A FISH probe combination for three-gene joint detection and its application

Abstract

The present invention provides a FISH probe combination for three-gene joint detection and its application, the FISH probe combination for three-gene joint detection simultaneously detects the translocation of MYC gene, BCL-2 gene and BCL-6 gene, wherein the fluorescein labeling methods of the probe for detecting MYC gene, the probe for detecting BCL-2 gene and the probe for detecting BCL-6 gene are all different, and the fluorescein labeling method includes using red fluorescein labeling, using green fluorescein labeling or using any one of red fluorescein and green fluorescein labeling simultaneously. The present invention improves the labeling method of the probe, realizes the simultaneous interpretation of three groups of probes on a single sample, and the operation is simpler, and the efficiency and flux of the detection are improved; the accuracy, sensitivity and specificity are all good, and the detection cost is reduced, and the value of practical application is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a FISH probe combination for three-gene joint detection and an application thereof. Background Art

[0002] High-grade B-cell lymphoma is accompanied by MYC, BCL-2, and BCL-6 gene translocations, and its genetic characteristics are the simultaneous presence of MYC and BCL-2 (or BCL-6) gene rearrangements (double hits), or the simultaneous presence of MYC, BCL-2, and BCL-6 gene rearrangements (triple hits). Its treatment is different from traditional B-cell lymphoma. It is insensitive to traditional chemotherapy, and the effect of enhanced R-CHOP is not ideal. Triple-target B-cell lymphoma represents an independent pathological subtype. With the demand for precise diagnosis and molecular targeted therapy, research on MYC inhibitors (such as PI3K kinase inhibitors, etc.), BCL2 small molecule inhibitors, and BCL6 inhibitors are being carried out one after another. Detection of these three targets can help determine the patient's prognosis and choose treatment methods.

[0003] Fluorescence in situ hybridization (FISH) is a cytogenetic detection method based on nucleic acid hybridization. It uses labeled nucleic acid probes to perform in situ hybridization with sample nucleic acids fixed on a solid phase carrier. Under the stimulation of a suitable light source, microscopic signal recognition is performed to complete the detection of genetic abnormalities. FISH achieves multi-color detection through filter blocks and different fluorescent groups. In the FISH detection of triple-hit targets of lymphoma, three sets of two-color probes are generally used for the rearrangement detection of MYC, BCL2 and BCL6 genes. Generally, three consecutive sections are used to hybridize different probes respectively, and the detection status of one probe in each sample area is observed to make a comprehensive judgment.

[0004] Commonly used fluorescent dyes for FISH include red and green, and the signal can be directly observed through a three-way or two-way filter block. Multicolor FISH (M-FISH) can perform up to four-color detection (red, green, cyan and gold), but the cyan group (such as coumarin) is easily quenched, and there are few commercial manufacturers of gold groups, so clinical applications are not widespread. In addition, due to the wide excitation / emission spectrum and the lack of suitable filter block components, it is impossible to observe four-color signals in one field of view at the same time.

[0005] Therefore, how to realize the simultaneous interpretation of multiple sets of probes and then quickly detect multiple genes has become a problem that needs to be solved urgently. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides a FISH probe combination for the joint detection of three genes and its application. By simultaneously labeling one of the probes with red fluorescent dye and green fluorescent dye, which appears yellow after mixing, the joint detection of three genes can be completed under a single filter, which is simple to operate, takes less time for detection, and is more efficient.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a FISH probe combination for three-gene combined detection, wherein the FISH probe combination for three-gene combined detection simultaneously detects the translocation of MYC gene, BCL-2 gene and BCL-6 gene;

[0009] Among them, the probe for detecting the MYC gene, the probe for detecting the BCL-2 gene, and the probe for detecting the BCL-6 gene are all labeled with different fluorescein methods, and the fluorescein labeling method includes using red fluorescein labeling, using green fluorescein labeling, or using both red fluorescein and green fluorescein labeling.

[0010] In the present invention, one of the probes is labeled with red fluorescent dye and green fluorescent dye at the same time, and appears yellow after mixing, thereby realizing the simultaneous interpretation of three groups of probes on a single sample, thereby performing low-cost rapid detection of the status of MYC gene, BCL-2 gene and BCL-6 gene, and improving the target flux of the detection; the signal interpretation of the three genes can be completed under a single filter, the operation is simpler, the detection is faster, the efficiency is higher, and the clinical detection cost is reduced; the detection results are consistent with expectations, and the sensitivity and specificity are excellent.

[0011] Preferably, the template for preparing the probe for detecting the MYC gene includes RP11-524D19, RP11-1039B2, CTD-2533C10, RP11-440N18, CTD-2205H22, RP11-55J15, CTD-2160F24 and RP11-485I14 cloning plasmids.

[0012] Preferably, the preparation template for detecting the BCL-2 gene includes CTD-2145C18, RP11-280C1, CTD-2270P21, CTD-2166H2 and RP11-977F4 cloning plasmids.

[0013] Preferably, the template for preparing the probe for detecting the BCL-6 gene includes RP11-1058L21, RP11-58M14, RP11-116N23, RP11-465M10, RP11-44H4 and RP11-464D9 cloning plasmids.

[0014] Preferably, the fluorescein is labeled with dUTP.

[0015] Preferably, the red fluorescent pigment comprises TRITC.

[0016] Preferably, the green fluorescent pigment comprises Fluorescein.

[0017] In a second aspect, the present invention provides a method for preparing the FISH probe combination for three-gene joint detection according to the first aspect, the preparation method comprising:

[0018] The preparation template of the probe is mixed with a buffer, dNTPs, fluorescein-labeled dUTP, DNA polymerase and DNaseI respectively, and after labeling, the FISH probe combination for the three-gene joint detection is obtained.

[0019] Preferably, in a reaction system using both red fluorescent and green fluorescent labeled probes, the molar ratio of dUTP labeled with Fluorescein to dUTP labeled with TRITC is (1.3-1.5):1, for example, it can be 1.3:1, 1.35:1, 1.4:1, 1.45:1 or 1.5:1, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0020] In the present invention, the preparation method of the probe labeled with red fluorescein and green fluorescein is improved to make the yellow signal of the probe more uniform. The experimental results show that the effect of double fluorescein mixed labeling of the probe is better than that of labeling the probe with single fluorescein and then mixing. When the molar ratio of dUTP labeled with Fluorescein and dUTP labeled with TRITC is within the range of (1.3-1.5):1, the yellow signal is clearer and more specific, and the detection effect is better.

[0021] In a third aspect, the present invention provides a FISH kit for three-gene combined detection, wherein the FISH kit for three-gene combined detection comprises the FISH probe combination for three-gene combined detection described in the first aspect.

[0022] Preferably, the FISH kit for three-gene combined detection further comprises a hybridization buffer, a permeabilization agent, a digestion agent and an in situ hybridization staining solution.

[0023] Preferably, the hybridization buffer comprises sodium citrate buffer (SSC).

[0024] Since the fluorescent marker in the FISH probe has the characteristic of random incorporation, the probe will hybridize competitively during hybridization, resulting in poor uniformity of nucleic acid hybridization signals. The hybridization buffer in the present invention increases the stability of the system, promotes system uniformity, and improves the effect of hybridization.

[0025] Preferably, the mass fraction of the tetraacetic acid in the permeabilizing agent is 1.5% to 3%, for example, 1.5%, 2%, 2.5% or 3%, etc. Other specific values ​​within this numerical range can be selected and will not be described in detail here.

[0026] Preferably, the mass fraction of the polyethylene glycol in the permeabilizing agent is 6% to 8%, for example, 6%, 6.5%, 7%, 7.5% or 8%, etc. Other specific points within this numerical range can be selected and will not be described in detail here.

[0027] Preferably, the mass fraction of Tween in the permeabilizing agent is 1% to 3%, for example, 1%, 1.5%, 2%, 2.5% or 3%, etc. Other specific points within this numerical range can be selected and will not be described in detail here.

[0028] As a preferred technical solution, the permeabilizing agent of the present invention includes, by mass fraction, 1.5% to 3% tetraacetic acid, 6% to 8% polyethylene glycol and 1% to 3% Tween.

[0029] In the present invention, a permeabilizing agent is used to pretreat the sample, which increases the permeability of the sample and improves the hybridization and staining effects; compared with the traditional high-temperature boiling pretreatment method, it can avoid nuclear lysis or nucleic acid breakage caused by high temperature, improve the signal-to-noise ratio, and enhance the sensitivity and accuracy of detection.

[0030] Preferably, the digestive agent comprises pepsin.

[0031] In a fourth aspect, the present invention provides a method for using the FISH kit for three-gene combined detection according to the third aspect for the purpose of non-disease diagnosis and / or treatment, the method comprising:

[0032] The samples were digested with a digestive agent, and the FISH probe combination for three-gene combined detection was added for denaturation and hybridization, followed by staining and observation.

[0033] Preferably, the final concentration of the digestive agent is 3 to 5 mg / mL, for example, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL or 5 mg / mL, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0034] Preferably, the digestion time of the digestive agent is 5 to 8 minutes, for example, it can be 5 minutes, 5.5 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes or 8 minutes, and other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0035] Preferably, the final concentration of the FISH probe combination for the three-gene combined detection is 10-20 ng / μL, for example, it can be 10 ng / μL, 11 ng / μL, 12 ng / μL, 13 ng / μL, 14 ng / μL, 15 ng / μL, 16 ng / μL, 17 ng / μL, 18 ng / μL, 19 ng / μL or 20 ng / μL, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0036] Preferably, the denaturation condition is incubation at 75-90°C for 2-8 min. The temperature can be, for example, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C or 90°C, and the time can be, for example, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min or 8 min. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0037] Preferably, the hybridization conditions are incubation at 36.5-37.5°C for 4-8h. The temperature can be, for example, 36.5°C, 37°C or 37.5°C, and the time can be, for example, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h or 8h. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0038] Preferably, the method of use further comprises the step of permeabilizing using a permeabilizing agent.

[0039] Preferably, the permeability time is 3 to 8 minutes, for example, it can be 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes or 8 minutes, and other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0040] As a preferred technical solution, the method of using the FISH kit for three-gene combined detection of the present invention for the purpose of non-disease diagnosis and / or treatment includes:

[0041] Add permeabilization agent to the sample and incubate for 3 to 8 minutes;

[0042] Digest with pepsin at a final concentration of 3 to 5 mg / mL for 5 to 8 min and wash;

[0043] Add FISH probe combination for three-gene joint detection at a final concentration of 10-20 ng / μL and hybridization buffer, denature at 75-90°C for 2-8 min, then hybridize at 36.5-37.5°C for 4-8 h, and remove unhybridized probes;

[0044] Use in situ hybridization staining solution for staining and observation.

[0045] In a fifth aspect, the present invention provides a device for three-gene combined detection, wherein the device for three-gene combined detection simultaneously detects the translocation of MYC gene, BCL-2 gene and BCL-6 gene;

[0046] The device for three-gene combined detection comprises:

[0047] Pretreatment module: permeabilize and digest the samples;

[0048] Hybridization module: denaturation and hybridization of samples using FISH probe combination for three-gene joint detection;

[0049] Color development and analysis module: Use in situ hybridization staining solution to stain the hybridized samples and perform analysis.

[0050] In a sixth aspect, the present invention provides the use of any one or a combination of at least two of the FISH probe combination for three-gene combined detection described in the first aspect, the FISH kit for three-gene combined detection described in the third aspect, or the device for three-gene combined detection described in the fifth aspect in the preparation of a three-gene combined detection product.

[0051] Preferably, the three-gene combined detection product simultaneously detects the translocation of the MYC gene, the BCL-2 gene and the BCL-6 gene.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The present invention improves the labeling method of the probes to achieve simultaneous interpretation of three groups of probes on a single sample, thereby improving the detection throughput, making the operation simpler, taking less time, being more efficient, and reducing the cost;

[0054] (2) The present invention improves the preparation method of the probe labeled with red and green fluorescent pigments, uses dual fluorescent pigments for mixed labeling, and optimizes the ratio between green and red fluorescent pigments. The yellow signal of the probe is more uniform, and the image is clearer, which is convenient for observation.

[0055] (3) The present application uses a permeabilizing agent to pre-treat the sample, thereby avoiding the damage to the sample nucleic acid by traditional high-temperature boiling, improving the signal-to-noise ratio, and also improving the sensitivity and specificity of the detection;

[0056] (4) The samples were tested using the FISH probe combination of the present invention, and the results were in line with expectations, with accuracy and specificity reaching 100%. Compared with conventional dual-color break probes, the detection accuracy was higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1A This is a picture of the staining results of the FISH kit for three-gene combined detection prepared in Example 4 in Test Example 1 (magnification is 1000×);

[0058] Figure 1B This is a picture of the staining results of the FISH kit for three-gene combined detection prepared in Example 5 in Test Example 1 (magnification is 1000×);

[0059] Figure 1C This is a picture of the staining results of the FISH kit for three-gene combined detection prepared in Example 6 in Test Example 1 (magnification is 1000×);

[0060] Figure 2A This is a picture of the result of staining human peripheral blood culture cells using the FISH kit for three-gene combined detection prepared in Example 4 in Test Example 2 (magnification is 1000×);

[0061] Figure 2B This is a picture of the result of staining a tissue section sample using the FISH kit for three-gene combined detection prepared in Example 4 in Test Example 2 (magnification is 1000×);

[0062] Figure 2C This is a picture of the result of staining human peripheral blood culture cells using the MYC gene breakage detection probe in Comparative Example 2 in Test Example 2 (magnification is 1000×);

[0063] Figure 2D This is a picture of the result of staining a tissue section sample using the MYC gene break detection probe in Comparative Example 2 in Test Example 2 (magnification is 1000×);

[0064] Figure 2E This is a picture of the result of staining human peripheral blood culture cells using the BCL-2 gene breakage detection probe in Comparative Example 2 in Test Example 2 (magnification is 1000×);

[0065] Figure 2FThis is a picture of the result of staining a tissue section sample using the BCL-2 gene breakage detection probe in Comparative Example 2 in Test Example 2 (magnification is 1000×);

[0066] Figure 2G This is a picture of the result of staining human peripheral blood culture cells using the BCL-6 gene breakage detection probe in Comparative Example 2 in Test Example 2 (magnification is 1000×);

[0067] Figure 2H This is a picture of the result of staining a tissue section sample using the BCL-6 gene breakage detection probe in Comparative Example 2 in Test Example 2 (magnification is 1000×);

[0068] Figure 3A This is a picture of the staining result of the sample pre-treated using the traditional method in Test Example 3 (magnification is 1000×);

[0069] Figure 3B This is a picture of the staining result of the sample pretreated with a permeabilizing agent in Test Example 3 (magnification is 1000×);

[0070] Figure 4A This is the test result image of the MYC positive sample in test example 4 (magnification is 1000×);

[0071] Figure 4B This is the test result image of the MYC positive sample in test example 4 (magnification is 1000×);

[0072] Figure 4C This is the test result image of the BCL2 positive sample in test example 4 (magnification is 1000×);

[0073] Figure 4D This is the test result image of the BCL2 positive sample in test example 4 (magnification is 1000×);

[0074] Figure 4E This is the test result image of the BCL6 positive sample in test example 4 (magnification is 1000×);

[0075] Figure 4F This is a picture of the detection results of the BCL6 positive sample in Test Example 4 (magnification is 1000×). DETAILED DESCRIPTION

[0076] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0077] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0078] Example 1

[0079] This embodiment provides a FISH probe combination for three-gene joint detection, wherein the FISH probe combination for three-gene joint detection simultaneously detects the translocation of MYC gene, BCL-2 gene and BCL-6 gene;

[0080] Among them, the probes for detecting the MYC gene, the probes for detecting the BCL-2 gene, and the probes for detecting the BCL-6 gene are labeled with different fluorescein labeling methods. The probe for detecting the MYC gene is labeled with red fluorescein, the probe for detecting the BCL-2 gene is labeled with green fluorescein, and the probe for detecting the BCL-6 gene is labeled with both red and green fluorescein.

[0081] Among them, the preparation system of the probe for detecting the MYC gene is as follows:

[0082]

[0083]

[0084] The DNA template consisted of RP11-524D19, RP11-1039B2, CTD-2533C10, RP11-440N18, CTD-2205H22, RP11-55J15, CTD-2160F24 and RP11-485I14 cloned plasmids (purchased from Invitrogen), and the plasmids were extracted according to the conventional process, with OD260 / 280>1.9. The probe length was about 1.4Mbp (chr8:127,995,358-129,396,342, GRCh37 / hg19).

[0085] After plasmid labeling, use sodium acetate to precipitate nucleic acid, and obtain labeled probe after high-speed centrifugation. The probe is reconstituted with 5 μL purified water, the concentration is determined, and stored in the dark.

[0086] The preparation system of the probe for detecting the BCL-2 gene is as follows:

[0087]

[0088]

[0089] The DNA template consisted of CTD-2145C18, RP11-280C1, CTD-2270P21, CTD-2166H2 and RP11-977F4 cloned plasmids (purchased from Invitrogen), and the plasmids were extracted according to the conventional process, with OD260 / 280>1.9. The probe length was about 768 kbp (chr18: 60,531,844-61,300,107, GRCh37 / hg19).

[0090] After plasmid labeling, use sodium acetate to precipitate nucleic acid, and obtain labeled probe after high-speed centrifugation. The probe is reconstituted with 5 μL purified water, the concentration is determined, and stored in the dark.

[0091] The preparation system of the probe for detecting the BCL-6 gene is as follows:

[0092]

[0093] The DNA template consisted of RP11-1058L21, RP11-58M14, RP11-116N23, RP11-465M10, RP11-44H4 and RP11-464D9 cloned plasmids (purchased from Invitrogen), and the plasmids were extracted according to the conventional process, with OD260 / 280>1.9. The probe length was about 858 kbp (chr3:187,010,704-187,869,346, GRCh37 / hg19).

[0094] After plasmid labeling, use sodium acetate to precipitate nucleic acid, and obtain labeled probe after high-speed centrifugation. The probe is reconstituted with 5 μL purified water, the concentration is determined, and stored in the dark.

[0095] In the above process, Fluorescein (fluorescein) labeled dUTP was purchased from Roche (Cat. No. 11373242910), TRITC (tetramethylrhodamine) labeled dUTP was purchased from Roche (Cat. No. 11534378910), DNaseI was purchased from NEB (Cat. No. M0303), and DNA polymerase I was purchased from NEB (Cat. No. M0209).

[0096] Example 2

[0097] This embodiment provides a FISH probe combination for three-gene combined detection, which is different from Embodiment 1 only in that the preparation system of the probe for detecting the BCL-6 gene is different, and the other materials and preparation methods are the same as those in Embodiment 1.

[0098] In this embodiment, the preparation system of the probe for detecting the BCL-6 gene is as follows:

[0099]

[0100]

[0101] Example 3

[0102] This embodiment provides a FISH probe combination for the combined detection of three genes. The only difference from Example 1 is that when preparing the probe of the BCL-6 gene, the probes are labeled with Fluorescein-labeled dUTP and TRITC-labeled dUTP, respectively, and then mixed. The remaining materials and preparation methods are the same as those in Example 1.

[0103] The preparation system of the probe (component A) for detecting the BCL-6 gene is as follows:

[0104]

[0105] The preparation system of the probe (B component) for detecting the BCL-6 gene is as follows:

[0106]

[0107]

[0108] Example 4

[0109] This embodiment provides a FISH kit for three-gene combined detection, and the FISH kit for three-gene combined detection includes the FISH probe combination for three-gene combined detection in Example 1, a hybridization buffer, a permeabilization agent, a digestion agent, and an in situ hybridization blue dye solution (purchased from Guangzhou Anping Pharmaceutical Technology Co., Ltd.);

[0110] Wherein, the hybridization buffer is sodium citrate buffer;

[0111] In terms of mass fraction, the permeabilizing agent contains 2% tetraacetic acid, 7% polyethylene glycol and 2% Tween, and the balance is water;

[0112] The digestive agent is pepsin (purchased from Sangon Biotechnology (Shanghai) Co., Ltd.).

[0113] Among them, the FISH probe combination for the combined detection of three genes was dissolved in sodium citrate buffer to prepare a hybridization solution. In the hybridization solution, the concentration of the probe for detecting the MYC gene was 10 ng / μL, the concentration of the probe for detecting the BCL-2 gene was 16 ng / μL, and the concentration of the probe for detecting the BCL-6 gene was 20 ng / μL.

[0114] Example 5

[0115] This embodiment provides a FISH kit for three-gene combined detection, which is different from Example 4 only in that the FISH probe combination for three-gene combined detection in Example 2 is used to replace the FISH probe combination for three-gene combined detection in Example 1, and the other materials and conditions are the same as those in Example 4.

[0116] Example 6

[0117] This embodiment provides a FISH kit for three-gene combined detection, which is different from Example 4 only in that the FISH probe combination for three-gene combined detection in Example 3 is used to replace the FISH probe combination for three-gene combined detection in Example 1, wherein the concentration of the probe prepared by component A is 16 ng / μL, and the concentration of the probe prepared by component B is 10 ng / μL, and the other materials and conditions are the same as those in Example 4.

[0118] Test Example 1

[0119] The FISH kits for three-gene combined detection prepared in Examples 4 to 6 were used to perform hybridization detection with human peripheral blood culture cells respectively, and the steps were as follows:

[0120] (1) Pretreatment:

[0121] After resuspending the cells, 3 μL of the cell suspension was added to the slide and allowed to dry at room temperature;

[0122] With the front side of the slide facing up, add 200 μL of pepsin working solution (final concentration 4 mg / mL) and digest for 5 min;

[0123] Add 2×SSC and incubate at room temperature for 5 min;

[0124] Dehydrate with 70%, 90%, and 100% ethanol gradients, incubating for 2 min each;

[0125] Remove the slides and let them dry at room temperature.

[0126] (2) Denaturation hybridization:

[0127] Add 10 μL of hybridization solution to the hybridization area and seal the slide;

[0128] Denature at 78°C for 2 min and hybridize at 37°C for 6 h.

[0129] (3) Re-staining (avoid light):

[0130] Incubate with 0.3% NP-40 (1× SSC configuration) at 72 ± 1°C for 2 min;

[0131] Incubate with 0.1% NP-40 (2×SSC configuration) at room temperature for 30 s;

[0132] Wash with 70% ethanol at room temperature for 2 min;

[0133] Take out the slide and dry it in the dark;

[0134] Add 10 μL of in situ hybridization blue staining solution and store in the dark until observation.

[0135] The staining picture of the FISH kit for three-gene combined detection prepared in Example 4 is as follows: Figure 1A As shown in the figure, the cells shown in the picture contain 2 green signals, 2 red signals and 2 yellow signals, and the overall signal uniformity is good and the signal is clear; compared with the result picture of Example 4, the result picture of Example 5 (as shown in Figure 1B As shown in the figure, the arrow indicates the green signal point) the background is green, which affects the overall observation and is easily confused with the green signal of BCL-2; in Example 6, the method of labeling the probes separately and then mixing them is adopted, the probes are unevenly distributed, there are obvious differences between different cell nuclei, and false signals are easy to appear (such as Figure 1C As shown, the green signal indicated by the long arrow is weak, thus appearing as a false red signal; the short arrow indicates a normal yellow signal). The above results show that the preparation method of the probe labeled with red and green fluorescent pigments is closely related to the detection results. Only when dual fluorescent pigments are mixed and labeled at the same time, and the molar ratio of green and red fluorescent pigments is within the range of (1.3-1.5):1, the yellow signal is clear and the detection effect is good.

[0136] Comparative Example 1

[0137] This comparative example provides a FISH probe combination for simultaneously detecting the MYC gene, the BCL-2 gene and the BCL-6 gene, wherein the probe for detecting the MYC gene comes from the MYC gene break detection probe (fluorescence in situ hybridization method) (item number F.01054-01) of Guangzhou Anping Pharmaceutical Technology Co., Ltd., the probe for detecting the BCL2 gene comes from the BCL2 gene break detection probe (fluorescence in situ hybridization method) (item number F.01172-01) of Guangzhou Anping Pharmaceutical Technology Co., Ltd., and the probe for detecting the BCL6 gene comes from the BCL26 gene break detection probe (fluorescence in situ hybridization method) (item number F.01069-01) of Guangzhou Anping Pharmaceutical Technology Co., Ltd.

[0138] Comparative Example 2

[0139] This comparative example provides a FISH kit for combined detection of three genes, which is different from Example 4 only in that the FISH probe combination for simultaneous detection of MYC gene, BCL-2 gene and BCL-6 gene in Comparative Example 1 is used to replace the FISH probe combination for combined detection of three genes in Example 1, and the other materials and conditions are the same as those in Example 4.

[0140] Test Example 2

[0141] The FISH kits for three-gene combined detection prepared in Example 4 and Comparative Example 2 were used to perform hybridization detection with human peripheral blood culture cells and tissue section samples, respectively.

[0142] The steps of hybridizing with human peripheral blood culture cells using the FISH kit for three-gene combined detection in Example 4 are the same as those in Test Example 1, and the steps of hybridizing with tissue section samples are as follows:

[0143] (1) Pretreatment:

[0144] Dewax using xylene (or xylene substitute) at room temperature for 10 min;

[0145] Incubate with 100% ethanol, 90% ethanol, and 70% ethanol for 3 min each at room temperature;

[0146] Wash with deionized water at room temperature for 3 minutes and shake off excess water;

[0147] Add permeabilization agent and incubate for 5 min;

[0148] Wash with purified water at 80-95°C for 5 minutes;

[0149] Wash with 2×SSC at room temperature for 3 min and dry at room temperature;

[0150] Add 200 μL of pepsin (final concentration 4 mg / mL) and digest at 37°C for 6 min;

[0151] Wash with 2×SSC at room temperature for 3 min;

[0152] Wash with 70% ethanol at room temperature for 2 min and dry at room temperature.

[0153] (2) Re-staining (avoid light):

[0154] Add 10 μL of in situ hybridization blue staining solution and store in a dark place until observation.

[0155] When using the FISH kit for three-gene combined detection prepared in Comparative Example 2, the MYC gene break detection probe, the BCL-2 gene break detection probe and the BCL-6 gene break detection probe are hybridized with three human peripheral blood culture cell samples and three tissue section samples, respectively, and observed separately. For the specific hybridization steps, please refer to the instructions in the product manual.

[0156] The staining pictures of human peripheral blood culture cells and tissue section samples using the FISH kit for three-gene combined detection prepared in Example 4 are as follows: Figure 2A and Figure 2BAs shown in the figure, the cells shown in the picture contain 2 green signals, 2 red signals and 2 yellow signals, and the signals are uniform and obvious; the staining pictures of the MYC gene break detection probe on human peripheral blood culture cells and tissue section samples in Comparative Example 2 are as follows Figure 2C and Figure 2D As shown, the staining images of BCL-2 gene break detection probe on human peripheral blood culture cells and tissue section samples are shown in Figure 2E and Figure 2F As shown, the staining images of BCL-6 gene break detection probe on human peripheral blood culture cells and tissue section samples are shown in Figure 2G and Figure 2H As shown. In the above pictures, fluorescent signals can be observed at the target site, and non-specific hybridization does not occur at other positions. In addition, gene fusion signals in the cell nucleus can be observed in some pictures, but only one gene can be detected and observed in one field of view. The above results show that compared with conventional two-color break probes, the FISH probe combination for the combined detection of three genes in this application shows a brighter signal in the sample, and the three genes can be interpreted at the same time, and accurate interpretation can be achieved under double-pass, the detection effect is better, the results are in line with expectations, and the detection accuracy is high.

[0157] Test Example 3

[0158] This test case verifies the impact of different preprocessing methods on samples.

[0159] The steps of the traditional method are as follows:

[0160] (1) Pretreatment:

[0161] Dewax using xylene (or xylene substitute) at room temperature for 10 min;

[0162] Incubate with 100% ethanol, 90% ethanol, and 70% ethanol for 3 min each at room temperature;

[0163] Wash with deionized water at room temperature for 3 minutes and shake off excess water;

[0164] Boil purified water at 95-100°C for 10 minutes;

[0165] Wash with 2×SSC at room temperature for 3 min and dry at room temperature;

[0166] Add 200 μL of pepsin (final concentration 4 mg / mL) and digest at 37°C for 6 min;

[0167] Wash with 2×SSC at room temperature for 3 min;

[0168] Wash with 70% ethanol at room temperature for 2 min and dry at room temperature.

[0169] (2) Re-staining (avoid light):

[0170] Add 10 μL of in situ hybridization blue staining solution and store in a dark place until observation.

[0171] The steps for using a permeabilizing agent are as follows:

[0172] (1) Pretreatment:

[0173] Dewax using xylene (or xylene substitute) at room temperature for 10 min;

[0174] Incubate with 100% ethanol, 90% ethanol, and 70% ethanol for 3 min each at room temperature;

[0175] Wash with deionized water at room temperature for 3 minutes and shake off excess water;

[0176] Add permeabilization agent and incubate for 5 min;

[0177] Wash with purified water at 80-95°C for 5 minutes;

[0178] Wash with 2×SSC at room temperature for 3 min and dry at room temperature;

[0179] Add 200 μL of pepsin (final concentration 4 mg / mL) and digest at 37°C for 6 min;

[0180] Wash with 2×SSC at room temperature for 3 min;

[0181] Wash with 70% ethanol at room temperature for 2 min and dry at room temperature.

[0182] (2) Re-staining (avoid light):

[0183] Add 10 μL of in situ hybridization blue staining solution and store in a dark place until observation.

[0184] The staining results are as follows Figure 3A and Figure 3B As shown. Figure 3A It can be seen that the cell nucleus disintegrated, and the chromatin decondensed, stacked, and fused, indicating that high-temperature boiling caused damage to the cell nucleus; Figure 3B The nuclear membrane of the cell nucleus is intact and the nuclear segmentation is clear, indicating that the treatment method using the permeabilization agent is relatively mild and avoids the damage of nucleic acid to high temperature.

[0185] Lymphoma samples have their own particularity, with small and densely arranged nuclei. Improper handling will lead to internuclear fusion, affecting the interpretation of subsequent signals. Tissue permeabilization before pepsin digestion is an important step. The permeabilization process opens the cross-linking during fixation, loosens the protein structure, and facilitates pepsin digestion. The above results show that the use of permeabilization agents has a better de-crosslinking effect and is relatively mild, without damaging the cell nucleus or nucleic acid.

[0186] Test Example 4

[0187] This test example uses the FISH kit for three-gene combined detection prepared in Example 4 for detection.

[0188] Twenty B-cell lymphoma FFPE samples (from Ambiping Laboratory) were collected, and the case information showed that the samples were positive for CD10, BCL2, and FOXP1. 18 of the 20 samples were CD20 positive, 19 samples showed high expression of MYC (19 / 20, 95%), 14 samples showed high expression of BCL6 (14 / 20, 70%), and MUM1 / IRF4 was positive in 10 samples (10 / 20, 50%). T cell antigens, TdT, and EBV-encoded RNA were negative in all samples. 10 of the 20 cases showed a high proliferation index (70%-100%), and the proliferation rate of follicular lymphoma was 30%. Another 10 breast cancer / lung cancer FFPE samples (from Ambiping Laboratory) were collected as negative controls.

[0189] The sample sections were 5 μm thick, and sample pretreatment, denaturation hybridization, and counterstaining were performed according to the following methods.

[0190] (1) Pretreatment:

[0191] Dewax using xylene (or xylene substitute) at room temperature for 10 min;

[0192] Incubate with 100% ethanol, 90% ethanol, and 70% ethanol for 3 min each at room temperature;

[0193] Wash with deionized water at room temperature for 3 minutes and shake off excess water;

[0194] Add permeabilization agent and incubate for 5 min;

[0195] Wash with purified water at 80-95°C for 5 minutes;

[0196] Wash with 2×SSC at room temperature for 3 min and dry at room temperature;

[0197] Add 200 μL of pepsin (final concentration 4 mg / mL) and digest at 37°C for 6 min;

[0198] Wash with 2×SSC at room temperature for 3 min;

[0199] Wash with 70% ethanol at room temperature for 2 min and dry at room temperature.

[0200] (2) Denaturation hybridization:

[0201] Add 10 μL of hybridization solution to the sample area;

[0202] Denaturation at 85°C for 5 min and hybridization at 37°C for 6 h.

[0203] (3) Re-staining (avoid light):

[0204] Wash in 2× SSC at 37 ± 1°C for 10 min;

[0205] Incubate with 0.1% NP-40 (2× SSC configuration) at 37 ± 1°C for 5 min;

[0206] Wash with 70% ethanol at room temperature for 3 min and dry in the dark;

[0207] Add 10 μL of in situ hybridization blue staining solution and store in a dark place until observation.

[0208] The results are as follows Figure 4A , Figure 4B , Figure 4C , Figure 4D , Figure 4E and Figure 4F As shown. Among them, Figure 4A and Figure 4B This is the test result picture of the MYC-positive sample, which clearly shows 2 green signals, 3 red signals and 2 yellow signals; Figure 4C and Figure 4D This is the test result picture of the BCL2 positive sample, which clearly shows 3 green signals, 2 red signals and 2 yellow signals; Figure 4E and Figure 4F This is a picture of the test results of a BCL6 positive sample. It can be clearly seen that there are 2 green signals, 2 red signals and 3 yellow signals. The above results show that the test results are consistent with the actual sample type, with a compliance rate of 100%, and the test accuracy is high.

[0209] In summary, the present invention uses a combination of FISH probes for three-gene joint detection in conjunction with a double-pass filter to directly observe the detection signals of three genes (MYC, BCL2, and BCL6) under a microscope, and can effectively detect target gene abnormalities. Compared with the conventional method of using three sets of probes to hybridize with three regions and observe three times, it saves detection time, reduces detection costs, and has higher application value.

[0210] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A FISH probe combination for three-gene joint detection, characterized in that: The FISH probe combination for the three-gene combined detection simultaneously detects the translocation of the MYC gene, the BCL-2 gene, and the BCL-6 gene; The fluorescein labeling methods of the probe for detecting the MYC gene, the probe for detecting the BCL-2 gene, and the probe for detecting the BCL-6 gene are all different, and the fluorescein labeling method is selected from any one of labeling with red fluorescein, labeling with green fluorescein, or labeling with both red fluorescein and green fluorescein; The fluorescein is labeled with dUTP; The red fluorescent pigment includes TRITC; The green fluorescent pigment includes Fluorescein; In the reaction system using red fluorescent and green fluorescent labeled probes at the same time, the molar ratio of dUTP labeled with Fluorescein to dUTP labeled with TRITC is (1.3-1.5):

1.

2. A method for preparing the FISH probe combination for three-gene joint detection according to claim 1, characterized in that: The preparation method comprises: The probe preparation template is mixed with a buffer, dNTPs, fluorescein-labeled dUTP, DNA polymerase and DNaseI respectively, and after labeling, a FISH probe combination for the three-gene joint detection is obtained; In the reaction system using both red fluorescent and green fluorescent labeled probes, the molar ratio of Fluorescein labeled dUTP to TRITC labeled dUTP is (1.3~1.5):

1.

3. A FISH kit for three-gene combined detection, characterized in that: The FISH kit for three-gene combined detection comprises the FISH probe combination for three-gene combined detection according to claim 1.

4. The FISH kit for three-gene combined detection according to claim 3, characterized in that: The FISH kit for the combined detection of three genes also includes a hybridization buffer, a permeabilization agent, a digestion agent and an in situ hybridization staining solution.

5. The FISH kit for three-gene combined detection according to claim 4, characterized in that: The hybridization buffer includes sodium citrate buffer.

6. The FISH kit for three-gene combined detection according to claim 4, characterized in that: In terms of mass fraction, the permeabilizing agent includes 1.5% to 3% tetraacetic acid, 6% to 8% polyethylene glycol and 1% to 3% Tween.

7. The FISH kit for three-gene combined detection according to claim 4, characterized in that: The digestive agent includes pepsin.

8. A device for three-gene joint detection, characterized in that: The three-gene combined detection device simultaneously detects the translocation of the MYC gene, the BCL-2 gene, and the BCL-6 gene; The device for three-gene combined detection comprises: Pretreatment module: permeabilize and digest the samples; Hybridization module: denaturation and hybridization of samples using FISH probe combination for three-gene joint detection; Color development and analysis module: Use in situ hybridization staining solution to stain the hybridized samples and perform analysis; The probe for detecting the MYC gene was labeled with red fluorescent dye TRITC; The probe for detecting the BCL-2 gene was labeled with green fluorescent protein (Fluorescein); The probe for detecting the BCL-6 gene is labeled with both red fluorescent dye TRITC and green fluorescent dye Fluorescein, and the molar ratio of Fluorescein-labeled dUTP to TRITC-labeled dUTP is (1.3~1.5):

1.

9. Use of the FISH probe combination for three-gene combined detection according to claim 1 in the preparation of a three-gene combined detection product; the three-gene combined detection product simultaneously detects the translocation of the MYC gene, the BCL-2 gene, and the BCL-6 gene.

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