CN111269983A - FISH detection method, probe and kit for bladder cancer gene amplification - Google Patents

FISH detection method, probe and kit for bladder cancer gene amplification Download PDF

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CN111269983A
CN111269983A CN202010108375.5A CN202010108375A CN111269983A CN 111269983 A CN111269983 A CN 111269983A CN 202010108375 A CN202010108375 A CN 202010108375A CN 111269983 A CN111269983 A CN 111269983A
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frs2
bladder cancer
gene
amplification
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吴松
欧铜
蒋文祺
徐银燕
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Shenzhen Following Precision Medicine Research Institute Technical Director
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    • 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
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Abstract

The invention discloses a FISH detection method, a probe and a kit for bladder cancer Gene amplification, wherein a group of bicolor FISH probes for detecting bladder cancer related FRS2 Gene (NCBI Gene ID:10818) amplification are (1) an FRS2 Gene probe marked red and (2) a chromosome 12 centromere probe marked green. The FISH detection method for bladder cancer related FRS2 gene amplification comprises the steps of carrying out conventional pretreatment on urine exfoliated cells and bladder cancer tissue paraffin sections, carrying out in-situ hybridization by adopting a group of bicolor FISH probes, and judging the amplification condition of the bladder cancer related FRS2 gene according to a detected fluorescence signal. The method is simple to operate, high in specificity and sensitivity, capable of rapidly and intuitively detecting the amplification state of the FRS2 gene related to the bladder cancer, accurate and reliable, and therefore capable of assisting bladder cancer patients in clinical application in the aspects of early urine noninvasive diagnosis, postoperative recurrence prediction and detection and the like.

Description

FISH detection method, probe and kit for bladder cancer gene amplification
Technical Field
The invention relates to the technical field of gene detection, in particular to a FISH detection method, a probe and a kit for bladder cancer gene amplification.
Background
Bladder cancer is a common malignant tumor of the urinary system. Currently, clinical diagnostic methods for bladder cancer involve cystoscopy, routine examination of urine, cytological analysis of urine shedding, examination of urine tumor markers, abdominal and pelvic B-ultrasonography, and the like. Research on urinary bladder cancer markers provides multiple detection indexes for noninvasive diagnosis of bladder cancer, and BTastat, NMP22, Fluorescence In Situ Hybridization (FISH) and the like are approved by the U.S. food and drug administration for auxiliary diagnosis of bladder cancer. Research has shown that the FISH technology has higher sensitivity and specificity. At present, FISH is mainly used for detecting chromosome 3, 7 and 17 aneuploidy and p16 gene amplification conditions to assist in diagnosing bladder cancer clinically. With the deep research of tumor genomics, a series of gene detection methods developed based on FISH technology play an important role in the field of tumor diagnosis and treatment. Recent bladder cancer omics studies have revealed that FRS2 (fibroblast growth factor receptor substrate 2) gene has specific copy number amplification and high mutation frequency in bladder cancer. Therefore, the detection of FRS2 amplification by FISH method can be used as an important index for auxiliary diagnosis of bladder cancer.
FRS2 is an adaptor protein, which can bind to various receptors related to cell growth regulation and mediate the activation process of downstream signaling pathways, thereby playing important biological functions. Research has revealed that FRS2 copy number amplification leads to high expression of its encoded protein in tumor tissues and poor prognosis association. Therefore, the FRS2 amplification condition detected by FISH can be used as an important target molecule for pathological diagnosis of bladder cancer.
Disclosure of Invention
In view of the above important significance of detecting FRS2 amplification in bladder cancer diagnosis and treatment and the lack of a detection method with simple operation and stable and intuitive result, the invention aims to provide an FRS2 gene amplification detection method based on FISH technology, which is used for urine noninvasive diagnosis of bladder cancer and postoperative recurrence monitoring of bladder cancer.
The technical scheme of the invention is as follows:
a set of two-color FISH probes for detecting bladder cancer associated FRS2 gene amplification, wherein the set of two-color FISH probes are (1) FRS2 gene probe labeled red and (2) centromere probe of chromosome 12 labeled green. Wherein FRS 2: NCBI Gene ID 10818; sequence reference: NC-000012.12; website address: https:// www.ncbi.nlm.nih.gov. Further, the FRS2 gene probe labeled red was prepared by nick translation method, and the centromere probe of chromosome 12 labeled green was prepared by PCR method.
A FISH detection kit for detecting bladder cancer-associated FRS2 gene amplification, wherein the FISH detection kit comprises a set of bi-color FISH probes, wherein the set of bi-color FISH probes is (1) FRS2 gene probe labeled red and (2) centromere 12 probe labeled green.
A FISH detection method of bladder cancer-associated FRS2 gene amplification, wherein the FISH detection method comprises: pretreating a urine cast-off cell and a paraffin section of a bladder cancer tissue, carrying out in-situ hybridization by adopting a group of bicolor FISH probes, and judging whether the amplification of the FRS2 gene related to the bladder cancer exists or not according to a detected fluorescent signal; wherein the set of bi-color FISH probes are (1) FRS2 gene probe labeled red and (2) chromosome 12 centromere probe labeled green.
Has the advantages that: the FISH detection method for bladder cancer related FRS2 gene amplification comprises the steps of carrying out conventional pretreatment on urine exfoliated cells and bladder cancer tissue paraffin sections, carrying out in-situ hybridization by adopting a group of two-color FISH probes, and judging the amplification condition of the bladder cancer related FRS2 gene according to a detected fluorescence signal. The method is simple to operate, high in specificity and sensitivity, capable of rapidly and intuitively detecting the amplification state of the FRS2 gene related to the bladder cancer, accurate and reliable, and therefore capable of assisting bladder cancer patients in clinical application in the aspects of early urine noninvasive diagnosis, postoperative recurrence prediction and detection and the like.
Drawings
FIG. 1 is a diagram of the expansion result of a paraffin section FRS2 of a tumor tissue of a patient with bladder cancer.
Detailed Description
The invention provides a FISH detection method, a probe and a kit for bladder cancer gene amplification, and the invention is further detailed below in order to make the purpose, the technical scheme and the effect of the invention clearer and more clear. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In order to facilitate the understanding of the technical scheme of the invention, the FISH detection method for detecting the amplification condition of the bladder cancer related gene FRS2 is further described by combining specific test examples, which comprises the following specific steps:
firstly, preparation of probe working reagent
1. Preparation method of dATP \ TTP \ CTP \ GTP (10mM dATP/CTP/GTP, 5mM dTTP):
Figure BDA0002389155250000031
mixing, packaging, and storing at-20 deg.C at 5 μ L/tube.
2. Preparation method of dATP \ TTP \ CTP \ GTP (1mM dATP/CTP/GTP, 0.5mM dTTP):
10mM dATP/CTP/GTP、5mM dTTP 10μL
0.1M Tris-HCl(pH 8.0) 90μL
quickly mixing and centrifuging, and using the preparation when in use.
3. Preparation method of 10 × TE:
Tris 1.21g
EDTA 0.292g
80mL of deionized water
Adjusting pH to 8.0 with 1M HCl, diluting to 100mL with deionized water, filtering with 0.45 micrometer filter membrane, and storing at 4 deg.C.
4. Preparation method of 1 × TE:
10×TE 1mL
deionized water 9mL
Mixing, centrifuging, packaging, and storing at 4 deg.C with 1 mL/tube.
5. The preparation method of the hybrid Buffer comprises the following steps:
Figure BDA0002389155250000041
during preparation, the dextran sulfate is dissolved firstly, 20 XSSC is added, the mixture is dissolved in water bath at 60 ℃, then other reagents are added, finally the volume is determined to be 40mL, the mixture is subpackaged, and the mixture is stored at minus 20 ℃ in 1 mL/tube.
6. Primer and method for producing the same
The sequence of the upstream primer is as follows: GAACGCTAGAAAGAAGAATACTG
The sequence of the downstream primer is as follows: CATTTGGCCTAAAAGCGCTT
Dissolving with TE for later use.
Second, obtaining Probe DNA
1. And (3) bacterial liquid amplification culture: 10uL of BAC strain carrying FRS2 gene was added to 20mL LB medium containing chloramphenicol (25ug/mL), and cultured overnight at 200rpm on a shaker at 37 ℃.
2. Extraction of FRS2 plasmid DNA: mass extraction and purification of plasmid DNA was carried out according to the instructions of the OMEGA D2156 kit.
3. Extracting cell genome DNA of the bladder cancer cell line BIU-87: the extraction and purification of the cell genome DNA was carried out according to the protocol of MAGEN Universal DNA extraction kit.
Third, probe labeling
1. Preparation of FRS2 gene probe: the reaction system was prepared on ice using the nick translation method under strict light-shielding conditions according to the following protocol.
Figure BDA0002389155250000042
Figure BDA0002389155250000051
After the preparation, the mixture is blown and beaten by a pipette, is centrifuged, and is reacted for 2 hours at 15 ℃ (precooling) and 10 minutes at 80 ℃ on a PCR instrument.
2. Preparing a chromosome 12 centromere probe: the reaction system was prepared on ice under stringent conditions in the absence of light according to the following protocol.
Figure BDA0002389155250000052
After the preparation is finished, uniformly mixing the materials by blowing with a gun head, centrifuging, and reacting on a PCR instrument, wherein the reaction procedure is as follows:
Figure 1
Figure BDA0002389155250000061
3. probe purification
Taking out the reaction solution, sequentially adding 50uL of sterile water and 10uL of 3M NaAc, mixing, performing instantaneous centrifugation, adding 250uL-20 ℃ precooled 100% absolute ethyl alcohol, mixing, performing instantaneous centrifugation, and standing at-20 ℃ overnight.
The next day, the reaction solution was taken out, centrifuged at 12000r/min for 15mins at 4 ℃, and after centrifugation, the supernatant was removed, leaving a precipitate. Adding 600uL of 70% ethanol precooled at 4 ℃, inverting for several times, centrifuging for 12000g/min, 12mins at 4 ℃, finishing the centrifugation, removing supernatant, and leaving precipitate (repeating the step once). And placing the precipitate in a dark condition, and naturally drying for about 10 mins. The precipitate was dissolved in 10uL of 1 XTE Buffer and stored at-20 ℃ in the dark.
4. Probe mixture preparation
The formula of each 2 persons is as follows:
Figure BDA0002389155250000062
example one (detecting the FRS2 expansion of urine exfoliated cells in one month after operation of bladder cancer patients; the collection and detection of clinical samples were examined and informed patient consent was obtained by the medical ethics committee of Renshen's lake region people Hospital):
1. sample collection and slide preparation
1.1, taking urine in a 50mL centrifuge tube, and centrifuging for 20mins at room temperature and 1500 g.
1.2 discard part of the supernatant, leave about 1mL of supernatant in the tube, and take care not to agitate the cell pellet.
1.3, resuspend the cell pellet from the remaining 1mL of supernatant and transfer it to a 15mL centrifuge tube, rinse the 50mL centrifuge tube with 10mL of 1 XPBS, and transfer the rinse to the cell suspension.
1.4, centrifuging at 1500g for 10mins at room temperature.
1.5 discard part of the supernatant, leave about 500uL of supernatant in the tube, taking care not to agitate the cell pellet.
1.6, after the cell precipitation is resuspended, 5mL of KCl hypotonic buffer solution is added, the cells are blown gently, and the mixture is bathed for 20mins at 37 ℃.
1.7, 3mL of fresh fixative (methanol: glacial acetic acid ═ 3:1) was added slowly, dropwise and with stirring, -20 ℃ for at least 30 mins.
At room temperature, 1500g were centrifuged for 5mins and the supernatant carefully removed.
1.9 resuspend the cell pellet with 3mL of fixative.
At room temperature, 1500g were centrifuged for 5mins, 1.10, and the supernatant carefully removed. Step 1.8 and step 1.9 are repeated twice.
1.11, after centrifugation of the fixed cell suspension: if the cell particles are small and barely visible, then as much fixative as possible is removed with care, leaving approximately 100uL of solution; if the cell particles are readily visible, as much fixative is removed as possible and 0.8mL of fresh fixative is added to the cell pellet.
1.12 resuspend the cell particles and drop 3uL, 10uL, 30uL of the cell suspension onto different slides (numbers 1, 2, 3).
1.13, drying the glass slide at room temperature.
1.14, observing the slide by using a 20-fold optical microscope, and selecting a hybridization area which is visible by about 100-200 cells in a visual field range; if the cell density on the slide glass with the number 3 is too low, then dripping 30uL of cell suspension, drying at room temperature, observing the cell density under an optical microscope, and repeatedly dripping when necessary; if the cell density on slide number 1 is too high, the cell suspension is diluted and steps 1.12-1.14 are repeated.
2. Slide pretreatment procedure:
2.1 pretreatment of urine exfoliated cell slides
2.1.1, preparing a pepsin solution: 50mL of pepsin buffer solution is placed in a water bath kettle at the temperature of 37 +/-1 ℃, 0.1g of pepsin is dissolved by the prepared pepsin solution 10 minutes before use, and then the dissolved pepsin solution is completely added into 50mL of pepsin solution and mixed evenly.
2.1.2 and baking at 56 ℃ for 20 mins.
2.1.3 slides were placed in 2 XSSC buffer for 2mins and repeated once.
2.1.4, placing the slide into a pepsin solution with the temperature of 37 +/-1 ℃ for digestion for 5 mins.
2.1.5, taking out the slide, putting the slide into 70 percent, 90 percent and 100 percent gradient ethanol for 2mins respectively, taking out the slide, and airing the slide at room temperature in a dark place.
2.1.6, denaturing hybridization: taking out the probe from the refrigerator, returning the temperature to room temperature, instantaneously centrifuging to enable the probe to sink to the bottom of the tube, then uniformly mixing in a vortex mode, and centrifuging. Add 10uL of probe to the hybridization region.
2.1.7, cover the glass slide (care to avoid air bubbles), and seal the glass slide edge with coverslipping glue.
2.1.8, soaking the moisture-keeping strip in pure water and placing the moisture-keeping strip in a corresponding clamping groove of the hybridization instrument.
2.1.9, the procedure for setting up the denaturing hybridization was 73 ℃, 3mins, 37 ℃, overnight.
2.1.10, post-hybridization wash: 50mL of Wash Buffer I (0.3% NP40/0.7 XSSC) was poured into a Cooprin bottle and used at room temperature (Wash Buffer I was used only on the day and discarded).
2.1.11 50mL of Wash Buffer II (0.1% NP40/2 XSSC) was poured into a Cooprine bottle, and the Cooprine bottle was placed in a water bath, and then heated to 74. + -. 1 ℃ with heating in the water bath, ensuring that the Wash Buffer II reached 74. + -. 1 ℃ for at least 30mins before use. (Wash Buffer II is used only on the day, and is discarded after use).
2.1.12 the coverslip was gently removed from the section by forceps and the section was placed in a Wash Buffer II at room temperature for 10mins to allow the coverslip to slide off gently.
2.1.13 the slices are put into a Wash Buffer I at 72 +/-1 ℃, pulled gently for 1-3 seconds, timed for 2mins and taken out.
2.1.14 putting the slices into Wash Buffer II, gently pulling for 1-3 seconds, timing for 2mins, and taking out
2.1.1570%, 85% gradient ethanol for 2mins each.
2.1.16 the slide is removed and the section is left to air dry vertically in the dark.
2.1.17 counterstaining and Observation: and (3) dropwise adding 10uLDAPI repolarization solution into the target area on the section, and covering a cover glass. The sections were placed in a refrigerator at-20 ℃ for 30mins and observed again. As a result, as shown in table 1 below, since the probe signal FRS2/12 chromosome centromere is 0.93<2.0, the sample FRS2 was amplified negatively.
TABLE 1 FISH DETECTION STATISTATIONAL TABLE
Figure BDA0002389155250000091
Example two (detection of FRS2 amplification in paraffin section of tumor tissue of bladder cancer patients; clinical sample collection and detection were reviewed and informed patient consent was obtained by the medical ethics committee of Renshen's Hospital, Roche, Shenzhen, Inc.:
1. paraffin section processing
Baking at about 1.1 deg.C and 65 deg.C for more than three hours.
1.2, preparing a pepsin treatment solution in advance, adding 0.1g of pepsin powder into 50mL of the pepsin treatment solution, and preheating to ensure that the temperature reaches 37 ℃ for later use.
1.3, placing the slices in xylene for 10mins at room temperature, and repeating the step twice.
1.4, 100% ethanol at room temperature for 5mins, repeat the procedure once.
1.5, processing the slices in 85 percent ethanol and 70 percent ethanol in sequence at room temperature for 3 mins.
1.6, placing the mixture in deionized water for 2mins at room temperature, and repeating for 3 times.
1.7, boiling a proper amount of pretreatment buffer solution by using a microwave oven, keeping the pretreatment buffer solution in a heat preservation state, and putting the pretreatment buffer solution into slices for high-pressure repair for 5 mins.
1.8, take out the slide, wash with deionized water three times. The slices were directly placed in pepsin digestion solution and digested for 25 mins.
1.9, dehydration: sequentially treating the raw materials in 70%, 85% and 100% ethanol solution for 2mins, and air drying.
1.10, denaturing hybridization: the hybridization solution containing the FRS2/12 chromosome centromere probe was removed from the refrigerator, centrifuged instantaneously, 10uL of the resulting solution was pipetted into each sample, and added dropwise to the target region.
1.11, the sample is covered with a 18mm × 18mm cover glass to avoid air bubbles, and the sample is mounted with mounting glue.
1.12, putting the slide into a hybridization instrument, denaturing at 80 ℃ for 6mins, and hybridizing at 40 ℃ overnight.
Wash I was heated to 73 ℃ in a water bath 30mins earlier 1.13 and the next day, and the mounting gel was carefully removed with tweezers.
1.14, placing the section in a washing solution II, placing for 10mins, and removing the cover glass.
1.15, placing the slices in a washing solution I (heated to 73 ℃ in a water bath), pulling the slices up and down for 3s, and placing for 2 mins.
And (5) placing the slices in a washing solution II at room temperature, pulling up and down for 3s, and treating the slices for 3 mins.
1.17, placing the mixture in 70 percent and 85 percent ethanol solution for 2mins in sequence.
1.18, drying the slices in the dark.
1.19, dripping 10uL of DAPI compound dyeing solution on the sliced tissues, covering a cover glass to avoid bubbles, and treating for 15mins at-20 ℃ in a dark place.
1.20, and observing and counting under a fluorescence microscope. The results are shown in FIG. 1 (in the figure, the long arrow indicates FRS2 probe signal, the short arrow indicates chromosome 12 probe signal, and the dotted line indicates one cell), and since the picture is black and white, FRS2 indicates a gray point in the figure, and the long arrow indicates cluster-like amplification. Chromosome 12 centromere is a round highlight in the figure, indicated by the short arrow. A nucleus is shown within the dotted line. According to the microscopic photographing result, the FRS2 of the sample is amplified in a cluster shape, and the FRS2 is amplified positively.
Analysis of FISH assay results
Counting 25 cells, and counting the Ratio value (25 total red signals in cell nucleus/═ 25 total green signals in cell nucleus)
Common types of anomalies: FRS2 site amplification.
1. Normal cells: red and green signals were 2 each in a single interphase nucleus.
FRS2 gene amplification abnormal cells: the red signal in the nucleus of a single interphase cell is more than 2, and the green signal is not less than 2.
Reference ranges: when the ratio of the FRS2/12 chromosome centromere is more than or equal to 2.0, the positive result indicates that the FRS2 gene of the sample is amplified;
when the ratio of the FRS 2/chromosome 12 centromere is less than 2.0, the result is positive when the average FRS2 copy number/cell is more than or equal to 6.0;
a negative result when the chromosome centromere ratio of FRS2/12 <2.0 and the average FRS2 copy number/cell < 4.0;
when the chromosome centromere ratio of FRS2/12 is less than 2.0 and the average FRS2 copy number/cell is less than 6.0, but not less than 4.0, the result of FRS2 FISH is uncertain. One can choose to increase the counted cells to 100 re-counts.
In conclusion, the invention provides a FISH detection method, a probe and a kit for bladder cancer gene FRS2 amplification. The FISH detection method for bladder cancer related FRS2 gene amplification comprises the steps of carrying out conventional pretreatment on urine exfoliated cells and bladder cancer tissue paraffin sections, carrying out in-situ hybridization by adopting a group of two-color FISH probes, and judging the amplification condition of the bladder cancer related FRS2 gene according to a detected fluorescence signal. The method is simple to operate, high in specificity and sensitivity, capable of rapidly and intuitively detecting the amplification state of the FRS2 gene related to the bladder cancer, accurate and reliable, and therefore capable of assisting in clinical application of bladder cancer urine noninvasive diagnosis, postoperative recurrence monitoring and the like.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.

Claims (5)

1. A group of double-color FISH probes for detecting bladder cancer related FRS2 gene amplification, wherein the group of double-color FISH probes is (1) FRS2 gene probe marked with red and (2) chromosome 12 centromere probe marked with green.
2. The set of bi-color FISH probes for detecting bladder cancer-associated FRS2 gene amplification of claim 1, wherein the FRS2 gene probe labeled red is made using nick translation method.
3. The set of bi-color FISH probes for detecting amplification of bladder cancer-associated FRS2 gene according to claim 1, wherein the centromere 12 chromosome labeled green is prepared by PCR method.
4. A FISH detection kit for detecting bladder cancer-associated FRS2 gene amplification, comprising a set of bi-color FISH probes, wherein the set of bi-color FISH probes is (1) an FRS2 gene probe labeled red and (2) a centromere 12 chromosome probe labeled green.
5. A FISH detection method of bladder cancer associated FRS2 gene amplification, comprising: pretreating a urine cast-off cell and a paraffin section of a bladder cancer tissue, carrying out in-situ hybridization by adopting a group of bicolor FISH probes, and judging whether the amplification of the FRS2 gene related to the bladder cancer exists or not according to a detected fluorescent signal; wherein the set of bi-color FISH probes are (1) FRS2 gene probe labeled red and (2) chromosome 12 centromere probe labeled green.
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