Zika virus nucleic acid detection method based on electrochemical luminescence amplification principle

A Zika virus and detection method technology, which is applied in the field of Zika virus nucleic acid detection based on the principle of electrochemiluminescence amplification, can solve the problems of Zika virus detection mode, time-consuming, and cumbersome detection process, etc.

Active Publication Date: 2017-12-15
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above methods have shortcomings such as time-consuming, cumbersome detection process, high price, timeliness and strong cross-reactivity in antibody detection, and cannot achieve a simple and rapid detection mode of Zika virus

Method used

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  • Zika virus nucleic acid detection method based on electrochemical luminescence amplification principle
  • Zika virus nucleic acid detection method based on electrochemical luminescence amplification principle
  • Zika virus nucleic acid detection method based on electrochemical luminescence amplification principle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1 Zika virus detection technical route

[0111] (1) Zika virus nucleic acid extraction

[0112] ①Collect certain tissue or cell samples. The tissue and cell samples must be samples infected with Zika virus. The weight of a single extracted tissue should be less than 0.1g. 7 cells, to avoid too many samples resulting in ineffective cell lysis;

[0113] ② Sample pretreatment:

[0114] a. Tissue samples

[0115] Grind the tissue into powder in liquid nitrogen, add 1mL of sample protector, shake it 3-5 times with an oscillator (1-2 seconds each time), let it rest for 30 minutes, remove the RNase in the cells, centrifuge for 30 minutes, and remove the supernatant. clear;

[0116] b. Monolayer adherent cell sample

[0117] Remove the liquid medium in the culture plate, add trypsin to the culture plate, digest the cells for 1 min, add the medium to stop the digestion, collect the cells by centrifugation and discard the supernatant, add 1 mL of sample protector, sh...

Embodiment 2

[0148] Example 2 Probe Characterization and Detection Principle Verification

[0149] In order to verify the feasibility of the construction of the probe of the present invention, the inventor characterized the labeling of the probe and the DNA connection (experimental results such as image 3 A, B shown). The inventor first detected the excitation and emission spectra of the terpyridine ruthenium polymer ( image 3 A), the experimental results show that the ruthenium terpyridine polymer has a maximum absorption peak at 460nm, and its emission peak is at 650nm, which is consistent with a single ruthenium terpyridine molecule. At the same time, the inventor characterized the product after the polymer was linked to the DNA nucleic acid, and the experimental results were as follows: image 3 As shown in B, a single ruthenium terpyridine polymer molecule has an absorption peak at 460nm, and when the ruthenium terpyridine polymer is connected with a DNA probe to construct an elec...

Embodiment 3

[0151] Example 3 Detection condition optimization and performance evaluation

[0152] The key point of the probe construction system of the present invention lies in the ratio and purification of the ruthenium terpyridine polymer and the DNA recognition domain when the polymer probe is constructed. The inventors set different molecular ratios and purified the product by ultrafiltration .

[0153] First, the inventors set different molecular ratio polymers, polymer: DNA recognition domain = 1:1, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1: 70, and in the case of sufficient reaction time, the electrochemiluminescent intensity after the probe captured the target nucleic acid was compared (the experimental results are as follows Figure 4 Shown in A), increases with the concentration of the DNA recognition domain and reaches a plateau when the ratio of polymer:DNA recognition domain is 1:50. Therefore, the present invention regards polymer:DNA recognition domain=1:50 as the optimal mo...

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Abstract

The invention discloses a zika virus nucleic acid detection method based on an electrochemical luminescence amplification principle and belongs to the technical field of zika virus molecular diagnosis. The invention respectively provides the zika virus nucleic acid detection method based on a linear and tree-shaped terpyridyl ruthenium polymer electrochemical luminescence amplification method. The method has the following advantages: 1) high sensitivity; 2) stable probe: the linear and tree-shaped terpyridyl ruthenium polymer used as an electrochemical luminescence amplification part of the probe, stable performance, and uniform degree of polymerization and luminous intensity; 3) simple and quick detection process: the zika virus detection is performed through simple sample pre-treatment and nucleic acid extraction process, time consumption is less, the amplification step is avoided and the detection is quick; 4) low cost. The method disclosed by the invention is a technical system for detecting zika virus nucleic acid; a novel molecular diagnosis method based on the electrochemical luminescence method technique is provided; the defect of single technique for detecting zika virus at present is effectively made up; a zika virus detection method requiring no amplification is supplied.

Description

technical field [0001] The invention belongs to the technical field of Zika virus molecular diagnosis, in particular to a Zika virus nucleic acid detection method based on the principle of electrochemiluminescence amplification. Background technique [0002] Zika virus (Zika virus), belonging to the Flaviviridae, is a single positive-sense RNA virus. First isolated from a febrile rhesus monkey in the Zika forest near Lake Victoria, Uganda (1947), it was transmitted primarily by the mosquito-borne route. In 2015, Zika virus broke out in a large area in Brazil, causing 1.5 million infections and more than 4,000 newborns with cerebellar malformation. As of March 10, 2017, Zika virus infection cases had occurred in 84 countries around the world. After Zika virus infection, most patients have flu-like manifestations, and generally can recover on their own. A small number of patients develop Guillain-Barré syndrome. Zika virus infection in pregnant women is related to neonatal mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6806C12Q1/682C12Q2563/103C12Q2563/143C12Q2563/149C12Q2521/537
Inventor 黄曦廖玉辉赵钊艳谭青琴
Owner SUN YAT SEN UNIV
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