CN117247995A - Method for carrying out molecular fluorescence in-situ hybridization treatment on G-banding glass slide - Google Patents

Method for carrying out molecular fluorescence in-situ hybridization treatment on G-banding glass slide Download PDF

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CN117247995A
CN117247995A CN202311179445.6A CN202311179445A CN117247995A CN 117247995 A CN117247995 A CN 117247995A CN 202311179445 A CN202311179445 A CN 202311179445A CN 117247995 A CN117247995 A CN 117247995A
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slide
banding
immersing
ethanol
tape
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聂丽萍
崔妮妮
苏燕辉
邓小净
黄士昂
朱晓燕
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Shanghai Simplegen Medical Inspection And Verification Institute Co ltd
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Shanghai Simplegen Medical Inspection And Verification Institute Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Abstract

The application relates to the field of biotechnology, in particular to a method for performing molecular fluorescence in situ hybridization treatment on a G-banding slide. The application provides a treatment method for molecular fluorescence in situ hybridization on a G-banding slide, which comprises the following steps: 1) Immersing the G-tape slide in an organic solvent, immersing and washing the G-tape slide in a fixing solution and immersing the G-tape slide in a buffer solution; 2) Dehydrating and drying the G-banding slide processed in the step 1), adding a probe for hybridization reaction, washing the slide, drying, and adding a coloring agent for microscopic examination. The treatment method can carry out molecular fluorescence in-situ hybridization on the G-banding slide, avoid repeatedly collecting bone marrow fluid and reduce pain caused by patient puncture. And for patients with less bone marrow fluid taking amount, dry pumping or need to do a plurality of detection items caused by certain diseases, the sample amount is saved, and the detection items are enlarged.

Description

Method for carrying out molecular fluorescence in-situ hybridization treatment on G-banding glass slide
Technical Field
The application relates to the field of biotechnology, in particular to a method for performing molecular fluorescence in situ hybridization treatment on a G-banding slide.
Background
The chromosome banding technology is to treat chromosome specimen and dye it with specific dye to make the chromosome show the alternate light and shade and the horizontal lines with lines. Each chromosome is made to show unique banding through banding technology, which constitutes the banding pattern of the chromosome. The banding patterns of each pair of homologous chromosomes are substantially identical and stable, with the banding patterns of different pairs of chromosomes being different.
The method can be classified into Q development, G development, C development, T development, etc. according to the development method, wherein G development is the most commonly used technique. The chromosome specimen is treated by alkali, trypsin or other salt solution to denature protein on the chromosome, and then is dyed by Giemsa dye liquor, so that alternate bands of depth can be observed under a common microscope, positive bands (Positive bands) which are easy to color are chromosome segments rich in A-T, and conversely, segments with high G-C content are difficult to color and are Negative bands (Negative bands). The G banding method is simple and convenient, has clear banding, and can preserve chromosome specimens for a long time, so the G banding method is widely used for diagnosis and research of chromosome diseases.
Fluorescent in situ hybridization (fluorescent in situ hybridization: FISH), a fluorescent dye is labeled on a nucleic acid fragment (probe) paired with a target DNA according to the principle of base complementary pairing, the probe is specifically bound (hybridized) with a corresponding nucleic acid fragment in a specific detection material under a certain condition to form double-stranded nucleic acid, and the type and the number of the formed hybridized double strand are observed and recorded by means of a fluorescent microscope, so that the detection method for judging whether the target DNA in a sample to be detected is normal or not. Is a molecular cytogenetic diagnosis technology widely applied to clinical pathology detection for more than ten years.
For bone puncture, only conventional banding pattern analysis is sent out, but no relevant disease FISH detection is carried out, no cell suspension or bone marrow liquid remains, the patient is not willing to collect the bone marrow liquid again, or a chemotherapy scheme is used, and the FISH detection is very difficult under the condition that a living cell sample cannot be obtained again. When peripheral blood cells are detected by the fluorescence in situ hybridization technique, abnormality is not detected under the condition that blood diseases are not infiltrated into peripheral blood or abnormal cells in the peripheral blood are very few.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present application to provide a method for performing molecular fluorescence in situ hybridization on a G-banded slide, which is used to solve the problems of the prior art.
The inventors of the present application studied and found that: the following drawbacks exist due to conventional banding pattern analysis: the microscopic deletion, the three-position or more than three-position translocation or the hidden translocation is difficult to judge, the sensitivity of the FISH technology is higher, the microscopic deletion or the hidden translocation can be detected, the deficiency of the chromosome can be made up by the FISH detection, the chromosome detection is negative, and the FISH can be positive. The requirement on the middle split phase in conventional banding pattern analysis is high: the conventional banding pattern analysis can only analyze metaphase cells, and requires that metaphase division is dispersed, the bands are clear, and the number of the bands reaches an analysis standard; while most cells of the human body do not divide, it is difficult to obtain metaphase chromosome division phase by culture, and interphase FISH is not limited by this, and metaphase division phase cells are not required.
Therefore, the conventional banding pattern analysis is the preferred technology for screening chromosomal abnormalities of hematological malignancies, and the FISH is an important supplement to the conventional banding technology, and the FISH detection is performed on a glass slide subjected to the conventional banding core analysis such as the G-banding, so that the method can become a great improvement of the detection technology, can avoid repeated collection of bone marrow fluid, reduce pain caused by patient puncture, save sample quantity and enlarge detection projects.
To achieve the above and other related objects, a first aspect of the present application provides a method for performing molecular fluorescence in situ hybridization on a G-banded slide, comprising the steps of:
1) Immersing the G-tape slide in an organic solvent, immersing and washing the G-tape slide in a fixing solution and immersing the G-tape slide in a buffer solution;
2) Dehydrating and drying the G-banding slide processed in the step 1), adding a probe for hybridization reaction, washing the slide, drying, and adding a coloring agent for microscopic examination.
In a second aspect, the present application provides a G-tape slide, which is processed by the aforementioned processing method.
In a third aspect the present application provides the use of the aforementioned treatment method, or the aforementioned G-banding slide, in the preparation of a chromosome detection product.
Compared with the prior art, the beneficial effects of this application are:
1. the treatment method can carry out molecular fluorescence in-situ hybridization on the G-banding slide, avoid repeatedly collecting bone marrow fluid and reduce pain caused by patient puncture. And for patients with less bone marrow fluid taking amount, dry pumping or need to do a plurality of detection items caused by certain diseases, the sample amount is saved, and the detection items are enlarged.
2. The method can make up for the defect of performing karyotyping analysis by conventional banding, and can detect microdeletion or hidden translocation.
3. The application realizes the reutilization of the waste glass slide of the G-developing glass slide, and further expands the sample possibility of biological detection.
Drawings
FIG. 1 is a graph showing the results of Acute Lymphoblastic Leukemia (ALL) in example 1. Wherein, FIG. 1A shows the G-banding result, and FIG. 1B shows the hybridization microscopic examination result of TEL/AML1 bicolor ES probe on the G-banding slide: 85% of the cells had a TEL gene deletion.
FIG. 2 is a graph showing the results of Acute Myeloid Leukemia (AML) in example 2. Wherein, FIG. 2A shows the G-banding result, and FIG. 2B shows the hybridization microscopic examination result of AML1/ETO double-color double-fusion probe on the G-banding slide: 88% of the cells have AML1/ETO fusion genes present and have three or more chromosomal complex translocations.
FIG. 3 is a graph showing the results of high grade B cell lymphoma (HGBL) of example 3. Wherein, FIG. 3A is the G-banding result, and FIG. 3B is the hybridization microscopy result of MYC and BCL2 two-color separation probe on the G-banding slide: 92% MYC gene rearrangement and 90% bcl2 gene rearrangement.
FIG. 4 is a graph showing the results of Chronic Lymphocytic Leukemia (CLL) of example 4. Wherein, FIG. 4A shows the G-banding result, and FIG. 4B shows the hybridization microscopy result of D13S25 monochromatic probe on the G-banding slide: 89% of the D13S25 gene was deleted.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application is further described below with reference to examples. It should be understood that the examples are presented by way of illustration only and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, unless otherwise indicated, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.
The inventors of the present application have found a treatment method for performing molecular fluorescence in situ hybridization on a G-banded slide through extensive research and study, and completed the present application on the basis of the treatment method. The treatment method can carry out molecular fluorescence in-situ hybridization on the G-banding slide, avoid repeatedly collecting bone marrow fluid and reduce pain caused by patient puncture. And for patients with less bone marrow fluid taking amount, dry pumping or need to do a plurality of detection items caused by certain diseases, the sample amount is saved, and the detection items are enlarged.
In one aspect, the present application provides a method for performing molecular fluorescence in situ hybridization on a G-banded slide, comprising the steps of:
1) Immersing the G-tape slide in an organic solvent, immersing and washing the G-tape slide in a fixing solution and immersing the G-tape slide in a buffer solution;
2) Dehydrating and drying the G-banding slide processed in the step 1), adding a probe for hybridization reaction, washing the slide, drying, and adding a coloring agent for microscopic examination.
In the treatment method provided by the application, the step 1) refers to immersing the G-tape slide in an organic solvent, immersing in a fixing solution, and immersing in a buffer solution. Wherein the organic solvent comprises one or more of xylene, diethyl ether and ethanol. The organic solvent serves to remove the microscope oil from the G-tape slide. The time for immersing the organic solvent is 1-3 min; specifically, the time period may be 1 to 2 minutes or 2 to 3 minutes.
In the step 1), the fixing solution comprises methanol and glacial acetic acid; further, the volume ratio of methanol to iceethanol is 3:1. the addition amount of the fixing liquid is based on the immersion of the G-tape slide; preferably, the amount of the fixative to be added to each G-tape slide is 40 to 50mL. In some embodiments, the fixative dip may be dip washed 3 times. The fixing solution is used for cleaning the organic solvent and removing the color on the G-band slide.
In step 1), the buffer is selected from SSC, HEPES, SSPE, PIPES, TMAC, TRIS, SET, potassium phosphate, citric acid, or sodium pyrophosphate. The concentration of the buffer solution is 0.5-50 x; preferably, the concentration of the buffer is 2x. In one embodiment of the present application, the buffer is SSC at 2x. SSC is composed of 0.03M trisodium citrate C 6 H 5 Na 3 O 7 ·2H 2 O and 0.3M NaCl. The buffer solution needs to be preheated before use; preferably, the temperature of the preheating is 37 ℃. The buffer is immersed in a kohlrabi jar. The time for immersing the buffer solution is 30-40 min; specifically, the time period may be 30 to 35 minutes, 35 to 38 minutes, 38 to 40 minutes, or the like. The effect of the buffer addition is to age the G-tape slide, which helps to preserve the morphology of the cells.
In step 1), dehydration is carried out by soaking in ethanol with different concentrations. The concentration of ethanol is selected from 70% (v/v), 85% (v/v), or 100% (v/v). Dewatering, namely soaking in the ethanol with low concentration to the ethanol with high concentration in sequence; when dehydrating, soaking in ethanol with each concentration for 2-5 min. In one embodiment of the present application, dehydration is performed by immersing the G-tape slide in 70%,85% and 100% alcohol for 2 minutes each.
In the processing method provided by the application, the step 2) refers to that the G-banding glass slide processed in the step 1) is dehydrated and dried, a probe is added for hybridization reaction, the glass slide is dried after being washed, and a coloring agent is added for microscopic examination. Wherein, in the step 2), the probe is selected from one or a combination of a plurality of TEL/AML1, AML1/ETO, MYC, BCL-2 and D13S 25.
In the step 2), the melting temperature of the hybridization reaction is 72-95 ℃; specifically, the temperature may be 72 to 85 ℃,85 to 92 ℃, 92 to 95 ℃, or the like. The melting time of the hybridization reaction is 2-5 min; specifically, the time period may be 2 to 3 minutes, 3 to 4 minutes, or 4 to 5 minutes. In the hybridization reaction, the melting temperature is insufficient, the denaturation is insufficient, the hybridization rate is low, and the signal is weak; too high a temperature may lead to erosion of the nucleus. The melting temperature of the hybridization reaction of the application is slightly higher than that of the conventional FISH, and the denaturation temperature of the conventional FISH Vysis probe in the specification is 72 ℃.
In step 2), the film is washed with an eluent including SSC and NP40. In some embodiments, the washing comprises two elution steps, a first elution and a second elution, respectively. The eluent used in the first elution is 0.4 XSSC/0.3% NP-40 eluent, the first elution is carried out in a water bath kettle, the temperature of the water bath kettle is 72+/-1 ℃ water bath kettle, and the first elution time is 2min. The second elution was performed at room temperature using 2 XSSC/0.1% NP-40 eluent for a second elution time of 1min. The purpose of the elution in two steps is to wash away excess unbound and non-specifically bound probe fragments, effectively reducing the hybridization background. .
In step 2), the drying is performed in the dark, since the fluorescent signal in the probe cannot be exposed, otherwise the fluorescent signal is quenched.
In step 2), the staining agent is selected from DAPI. The stain is used to stain cells for ease of observation under a microscope.
In step 2), the proportion of positive cells was counted by microscopic examination.
In another aspect, the present application provides a G-tape slide, which is processed by the aforementioned processing method. The G-tape slide processed by the method can be combined with the FISH detection characteristic, so that the reutilization of waste glass of the G-tape slide is realized, and the possibility of a biological detection sample is further enlarged.
In another aspect, the present application provides the use of the aforementioned treatment method, or the aforementioned G-tape slide, in the preparation of a chromosome detection product. According to the method, the defect of nuclear analysis of the conventional tape can be overcome by carrying out the FISH detection on the G-tape slide, the subtle deletion or hidden translocation is detected, the repeated collection of bone marrow fluid is avoided, and the pain brought to patient puncture is reduced. And for patients with less bone marrow fluid taking amount, dry pumping or need to do a plurality of detection items caused by certain diseases, the sample amount is saved, and the detection items are enlarged.
The present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.
Example 1
Acute Lymphoblastic Leukemia (ALL): as shown in FIG. 1A, the conventional banding nuclear analysis suspected of chromosome 12 structural abnormalities, and no cell suspension remained, was used for molecular fluorescence in situ hybridization with a G-banding slide, as follows:
1) Taking a G-banding slide;
2) The slide was put in xylene to remove the microscope oil and immersed for 2 minutes;
3) Preparing 160ml of 3:1 methanol/glacial acetic acid fixing solution;
4) Preheating 2 XSSC in a water bath kettle at 37 ℃;
5) Removing xylenes from the slide but preventing the slide from drying out;
6) The slides were placed on small petri dishes and rinsed with 3:1 fixative for 2 minutes;
7) Pouring the fixing liquid out, and then pouring fresh fixing liquid for washing for 3 times;
8) The slide was placed in a Colprin cylinder of 2 XSSC at 37℃for 30 minutes;
9) Soaking the slide in 70%,85% and 100% ethanol for 2min, dehydrating, and air drying;
10 Adding probe TEL/AML1 (Vysis LSI ETV6 (TEL)/RUNX 1 (AML 1) ESDual Color Translocation Probe), hybridizing in a hybridization instrument, and selecting a procedure of denaturation at 85 ℃ for 2 minutes and hybridization at 37 ℃ for 10-18 hours;
11 Film-washing: a water bath kettle at 72+/-1 ℃, 0.4 XSSC/0.3% NP-40 eluent, and gently soaking and washing for 2 minutes, and 2 XSSC/0.1% NP-40 eluent at room temperature and gently soaking and washing for 1 minute;
12 Taking out the slide, naturally drying in dark, adding DAPI staining solution, performing microscopic examination, and counting the proportion of positive cells.
The results are shown in FIG. 1B, which shows the results of hybridization microscopy of TEL/AML1 bicolor ES probes on a G-tape slide: 85% of the cells had a TEL gene deletion.
Example 2
Acute Myeloid Leukemia (AML): as shown in fig. 2A, a conventional banding pattern analysis suspected of complex three-position chromosomal translocations, and no cell suspension remained, was performed using G-banding slides and molecular fluorescence in situ hybridization as follows:
1) Taking a G-banding slide;
2) The slide was put in xylene to remove the microscope oil and immersed for 2 minutes;
3) Preparing 160ml of 3:1 methanol/glacial acetic acid fixing solution;
4) Preheating 2 XSSC in a water bath kettle at 37 ℃;
5) Removing xylenes from the slide but preventing the slide from drying out;
6) The slides were placed on small petri dishes and rinsed with 3:1 fixative for 2 minutes;
7) Pouring the fixing liquid out, and then pouring fresh fixing liquid for washing for 3 times;
8) The slide was placed in a Colprin cylinder of 2 XSSC at 37℃for 30 minutes;
9) Soaking the slide in 70%,85% and 100% ethanol for 2min, dehydrating, and air drying;
10 Probe AML1/ETO (Vysis LSI AML1/ETO Dual Color, dual Fusion Translocation Probe) was added and hybridized in a hybridization apparatus using a melting procedure of 85 ℃/2 minutes;
11 Film-washing: a water bath kettle at 72+/-1 ℃, 0.4 XSSC/0.3% NP-40 eluent, and gently soaking and washing for 2 minutes, and 2 XSSC/0.1% NP-40 eluent at room temperature and gently soaking and washing for 1 minute;
12 Taking out the slide, naturally drying in dark, adding DAPI staining solution, performing microscopic examination, and counting the proportion of positive cells.
The results are shown in FIG. 2B, which shows the results of hybridization microscopy of AML1/ETO bicolor dual-fusion probes on a G-tape slide: 88% of the cells have AML/ETO fusion genes present and have three or more chromosomal complex translocations.
Example 3
High grade B cell lymphoma (HGBL): as shown in FIG. 3A, conventional banding pattern analysis suspected of MYC and BCL2 gene locus abnormalities, and no cell suspension remained, using a G-banding slide as MYC and BCL2 rearrangement probes.
1) Taking two G-banding slides;
2) The slide was put in xylene to remove the microscope oil and immersed for 2 minutes;
3) Preparing 160ml of 3:1 methanol/glacial acetic acid fixing solution;
4) Preheating 2 XSSC in a water bath kettle at 37 ℃;
5) Removing xylenes from the slide but preventing the slide from drying out;
6) The slides were placed on small petri dishes and rinsed with 3:1 fixative for 2 minutes;
7) Pouring the fixing liquid out, and then pouring fresh fixing liquid for washing for 3 times;
8) The slide was placed in a Colprin cylinder of 2 XSSC at 37℃for 30 minutes;
9) Soaking the slide in 70%,85% and 100% ethanol for 2min, dehydrating, and air drying;
10 Two slides were hybridized with probes MYC (Vysis LSIMYC Dual Color, break Apart Rearrangement Probe) and BCL2 (Vysis LSI BCL2 Dual Color, break Apart Rearrangement Probe), respectively, using a melting procedure of 85 ℃/2 minutes in a hybridization apparatus;
11 Film-washing: a water bath kettle at 72+/-1 ℃, 0.4 XSSC/0.3% NP-40 eluent, and gently soaking and washing for 2 minutes, and 2 XSSC/0.1% NP-40 eluent at room temperature and gently soaking and washing for 1 minute;
12 Taking out the slide, naturally drying in dark, adding DAPI staining solution, performing microscopic examination, and counting the proportion of positive cells.
The results are shown in FIG. 3B, which shows the results of hybridization microscopy of MYC and BCL2 two-color separation probes on a G-tape slide: 92% MYC gene rearrangement and 90% BCL2 gene rearrangement.
Example 4
Chronic Lymphocytic Leukemia (CLL): as shown in FIG. 4A, the conventional banding pattern analysis had only 3 split phases, few split phases, and no cell suspension remained, and the G-banding slide was used as the D13S25 probe.
1) Taking a G-banding slide;
2) The slide was put in xylene to remove the microscope oil and immersed for 2 minutes;
3) Preparing 160ml of 3:1 methanol/glacial acetic acid fixing solution;
4) Preheating 2 XSSC in a water bath kettle at 37 ℃;
5) Removing xylenes from the slide but preventing the slide from drying out;
6) The slides were placed on small petri dishes and rinsed with 3:1 fixative for 2 minutes;
7) Pouring the fixing liquid out, and then pouring fresh fixing liquid for washing for 3 times;
8) The slide was placed in a Colprin cylinder of 2 XSSC at 37℃for 30 minutes;
9) Soaking the slide in 70%,85% and 100% ethanol for 2min, dehydrating, and air drying;
10 Adding probe D13S25 (Vysis LSID13S25 (13 q 14) SpectrumOrange Probe), hybridizing in a hybridization instrument, and adopting a melting program of 85 ℃/2 minutes;
11 Film-washing: a water bath kettle at 72+/-1 ℃, 0.4 XSSC/0.3% NP-40 eluent, and gently soaking and washing for 2 minutes, and 2 XSSC/0.1% NP-40 eluent at room temperature and gently soaking and washing for 1 minute;
12 Taking out the slide, naturally drying in dark, adding DAPI staining solution, performing microscopic examination, and counting the proportion of positive cells.
The results are shown in FIG. 4B, which shows the hybridization microscopy results of D13S25 single-color probes on G-tape slides: 89% of the D13S25 gene was deleted.
In conclusion, the treatment method can carry out molecular fluorescence in-situ hybridization on the G-banding slide, avoid repeatedly collecting bone marrow fluid and reduce pain caused by patient puncture. And for patients with less bone marrow fluid taking amount, dry pumping or need to do a plurality of detection items caused by certain diseases, the sample amount is saved, and the detection items are enlarged. The method can make up for the defect of performing karyotyping analysis by conventional banding, and can detect microdeletion or hidden translocation. The application realizes the reutilization of the waste glass slide of the G-developing glass slide, and further expands the sample possibility of biological detection.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those of ordinary skill in the art without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications and variations which can be accomplished by persons skilled in the art without departing from the spirit and technical spirit of the present disclosure shall be covered by the claims of this application.

Claims (10)

1. A method of performing molecular fluorescence in situ hybridization on a G-banded slide, comprising the steps of:
1) Immersing the G-tape slide in an organic solvent, immersing and washing the G-tape slide in a fixing solution and immersing the G-tape slide in a buffer solution;
2) Dehydrating and drying the G-banding slide processed in the step 1), adding a probe for hybridization reaction, washing the slide, drying, and adding a coloring agent for microscopic examination.
2. The process according to claim 1, wherein in step 1), the time of immersion of the organic solvent is 1 to 3 minutes;
and/or, in step 1), the organic solvent comprises one or more of xylene, diethyl ether, ethanol.
3. The process of claim 1, wherein in step 1), the fixative solution comprises methanol and glacial acetic acid;
further, the volume ratio of the methanol to the glacial ethanol is 3:1, a step of;
and/or, the fixing liquid submerges the G-tape slide; preferably, the fixative solution is 40-50 mL for each G-tape slide.
4. The method of claim 1, wherein in step 1), the buffer is selected from SSC, HEPES, SSPE, PIPES, TMAC, TRIS, SET, potassium phosphate, citric acid, or sodium pyrophosphate;
and/or, in the step 1), the concentration of the buffer solution is 0.5-50 x; preferably, the concentration of the buffer is 2x;
and/or, in step 1), the buffer solution needs to be preheated before use; preferably, the temperature of the preheating is 37 ℃;
and/or, in the step 1), immersing the buffer solution in a Columba cylinder;
and/or, in the step 1), the time for immersing the buffer solution is 30-40 min.
5. The process of claim 1, wherein in step 2) the dehydration is soaking in ethanol of different concentrations;
preferably, the concentration of ethanol is selected from 70% (v/v), 85% (v/v), or 100% (v/v); the dehydration is carried out by soaking in the ethanol with low concentration to the ethanol with high concentration in sequence; when dehydrating, soaking in ethanol with each concentration for 2-5 min.
6. The method of claim 1, wherein in step 2) the probe is selected from the group consisting of one or more of TEL/AML1, AML1/ETO, MYC, BCL2, D13S 25.
7. The method according to claim 1, wherein in step 2), the melting temperature of the hybridization reaction is 72 to 95 ℃; the melting time of the hybridization reaction is 2-5 min.
8. The process of claim 1, wherein in step 2) the film is washed with an eluent comprising SSC and NP40;
and/or, in step 2), the drying is performed in the dark;
and/or, in step 2), the staining agent is selected from DAPI;
and/or, in step 2), the proportion of positive cells is counted by microscopic examination.
9. A G-tape slide treated by the treatment method according to any one of claims 1 to 8.
10. Use of the treatment method according to any one of claims 1 to 8, or the G-banding slide according to claim 9, in the preparation of a chromosome detection product.
CN202311179445.6A 2023-09-12 2023-09-12 Method for carrying out molecular fluorescence in-situ hybridization treatment on G-banding glass slide Pending CN117247995A (en)

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