and react at room temperature for 30 minutes to make the ochratoxin bind to the ochratoxin aptamer in the double strand and open the double strand structure; make it compatible with the nucleic acid The cDNA whose ligand is not completely complementary is released into single strands;

[0072] (3) Add the mixed solution of hairpin sequence H1 and H2 to the reaction system in 2), react at room temperature for 1 hour, and per...

Example Embodiment

[0075] Example 3 Detection of target ochratoxin at different concentrations

[0076] In this example, different concentrations of target ochratoxin were detected, which specifically included the following:

[0077] Prepare the standard solution of ochratoxin, the final concentration is 10μg / mL, 100μg / mL, 500μg / mL, 1mg / mL and stored at room temperature.

[0078] The different concentrations of ochratoxin were detected according to the method in Example 2, and the reaction was carried out fully, and the light absorption intensity at 450 nm was detected and the color change was observed by naked eyes.

[0079] From figure 2 It can be seen from the test results that when ochratoxin is present, the color changes from colorless to blue (not shown in the figure), and the absorbance value increases significantly; and as the concentration of ochratoxin increases, the absorbance value increases. Among them, the Hemin blank group has only methemoglobin plus magnetic beads and no pre-self-assemb...

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Abstract

The invention discloses a detection kit of ochratoxin and a method thereof for detecting the ochratoxin, and belongs to the technical field of detection of harmful substances. The detection kit of ochratoxin disclosed by the invention comprises a nucleic acid aptamer of the ochratoxin, a cDNA which is not completely complementary to the nucleic acid aptamer, and two hairpin sequences H1 and H2, wherein the sequence of the nucleic acid aptamer of the ochratoxin is as shown in a sequence table SEQ ID NO. 1; the two hairpin sequences H1 and H2 can be self-assembled into a long chain; and the cDNAwhich is not completely complementary to the nucleic acid aptamer of the ochratoxin can trigger the self-assembly of the hairpin sequences H1 and H2. The kit provided by the invention has very high sensitivity and specificity, simple operation and rapid detection, the result is visible to naked eyes, the kit is suitable for promotion in all laboratories, and real-time onsite analysis of the ochratoxin can be realized without professional training.

Description

technical field [0001] The invention belongs to the technical field of detection of harmful substances, and in particular relates to a detection kit for ochratoxin and a method for detecting ochratoxin. Background technique [0002] Ochratoxins are natural contaminants belonging to the mycotoxin group, produced by various fungal types Aspergillus and Penicillium. Ochratoxin A (OTA), a mycotoxin, was discovered in South African corn flour in the 1960s. Years of studies have shown that OTA has nephrotoxicity, hepatotoxicity, embryotoxicity, teratogenicity, neurotoxicity, immunotoxicity, genotoxicity and carcinogenicity. Of the 6 known ochratoxin types, ochratoxin A exhibits the highest toxicity. The International Agency for Research on Cancer (IARC) has classified OTA as a probable human carcinogen (category 2B) based on substantial evidence of carcinogenicity found in several animal studies. Because OTA exists widely in nature and is very stable and not easy to metabolize,...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 吴薇杨庆利于春娣
Owner QINGDAO AGRI UNIV
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