A paper-based visual detection method for nucleic acid amplification for non-diagnostic purposes

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

Active Publication Date: 2021-06-22
QINGDAO UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the integrated method of amplification and detection based on paper-based materials is rarely reported.

Method used

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  • A paper-based visual detection method for nucleic acid amplification for non-diagnostic purposes
  • A paper-based visual detection method for nucleic acid amplification for non-diagnostic purposes
  • A paper-based visual detection method for nucleic acid amplification for non-diagnostic purposes

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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 GreenI dye were 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.

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Abstract

The invention provides a nucleic acid amplification paper-based visual detection platform, which directly adds amplification products and nucleic acid dyes to the paper-based material, and only uses a small ultraviolet lamp to realize the judgment of negative and positive results. Through the technical scheme of the present invention, based on the amplification reaction in the silica gel membrane, SYBRGreen I dye is added, and simple ultraviolet light is used to perform visual judgment, and the answer of "yes or not" can be obtained conveniently and intuitively. Silica gel membrane is a compatible matrix for DNA and SYBRGreen I signal display, which can provide an effective platform for nucleic acid amplification detection. The use of silica gel membrane will not interfere with the amplification process. The detection sensitivity of the amplification platform and the use of fluorescence The detection sensitivity of quantitative PCR is consistent, and the size of the amplified product is not affected. The silica gel membrane not only provides a platform for result interpretation, but its multi-aperture property provides more space for the reaction, allowing the reaction components to fully mix and diffuse, and provides a good container for the amplification reaction.

Description

technical field [0001] The present invention relates to the technical field of nucleic acid detection, in particular to a non-diagnostic nucleic acid amplification paper-based visual detection method. 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., ...

Claims

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Application Information

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Patent Type & Authority Patents(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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