CN107904283A - A kind of instant multiprobe hybridized slides and its preparation method and application - Google Patents

Abstract

The invention belongs to fluorescence in situ hybridization technique field, and in particular to a kind of instant multiprobe hybridized slides and its preparation method and application.The multiprobe hybridized slides are made of probe attachment slide, each probe, hybridization buffer and sample attachment slide；Each probe is respectively attached on each mutually independent probe attachment region in the form of lyophilized；Sample drop is added on sample attachment region, and when sample attachment region is bonded with probe attachment region, the probe of the sample of sample attachment region and corresponding probe attachment region realizes that hybridization combines.Tested compared to traditional FISH, the present invention can further open preset multigroup probe, and can complete the detection of a variety of probes on same slide again at the same time on slide, enormously simplify the operating procedure of traditional FISH；Reduce the use cost that the usage amount of probe reduces probe；Instant multiprobe hybridized slides of the present invention have high signal-to-noise ratio, specificity and sample recall rate.

Description

A kind of ready-to-use multi-probe hybridization glass slide and its preparation method and application

technical field

The invention belongs to the field of molecular biology, and in particular relates to a ready-to-use multi-probe hybridization glass slide and a preparation method and application thereof.

Background technique

Fluorescence in situ hybridization is a non-radioactive molecular genetic technique developed on the basis of radioactive in situ hybridization in the 1980s. It is a new in situ hybridization method formed by replacing radioactive isotope labels with fluorescein labels. FISH has the advantages of safety, rapidity, high sensitivity and simultaneous display of multiple colors.

At present, in the detection of hematology FISH, conventional slides are usually used, and the probes and slides are used separately. Usually, it is necessary to use blood samples to make slices first, and then perform a series of pretreatments on the slides. , then add the probe dropwise and seal the slide with rubber glue, and then carry out probe sample consistency and hybridization after sealing the slide; at present, there are dozens of FISH detection items for common blood diseases such as leukemia, such as CLL (chronic granulocyte Leukemia) requires detection of cMYC gene breaks, P16 deletions, E2A breaks, hyperdiploidy detection (CEP10/CEP17/CEP4), TEL/AML1 fusions, MLL breaks, BCR/ABL fusions, and IGH breaks to assess the type and process of the disease. If the existing hybridization technology needs to repeat multiple detections for diseases that require the detection of multiple probes, it will be time-consuming and labor-intensive. In addition, traditional FISH experiments use traditional FISH probes that require overnight hybridization and testing multiple items at one time is not only time-consuming, but also high costs increase the financial burden on patients.

Contents of the invention

The invention aims at the deficiencies of the prior art, and aims to provide a ready-to-use multi-probe hybridization glass slide and its preparation method and application.

For realizing above-mentioned purpose of the invention, the technical scheme that the present invention adopts is:

A ready-to-use multi-probe hybridization slide kit consisting of a probe-attached slide, each probe, a hybridization buffer, and a sample-attached slide; the probe-attached slide contains several independent probes In the attachment area, each probe is attached to each independent probe attachment area in a freeze-dried form; the sample attachment slide contains several independent sample attachment areas, and the sample attachment area and the probe attachment area are one by one. Correspondingly, the sample is dropped on the sample attachment area, and when the sample attachment area is attached to the probe attachment area, the sample in the sample attachment area is hybridized with the probe in the corresponding probe attachment area.

In the above scheme, the probe attachment slide is composed of a probe information identification area, a probe attachment area, and a hybridization slide junction area, and the probe information identification area records the corresponding The probe type, the probe attachment region contains several independent probe attachment regions, the probe is attached to the probe attachment region in a lyophilized form, and the surface of the hybridization glass junction region is attached with acrylic acid Glue for probe-attached slide packaging and bonding to sample-attached slides.

In the above solution, the probe attachment area is a groove, and the groove contains several independent probe attachment blocks, and each independent probe attachment block is composed of a probe attachment area and a peripheral isolation area; There is a small isolation groove between each independent probe attachment block.

In the above scheme, the sample attachment slide is composed of a sample information recording area, a sample attachment area and a sample isolation area, the sample information is recorded in the sample information recording area, the sample is dropped on the sample attachment area, and the sample isolation area Zones are used to prevent cross-contamination of samples.

In the above scheme, the sample attachment area is made of hydrophilic material; the sample isolation area is attached with a hydrophobic coating.

In the above scheme, the components of the hybridization buffer are: 10wt% ethylene carbonate, 30wt% dextran sulfate, 1ugRNaseA, 0.6M sodium chloride, 10mM citrate buffer.

In the above scheme, the probe is a FISH gene locus probe or a FISH gene centromere/telomere probe.

In the above scheme, each probe is selected from the following combination A or combination B:

Combination A: BCR/ABL, IGH/BCL2, AML/ETO;

Combination B: PML/RARA, BCR/ABL, P53, D13S319, 1Q21, IGH/BCL2, CBFB, AML/ETO.

The preparation method of the above-mentioned ready-to-use multi-probe hybridization glass slide comprises the steps:

(1) Prepare probe-attached glass slides with each probe attached:

a. Prepare a probe-attached glass slide having the above-mentioned structure;

b. use deionized water to prepare excipients, the formulation of the excipients is: levoside 0.5wt%, acacia 3wt%, mannitol 1wt%, polyacrylate 0.3wt%;

c. Prepare each probe solution separately, mix each probe solution with excipients evenly, drop them respectively and smear them evenly on each independent probe attachment area on the probe attachment glass slide; then attach the probe attachment glass The slices were freeze-dried and sealed to prepare probe-attached glass slides with each probe attached;

(2) preparing a sample-attached glass slide having the above-mentioned structure;

(3) prepare hybridization buffer;

(4) A ready-to-use multi-probe hybridization slide is composed of the prepared probe-attached slide to which the probe is attached, the hybridization buffer, and the sample-attached slide.

The application of the above-mentioned ready-to-use multi-probe hybridization slide specifically includes the following steps:

(1) Sample processing: Add the fixed peripheral blood cells dropwise to the sample attachment area of the sample attachment slide, immerse the sample attachment slide in a container containing 2XSSC at 37°C for 30 minutes, heat it in a microwave oven for 3 minutes until liquid Boil, and then continue to heat for 10 minutes at medium-low heat; immediately after the treatment, place the slides in gradient alcohol pre-cooled at -20 degrees Celsius to dehydrate and dry, and set aside;

(2) Probe-sample covariance: Add hybridization buffer dropwise to the probe attachment area on the probe-attached glass slide, and reverse the sample-attached glass slide so that the sample attachment area and the probe attachment area fit together. After merging, invert the entire hybridization slide together with the attached sample slide, place in a metal bath at 73°C for denaturation for 5 minutes, and after the deformation is complete, place the hybridization device in a 37°C wet box for hybridization for 1 hour;

(3) Washing after hybridization: After the hybridization is completed, remove the probe-attached slide, and place the sample-attached slide in 60°C preheated washing solution (containing 0.3M sodium chloride, 0.03M sodium citrate and 0.1% Tween Wash in -20) to remove unbound probes; after washing, place the slide in distilled water at room temperature and wash again, and dry at room temperature;

(4) Counterstaining and microscopic examination: Add anti-fade mounting agent dropwise to the sample attachment area of the dried sample attached to the glass slide, seal the slide with a cover slip and observe the hybridization result under a fluorescent microscope.

The preparation method of FISH gene locus probe described in the present invention is as follows:

(1) Download the non-repeated genome sequence of the region covered by the probe from UCSC Genome Browser, and use the perl plug-in program chunks.pl to split the non-repeated sequence of the genome covered by the probe into 1kb blocks; Import blocks into OligoArray software in batches for probe design and probe screening, and then export the screened probes to the EXCEL table, and then add 18bp tag sequences to the 5' and 3' ends of each probe , to obtain a series of probe sequences with tag sequences;

(2) Use a DNA synthesizer to chemically synthesize the probe sequence with the tag sequence obtained in step (1), mix the chemically synthesized probes to prepare a probe library; synthesize the 5' end with a green fluorescent group respectively The M13 universal primer with a group and the M13 universal primer with a fluorescent group at the 5' end, use the M13 universal primer with a fluorescent group at the 5' end to amplify and label the probe library;

(3) The amplified labeled product of the probe library obtained in step (2) is purified and diluted to obtain a fluorescently labeled probe library.

In the above-mentioned scheme, the conditions for screening the probes described in step (1) are: probe length 50bp, TM value 85-99°C, GC ratio 40-80%, TTTT/GGGG/AAAA/CCCC not included, between probes The minimum interval is 5bp; the base sequence of the tag sequence in step (1) is as follows:

The 5' end tag sequence is: TGTAAAACGACGGCCAG (M13 upstream sequence)

The 3' end tag sequence is: GGTCATAGCTGTTTCCTG (M13 downstream sequence).

In the above scheme, the universal primer sequence described in step (2) is: M13-F TGTAAAACGACGGCCAGT; M13-R CAGGAAACAGCTATGACC.

In the above scheme, the PCR reaction system of the amplification labeling reaction described in step (2) is as follows:

In the above scheme, the PCR reaction conditions for the amplification and labeling reaction in step (2) are as follows: 95°C for 5 minutes; 94°C for 30s, 58°C for 30s, and 72°C for 30s for a total of 20 cycles; 72°C for 10 minutes.

The above method for preparing FISH gene locus probes is applicable to the preparation of FISH probes listed below, specifically including: ALK, BCL2, BCL3, BCL6, BCL10, BCL12, BCR, CCND1, E2A, EGFR, ETV6, FIP1L1, HER2, IGH, IGK, IGL, MALT1, MLL (ALL-1, HTRX1, HRX), MYC (c-Myc), PAX5, PDGFRA, PDGFRB, SIL, TCF3 (E2A, ITF1), TCL1A, TCRAD, TCRB, TCRG, TLX1 , TLX3(HOX11L2, RNX) or TOP2A, BASE, BRCA1, CCND1, CCNE1, DCD, E2F3, n-MYC/MYCN, COX-2/PTGS2, LRIG1, ERa, hTERT, MLN64/STARD3, PGR, SNAI1, SRC, TOP1, TUBB1, AIB1, DLC-1, EDD, Pip4k2b/5k, Sil, TBX2, c-Kit, VEGF, VCAM-1, Tie-1, Ts/TYMS, PSMA, PSA, PAP, P15, P16, BCL1, BCL2, MTOR, TIMP1, ESR1, PTEN, MDM2/CDK4, MET, C-MET, ERB1, FGFR1, IGF1R, NET, FGFR3, ABCB1, TMPRSS2, BRCA2, TOP2B, ERCC1, AKT1, AKT2, AKT3, HRAS, NRAS, RAF1, HER3, HER4, ENT1, RRM1, RRM2, RRM2B, PIK3CA, AURK4, AURKB, AURKC, MAPT/tau, TTBK1, TUBB, VEGFR, CCND3, CDK6, CDK2, CDC2, HDAC, ESR2, SCUBE2, BIRC5, FASN, DHFR, TP/ECGF1, TYMP, DPYD, TK1, HMGIC, ABCA2, ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCG2, MVP, ATP7A, ATP7B, SLC29A1, SLC28A1, SLC19A1, TUBB4, TUBA, MAP4, MAP7, STMN1, KIF5B, HSPA5, PSMD14, FPGS, GSTP1, GPX, GCLC, :ABL t(9;22)(q34;q11), PRDM16del(1p36.32)del(21q22.12), RUNX1/AML1del(1p36.32 )del(21q2 2.12), CEP8, PDGFRB, NUP98, FGFR1, ASS, ETO t(8;21)(q22;q22), AML1t(8;21)(q22;q22), CBFbeta inv(16)(p13q22)t(16; 16)(p13;q22), MYH11inv(16)(p13q22)t(16;16)(p13;q22), AF9t(9;11), PML t(15;17)(q22;q21), PLZF t( 11;17)(q23;q21), NuMAt(11;17)(q13;q21), NPMt(5;17)(q23;q12), RAR αt(15;17)(q22;q21)t(11; 17)(q23;q21)t(11;17)(q13;q21)t(5;17)(q23;q21), EVI1t(3;v)(q26;v).

The preparation method of FISH gene centromere and telomere probe described in the present invention is as follows:

(1) PCR amplification of specific fragments of centromere probes or telomeric probes;

(2) The above-mentioned specific fragments are marked by the nick translation method (the labeling system is a conventional nick translation body);

(3) The prepared centromere/telomere probe was purified by ethanol sodium acetate method, dissolved in deionized water and diluted to 100 ng/ul for later use.

The preparation method of the FISH gene centromere and telomere probes is applicable to the preparation of the following centromere/telomere probes, specifically: CEP1, CEP2, CEP3, CEP4, CEP5, CEP6, CEP7, CEP8, CEP9, CEP10, CEP11, CEP12, CEP13, CEP14, CEP15, CEP16, CEP17, CEP18, CEP19, CEP20, CEP21, CEP22, CEP23, CEP X, CEPY and telomeres.

In the present invention, the probes used are dissolved in the excipient composition and applied to the corresponding independent probe attachment areas with a pipette. The specific formulation of the excipient composition is: 0.5% levoside, 3% acacia, 1% mannitol, 0.3% polyacrylate, dissolved in deionized water, and stored at 4°C for later use.

When the multi-probe hybridization slide of the present invention is used specifically, 2 μl of hybridization buffer solution needs to be added dropwise to each probe attachment area of the probe attachment slide, and then the sample attachment slide is buckled upside down on the probe attachment slide , the sample and the probe were combined, and the combined hybridization slide was denatured on a metal bath at 73°C for 2min, and then hybridized in a wet box at 37°C for 2h.

Beneficial effects of the present invention:

(1) Compared with the traditional FISH experiment, the present invention can preset multiple groups of probes on one glass slide at the same time, and can complete the detection of multiple probes on the same slide, which greatly simplifies the operation steps of traditional FISH ;

(2) Compared with all existing technologies, the present invention reduces the usage of probes and reduces the usage cost of probes through unique design, combined with specific hybridization buffer and unique detection method;

(3) The ready-to-use multi-probe hybridization slide of the present invention has extremely high signal-to-noise ratio, specificity and sample detection rate;

(4) Compared with the preparation method of rapid fluorescent in situ hybridization probes without repetitive sequences established in recent years, the probe screening used in the present invention mainly depends on the completion of the program, and uses a more stable PCR method to amplify And labeled probes, and the probe labeling rate is higher and more uniform, only fluorescent labeling is carried out at the 5-end of the probe, so it does not affect the hybridization and pairing reaction of the probe; in addition, the present invention does not use a gap translation reaction that is easily affected by the environment or operating methods React with random primers to label the probes, the length of the labeled probes is consistent, the labeling yield is high and the stability between batches is increased.

Description of drawings

Fig. 1 is a plan view of the probe-attached slide of the multi-probe hybridization slide of the present invention.

Fig. 2 is the three-dimensional structure and size data of the probe-attached glass slide of the multi-probe hybridization slide of the present invention.

Fig. 3 is a plane view of a sample-attached slide of the multi-probe hybridization slide of the present invention.

Among them, 1 is the probe information identification area, 2 is the probe attachment area, 3 is the hybridization slide junction area, 4 is the probe attachment area, 5 is the peripheral isolation area, 6 is the isolation groove, and 7 is the sample information recording area , 8 is the sample attachment area, and 9 is the sample isolation area.

Fig. 4 is the code figure that chunks.pl program uses in embodiment 1.

Fig. 5 is a sequence diagram of part of the probes of the CBFB gene breakage detection kit in Example 1 after removing repetitive sequences.

Figure 6 is a schematic diagram of probe preparation.

7 is a schematic diagram of the ready-to-use multi-probe hybridization slide described in Example 3.

Figure 8 is a diagram of the detection results of the present invention.

Detailed ways

In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

As shown in accompanying drawings 1 to 3, a multi-probe hybridization slide is composed of two parts: a probe-attached slide (A) and a sample-attached slide (B). The probe attachment slide (A) is composed of a probe information identification area (1), a probe attachment area (2) and a hybridization slide junction area (3), and the probe attachment area (2) is a concave Groove, the groove contains several independent probe attachment blocks, each independent probe attachment block is composed of a probe attachment area (4) and a peripheral isolation area (5), each independent probe attachment area There are isolated small grooves (6) between the small blocks, and the probes are attached to the probe attachment area in a freeze-dried form; the probe information identification area (1) records the corresponding value of each independent probe attachment area. Acrylate glue is attached to the surface of the hybridization glass slide bonding area (3), which is used for the packaging sealing film of the probe-attached glass slide and the bonding with the sample-attached glass slide.

The sample attachment slide (B) is composed of a sample information recording area (7), several mutually independent sample attachment areas (8) and a sample isolation area (9), and the sample is dropped on the sample attachment area (8), The sample attachment area is made of a hydrophilic material, the sample isolation area is attached with a hydrophobic coating, and the sample information is recorded on the sample information recording area, and the sample attachment area (8) and the probe attachment area (4) are arranged one by one. Correspondingly, when the sample-attached glass slide is upside down on the probe-attached glass slide, the sample in the sample-attached area binds to the probe in the corresponding probe-attached area, thereby realizing hybridization.

The preparation of embodiment 1FISH probe

This example demonstrates the preparation process of the rapid detection probe for CBFB gene breakage, which specifically includes the following steps:

(1) Download the genome non-repeated sequence of the region covered by the CBFB gene probe from UCSC Genome Browser, where the CBFB upstream probe coverage area is: chr16:66885331-67101057, and the CBFB downstream probe coverage area is: chr16:67122220-67337946; The obtained sequence is saved in fasta format, and the repeated sequence region in the genome is replaced by N;

(2) The genomic non-repetitive sequence of the region covered by the CBFB gene upstream probe obtained in step (1) is divided into blocks of 1 kb size using the perl plug-in program chunks.pl (the code map of the chunks.pl program is shown in Figure 4); Import the divided blocks into OligoArray software in batches for probe design and probe screening. The conditions for probe screening are: probe length 50bp, TM value 85-99°C, GC ratio 40-80%, TTTT/GGGG free /AAAA/CCCC, the minimum interval between probes is 5bp; then export the screened probes to the EXCEL table, and add 18bp tag sequences to the 5' end and 3' end of each probe respectively to obtain A series of CBFB gene upstream probe sequences of the tag (Figure 5 shows the CBFB gene probe pool, which will not be shown one by one due to too many sequences); the base sequence of the tag sequence is as follows: the 5' end tag sequence is: TGTAAAACGACGGCCAG (M13 upstream sequence), the 3' end tag sequence is: GGTCATAGCTGTTTCCTG (M13 downstream sequence);

(3) The genomic non-repetitive sequence of the region covered by the CBFB gene downstream probe obtained in step (1) is divided into blocks of 1 kb size using the perl plug-in program chunks.pl (the code map of the chunks.pl program is shown in Figure 4); Import the divided blocks into OligoArray software in batches for probe design and probe screening. The conditions for probe screening are: probe length 50bp, TM value 85-99°C, GC ratio 40-80%, excluding TTTTT/GGGG /AAAAA/CCCC, the minimum interval between probes is 5bp; then export the screened probes to the EXCEL table, and then add 18bp tag sequences (base sequence As above), obtain a series of CBFB gene downstream probe sequences with labels;

(4) Use a DNA synthesizer to chemically synthesize the CBFB gene upstream probe sequence with the tag sequence obtained in step (2), mix the chemically synthesized probes, and prepare the CBFB gene upstream probe library, the CBFB gene The upstream probe library contains 1109 CBFB gene upstream probes with tag sequences; similarly, use a DNA synthesizer to chemically synthesize the CBFB gene downstream probe sequences with tag sequences obtained in step (3), and the chemically synthesized The probes are mixed to prepare a CBFB gene downstream probe library, which includes 1238 CBFB gene downstream probes with tag sequences;

(5) Synthesize M13 universal primers with a green fluorescent group and an orange-red fluorescent group at the 5' end respectively. The universal primer sequence is: M13-F TGTAAAACGACGGCCAGT, M13-R CAGGAAACAGCTATGACC; use a green fluorescent group at the 5' end The M13 universal primer of the CBFB gene upstream probe library is used for amplification and labeling reaction; the M13 universal primer with an orange-red fluorescent group is used for the amplification and labeling reaction of the CBFB gene downstream probe library at the 5' end; the amplification labeling The PCR reaction system of the reaction is as follows:

The PCR reaction conditions of the amplification labeling reaction are as follows: 95° C. for 5 min; 94° C. for 30 s, 58° C. for 30 s, and 72° C. for 30 s for a total of 20 cycles; 72° C. for 10 min.

(6) Purify the amplified labeled product of the CBFB gene upstream probe library and the amplified labeled product of the CBFB gene downstream probe library obtained in step (5) respectively using the ethanol sodium acetate method, and obtain after purification by dilution: the concentration is 100ng/ul fluorescently labeled CBFB gene upstream probe library and 100ng/ul fluorescently labeled CBFB gene downstream probe library.

The preparation of embodiment 2 ready-to-use multi-probe hybridization slides

The present embodiment has invented a kind of preparation method of ready-to-use multi-probe hybridization glass slide

The probe combinations used in this example were: PML/RARA, BCR/ABL, P53, D13S319, 1Q21, IGH/BCL2, CBFB, AML/ETO; in the corresponding probe attachment area. (Figure 7)

The hybrid slides used in this embodiment are made by entrusting the manufacturer according to the design drawing of the present invention.

The following is the preparation process of the ready-to-use multi-probe hybridization slide (M-probe Slide):

(1) Entrust the manufacturer to prepare probe-attached slides and sample-attached slides as shown in Figures 1 to 3;

(2) Carrying out probe attachment in the probe attachment region of the probe attachment glass slide;

(2.1) According to the formula: levoside 0.5% acacia 3% mannitol 1% polyacrylate 0.3%, use deionized water to dissolve the above excipient composition, and store it at 4°C for later use;

(2.2) Prepare each probe composition: the final concentration of the probe aqueous solution is 100ng/ul;

(2.3) Take 10ul of the probe composition and 90ul of the excipient composition, mix them evenly, use a pipette to draw 5ul of the mixed probe excipient composition, add it dropwise and evenly spread it on each probe attachment area;

(2.4) Place the probe-attached glass slide with the probe excipient composition attached in a lyophilizer to freeze-dry, and seal it with a special sealing film for later use. Note: Avoid light during operation

Example 3

The method for using the ready-to-use multi-probe hybridization slide comprises the following steps:

This example demonstrates the usage method and hybridization effect of the ready-to-use multi-probe hybridization slide.

The probes included in the ready-to-use multi-probe hybridization slide used in this example are: PML/RARA, BCR/ABL, P53, D13S319, 1Q21, IGH/BCL2, CBFB, AML/ETO;

The sample detected in this embodiment is bone marrow blood lymphocytes fixed by Carnoy's fixative:

(1) Sample treatment: Add the fixed peripheral blood cells dropwise to the corresponding areas of the sample-attached slides, immerse the sample-attached slides in a container filled with 2×SSC at 37°C for 30 minutes, and pass 70% after the aging is completed. 85%, 100% gradient alcohol dehydration, dry, set aside.

(2) Probe sample covariance: Add 2ul of hybridization buffer dropwise to the probe attachment area of the hybridization slide, and turn the sample attachment slide upside down so that the sample attachment area and the probe attachment area are pasted together. After combining, invert the entire hybridization slide together with the attached sample slide, place in a metal bath at 73°C for denaturation for 5 minutes, and after the deformation is complete, place the hybridization device in a 37°C wet box for hybridization for 1 hour.

(3) Washing after hybridization: Remove the hybridized slide after the hybridization is completed, and place the slide in a preheated washing solution (containing 0.3M sodium chloride, 0.03M sodium citrate and 0.1% Tween-20) at 60°C Wash to remove unbound probe. After washing, the slides were washed again in distilled water at room temperature and dried at room temperature.

(4) Counterstaining and microscopic examination: Add 5ul of anti-fade mounting medium to each area of the corresponding area of the dried sample attached glass slide, use a suitable cover glass to seal the slide, and observe the hybridization results under a fluorescent microscope; the results are as follows: Figure 8 shows.

It can be seen from Fig. 8 that the present invention has excellent hybridization effect when used for multi-probe hybridization, and has extremely high signal-to-noise ratio, specificity and sample detection rate.

Apparently, the above-mentioned embodiments are only examples for clear illustration, rather than limiting the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or modifications thus extended are still within the scope of protection of the present invention.

Claims (10)

1. a kind of instant multiprobe hybridized slides kit, it is characterised in that slow by probe attachment slide, each probe, hybridization Fliud flushing and sample attachment slide composition；Several mutually independent probe attachment regions are included on the probe attachment slide, it is each to visit Pin is respectively attached on each mutually independent probe attachment region in the form of lyophilized；Several are included on the sample attachment slide Mutually independent sample attachment region, sample attachment region are corresponded with probe attachment region, and sample drop is added on sample attachment region, when When sample attachment region is bonded with probe attachment region, the probe of the sample of sample attachment region and corresponding probe attachment region is realized miscellaneous Knot is closed.

2. instant multiprobe hybridized slides kit according to claim 1, it is characterised in that the probe adheres to slide It is made of detecting probe information tag slot, probe adhering zone and hybridized slides bonding land, have recorded on the detecting probe information tag slot Corresponding probe species on each independent probe attachment region, the probe adhering zone is interior to include several mutually independent spies Pin attachment region, probe are attached on probe attachment region in the form of lyophilized, and the surface of the hybridized slides bonding land has propylene Sour fat glue, the bonding for the packaging sealer of probe attachment slide and with sample attachment slide.

3. instant multiprobe hybridized slides kit according to claim 2, it is characterised in that the probe adhering zone For a groove, groove includes several mutually independent probe attachment block of cells, and each independent probe adheres to block of cells by visiting Pin attachment region and peripheral isolated area form；There is isolation ditch between each independent probe attachment block of cells.

4. instant multiprobe hybridized slides kit according to claim 1, it is characterised in that the sample adheres to slide It is made of sample message recording areas, sample attachment region and sample isolated area, sample letter is have recorded on the sample message recording areas Breath, sample drop are added on sample attachment region, and the sample isolated area is used to prevent sample cross contamination.

5. instant multiprobe hybridized slides kit according to claim 4, it is characterised in that the sample attachment region is adopted With hydrophilic material；The sample isolated area has hydrophobic coating.

6. instant multiprobe hybridized slides kit according to claim 1, it is characterised in that the hybridization buffer Component is：10wt% ethylene carbonates, 30wt% dextran sulfates, 1ugRNaseA, 0.6M sodium chloride, 10mM citrate buffer solutions.

7. instant multiprobe hybridized slides kit according to claim 1, it is characterised in that each probe is FISH Gene loci probe or FISH genes centromere/telomere probe.

8. instant multiprobe hybridized slides kit according to claim 7, it is characterised in that each probe is selected from such as the following group Close A or combination B：

Combine A：BCR/ABL, IGH/BCL2, AML/ETO；

Combine B：PML/RARA, BCR/ABL, P53, D13S319,1Q21, IGH/BCL2, CBFB, AML/ETO.

9. the preparation method of any instant multiprobe hybridized slides of claim 1 ~ 8, it is characterised in that including following step Suddenly：

（1）Prepare the probe attachment slide for being attached with each probe：

A. the probe attachment slide with any structure of claim 1 ~ 8 is prepared；

B. excipient is prepared using deionized water, the formula of the excipient is：Left-handed glucosides 0.5wt%, acacia

3wt%, mannitol 1wt%, polyacrylate 0.3wt%；

C. each probe solution is prepared respectively, each probe solution is uniformly mixed with excipient respectively, is added dropwise and is uniformly smeared respectively On probe attachment slide on each independent probe attachment region；Then probe attachment slide is freezed, sealer, be prepared attached The probe attachment slide of each probe；

（2）Prepare the sample attachment slide with any structure of claim 1 ~ 8；

（3）Preparing hybrid buffer solution；Probe attachment slide, hybridization buffer and the sample that each probe is attached with obtained by preparing are attached Slide composition instant multiprobe hybridized slides.

10. the application of any instant multiprobe hybridized slides of claim 1 ~ 8, specifically comprises the following steps：

（1）Sample process：Sample is added drop-wise to each sample attachment region of sample attachment slide respectively, it is after pretreatment, spare；

（2）Probe sample co-variation：Hybridization Buffer is added dropwise to each mutually independent probe attachment region on probe attachment slide respectively Liquid, and sample attachment slide is tipped upside down on probe attachment slide, sample attachment region is fit together with probe attachment region, paste Whole probe attachment slide is inverted together with sample attachment slide after conjunction, is placed in 73 DEG C of denaturation 5min of metal bath, deformation is completed It is placed in 37 DEG C of wet box hybridization 1h again afterwards；

（3）Post-hybridization washing：Wait to remove probe attachment slide after the completion of hybridizing, room temperature after sample attachment slide washing is dried；

（4）Redye and microscopy：Anti-cancellation mountant is added dropwise to the sample attachment region of the sample attachment slide dried, uses lid fragmentation Mounting and in fluorescence microscopy Microscopic observation results of hybridization.