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Watermelon mosaic virus detection sensor based on t4 DNA polymerase and its assembly method

A watermelon mosaic virus, detection sensor technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problem that target DNA has no advantage in selectivity, hinder target detection performance, and prolong reaction time. and other problems, to achieve the effect of high sensitivity and rapid detection, high sensitivity and fast response speed

Active Publication Date: 2021-10-15
LINYI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, DNA templates without secondary structures are usually used, which has no advantage in the selectivity of target DNA, and the recognition sites of nicking enzymes and polymerases overlap, which will prolong the reaction time
In addition, the generated DNA replicons can hybridize to the free DNA template, hindering the detection performance of the target to some extent.

Method used

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  • Watermelon mosaic virus detection sensor based on t4 DNA polymerase and its assembly method
  • Watermelon mosaic virus detection sensor based on t4 DNA polymerase and its assembly method
  • Watermelon mosaic virus detection sensor based on t4 DNA polymerase and its assembly method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] A watermelon mosaic virus detection sensor based on T4 DNA polymerase, including: probe P1, probe P2, probe P3, hairpin probe HP1, Nb.BbvCl, T4 DNA polymerase, dGTP, dNTP, hairpin probe Needle HP2, gold electrode;

[0059] Each DNA sequence is as follows:

[0060] The gene sequence of the probe P1 is shown in SEQ ID No.1:

[0061] SEQ ID No.1: 5'-ACACACAGCGATCACCCATGCCTCAGCTTTT-3';

[0062] The gene sequence of the probe P2 is shown in SEQ ID No.2:

[0063] SEQ ID No.2: 5'-GGGTTTAACATGGGTGATCGAAATAGTGGGTG-3';

[0064] The gene sequence of the probe P3 is shown in SEQ ID No.3:

[0065] SEQ ID No.3: 5'-CACCCACTATTTCGATCACCCATGTTAAACCC-3';

[0066] The gene sequence of the hairpin probe HP1 is shown in SEQ ID No.4:

[0067] SEQ ID No.4:

[0068] 5'-ATGCCTCAGATAAGCCACAAAAAAAAGCTGAGGCAT-3';

[0069] The gene sequence of the hairpin probe HP2 is shown in SEQ ID No.5:

[0070] SEQ ID No.5:

[0071] 5'-SH5'-SH-(CH2)6-TTTTGGGTTGGGCGGGATGGGTTTATrAGGTGTGTATCCCGCCC-3'.

...

Embodiment 2

[0086] With the gold electrode obtained at last in embodiment 1 as the working electrode, the Pt electrode as the counter electrode, and the SCE electrode as the reference electrode, the current is detected by differential pulse voltammetry, and the i-t curve is recorded; the result is as follows figure 2 As shown, there is a positive correlation between the magnitude of the current value and the concentration of the target DNA (WMV cDNA).

Embodiment 3

[0088] According to the current value of different concentration target DNA (WMV cDNA) solutions in implementation 1, the ordinate is taken as the abscissa with the corresponding target DNA solution concentration, and the concentration standard curve is established; the results are as follows image 3 As shown, a straight line is obtained by linearly fitting the current value and the concentration of the target DNA solution, and the linear correlation is above 0.99. The detection limit is calculated by the 3σ method, and the linear range is 50fM-1nM, and the minimum detection limit is 50fM.

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Abstract

The invention discloses a watermelon mosaic virus detection sensor based on T4 DNA polymerase and an assembly method thereof. The sensor includes probe P1, probe P2, probe P3, hairpin probe HP1, Nb.BbvC I, T4 DNA polymerase, dGTP, dNTP, hairpin probe HP2, gold electrode; use the target DNA and P1 to open HP1 together, and then use the Nb.BbvC Ⅰ recognition site to digest at 37°C to obtain two fragments; add P2 and one of them Fragment hybridization, exocution reaction under the action of T4 DNA polymerase / dGTP, adding dNTP for polymerization reaction, and then adding P3 to make it completely complementary to P2, that is, to obtain free Mg 2+ ‑Rely on the DNAzyme mixed solution; add it dropwise to the gold electrode modified by HP2 for reaction, add Hemin, K in turn + 、H 2 o 2 Perform current signal detection. The detection system of the present invention has a linear range of 50fM-1nM, a minimum detection limit of 50fM, and can realize high-sensitivity and rapid detection of WMV in the field.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a T4 DNA polymerase-based watermelon mosaic virus detection sensor and an assembly method thereof. Background technique [0002] Watermelon mosaic virus-2 (WMV-2) is an important member of the Potyviridae family Potyvirus. It is one of the important viruses in Cucurbitaceae and leguminous crops and can Infected plants have severely reduced yields. [0003] At present, the detection methods of plant viruses mainly use molecular biology and serological detection. Molecular biology detection technology has high sensitivity, but there are problems of tedious RNA extraction and easy degradation. Serological testing is widely used due to its simple operation and the ability to process a large number of samples at the same time, but expensive serological testing kits need to be purchased, and the accuracy of the test results depends on the quality of the antibody. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6825C12R1/94
CPCC12Q1/6825C12Q1/701C12Q2531/113C12Q2565/607
Inventor 王莹
Owner LINYI UNIVERSITY
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