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Pretreatment solution, kit and pretreatment method for fluorescence in situ hybridization (FISH) of cell sample

A technology of fluorescence in situ hybridization and pretreatment solution, which is applied in the direction of biochemical equipment and methods, measurement/inspection of microorganisms, etc., and can solve problems such as decreased activity and incomplete enzyme digestion

Active Publication Date: 2021-01-22
JIAXING ACCB DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different protease batches have different activities, and after long-term use, the activity will gradually decrease, which can lead to incomplete enzyme digestion

Method used

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  • Pretreatment solution, kit and pretreatment method for fluorescence in situ hybridization (FISH) of cell sample
  • Pretreatment solution, kit and pretreatment method for fluorescence in situ hybridization (FISH) of cell sample
  • Pretreatment solution, kit and pretreatment method for fluorescence in situ hybridization (FISH) of cell sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] After cytological preparation with normal human blood, pretreatment was carried out according to the method of the present invention (the steps shown in Table 1). postnatal results. After pretreatment, observe under a bright-field microscope whether there are still cell outlines. Then FISH hybridization and DAPI staining were performed. Observe under a fluorescent microscope whether there are DAPI-stained nuclei, and the signal points of the orange gene-specific probe and the green chromosome CEP probe.

[0051] The test results are shown in Table 1 and figure 1 and figure 2 As shown, among them, figure 1 It shows the profile of normal human blood cell droplet under bright field microscope after pretreatment with 100mM NaOH, 0.5% Triton x-100 solution at different temperatures; figure 2 Shown is the FISH signal of the normal human blood cell droplet under the fluorescence microscope after pretreatment with 100mM NaOH, 0.5% Triton x-100 solution.

[0052] figure...

Embodiment 2

[0057] In Example 2, the same blood cell sample as in Example 1 was used for fluorescence in situ hybridization, except that the steps shown in Table 2 were used for pretreatment, and more experimental conditions were tried. The test results are shown in Table 2.

[0058] Table 2:

[0059]

[0060]

[0061]

[0062]It can be seen from Table 2 that after the blood cells are treated with the pretreatment solution containing 15-150 mM lye and 0.1%-1% non-ionic surfactant, FISH signals can be detected. Wherein, the fluorescence in situ detection effect of the blood cells after the pretreatment solution containing 100mM KOH and 0.5% NP-40 is equivalent to the treatment effect of the pretreatment solution containing 100mM NaOH and 0.5% TritonX-100 in Example 1, in The fluorescence images at 25°C are relatively clear. The second is the pretreatment solution containing 15mM NaOH and 0.8% Triton X-100, and the pretreatment solution containing 150mM NaOH and 0.3% Triton X-100...

Embodiment 3

[0064] According to the conventional method, exfoliated cells in the urine of normal people were used for preparation. After sheeting, pretreatment was carried out according to the method of the present invention (shown in Table 3). In this example, different treatment fluid formulations were tested (as shown in Table 3 below). Observation by bright field microscopy was performed after pretreatment. Then, FISH hybridization and DAPI staining were performed according to conventional methods, and observed under a fluorescent microscope.

[0065] table 3:

[0066]

[0067] The test results are shown in Table 3 and image 3 and Figure 4 As shown, among them, image 3 It shows the profile of the exfoliated cell droplet in normal human urine under bright field microscope after the solution of different formulations pretreated the exfoliated cells in urine at room temperature; Figure 4 It shows the FISH signal under the fluorescence microscope after the normal human urine ...

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Abstract

The invention provides a pretreatment solution, kit and pretreatment method for fluorescence in situ hybridization (FISH) of a cell sample. The pretreatment solution comprises the following components: 20-110 mM of NaOH or KOH; and 0.1-1.0% of a non-ionic surfactant. By adopting the non-ionic surfactant with the concentration range, cell membranes are destroyed to a certain extent, and the basic form and structure of cell nucleuses can be maintained. Alkali liquor with the concentration range is used for treating the cell sample, so that DNA and protein in cells are denatured, and nucleic acidcombined with the protein is also exposed. In addition, the non-ionic surfactant is also beneficial to increase of cell surface tension so as to promote loosening of the whole cell structure, and subsequent effective hybridization of DNA in the cell nucleuses with a fluorescent probe is facilitated. The pretreatment solution is short in treatment time, low in cost and good in treatment effect stability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a pretreatment liquid, a kit and a pretreatment method for fluorescence in situ hybridization of cell samples. Background technique [0002] Fluorescence in situ hybridization (FISH) is a new technology developed in the 1980s that combines molecular biology and cytogenetics. Its principle is to use fluorescently labeled nucleic acid probes to hybridize with complementary regions of nucleic acids in samples. At present, it has been widely used in many fields such as genetic disease diagnosis, virus infection analysis, prenatal diagnosis, tumor genetics and genome research. [0003] Cytological samples include blood cells and various exfoliated cells, including urine, cervical samples, pleural fluid, amniotic fluid, etc., which contain exfoliated cells from various tissues and are widely used in clinical diagnosis and scientific research. For example, exfoliated cells in u...

Claims

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Application Information

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IPC IPC(8): C12Q1/6841C12Q1/6806
CPCC12Q1/6841C12Q1/6806C12Q2543/10C12Q2563/107C12Q2527/125C12Q2565/50
Inventor 丁凤王芳高梦娇凌萧洁李轶亢陈皓媛
Owner JIAXING ACCB DIAGNOSTICS
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