Nano-gold biosensor for simultaneously detecting four hemorrhagic fever viruses and detection method thereof

A biosensor and nano-gold technology, applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of insufficient sensitivity, poor repeatability, complicated process, etc., and achieve high sensitivity and short detection time Short, cost-effective detection

Inactive Publication Date: 2019-07-23
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At home and abroad, there are mainly methods such as isolation and culture, electron microscope observation, immunofluorescence and RT-PCR for the detection of h

Method used

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  • Nano-gold biosensor for simultaneously detecting four hemorrhagic fever viruses and detection method thereof
  • Nano-gold biosensor for simultaneously detecting four hemorrhagic fever viruses and detection method thereof
  • Nano-gold biosensor for simultaneously detecting four hemorrhagic fever viruses and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The present embodiment provides a nano-gold biosensor for simultaneously detecting four kinds of hemorrhagic fever viruses, comprising a substrate 1, a sample pad 2, an absorbent pad 3, a nitrocellulose film 4 and a binding pad 5, and the sample pad 2 and the absorbent pad 3 are fixed At both ends of the base 1, the nitrocellulose film 4 is fixed in the middle of the base 1; there is an overlap of 2-3mm between the absorbent pad 3 and the nitrocellulose film 4, and the nitrocellulose film There is an overlap of 2-3 mm between 4 and the binding pad 5, and there is an overlap of 2-3 mm between the binding pad 5 and the sample pad 2; there is a gold nanoparticle DNA probe on the binding pad 5; The gold nanoparticle DNA probe comprises:

[0025] Hantavirus gold nanoparticle DNA probe: S-n-GGACAACTTCCACTTAGGGGA;

[0026] Chikungunya virus gold nanoparticle DNA probe: S-n-CTACAGCCCCATGGTACTGG;

[0027] Dengue virus gold nanoparticle DNA probe: S-n-GCTGAAACGCGAGAGAAACC;

[...

Embodiment 2

[0039] The present embodiment provides a detection method using the nano-gold biosensor for simultaneous detection of four hemorrhagic fever viruses described in Embodiment 1, comprising the following steps:

[0040] S1. After the pathogen is lysed, RNA extraction uses magnetic nanoparticles to enrich nucleic acid, and further reverse transcription after elution;

[0041] S2. The reverse transcription product was screened again using magnetic nanoparticles, and the finally eluted nucleic acid was added to the isothermal strand displacement amplification (strand displacement amplification, SDA) system and placed in a 37°C water bath for 30 minutes of reaction Finally, the amplified product is dropped onto the sample pad as a test sample, and the result can be observed with the naked eye after 5 minutes.

Embodiment 3

[0043] In this example, the performance of the nano-gold biosensor provided in Example 1 is verified through experiments.

[0044] 1. Detection principle Amplified product electrophoresis gel verification

[0045] In order to verify the feasibility of the isothermal strand displacement amplification system, the recombinant plasmids of four viruses were used here, and the plasmids were diluted into 6 concentration gradients of 1×10 12 , 1×10 8 , 1×10 6 , 1×10 4 , 1×10 2 , 1×10 1 copies / μL, added to the isothermal strand displacement amplification reaction system and reacted for 30 minutes, the amplified product was added to a 3% agarose gel, and the result was observed with a gel imager after 30 minutes at 100V (such as figure 2 shown).

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Abstract

The invention discloses a nano-gold biosensor for simultaneously detecting four hemorrhagic fever viruses and a detection method thereof. The biosensor comprises a substrate, a sample pad, an absorption pad, a nitrocellulose film and a bonding pad, wherein the sample pad and the absorption pad are fixed at two ends of the substrate, and the nitrocellulose film is fixed at the middle part of the substrate; a 2-3mm overlap is formed between the absorption pad and the nitrocellulose film, a 2-3mm overlap is formed between the nitrocellulose film and the binding pad, and a 2-3mm overlap is formedbetween the binding pad and the sample pad; and gold nanoparticle DNA probes are arranged on the binding pad. The biosensor has the advantages of high sensitivity, high specificity, short detection time, capability of completing detection within 60 minutes and low detection cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nano-gold biosensor and a detection method for simultaneously detecting four hemorrhagic fever viruses. Background technique [0002] Blood fever viruses are diverse, easily contagious and extremely harmful. The Ebola virus that broke out in 2014 has killed more than 7,000 people, with a combined fatality rate of over 50%. In the same year, the number of dengue virus infections in Guangdong Province exceeded 40,000. At home and abroad, there are mainly methods for detection of hemorrhagic fever virus such as isolation and culture, electron microscope observation, immunofluorescence and RT-PCR. The previous diagnostic methods have disadvantages such as complicated process, poor repeatability, insufficient sensitivity and high cost. Contents of the invention [0003] Aiming at the deficiencies in the prior art, the present invention aims to provide a nano-gold biosensor and detect...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6825C12N15/11
CPCC12Q1/701C12Q1/6825
Inventor 曾令文赵锦
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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