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Rapid virus detection method based on single-molecule total internal reflection fluorescence imaging technology

A technology of total internal reflection and fluorescence imaging, which is applied in the direction of fluorescence/phosphorescence, measurement devices, analysis materials, etc., can solve the problem of high cost

Active Publication Date: 2020-08-14
MINZU UNIVERSITY OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This type of method requires dyeing treatment, which takes at least 30 minutes, and the cost is relatively high

Method used

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  • Rapid virus detection method based on single-molecule total internal reflection fluorescence imaging technology
  • Rapid virus detection method based on single-molecule total internal reflection fluorescence imaging technology
  • Rapid virus detection method based on single-molecule total internal reflection fluorescence imaging technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Total Internal Reflection Fluorescence Imaging Device for SARS-COV-2 Detection

[0071] The invention provides a total internal reflection fluorescence imaging device for detecting SARS-COV-2, such as figure 1 As shown, it includes: an optical microscope system, a fluorescence excitation system, a signal acquisition system, and a microfluidic sample cell. As shown in the figure, the optical microscope system includes 11: a cover glass, 12: an objective lens, 13: a dichroic mirror, 14: an emission filter, and 15: a reflecting mirror. The fluorescence excitation system includes 21: a laser. The signal acquisition system includes 31: a charge-coupled device, and 32: a computer. A fluorescence excitation system is used to provide an excitation light source. The signal acquisition system is used for receiving fluorescent signals and converting the fluorescent signals into corresponding data signals. The microfluidic sample pool is used for storing the solution ...

Embodiment 2

[0075] Example 2 Rapid detection method of SARS-CoV-2 based on single-molecule fluorescence imaging technology

[0076] A method for rapid detection of novel coronavirus (SARS-CoV-2). The method includes the following steps: (1) fluorescently labeling short-chain ssDNA (single-stranded DNA, no specific requirement). (2) The fluorescently labeled ssDNA is added to the detection solution containing ionic halides, and the fluorescently labeled probe is electrostatically combined with the SARS-CoV-2 to be tested. (3) Place the solution to be tested on the total internal reflection fluorescence imaging device used for SARS-CoV-2 detection in Example 1 to detect fluorescent point signals. (4) Use fluorescent point coordinate positioning and least square method algorithm tracking to realize diffusion coefficient statistics. (5) The diffusion coefficient is used as the "unimolecular dynamics fingerprint signal" to calculate the average hydrodynamic diameter of the virus, and the dou...

Embodiment 3

[0084] Example 3 Tracking and quantitative detection of SARS-CoV-2 at the single-molecule level

[0085] The automatic target recognition and tracking system automatically extracts the target through the CCD image of the imaging system and performs identification and tracking. Its main algorithms include image data preprocessing, image segmentation, target detection, feature extraction, and target tracking. In this example, the realization of SARS-CoV-2 tracking at the single-molecule level is to use the CCD element in the total internal reflection fluorescence imaging device for SARS-COV-2 detection to image the fluorescent signal. For the identification of the fluorescent signal It is first based on effectively segmenting the local pixels of the target and the background, processing it into a grayscale image and binarizing it. For fluorescent signal targets of bright spots, the binarization process is:

[0086]

[0087] Among them, g(x, y) is the gray value at the coordi...

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Abstract

The invention provides a rapid virus detection method based on a single-molecule total internal reflection fluorescence imaging technology. The method comprises the following steps: step A, adding a fluorescent nucleic acid probe into a virus standard solution as a to-be-detected solution; step B, putting the to-be-detected solution on a total internal reflection fluorescence imaging device for fluorescence signal detection, calculating a diffusion coefficient according to the dynamic change of a tracked fluorescence signal phase point, taking the diffusion coefficient as a single molecular dynamics fingerprint signal of virus particles, and calculating the average hydrodynamic diameter of the virus by utilizing a Stocks-Einstein equation; and step C, replacing the virus standard substancewith a to-be-detected virus sample, obtaining a monomolecular kinetic fingerprint signal and a hydrodynamic diameter of the to-be-detected virus according to the same operation, and indicating that the to-be-detected virus sample contains the same type of virus if the monomolecular kinetic fingerprint signal and the hydrodynamic diameter conform to the monomolecular kinetic fingerprint signal andthe average hydrodynamic diameter. Quantitative detection can be realized by counting the two-dimensional paths of the fluorescence point signals conforming to the diffusion coefficient. The method has the advantages of simplicity, rapidness, high efficiency and economy, and is suitable for large-scale rapid detection of viruses.

Description

technical field [0001] The invention relates to virus detection technology, in particular to a rapid virus detection method based on single-molecule total internal reflection fluorescence imaging technology. Background technique [0002] The new coronavirus, the World Health Organization (WHO) has officially named it SARS-CoV-2, formerly known as 2019-nCoV. SARS-CoV-2 is different from SARS-CoV in that it is a new strain with special clinical, virological and epidemiological characteristics. The disease it causes is called COVID-19. [0003] SARS-CoV-2 belongs to the Betacoronavirus genus (Betacoronavirus), has an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60-140nm. SARS-CoV-2 is a positive-sense single-stranded RNA virus with a genetic material of about 30,000 bases in length. The protruding spike protein (S-Protein) on the surface of the protein is the key to infecting host cells. Like SARS-CoV virus, SARS-CoV-2 interacts with t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6458G01N2021/6439Y02A50/30
Inventor 耿照新马正泰吕晓庆
Owner MINZU UNIVERSITY OF CHINA
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