CN108949913B - Gene detection kit containing universal fluorescent reduction probe nucleic acid molecules and application thereof - Google Patents

Abstract

The invention discloses a gene detection kit and a gene detection method. The fluorescence reduction probe technology of the invention enables the fluorescence of the fluorescence labeling probe which is not hybridized with the target nucleic acid to be quenched by the quenching group of the general quenching probe, achieves the effect of removing the non-hybridized fluorescence signal only by controlling the hybridization temperature without the complicated and difficult-to-control washing step, greatly improves the nucleic acid hybridization efficiency, saves time and labor, can quickly complete the fluorescence in situ hybridization and detect the specific gene or the expression distribution thereof, is the improvement of the traditional FISH and has good application prospect.

Description

Gene detection kit containing universal fluorescent reduction probe nucleic acid molecules and application thereof

Technical Field

The invention relates to the field of gene detection, in particular to a gene detection kit containing a universal fluorescent subtraction probe nucleic acid molecule and application thereof.

Background

Nucleic acid molecular hybridization means that two nucleic acid single strands with certain homology can be combined into a double strand according to base complementarity under certain conditions (suitable temperature, humidity, ionic strength and the like). The hybridization of nucleic acid molecules can be further classified into Southern hybridization, Northern hybridization, In Situ Hybridization (ISH), Fluorescence In Situ Hybridization (FISH), chip hybridization (solid-liquid phase hybridization), and the like. Each hybridization technique has the characteristics that a plurality of related techniques can be derived due to different selection of probes, and the hybridization technique plays an important role in different fields.

FISH is a nonradioactive in situ hybridization technique. The basic principle is as follows: if the target DNA or RNA on the chromosome or tissue section to be detected is homologously complementary to the nucleic acid probe used, the target DNA or RNA is denatured, annealed, and renatured to form a hybrid of the target nucleic acid and the nucleic acid probe. The nucleic acid probe is labeled with reporter molecule such as biotin and digoxigenin, and the immunochemical reaction between the reporter molecule and fluorescein labeled specific avidin can be used to perform qualitative, quantitative or relative positioning analysis on the DNA to be detected under the mirror by using a fluorescent detection system. The FISH technology is not only applied to clinical and scientific research of cytogenetics, but also widely applied to tissue cell positioning research of gene expression. However, the traditional FISH technology is complex in operation, multiple in influencing factors, long in consumed time, high in technical difficulty and difficult to popularize in a large area. Other subtractive probe technologies have high cost and difficult standardization of hybridization conditions because each gene needs to synthesize a specific fluorescence-labeled probe and a specific fluorescence quenching probe.

Disclosure of Invention

In order to solve the above problems, the present invention provides a novel kit and method. The invention provides a technical method for carrying out fluorescence in situ hybridization by using a special double-chain fluorescence reduction probe, which is simple and rapid in operation, thereby obviously reducing the technical difficulty and the detection cost of in situ hybridization.

The gene detection kit comprises the following probes:

a sequence:

5′–CTGAAGCGCTTCGCGAGCCM₁M₂N₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀M₃M₄CTGAAGCGCTTCGCGAGCC-3'; wherein N is₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀For the complementary sequence of the gene to be examined or of a fragment thereof, M₁M₂And M₃M₄Is a linker sequence;

b sequence 5' -n₅n₄n₃n₂n₁m₂m₁TAGGCTCGCGAAGC-3′；n₁n₂n₃n₄n₅And N₁N₂N₃N₄N₅Is complementary to the sequence of (1), m₁m₂And M₁M₂The sequence of (a) is complementary;

c sequence 5' -GCGAAGCGCTTCAGm₄m₃n₁₀n₉n₈n₇n₆-3′；n₆n₇n₈n₉n₁₀And N₆N₇N₈N₉N₁₀Is complementary to the sequence of (1), m₃m₄And M₃M₄The sequence of (a) is complementary;

d sequence is 5 '-fluorescent molecule-GGCTCGCGAAGCGCTTCAG-fluorescent molecule-3'

The E sequence is 5 '-fluorescence quenching molecule-CTGAAGCGCTTCGCGAGC-fluorescence quenching molecule-3'.

In the kit, the B sequence and the C sequence are combined with the A sequence, and the dosage of the B sequence and the C sequence can be combined with all the A sequence; combining the D sequence with the A sequence, wherein the D sequence can be used in an amount capable of combining all the A sequences; the D sequence is combined with the E sequence, and the E sequence can be used in an amount capable of combining all the D sequences.

Preferably, the molar ratio of the A sequence to the B sequence is 1: (2-7); the molar ratio of the A sequence to the C sequence is 1: (2-7); the molar ratio of the A sequence to the D sequence is 1: (2-7); the molar ratio of the D sequence to the E sequence is 1: (2-5);

more preferably, the molar ratio of the A sequence, the B sequence, the C sequence, the D sequence and the E sequence is 1:5:5:3: 6.

The invention also provides a kit, which comprises the following probes:

a sequence:

5′–CTGAAGCGCTTCGCGAGCCM₁M₂N₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀M₃M₄CTGAAGCGCTTCGCGAGCC-3'; wherein N is₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀For the complementary sequence of the gene to be examined or of a fragment thereof, M₁M₂And M₃M₄Is a linker sequence;

the sequence B is 5' -n₅n₄n₃n₂n₁m₂m₁TAGGCTCGCGAAGC-3′；n₁n₂n₃n₄n₅And N₁N₂N₃N₄N₅Is complementary to the sequence of (1), m₁m₂And M₁M₂The sequence of (a) is complementary;

a C sequence of 5' -GCGAAGCGCTTCAGm₄m₃n₁₀n₉n₈n₇n₆-3′；n₆n₇n₈n₉n₁₀And N₆N₇N₈N₉N₁₀Is complementary to the sequence of (1), m₃m₄And M₃M₄The sequence of (a) is complementary;

and F sequence:

5′-CCAGAGCGAGCAGTGTCAAGGCTCGCGAAGCGCTTCAGAACCA GAGCGAGCAGTGTC-3′

sequence of fifth F-R1 5'-CGAGCCTTGACACTGCTCG-3'

Sixthly, a F-R2 sequence 5'-GCTCGCTCTGGTTCTGAAGC-3'

Bare G sequences

5′-GAACAGTCGTGAACATCTGACACTGCTCGCTCTGGTGAACAGTC GTGAACATC-3′

And G-R1 sequence:

5′-GCAGTGTCAGATGTTCAC-3′

sequence of the self-lifting G-R2:

5′-GACTGTTCACCAGAGCG-3′

(10) h sequence:

5 '-fluorescent molecule-GATGTTCACGACTGTTC-fluorescent molecule-3'

The following series I:

5 '-quenching molecule-GAACAGTCGTGAACAT-quenching molecule-3'.

In the kit, the B sequence and the C sequence are combined with the A sequence, and the dosage of the B sequence and the C sequence can be combined with all the A sequence; f sequence is combined with A sequence, and the dosage of the F sequence can be combined with all A sequences; F-R1 sequence and F-R2 sequence are combined with the F sequence, and the F-R1 sequence and the F-R2 sequence can be used in combination with all the F sequences; g sequence is combined with F sequence, and the dosage of G sequence can be combined with all F sequences; the G-R1 sequence and the G-R2 sequence are combined with the G sequence, and the G-R1 sequence and the G-R2 sequence can be combined with all the G sequences; h sequence is combined with G sequence, and the dosage of H sequence can be combined with all G sequences; the I sequence is combined with the H sequence, and the I sequence can be used in an amount capable of combining all the H sequences.

Preferably, the molar ratio of the A sequence to the B sequence is 1: (2-7); the molar ratio of the A sequence to the C sequence is 1: (2-7); the molar ratio of the A sequence to the F sequence is 1: (2-7); the molar ratio of the F sequence to the F-R1 sequence is 1: (2-7); the molar ratio of the F sequence to the F-R2 sequence is 1: (2-7); the molar ratio of the F sequence to the G sequence is 1: (2-7); the molar ratio of the G sequence to the G-R1 sequence is 1: (2-7); the molar ratio of the G sequence to the G-R2 sequence is 1: (2-7); the molar ratio of the G sequence to the H sequence is 1: (2-7); the molar ratio of the H sequence to the I sequence is 1: (2-5);

preferably, the molar ratio of the A sequence, the B sequence, the C sequence, the F-R1 sequence, the F-R2 sequence, the G-R1 sequence, the G-R2 sequence, the H sequence and the I sequence is 1:2: 2.4:4: 8:8: 16.

In the aforementioned kit, said M₁M₂Is TA or AC, and/or, the M₃M₄Is AT, CA or TC.

The gene to be detected or a partial fragment sequence of the gene to be detected, which is represented by "N1N 2N3N4N5 … N6N7N8N9N 10" in the present invention, may have any length, and preferably has a length of 10 bp or more.

The gene to be detected is any human body, rat and mouse gene.

The fluorescent molecule is FAM (6-carboxyfluorescein), or TET (tetrachloro-6-carboxyfluorescein), HEX (hexachloro-6-methylfluorescein), JOE (2, 7, -dimethyl-4, 5, dichloro-6-carboxyfluorescein).

The fluorescence quenching molecule is TAMRA (6-carboxyl-4 methyl rhodamine) or BHQ (black hole quenching group).

The kit further comprises a buffer.

The buffer solution comprises TE buffer solution and prehybridization solution; TE buffer (containing 10mmol/L tris (tromethamine), 1mmol/L EDTA, pH 8.0); prehybridization solution (salt solution containing no probe and the same components as the hybridization solution, including 60mmol/L tris (tromethamine), 50mmol/L NaCl,1mmol/L EDTA, 0.5% SDS (sodium dodecyl sulfate), 5% formamide, pH 7.4.

The invention also provides a gene detection method, which comprises the following steps (avoiding light in the whole process):

(1) dissolving the probes in the kit in a TE buffer solution to obtain a mixed solution PM1 containing all probe sequences;

(2) adding the mixed solution PM1 into the pre-hybridization solution to prepare a hybridizationbuffer solution PM 2;

(3) taking a hybridization buffer solution PM2 to be dripped on a chromosome dripping sheet or a tissue slice to be detected in a dark process, covering a cover glass, and sealing with an adhesive;

(4) heating the slide to 95-98 ℃ for denaturation for 3-5 min, then rapidly cooling to 45-70 ℃ and preserving heat for 20-60 min to complete molecular hybridization of the universal fluorescent probe and the target nucleic acid, and then cooling to 30-50 ℃ and preserving heat for 40-60 min;

(5) adding the quenching prevention blocking tablet, and observing a hybridization signal under a fluorescence microscope.

Preferably, step (4) is: denaturing the glass slide at 95 ℃ or 98 ℃ for 3min, rapidly cooling to 68 ℃, preserving heat for 30min, naturally cooling to 40 ℃, preserving heat for 50min, and naturally cooling to room temperature;

the step (5) is as follows: adding the anti-quenching sealing tablet, placing a cover glass, and observing and photographing under 488nm, 405nm and 670nm exciting lights under a fluorescence microscope.

The invention has the beneficial effects that: the fluorescence reduction probe technology of the invention enables the fluorescence of the fluorescence labeling probe which is not hybridized with the target nucleic acid to be quenched by the quenching group of the general quenching probe, so as to achieve the effect of removing the non-hybridized fluorescence signal only by controlling the hybridization temperature without complicated and uncontrollable washing steps, greatly improve the nucleic acid hybridization efficiency, save time and labor, rapidly complete the fluorescence in situ hybridization and detect the specific gene or the expression distribution thereof, the method needs 2 hours to complete, is improved by more than 10 hours compared with the traditional FISH method, and is an improvement of the FISH method.

The method comprises the steps of detecting a probe of a site, synthesizing a set of probes, wherein the total number of the probe bases is 240 bases, each two OD (optical density) is 1.5 yuan, labeling two fluorescein, each 600 yuan, labeling two quenching groups, each 500 yuan, the total number is 2560 yuan, and the total number is 2600 yuan with other conventional reagents of 40 yuan, so that the target gene can be detected 400 times, and the production of 4 kits (100 times/box) can be completed. The cost of each kit is 650 yuan, each gene detection is 6.5 yuan, 4000 yuan/25 times of in situ hybridization reagents sold on the market at present, and each gene detection is 160 yuan, so that the kit has obvious cost advantage.

The traditional FISH method has long consumption time, which needs more than ten hours, and the invention only needs 2 hours to complete the experiment, and the time cost of the invention is lower.

The invention can be used for detecting any gene segment of human body, rat and mouse by specific design and the fixed sequence in the kit can not be combined with any gene of human body, rat and mouse, thus overcoming the problem that the specific design is needed when each gene is detected by the traditional method.

The kit and the method can be suitable for detecting any gene of a human body, and in the kit, the fluorescent sequence playing a key role is a universal sequence, so the cost is low.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 principle of the Gene detecting method of the present invention

FIG. 2 principle of the Gene detecting method of the present invention

FIG. 3 negative control results of AQP4 gene assay in Experimental example 1;

FIG. 4 positive group results of AQP4 gene assay in Experimental example 1;

FIG. 5 negative control results of telomere gene detection in Experimental example 2;

FIG. 6 positive group results of telomere gene detection in Experimental example 2;

FIG. 7 negative control results of IL-6 gene assay in Experimental example 3;

FIG. 8 results of positive group of IL-6 gene assay in Experimental example 3;

FIG. 9 negative control results of IL-10 gene assay in Experimental example 4;

FIG. 10 results of positive group of IL-10 gene assay in Experimental example 4;

FIG. 11 negative control results of IL-1. beta. gene detection in Experimental example 5;

FIG. 12 positive panel results of IL-1. beta. gene detection in Experimental example 5;

FIG. 13 negative set of results of telomere gene detection in Experimental example 6;

FIG. 14 Positive group results of telomere gene detection in Experimental example 6.

Detailed Description

Example 1 Gene detection method of the present invention

1. Constitution of detection kit

The schematic diagram is shown in figure 1:

a sequence:

5′–CTGAAGCGCTTCGCGAGCCM₁M₂N₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀M₃M₄CTGAAGCGCTTCGCGAGCC-3'; wherein N is₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀For the complementary sequence of the gene to be examined or of a fragment thereof, M₁M₂And M₃M₄Is a linker sequence;

b sequence 5' -n₅n₄n₃n₂n₁m₂m₁TAGGCTCGCGAAGC-3′；n₁n₂n₃n₄n₅And N₁N₂N₃N₄N₅Is complementary to the sequence of (1), m₁m₂And M₁M₂The sequence of (a) is complementary;

c sequence 5' -GCGAAGCGCTTCAGm₄m₃n₁₀n₉n₈n₇n₆-3′；n₆n₇n₈n₉n₁₀And N₆N₇N₈N₉N₁₀Is complementary to the sequence of (1), m₃m₄And M₃M₄The sequence of (a) is complementary;

d sequence is 5 '-FAM-GGCTCGCGAAGCGCTTCAG-FAM-3'

The sequence E is 5 '-. TAMRA-CTGAAGCGCTTCGCGAGC-TAMRA-3';

wherein M is₁M₂Is TA or AC, and/or, M₃M₄Is AT, CA or TC.

Buffer solution: TE buffer (containing 10mmol/L tris (tromethamine), 1mmol/L EDTA, pH 8.0); prehybridization solution (salt solution containing no probe and the same components as the hybridization solution, including 60mmol/L tris (tromethamine), 50mmol/L NaCl,1mmol/L EDTA, 0.5% SDS (sodium dodecyl sulfate), 5% formamide, pH 7.4.)

2. Detection method

(1) Dissolving the probes in TE buffer solution respectively to obtain mixed solution PM1 with the concentration of 1umol/L, B sequence, 5umol/L, C sequence, 5umol/L, D sequence, 3umol/L, E sequence and 6umol/L sequence; adding 1ul of mixed solution PM1 into 49ul of prehybridization solution to prepare hybridizationbuffer solution PM 2;

(2) taking a sample to be detected, dripping 25ul of hybridization solution PM2, covering with a glass slide, and sealing with an adhesive;

(3) denaturing the glass slide at 95 ℃ for 3min, rapidly cooling to 68 ℃ and preserving heat for 30min, naturally and slowly cooling to 40 ℃ and preserving heat for 50min, and naturally cooling to room temperature;

(4) after the slide is taken out, the anti-quenching sealing tablet is added, a cover glass is put on the anti-quenching sealing tablet, and the anti-quenching sealing tablet is observed and photographed under 488nm, 405nm and 670nm exciting lights under a fluorescence microscope.

Example 2 Gene detecting method of the present invention

1. Constitution of detection kit

The schematic diagram is shown in fig. 2:

a sequence:

5'–CTGAAGCGCTTCGCGAGCCM₁M₂N₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀M₃M₄CTGAAGCGCTTCGCGAGCC-3'; wherein N is₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀For the complementary sequence of the gene to be examined or of a fragment thereof, M₁M₂And M₃M₄Is a linker sequence;

wherein M is₁M₂Is TA or AC, and/or, M₃M₄Is AT, CA or TC.

The sequence B is 5' -n₅n₄n₃n₂n₁m₂m₁TAGGCTCGCGAAGC-3′；n₁n₂n₃n₄n₅And N₁N₂N₃N₄N₅Is complementary to the sequence of (1), m₁m₂And M₁M₂The sequence of (a) is complementary;

a C sequence of 5' -GCGAAGCGCTTCAGm₄m₃n₁₀n₉n₈n₇n₆-3′；n₆n₇n₈n₉n₁₀And N₆N₇N₈N₉N₁₀Is complementary to the sequence of (1), m₃m₄And M₃M₄The sequence of (a) is complementary;

and F sequence:

5′-CCAGAGCGAGCAGTGTCAAGGCTCGCGAAGCGCTTCAGAACCA GAGCGAGCAGTGTC-3′

sequence of fifth F-R1 5'-CGAGCCTTGACACTGCTCG-3'

Sixthly, a F-R2 sequence 5'-GCTCGCTCTGGTTCTGAAGC-3'

Bare G sequences

5′-GAACAGTCGTGAACATCTGACACTGCTCGCTCTGGTGAACAGTC GTGAACATC-3′

And G-R1 sequence:

5′-GCAGTGTCAGATGTTCAC-3′

sequence of the self-lifting G-R2:

5′-GACTGTTCACCAGAGCG-3′

(10) h sequence:

FAM-GATGTTCACGACTGTTC-FAM

the following series I:

TAMRA-GAACAGTCGTGAACAT-TAMRA。

buffer solution: TE buffer (containing 10mmol/L tris (tromethamine), 1mmol/L EDTA, pH 8.0); prehybridization solution (salt solution containing no probe and the same components as the hybridization solution, including 60mmol/L tris (tromethamine), 50mmol/L NaCl,1mmol/L EDTA, 0.5% SDS (sodium dodecyl sulfate), 5% formamide, pH 7.4.)

2. Detection method

(1) Dissolving the probes in the 11 in TE buffer solution respectively to obtain probe solutions with the concentration of 10ummol/L, and taking 1uL of A sequence solution, 2uL of B sequence, 2uL of C sequence, 2uL of F sequence, 4uL of F-R1 sequence, 4uL of F-R2 sequence, 4uL of G sequence, 8uL of G-R1 sequence, 8uL of G-R2 sequence, 8uL of H sequence and 16uL of I sequence to obtainmixed solution PM 1; adding 1ul of mixed solution PM1 into 49ul of prehybridization solution to prepare hybridizationbuffer solution PM 2;

(2) taking a sample to be detected, dripping 25ul of hybridization solution PM2, covering with a glass slide, and sealing with an adhesive;

(3) denaturing the glass slide at 98 ℃ for 3min, rapidly cooling to 68 ℃ and preserving heat for 30min, naturally and slowly cooling to 40 ℃ and preserving heat for 50min, and naturally cooling to room temperature;

(4) after the slide glass was taken out, it was observed under a fluorescence microscope under excitation light of 488nm, 405nm, and 670nm, and photographed.

The main performance indexes are as follows:

1. the sensitivity of nucleic acid detection reaches fM/LGrade:

the nucleic acid detection technology can detect 10 copies/. mu.L of target genes, and the FM sensitivity is 0.01667 fM/L.

2. Method specificity, enabling efficient recognition of single base differences in nucleic acid sequences:

a fluorescent molecular probe is designed according to the nucleic acid base pairing principle strictly, in-situ hybridization is carried out, single base recognition is achieved, and the phenomenon that the probe A is closed by combining the probe and no fluorescent hybridization signal exists if no target base sequence exists.

The beneficial effects of the invention are illustrated by way of experimental examples as follows:

experimental example 1 detection of brain cell edema Gene AQP4 Gene by the method of the present invention

1. Detection method

The sample to be detected is brain tissue nerve cells with rat cerebral edema.

The method of the invention, according to the method of example 1, detects:

(1) firstly, a set of fluorescent reduction probes is designed and synthesized according to the mRNA sequence of AQP4 gene, and the sequence and the label are respectively:

a sequence:

5′-.CTGAAGCGCTTCGCGAGCCTAGCTACATGGAGGTGGAGGACAA CCGATCTGAAGCGCTTCGCGAGCC-3′

sequence B of 5'— CCATGTAGCTAGGCTCGCGAAGC-3'

C sequence of 5 '-GCGAAGCGCTTCAGATCGGTTGTC-3'

D sequence is 5 '-FAM-GGCTCGCGAAGCGCTTCAG-FAM-3'

The sequence E is 5 '-. TAMRA-CTGAAGCGCTTCGCGAGC-TAMRA-3';

(2) dissolving the probes in TE buffer solution respectively, and making the mixed solution PM1 with the concentration of 1umol/L, B sequence, 5umol/L, C sequence, 5umol/L, D sequence, 3umol/L, E sequence and 6umol/L sequence; adding 1ul of mixed solution PM1 into 49ul of prehybridization solution to prepare hybridizationbuffer solution PM 2;

(3) taking rat brain tissue slices, keeping the rat away from light in the whole process, dripping 25ul of hybridization solution PM2 containing a fluorescent reduction probe, covering a glass slide, and sealing by using an adhesive;

(4) denaturing the glass slide at 95 ℃ for 3min, rapidly cooling to 68 ℃ and preserving heat for 30min, naturally and slowly cooling to 40 ℃ and preserving heat for 50min, and naturally cooling to room temperature;

(5) the slide glass was taken out and observed under a fluorescence microscope under 670nm excitation light and photographed.

Method for negative control: the method of the present invention is the same except that the A sequence is not used.

2. As a result:

as shown in FIGS. 3 to 4, the imaging signal of the red portion of the fluorography was comparatively analyzed using a two-photon confocal fluorescence microscope and Image J (Fiji is just) software.

It can be seen that the negative control has no red hybridization signal, while the positive control has a relatively strong fluorescent hybridization signal, indicating that the method of the present invention can accurately detect, whereas the removal of the specific sequence cannot accurately detect.

Experimental example 2 detection of telomere Gene by the method of the present invention

1. Detection method

The sample to be detected is rat liver tissue.

Method for negative control: the method of the present invention is the same except that the A sequence is not used.

The method of the invention, according to the method of example 1, detects:

(1) the sequences and markers are respectively:

a sequence

HCR-A:5’CTGAAGCGCTTCGCGAGCCTAGGGTTAGGGTTAGGGTTAGGGTTAGGGATCTG AAGCGCTTCGCGAGCC-3’

B sequence: HCR-B CCTAACCCTAGGCTCGCGAAGC

C sequence: HCR-C: GCGAAGCGCTTCAGATCCCTAACC

D sequence of HCR-D:5 '-FAM-GGCTCGCGAAGCGCTTCAG-FAM-3'

The sequence E is HCR-E5 '-. TAMRA-CTGAAGCGCTTCGCGAGC-TAMRA-3';

(2) dissolving the probes in TE buffer solution respectively, and making the mixed solution PM1 with the concentration of 1umol/L, B sequence, 5umol/L, C sequence, 5umol/L, D sequence, 3umol/L, E sequence and 6umol/L sequence; adding 1ul of mixed solution PM1 into 49ul of prehybridization solution to prepare hybridizationbuffer solution PM 2;

(3) taking a liver tissue section of a mouse, dripping 25ul of hybridization solution PM2 containing a fluorescent reduction probe, covering a glass slide, and sealing by sealing glue;

(4) in dark, denaturing the glass slide at 95 ℃ for 3min, rapidly cooling to 68 ℃ and preserving heat for 30min, naturally and slowly cooling to 40 ℃ and preserving heat for 50min, and naturally cooling to room temperature;

(5) the slide was taken out and observed under excitation light of 405nm under a fluorescence microscope and photographed.

Method for negative control: the method of the present invention is the same except that the A sequence is not used.

2. Results

As shown in FIGS. 5 to 6, the developed signal of the blue fluorescence portion of the fluorography was compared and analyzed with a negative control using a two-photon confocal fluorescence microscope and Image J (Fiji is just) software,

it can be seen that the negative control has almost no signal, while the positive control has a relatively strong blue fluorescent hybridization signal, indicating that the method of the invention can detect accurately, whereas the removal of the specific sequence cannot detect.

Experimental example 3 detection of IL-6 Gene by the method of the present invention

1. Detection method

The positive sample was spleen tissue from a mouse with sepsis as a model 24 hours after the assay in example 1, and the negative control sample was spleen tissue from a normal mouse.

Sepsis causes an increase in the expression of the IL-6 gene in the spleen of a mouse, and the IL-6 gene of spleen tissue 24 hours after the sepsis mouse should be significantly increased compared to a normal mouse.

(1) The sequences and markers are respectively:

IL-10-A probe:

CTGAAGCGCTTCGCGAGCCACAGAGGGGAGAAATCGATGACAGCGCCTCACTGAAGCGCTTC GCGAGCC

IL-10-B Probe CTCCCCTCTGTGGCTCGCGAAGC

IL-10-C Probe CGAAGCGCTTCAGTGAGGCGCTG

D probe sequence of HCR-D5 '-FAM-GGCTCGCGAAGCGCTTCAG-FAM-3'

E probe sequence of HCR-E5 '-TAMRA-CTGAAGCGCTTCGCGAGC-TAMRA-3'

(2) Dissolving the probes in TE buffer solution respectively, and making the mixed solution PM1 with the concentration of 1umol/L, B sequence, 5umol/L, C sequence, 5umol/L, D sequence, 3umol/L, E sequence and 6umol/L sequence; adding 1ul of mixed solution PM1 into 49ul of prehybridization solution to prepare hybridizationbuffer solution PM 2;

(3) taking a liver tissue section of a mouse, dripping 25ul of hybridization solution PM2 containing a fluorescent reduction probe, covering a glass slide, and sealing by sealing glue;

(4) in dark, denaturing the glass slide at 95 ℃ for 3min, rapidly cooling to 68 ℃ and preserving heat for 30min, naturally and slowly cooling to 40 ℃ and preserving heat for 50min, and naturally cooling to room temperature;

(5) the slide was taken out and observed under 488nm excitation light under a fluorescence microscope and photographed.

2. Results

The results are shown in FIGS. 7 to 8, and the green part development signals of the fluorography were compared and analyzed using a two-photon confocal fluorescence microscope and ImageJ software.

It can be seen that the fluorescence signal of the negative control is very weak, while the green fluorescence signal of the positive control is strong,

the method can accurately detect, but cannot accurately detect when the specific sequence is removed.

Experimental example 4 detection of IL-10 Gene by the method of the present invention

1. Detection method

The positive sample was liver tissue from mice with sepsis (120 mg/kg of safflor yellow injection in the protected group) 24 hours after molding, and the negative control sample was liver tissue from normal mice, which were examined according to the method of example 1.

After sepsis, the injection of 120mg/kg of safflor yellow can maintain the high expression of IL-10 in the liver of the mice, and compared with normal mice, the IL-10 of spleen tissues 24 hours after the sepsis mice should be obviously improved.

(1) The sequences and markers are respectively:

IL-10-A probe:

CTGAAGCGCTTCGCGAGCCACAGAGGGGAGAAATCGATGACAGCGCCTCACTGAAGCGCTTC GCGAGCC

IL-10-B Probe CTCCCCTCTGTGGCTCGCGAAGC

IL-10-C Probe CGAAGCGCTTCAGTGAGGCGCTG

D probe sequence of HCR-D:5 '-FAM-GGCTCGCGAAGCGCTTCAG-FAM-3'

E probe sequence HCR-E5 '-. TAMRA-CTGAAGCGCTTCGCGAGC-TAMRA-3'

(2) Dissolving the probes in TE buffer solution respectively, and making the mixed solution PM1 with the concentration of 1umol/L, B sequence, 5umol/L, C sequence, 5umol/L, D sequence, 3umol/L, E sequence and 6umol/L sequence; adding 1ul of mixed solution PM1 into 49ul of prehybridization solution to prepare hybridizationbuffer solution PM 2;

(3) taking a liver tissue section of a mouse, dripping 25ul of hybridization solution PM2 containing a fluorescent reduction probe, covering a glass slide, and sealing by sealing glue;

(4) in dark, denaturing the glass slide at 95 ℃ for 3min, rapidly cooling to 68 ℃ and preserving heat for 30min, naturally and slowly cooling to 40 ℃ and preserving heat for 50min, and naturally cooling to room temperature;

(5) the slide was taken out and observed under excitation light of 405nm under a fluorescence microscope and photographed.

2. Results

The results are shown in FIGS. 9 to 10, and the fluorescence-photographed blue portion imaging signals were analyzed by comparison using a two-photon confocal fluorescence microscope and ImageJ software.

It can be seen that the fluorescence signal of the negative control is very weak, while the blue fluorescence signal of the positive control is strong, and the detection result is consistent with the property of the tissue, which shows that the method of the invention can effectively and accurately detect.

Experimental example 5 detection of IL-1. beta. Gene by the method of the present invention

1. Detection method

The positive sample was lung tissue from mice molded with sepsis 24 hours after the assay as in example 1, and the negative control sample was lung tissue from normal mice.

(1) The sequences and markers are respectively:

IL-1. beta. -A probe:

IL-1. beta. -B Probe CAGCACCACTAGGCTCGCGAAG

IL-1 beta-C Probe GAAGCGCTTCAGGAGTTCCCCAAC

D probe sequence of HCR-D:5 '-FAM-GGCTCGCGAAGCGCTTCAG-FAM-3'

E probe sequence HCR-E5 '-. TAMRA-CTGAAGCGCTTCGCGAGC-TAMRA-3'

(2) Dissolving the probes in TE buffer solution respectively, and making the mixed solution PM1 with the concentration of 1umol/L, B sequence, 5umol/L, C sequence, 5umol/L, D sequence, 3umol/L, E sequence and 6umol/L sequence; adding 1ul of mixed solution PM1 into 49ul of prehybridization solution to prepare hybridizationbuffer solution PM 2;

(3) taking a liver tissue section of a mouse, dripping 25ul of hybridization solution PM2 containing a fluorescent reduction probe, covering a glass slide, and sealing by sealing glue;

(4) in dark, denaturing the glass slide at 95 ℃ for 3min, rapidly cooling to 68 ℃ and preserving heat for 30min, naturally and slowly cooling to 40 ℃ and preserving heat for 50min, and naturally cooling to room temperature;

(5) the slide was taken out and observed under 488nm excitation light under a fluorescence microscope and photographed.

2. Results

As shown in FIGS. 11 to 12, the green part development signals of the fluorography were compared and analyzed using a two-photon confocal fluorescence microscope and ImageJ software.

It can be seen that the fluorescence signal of the negative control is very weak, the fluorescence signal of the positive control is stronger, the fluorescence intensity quantification has statistical significance, and the detection result is consistent with the property of the tissue, thus the method can effectively and accurately detect.

Experimental example 6 detection of telomere Gene by the method of the present invention

1. Detection method

The sample to be tested was rat liver tissue, which was examined according to the method of example 2.

Method for negative control: the method of the present invention is the same except that the A sequence is not used.

a. The sequences and markers are respectively:

⑴HCR-A:

5’

⑵HCR-A-R1:CCTAACCCTAGGCTCGCGAAGC

⑶HCR-A-R2:GCGAAGCGCTTCAGTTCCCTAACC

⑷HCR-B:

5’CCAGAGCGAGCAGTGTCAAGGCTCGCGAAGCGCTTCAGAACCAGAGCGAGCAGTGTC ⑸HCR-B-R1:CGAGCCTTGACACTGCTCG

⑹HCR-B-R2:GCTCGCTCTGGTTCTGAAGC

⑺HCR-C

GAACAGTCGTGAACATCTGACACTGCTCGCTCTGGTGAACAGTCGTGAACATC

⑻HCR-C-R1:

GCAGTGTCAGATGTTCA⑼HCR-C-R2:

GACTGTTCACCAGAGCG

(10)HCR-D:

FAM-GATGTTCACGACTGTTC-FAM

⑾HCR-E:

TAMRA-GAACAGTCGTGAACAT-TAMRA

b. detection method

(1) Dissolving the probes in the 11 in TE buffer solution respectively to obtain probe solutions with the concentration of 10ummol/L, and taking 1uL of A sequence solution, 2uL of B sequence, 2uL of C sequence, 2uL of F sequence, 4uL of F-R1 sequence, 4uL of F-R2 sequence, 4uL of G sequence, 8uL of G-R1 sequence, 8uL of G-R2 sequence, 8uL of H sequence and 16uL of I sequence to obtainmixed solution PM 1; adding 1ul of mixed solution PM1 into 49ul of prehybridization solution to prepare hybridizationbuffer solution PM 2;

(2) taking a sample to be detected, dripping 25ul of hybridization solution PM2, covering with a glass slide, and sealing with an adhesive;

(3) denaturing the glass slide at 98 ℃ for 3min, rapidly cooling to 68 ℃ and preserving heat for 30min, naturally and slowly cooling to 40 ℃ and preserving heat for 50min, and naturally cooling to room temperature;

(4) the slide was taken out and observed under 488nm excitation light under a fluorescence microscope and photographed.

2. Results

The results are shown in FIGS. 13 to 14, and the green part development signals of the fluorography were compared and analyzed using a two-photon confocal fluorescence microscope and ImageJ software.

It can be seen that the negative control has almost no signal, while the positive control has a relatively strong fluorescence hybridization signal, indicating that the method of the invention can accurately detect, while the removal of the specific sequence cannot accurately detect.

In conclusion, the kit and the method can effectively detect the gene and have good application prospect.

Sequence listing

<110> Sichuan university Hospital in western China

<120> Gene detection kit comprising universal fluorescent subtraction Probe nucleic acid molecule and use thereof

<130> GY026-18P1368

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> fixed sequence in the first half of A sequence (artificial sequence)

<400> 1

ctgaagcgct tcgcgagcc 19

<210> 2

<211> 19

<212> DNA

<213> fixed sequence in the latter half of the A sequence (artificial sequence)

<400> 2

ctgaagcgct tcgcgagcc 19

<210> 3

<211> 14

<212> DNA

<213> B sequence fixed sequence (artificial sequence)

<400> 3

taggctcgcg aagc 14

<210> 4

<211> 13

<212> DNA

<213> C sequence fixed sequence (artificial sequence)

<400> 4

gcgaagcgct tca 13

<210> 5

<211> 19

<212> DNA

<213> D sequence (artificial sequence)

<400> 5

ggctcgcgaa gcgcttcag 19

<210> 6

<211> 18

<212> DNA

<213> E sequence (artificial sequence)

<400> 6

ctgaagcgct tcgcgagc 18

<210> 7

<211> 57

<212> DNA

<213> F sequence (artificial sequence)

<400> 7

ccagagcgag cagtgtcaag gctcgcgaag cgcttcagaa ccagagcgag cagtgtc 57

<210> 8

<211> 19

<212> DNA

<213> F-R1 sequence (artificial sequence)

<400> 8

cgagccttga cactgctcg 19

<210> 9

<211> 20

<212> DNA

<213> F-R2 sequence (artificial sequence)

<400> 9

gctcgctctg gttctgaagc 20

<210> 10

<211> 53

<212> DNA

<213> G sequence (artificial sequence)

<400> 10

gaacagtcgt gaacatctga cactgctcgc tctggtgaac agtcgtgaac atc 53

<210> 11

<211> 18

<212> DNA

<213> G-R1 sequence (artificial sequence)

<400> 11

gcagtgtcag atgttcac 18

<210> 12

<211> 17

<212> DNA

<213> G-R2 sequence (artificial sequence)

<400> 12

gactgttcac cagagcg 17

<210> 13

<211> 17

<212> DNA

<213> H sequence (artificial sequence)

<400> 13

gatgttcacg actgttc 17

<210> 14

<211> 16

<212> DNA

<213> I sequence (artificial sequence)

<400> 14

gaacagtcgt gaacat 16

Claims (12)

Translated fromChinese

1.一种基因检测试剂盒，其特征在于：它包括如下探针：1. a gene detection kit, is characterized in that: it comprises following probe:A序列:A sequence:5'–CTGAAGCGCTTCGCGAGCCM₁M₂N₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀M₃M₄CTGAAGCGCTTCGCGAGCC-3'；其中，N₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀为待检基因或其片段的互补序列，M₁M₂和M₃M₄为接头序列；5'–CTGAAGCGCTTCGCGAGCCM₁ M₂ N₁ N₂ N₃ N₄ N₅ …N₆ N₇ N₈ N₉ N₁₀ M₃ M₄ CTGAAGCGCTTCGCGAGCC-3’; where N₁ N₂ N₃ N₄ N₅ … N₆ N₇ N₈ N₉ N₁₀ is the complementary sequence of the gene to be detected or its fragment, and M₁ M₂ and M₃ M₄ are the linker sequences;B序列: 5'-n₅n₄n₃n₂n₁m₂m₁TAGGCTCGCGAAGC-3'； n₁n₂n₃n₄n₅与N₁N₂N₃N₄N₅的序列互补，m₁m₂与M₁M₂的序列互补；B sequence: 5'-n₅ n₄ n₃ n₂ n₁ m₂ m₁ TAGGCTCGCGAAGC-3'; n₁ n₂ n₃ n₄ n₅ is complementary to the sequence of N₁ N₂ N₃ N₄ N₅ , m₁ m₂ is complementary to the sequence of M₁ M₂ ;C序列: 5'-GCGAAGCGCTTCAGm₄m₃n₁₀n₉n₈n₇n₆-3'；n₆n₇n₈n₉n₁₀与N₆N₇N₈N₉N₁₀的序列互补，m₃ m₄与M₃M₄的序列互补；C sequence: 5'-GCGAAGCGCTTCAGm₄ m₃ n₁₀ n₉ n₈ n₇ n₆ -3'; n₆ n₇ n₈ n₉ n₁₀ is complementary to the sequence of N₆ N₇ N₈ N₉ N₁₀ , m₃ m₄ is complementary to the sequence of M₃ M₄ ;D序列:5'-荧光分子-GGCTCGCGAAGCGCTTCAG–荧光分子-3'D Sequence: 5'-Fluorescent Molecule-GGCTCGCGAAGCGCTTCAG-Fluorescent Molecule-3'E序列:5'- 荧光淬灭分子- CTGAAGCGCTTCGCGAGC-荧光淬灭分子- 3'。E sequence: 5'-fluorescence quenching molecule-CTGAAGCGCTTCGCGAGC-fluorescence quenching molecule-3'.2.一种基因检测试剂盒，其特征在于：它包括如下探针：2. a gene detection kit, is characterized in that: it comprises following probe:⑴ A序列:⑴ A sequence:5'–CTGAAGCGCTTCGCGAGCCM₁M₂N₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀M₃M₄CTGAAGCGCTTCGCGAGCC-3'；其中，N₁N₂N₃N₄N₅…N₆N₇N₈N₉N₁₀为待检基因或其片段的互补序列，M₁M₂和M₃M₄为接头序列；5'–CTGAAGCGCTTCGCGAGCCM₁ M₂ N₁ N₂ N₃ N₄ N₅ …N₆ N₇ N₈ N₉ N₁₀ M₃ M₄ CTGAAGCGCTTCGCGAGCC-3’; where N₁ N₂ N₃ N₄ N₅ … N₆ N₇ N₈ N₉ N₁₀ is the complementary sequence of the gene to be detected or its fragment, and M₁ M₂ and M₃ M₄ are the linker sequences;⑵ B序列: 5'-n₅n₄n₃n₂n₁m₂m₁TAGGCTCGCGAAGC-3'； n₁n₂n₃n₄n₅与N₁N₂N₃N₄N₅的序列互补，m₁m₂与M₁M₂的序列互补；(2) B sequence: 5'-n₅ n₄ n₃ n₂ n₁ m₂ m₁ TAGGCTCGCGAAGC-3'; n₁ n₂ n₃ n₄ n₅ is complementary to the sequence of N₁ N₂ N₃ N₄ N₅ , m₁ m₂ is complementary to the sequence of M₁ M₂ ;⑶ C序列: 5'-GCGAAGCGCTTCAGm₄m₃n₁₀n₉n₈n₇n₆-3'；n₆n₇n₈n₉n₁₀与N₆N₇N₈N₉N₁₀的序列互补， m₃ m₄与M₃M₄的序列互补；(3) C sequence: 5'-GCGAAGCGCTTCAGm₄ m₃ n₁₀ n₉ n₈ n₇ n_6-3 '; n₆ n₇ n₈ n₉ n₁₀ is complementary to the sequence of N₆ N₇ N₈ N₉ N₁₀ , m₃ m₄ is complementary to the sequence of M₃ M₄ ;⑷F序列:⑷F sequence:5'-CCAGAGCGAGCAGTGTCAAGGCTCGCGAAGCGCTTCAGAACCAGAGCGAGCAGTGTC-3'5'-CCAGAGCGAGCAGTGTCAAGGCTCGCGAAGCGCTTCAGAACCAGAGCGAGCAGTGTC-3'⑸ F-R1序列: 5'-CGAGCCTTGACACTGCTCG-3'⑸ F-R1 sequence: 5'-CGAGCCTTGACACTGCTCG-3'⑹ F-R2序列: 5'-GCTCGCTCTGGTTCTGAAGC-3'⑹ F-R2 sequence: 5'-GCTCGCTCTGGTTCTGAAGC-3'⑺ G序列⑺ G sequence5'-GAACAGTCGTGAACATCTGACACTGCTCGCTCTGGTGAACAGTCGTGAACATC-3'5'-GAACAGTCGTGAACATCTGACACTGCTCGCTCTGGTGAACAGTCGTGAACATC-3'⑻ G-R1序列:⑻ G-R1 sequence:5'-GCAGTGTCAGATGTTCAC-3'5'-GCAGTGTCAGATGTTCAC-3'⑼ G-R2序列:⑼ G-R2 sequence:5'-GACTGTTCACCAGAGCG-3'5'-GACTGTTCACCAGAGCG-3'⑽ H序列:⑽ H sequence:5'-荧光分子-GATGTTCACGACTGTTC-荧光分子-3'5'-Fluorescent Molecule-GATGTTCACGACTGTTC-Fluorescent Molecule-3'⑾ I序列:⑾ I sequence:5'-荧光淬灭分子-GAACAGTCGTGAACAT-荧光淬灭分子-3'。5'-fluorescence quenching molecule-GAACAGTCGTGAACAT-fluorescence quenching molecule-3'.3.根据权利要求1所述的检测试剂盒，其特征在于：所述M₁M₂为TA或AC，和/或，所述M₃M₄为AT、CA或TC。The detection kit according to claim 1, characterized in that: the M₁ M₂ is TA or AC, and/or the M₃ M₄ is AT, CA or TC.4.根据权利要求1所述的检测试剂盒，其特征在于：所述A序列与B序列的摩尔比为1：（2~7）；所述A序列与C序列的摩尔比为1：（2~7）；所述A序列与D序列的摩尔比为1：（2~7）；所述D序列与E序列的摩尔比为1：（2~5）。4. The detection kit according to claim 1, wherein the molar ratio of the A sequence to the B sequence is 1:(2～7); the molar ratio of the A sequence to the C sequence is 1:( 2~7); the molar ratio of the A sequence to the D sequence is 1:(2~7); the molar ratio of the D sequence to the E sequence is 1:(2~5).5. 根据权利要求4所述的检测试剂盒，其特征在于：A序列、B序列、 C序列、 D序列与E序列的摩尔比为1:5:5:3:6。5. detection kit according to claim 4, is characterized in that: the mol ratio of A sequence, B sequence, C sequence, D sequence and E sequence is 1:5:5:3:6.6.根据权利要求2所述的检测试剂盒，其特征在于：所述A序列与B序列的摩尔比为1：(2～7)；所述A序列与C序列的摩尔比为1：(2～7)；所述A序列与F序列的摩尔比为1：(2～7)；所述F序列与F-R1序列的摩尔比为1：(2～7)；所述F序列与F-R2序列的摩尔比为1：(2～7)；所述F序列与G序列的摩尔比为1：(2～7)；所述G序列与G-R1序列的摩尔比为1：(2～7)；所述G序列与G-R2序列的摩尔比为1：(2～7)；所述G序列与H序列的摩尔比为1：(2～7)；所述H序列与I序列的摩尔比为1：(2～5)。6. The detection kit according to claim 2, wherein the molar ratio of the A sequence to the B sequence is 1:(2～7); the molar ratio of the A sequence to the C sequence is 1:( 2-7); the molar ratio of the A sequence to the F sequence is 1: (2-7); the molar ratio of the F sequence to the F-R1 sequence is 1: (2-7); the F sequence and The molar ratio of the F-R2 sequence is 1: (2-7); the molar ratio of the F sequence to the G sequence is 1: (2-7); the molar ratio of the G sequence to the G-R1 sequence is 1: (2-7); the molar ratio of the G sequence to the G-R2 sequence is 1:(2-7); the molar ratio of the G sequence to the H sequence is 1:(2-7); the H sequence The molar ratio to the I sequence is 1:(2-5).7. 根据权利要求6所述的检测试剂盒，其特征在于：所述A序列、B序列、C序列、F序列、F-R1序列、F-R2序列、G序列、G-R1序列、G-R2序列、H序列、I序列的摩尔比为1:2:2:2:4:4:4:8:8:8:16。7. detection kit according to claim 6, is characterized in that: described A sequence, B sequence, C sequence, F sequence, F-R1 sequence, F-R2 sequence, G sequence, G-R1 sequence, G -The molar ratio of R2 sequence, H sequence, and I sequence is 1:2:2:2:4:4:4:8:8:8:16.8.根据权利要求1～7任意一项所述的试剂盒，其特征在于：所述待检基因为任意人体、大鼠或小鼠基因。8 . The kit according to claim 1 , wherein the gene to be tested is any human, rat or mouse gene. 9 .9. 根据权利要求1～7任意一项所述的试剂盒，其特征在于：所述荧光分子为6-羧基荧光素、四氯-6羧基荧光素、六氯-6甲基荧光素或者2，7， -二甲基-4 ,5-二氯-6-羧基荧光素；9. The kit according to any one of claims 1 to 7, wherein the fluorescent molecule is 6-carboxyfluorescein, tetrachloro-6-carboxyfluorescein, hexachloro-6 methylfluorescein or 2 , 7, -dimethyl-4,5-dichloro-6-carboxyfluorescein;和/或，所述荧光淬灭分子为6-羧基-4甲基罗丹明或者黑洞淬灭基团。And/or, the fluorescence quenching molecule is 6-carboxy-4 methylrhodamine or a black hole quenching group.10.根据权利要求1～7任意一项所述的试剂盒，其特征在于：所述试剂盒还包括TE缓冲液和预杂交液；10. The kit according to any one of claims 1 to 7, wherein the kit further comprises a TE buffer and a pre-hybridization solution;所述TE缓冲液是含有10mmol/L tris和1mmol/L EDTA的溶液；The TE buffer is a solution containing 10 mmol/L tris and 1 mmol/L EDTA;所述预杂交液是含有60mmol/L tris、50mmol/L NaCl、1mmol/L EDTA、0 .5％SDS(w/v)和5％(w/v)的甲酰胺的溶液。The prehybridization solution was a solution containing 60 mmol/L tris, 50 mmol/L NaCl, 1 mmol/L EDTA, 0.5% SDS (w/v) and 5% (w/v) formamide.11.一种基因检测方法，其特征在于：步骤如下：11. A gene detection method, characterized in that: the steps are as follows:(1)取权利要求1～8任意一项所述试剂盒中的探针，溶解于TE缓冲液中，得到包含所有探针序列的混合液PM1；(1) taking the probes in the kit according to any one of claims 1 to 8 and dissolving them in TE buffer to obtain a mixed solution PM1 containing all the probe sequences;(2)取混合液PM1加入到预杂交液配制成杂交缓冲液PM2；(2) get mixed solution PM1 and add it to pre-hybridization solution to prepare hybridization buffer PM2;(3)全程避光，取杂交缓冲液PM2滴加到待测的染色体滴片或组织切片上，盖上盖玻片，用封胶封固；(3) Protect from light during the whole process, take the hybridization buffer PM2 dropwise and add it to the chromosome drop or tissue section to be tested, cover it with a cover glass, and seal it with sealant;(4)将玻片加热到95℃～98℃变性3到5min，再迅速降温至45℃ -70℃保温20到60min，使通用荧光探针与靶核酸完成分子杂交，然后再降温至30℃ -50℃保温40到60min；(4) Heat the glass slide to 95℃～98℃ for 3 to 5 minutes, and then rapidly cool down to 45℃-70℃ for 20 to 60 minutes to complete the molecular hybridization between the universal fluorescent probe and the target nucleic acid, and then cool down to 30℃ -50°C for 40 to 60 minutes;(5)加入防淬灭封片剂，置荧光显微镜下观察杂交信号。(5) Add anti-quenching mountant, and observe the hybridization signal under a fluorescence microscope.12.根据权利要求11所述的方法，其特征在于：步骤如下：步骤(4)为：将玻片在95℃或者98℃环境下变性3min，再迅速降温至68℃保温30min，自然降温至40℃保温50min，再自然降至室温；12. The method according to claim 11, characterized in that: the steps are as follows: step (4) is: denaturation of the glass slide at 95°C or 98°C for 3min, then rapidly cooled to 68°C for 30min, and naturally cooled to Incubate at 40°C for 50min, and then naturally drop to room temperature;步骤(5)为：加入防淬灭封片剂，放上盖玻片，置荧光显微镜下488nm、405nm、670nm激发光下观察并照相。Step (5) is as follows: adding anti-quenching mounting medium, placing a cover glass, observing and photographing under a fluorescence microscope under excitation light of 488 nm, 405 nm and 670 nm.

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