Nucleic acid amplification paper-based visual detection platform

A detection platform, paper-based technology, applied in the field of nucleic acid detection, to achieve the effect of a good container

Active Publication Date: 2020-06-26
QINGDAO UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the integrated platform of amplification and detection based on paper-based materials.

Method used

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  • Nucleic acid amplification paper-based visual detection platform
  • Nucleic acid amplification paper-based visual detection platform
  • Nucleic acid amplification paper-based visual detection platform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Cut the silica gel membrane into discs with a diameter of 3 mm with a hole puncher, and place them on the lid of a 0.2 mL centrifuge tube; take the paper-based material disc, and mix 15 µL of 2-fold gradient dilution of pMD 18-T carrier DNA and 1 µL of SYBR Green I dye was added to the surface of the material, and the disk was directly placed under a UV lamp to excite and photographed and recorded; the obtained image was processed in grayscale and analyzed with ImageJ software, and the average pixel value was recorded as the signal intensity. Record the mean value of the blank sample as N (noise) and the mean value of DNA samples from a given sample as S (signal). Normalized intensities were calculated as signal-to-noise ratio (SNR) values ​​between samples and blanks: SNR = N / S.

Embodiment 2

[0036] Cut the nitrocellulose membrane into discs with a diameter of 3 mm with a hole punch, and place them on the lid of a 0.2 mL centrifuge tube; take the paper-based material discs, and add 15 µL of 2-fold serially diluted pMD 18-T carrier DNA and 1 µL of SYBR Green I dye were added to the surface of the material, and the disc was directly placed under a UV lamp to excite and photographed for recording; the obtained image was processed in grayscale and analyzed with ImageJ software, and the average pixel value was recorded as the signal intensity . Record the mean value of the blank sample as N (noise) and the mean value of DNA samples from a given sample as S (signal). Normalized intensities were calculated as signal-to-noise ratio (SNR) values ​​between samples and blanks: SNR = N / S.

Embodiment 3

[0038] Cut Whatman No. 1 filter paper into discs with a diameter of 3 mm and place them on the lid of a 0.2 mL centrifuge tube; take the paper-based material disc and add 15 µL of 2-fold serially diluted pMD 18-T carrier DNA and 1 µL of SYBR Green I dye were added to the surface of the material, and the disc was directly placed under a UV lamp to excite and photographed for recording; the obtained image was processed in grayscale and analyzed with ImageJ software, and the average pixel value was recorded as the signal intensity . Record the mean value of the blank sample as N (noise) and the mean value of DNA samples from a given sample as S (signal). Normalized intensities were calculated as signal-to-noise ratio (SNR) values ​​between samples and blanks: SNR = N / S.

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Abstract

The present invention provides a nucleic acid amplification paper-based visual detection platform. An amplification product and a nucleic acid dye are added directly to a paper-based material, and judgment of positive and negative results can be realized by only using a small ultraviolet lamp. Through the technical scheme, an SYBRGreen I dye is added based on an amplification reaction in a silicagel film, and by using simple ultraviolet light, visual judgment is performed, so that an answer of yes or not can be conveniently and visually obtained. The silica gel film is a compatibility matrixof DNA and SYBRGreen I signal display, an effective platform can be provided for nucleic acid amplification detection, the use of the silica gel film does not produce interference to an amplificationprocess, the detection sensitivity of the amplification platform is consistent with that of fluorescent quantitative PCR, and the size of the amplification product is not affected. The silica gel filmprovides a result interpretation platform, and the multi-pore diameter property of the silica gel film provides more spaces for the reaction, so that reaction components are fully mixed and diffused,and a good container is provided for the amplification reaction.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid amplification paper-based visual detection platform. Background technique [0002] The presence of various pathogenic bacteria is one of the most serious health problems worldwide. To prevent large-scale outbreaks, early detection of pathogens and diagnosis of disease is critical. Nucleic acid amplification and real-time fluorescence detection play an important role in the field of molecular diagnosis. However, this detection method often requires a large-scale fluorescence amplification instrument, which cannot meet the needs of on-site or point-of-care detection (POCT). SYBRGreen I dye has been widely used in the development of real-time quantitative nucleic acid detection methods, such as real-time fluorescent quantitative PCR (RT-PCR) or real-time fluorescent loop-mediated isothermal amplification reaction (RT-LAMP), etc., in analytical and dia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12R1/63
CPCC12Q1/6844C12Q1/689C12Q2563/107C12Q2565/125
Inventor 王秀丹马翠萍焉春雨
Owner QINGDAO UNIV OF SCI & TECH
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