Method and kit for detecting ricin based on relative DNA walker initiation index amplification of gold nanoprobe constructed by freezing

A technology of ricin and exponential amplification, applied in the direction of recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of increased detection costs, unsuitable for on-site detection, and restrictions on large-scale promotion and application, and achieves Good stability, excellent stability, high sensitivity effect

Active Publication Date: 2021-07-23
INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Immunolabeling and radioimmunoassays have the characteristics of high detection sensitivity, but their requirements for relatively complicated antibody preparation and special markers limit the large-scale application of this method; although biological mass spectrometry relies on high-tech instruments and equipment, but Expensive instruments and equipment greatly increase the cost of testing and are not suitable for on-site testing

Method used

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  • Method and kit for detecting ricin based on relative DNA walker initiation index amplification of gold nanoprobe constructed by freezing
  • Method and kit for detecting ricin based on relative DNA walker initiation index amplification of gold nanoprobe constructed by freezing
  • Method and kit for detecting ricin based on relative DNA walker initiation index amplification of gold nanoprobe constructed by freezing

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Experimental program
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Effect test

Embodiment 1

[0067] (1) Cryoconstruction of gold nanoprobes. The gold nanoparticles of about 15nm were prepared by sodium citrate reduction method. At the same time, thiol (SH) and PolyA double-labeled DNA was incubated with tris-(2-formylethyl)phosphine hydrochloride (TCEP) at a molar ratio of 1:100 for 1 h at room temperature. Next, the prepared gold nanoparticles (4 nM) were mixed with DNA (10 μM) at a molar ratio of 1:200 and incubated at −20 °C for 1 hour. After thawing at room temperature, the solution was centrifuged with HEPES buffer (0.05M, pH 7.6) at 14000 rpm at 4°C for 20 minutes and washed 3 times. Finally the pellet was resuspended in HEPES buffer and stored at 4°C in the dark. AW (DNA Walker-gold nanoprobe) and AT (DNA Track-gold nanoprobe) can be synthesized respectively by this method.

[0068] (2) Competition reaction of ricin. Before the reaction, 1 μM ricin aptamer was incubated at 95°C for 5 minutes, and then gradually cooled to room temperature. Next, 0.8 nM AW, ...

Embodiment 2

[0075] (1) Cryoconstruction of gold nanoprobes. The gold nanoparticles of about 15nm were prepared by sodium citrate reduction method. At the same time, thiol (SH) and PolyA double-labeled DNA was incubated with tris-(2-formylethyl)phosphine hydrochloride (TCEP) at a molar ratio of 1:100 for 1 h at room temperature. Next, the prepared gold nanoparticles (4 nM) were mixed with DNA (10 μM) at a molar ratio of 1:200 and incubated at −20 °C for 1 hour. After thawing at room temperature, the solution was centrifuged with HEPES buffer (0.05M, pH 7.6) at 14000 rpm at 4°C for 20 minutes and washed 3 times. Finally the pellet was resuspended in HEPES buffer and stored at 4°C in the dark. AW (DNA Walker-gold nanoprobe) and AT (DNA Track-gold nanoprobe) can be synthesized respectively by this method.

[0076] (2) Competition reaction of ricin. Before the reaction, 1 μM ricin aptamer was incubated at 95°C for 5 minutes, and then gradually cooled to room temperature. 2 mL of milk / juic...

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Abstract

The invention belongs to the field of protein toxin detection, and relates to a method and a kit for detecting ricin based on relative DNA walker initiation index amplification of a gold nanoprobe constructed by freezing. The method mainly comprises the following four processes: performing freezing to construct the gold nanoprobe, competitively combining the ricin and the gold nanoprobe with an aptamer, and carrying out a relative DNA walker and index amplification reaction. The method and the kit can be used for detecting the ricin with relatively high sensitivity, good specificity and excellent stability in practical application.

Description

technical field [0001] The invention belongs to the field of detection of protein toxins, and in particular relates to a method for detecting ricin by using a relative DNA walker based on freezing to construct gold nanoprobes and triggering exponential amplification, and a relative DNA based on freezing to construct gold nanoprobes. A kit for the detection of ricin by walker-induced exponential amplification. Background technique [0002] Castor is a plant of the genus Castor in the Euphorbiaceae family, and is an oil crop with high oil content. However, the stems, leaves or seeds of castor plants contain toxic substances, mainly including ricin, ricinine and ricin. Among them, ricin, also known as ricin, is the most toxic plant toxin known so far, and its toxicity is about 6000 times that of cyanide. Ricin is mainly present in ricin (about 5% of ricin, molecular weight is about 64-66kD), is a type II heterodimeric ribosome inactivating protein, produced by ribosome inacti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q2563/137C12Q2563/155C12Q2563/107C12Q2537/161C12Q2521/101
Inventor 高志贤王瑜彭媛李双韩殿鹏任舒悦秦康韩铁李森孙璇
Owner INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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