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Cancer screening and diagnosis method via staining quantification of cellular DNA by fluorescent dye DAPI

A fluorescent dye and cell technology, applied in the field of DNA detection, can solve the problems of long time, low quantitative accuracy, complicated dyeing operation, etc., and achieve the effect of simple operation, accurate quantification, and fast dyeing time

Inactive Publication Date: 2019-06-28
贺权源
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These traditional staining methods in specimen preparation have the following disadvantages: the time is long, and the Feulgen staining method takes about 2 hours; the staining operation is complicated, and the quantitative accuracy is low

Method used

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  • Cancer screening and diagnosis method via staining quantification of cellular DNA by fluorescent dye DAPI
  • Cancer screening and diagnosis method via staining quantification of cellular DNA by fluorescent dye DAPI

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment one, real-time staining, comprises the following steps:

[0025] Step 1: Use a cervical brush to brush some cells from the female cervix, place the cervical brush in a fixative solution containing alcohol and methanol, and shake the cell sample for 1 min to suspend it.

[0026] Step 2, take the cell sedimentation chamber method to make slices: take the filter, add 2.5ml of cell separation solution, and cover the filter;

[0027] Step 3, take 20-100ul DAPI 10* working solution and 300ul cell samples, mix evenly for 1 minute, and stain for 3 minutes;

[0028] Step 4, transfer the sample to the production chamber;

[0029] Step 5, centrifuge, 3min;

[0030] Step 6, take slides, wash with water for 20s, and wash with PBS for 30s×3 times;

[0031] Step 7, dehydration with 95% and 100% ethanol for 1 min each;

[0032] Step 8, wait until dry, and seal with resin;

[0033] Step 9: Examine under a fluorescent microscope for quantitative analysis.

[0034] Step 1...

Embodiment 2

[0036] Embodiment 2, integration of fixation and dyeing, comprises the following steps:

[0037] Step 1, improve the cell fixative and add DAPI working solution to make the final concentration between (0.2-1.0ug / ml).

[0038] Step 2, use a cervical brush to brush some cells from the female cervix, place the cervical brush in the fixative solution containing DAPI, and shake the cell sample for 2 minutes to make it suspended.

[0039] Step 3, add 2.5ml of cell separation medium to the cell sedimentation chamber, and cover the filter;

[0040] Step 4, take a 300ul cell sample and transfer the sample to the production chamber;

[0041] Step 5, centrifuge, 3min;

[0042] Step 6, take slides, wash with water for 20s, and wash with PBS for 30s×3 times;

[0043] Step 7, 95% and 100% ethanol dehydration for 1 min each;

[0044] Step 8, wait until dry, and seal with resin;

[0045] Step 9, check under a fluorescent microscope for quantitative analysis;

[0046] Step 10, identifyin...

Embodiment 3

[0047] Embodiment three, paraffin section, comprises the following steps:

[0048] Step 1, generally use a constant temperature water bath, the temperature is controlled at about 37-40°C, which is conducive to the unfolding of paraffin sections, and the pasted slices are dried in a 60°C constant temperature box for 2 hours, and the protein can be stained after solidification.

[0049] Step 2, dewaxing and hydration of the slices: the dried slices were dewaxed in xylene, and then dewaxed with pure alcohol, 95%, 85%, and 70% in descending order from high concentration to low concentration. change.

[0050] Step 3, soak the slices in 2ug / ml DAPI staining solution for 10 minutes.

[0051] Step 4, take slides, wash with water for 20s, and wash with PBS for 30s×3 times;

[0052] Step 5, dehydration with 95% and 100% ethanol for 1 min each;

[0053] Step 6, wait until dry, and seal with resin;

[0054] Step 7, check under a fluorescent microscope for quantitative analysis;

[0055...

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Abstract

The invention discloses a cancer screening and diagnosis method via staining quantification of cellular DNA by a fluorescent dye DAPI. According to the method, cell nucleuses in cell smears or cells and tissue sections are stained with DAPI as a dye, pictures are taken under a fluorescence microscope, and the DNA in each nucleus is relatively quantified according to the fluorescence intensity of each nucleus. Cells with abnormal DNA content (non-integer ploidy or ploidy number being greater than 4.5 or above) are suspected cancer cells. The number and morphology of the suspected cancer cells can be used for cancer screening and diagnosis. The dyeing method provided by the invention has the advantages of short dyeing time, simple operation and accurate quantification, is wide in application, can quantify the DNA of cells in cell smears, tissue sections and cultured cells, and can be used for screening cancer samples.

Description

technical field [0001] The invention relates to the technical field of DNA detection, in particular to a method for quantitatively measuring cellular DNA by using fluorescent dye DAPI and performing cancer screening and diagnosis. Background technique [0002] In general, the DNA content of normal cells is basically the same (diploid or tetraploid), while the DNA content of cells in cancer or other disease states may change. There have been many reports on the screening of cervical precancerous lesions by quantitatively analyzing the DNA content of each cell using cell (nucleus) staining images. In North America and Europe, quantitative analysis of cell DNA image diagnostic methods has been used as one of the routine clinical detection methods. In China, many large cities have also begun to use automatic DNA quantitative analysis systems for cervical cancer screening, such as the journal article "The Application Value of Cervical Cell DNA Quantitative Analysis in Cervical Ca...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N1/28G01N1/30
Inventor 贺权源周明青
Owner 贺权源
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