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Electrochemical sensor based on triblock probe and application of electrochemical sensor in detection of transgenic double-stranded RNA

An electrochemical, triblock technology, applied in the direction of electrochemical variables of materials, scientific instruments, biochemical equipment and methods, etc., can solve the problems of weak specificity of RT-PCR, improve the capture ability, improve the detection sensitivity, improve the The effect of the signal-to-noise ratio

Active Publication Date: 2021-01-01
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the inverted repeat structure of transgenic double-stranded RNA, the specificity of RT-PCR is weak; in addition, RT-PCR requires expensive experimental equipment and can only be used in a laboratory environment, which has certain limitations

Method used

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  • Electrochemical sensor based on triblock probe and application of electrochemical sensor in detection of transgenic double-stranded RNA
  • Electrochemical sensor based on triblock probe and application of electrochemical sensor in detection of transgenic double-stranded RNA
  • Electrochemical sensor based on triblock probe and application of electrochemical sensor in detection of transgenic double-stranded RNA

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The preparation of embodiment 1 transgenic double-stranded RNA

[0057] This embodiment prepares transgenic double-stranded RNA as the detection target of the electrochemical sensor, including the following steps:

[0058] (1) Synthesizing a DNA fragment with a length of 852bp comprising 261bp inverted repeat sequence and 98bp spacer sequence, and cloning it into the pET-28a(+) vector with T7 promoter;

[0059] (2) PCR is used to amplify a DNA fragment of 1034bp in length containing the target RNA and the T7 promoter from the recombinant plasmid;

[0060] (3) After purification, use the 1034bp DNA fragment as a template, under the action of RNA polymerase, transcribe in vitro at 37°C for 1 hour, and synthesize the target RNA;

[0061] (4) The synthesized target RNA is purified and stored at -80°C until use.

Embodiment 2

[0062] The preparation of embodiment 2 electrochemical sensors

[0063] After the gold electrode was polished by alumina, it was placed in ethanol and water for ultrasonication for 2 minutes; then connected to the circuit of the electrochemical workstation, and electrochemically cleaned with 0.5M sulfuric acid aqueous solution;

[0064] After electrochemical cleaning, the gold electrode was washed with water and dried with nitrogen gas, and 3 μM triblock probe-1, triblock probe-2 or triblock probe-3 were added dropwise, and incubated overnight at 45°C;

[0065] The electrode was sealed with 0.1 mM mercaptoethanol (MCH) at room temperature for 1 h to prepare an electrochemical sensor modified with a triblock probe and stored at 4°C until use.

Embodiment 3

[0066] Embodiment 3 electrochemical detection

[0067] The principle of electrochemical detection is as figure 1 As shown, the triblock probe modified on the surface of the gold electrode is used as the capture probe to perform specific complementary hybridization with the target transgenic double-stranded RNA. After the biotin-labeled detection probe and spacer probe are combined with the target transgenic double-stranded RNA, the With the strong affinity of "biotin-avidin", HPR is labeled on the sandwich structure of triblock probe-target transgenic double-stranded RNA-detection probe, and TMB or H 2 o 2 , through the generated electrochemical signal to realize the detection of transgenic double-stranded RNA.

[0068] Proceed as follows:

[0069] (1) 10nM target transgenic double-stranded RNA prepared in Example 1, 100nM biotin-labeled detection probe (SEQ ID NO:4~20) and spacer probe (SEQ ID NO:21~22) were mixed in hybridization buffer (50mM Na 2 HPO 4 , 50mMNaH 2 PO...

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Abstract

The invention provides an electrochemical sensor based on a triblock probe and application of the electrochemical sensor in detection of transgenic double-stranded RNA. The electrochemical sensor comprises a gold electrode and a triblock probe; the triblock probe comprises PolyA, and a first capture probe and a second capture probe which are connected with the PolyA; the first capture probe and the second capture probe comprise a section of nucleic acid sequence complementary with the transgenic double-stranded RNA; and the triblock probe is modified on the surface of the gold electrode through the adsorption effect of PolyA and the gold electrode. The surface of the electrochemical sensor is modified with the triblock probe, specific, sensitive and stable transgenic double-stranded RNA detection is achieved, and the electrochemical sensor has the advantages of being high in sensitivity, short in consumed time, low in cost, portable and the like.

Description

technical field [0001] The invention belongs to the technical field of biological detection, relates to an electrochemical sensor based on a triblock probe and its application, in particular to an electrochemical sensor based on a triblock probe and its application in detecting transgenic double-stranded RNA . Background technique [0002] Currently, commercialized transgenic crops contain protein-coding traits, and protein expression levels are often quantitatively analyzed by immunological methods or LC-MS / MS. In addition to protein-coding properties, transgenic crops also have pest-resistant properties. This kind of transgenic pest-resistant traits developed based on RNAi technology has become a powerful means of controlling pests. Since RNA is the active molecule of RNAi, accurate quantification of RNA is crucial for the quality monitoring of downstream transgenic food and feed during the trait development of transgenic crops. [0003] Transgenic crops based on RNAi te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/48G01N27/327C12Q1/6825
CPCG01N27/48G01N27/3277C12Q1/6825C12Q2565/607C12Q2525/173C12Q2565/519C12Q2563/131Y02A50/30
Inventor 刘刚宋世平闻艳丽郑宇王乐乐李兰英杨雪许丽杨镇州梁文李妍徐勤罗超
Owner SHANGHAI INST OF MEASUREMENT & TESTING TECH
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