Probe combination for detecting amplification of MYCL gene outside chromosome and application

Abstract

The invention relates to the technical field of biology, in particular to a probe combination for detecting MYCL gene amplification outside chromosomes and application of the probe combination. The probe combination is composed of a BAC (Bacillus subtilis) cloning fragment RP11 to 318B17, a BAC cloning fragment RP11 to 204L3, a BAC cloning fragment RP11 to 378I20 and a BAC cloning fragment RP11 to 469B24. The probe combination is used for detecting the amplification of the MYCL gene outside the chromosome and has the hybridization efficiency of 100%. The kit is convenient, rapid and reliable, has high success rate, and can be used for preparing the kit for detecting the amplification of the MYCL gene outside the chromosome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a probe combination and application for detecting extrachromosomal MYCL gene amplification. Background Art

[0002] Lung cancer is the malignant tumor with the highest morbidity and mortality in my country, and the prevalence is on the rise. Small cell lung cancer (SCLC) is a subtype of lung cancer with high malignancy and poor prognosis. Previous literature has reported that MYCL gene amplification is a poor prognostic marker for SCLC (Qin, J., et al., MYCL1 Amplificationand Expression of L-Myc and c-Myc in Surgically Resected Small-Cell Lung Carcinoma. Pathology Oncology Research : POR, 2021. 27: p. 1609775). Foreign studies have shown that this MYCL amplification may be located on extrachromosomal circular DNA (ecDNA) and is associated with poor prognosis (Pongor, LS, et al., Extrachromosomal DNA Amplification Contributes to Small Cell Lung Cancer Heterogeneity and Is Ass℃iated with Worse Outcomes. Cancer Discovery, 2023.).

[0003] Recently, there are also literature reports that ecDNA-mediated MYC gene amplification (ecMYC), including MYC, MYCN, and MYCL, can promote chemotherapy resistance in SCLC. In addition, the applicant's previous work suggests that SCLC with ecMYC amplification has reduced immune cells, especially T cells, and exhibits significant immunosuppression (Zhang, J. et al. P3.13D.04 Extrachromosomal MYC-Paralogs Amplification Defines ImmunosuppressiveMicroenvironment with Metastatic Potential in Small Cell Lung Cancer. Journal of Thoracic Oncology, Volume 19, Issue 10, S360 - S361). Therefore, the amplification of the extrachromosomal MYC family, including MYCL, is expected to serve as a biomarker for immunotherapy combined with chemotherapy in extensive-stage SCLC.

[0004] At present, the gold standard for the amplification of the MYC gene is determined by FISH. However, most of the mature probes on the market are MYC or MYCN single-color probes (such as the MYC amplification probe in lymphoma and the MYCN single-color probe in neuroblastoma M, which have been approved for clinical diagnosis). The MYCL break probe (Break-Apart Probe) has been reported internationally for use in scientific research experiments, but there are no commercialized MYCL amplification probes for clinical diagnosis in China. Summary of the invention

[0005] Based on the defects of the prior art, the first aspect of the present invention provides a FISH probe combination for detecting extrachromosomal MYCL gene amplification.

[0006] In one embodiment, the FISH probe combination consists of BAC clone fragments RP11-318B17, RP11-204L3, RP11-378I20 and RP11-469B24, and the two BAC clone fragments RP11-318B17 and RP11-204L3 corresponding to MYCL are labeled with the same fluorescence of any color; RP11-378I20 and RP11-469B24 are both labeled with the same fluorescent marker but a different color from RP11-318B17 and RP11-204L3.

[0007] In a preferred embodiment, RP11-318B17 and RP11-204L3 are both labeled with red fluorescence, and RP11-378I20 and RP11-469B24 are both labeled with green fluorescence; or the labeled fluorescence colors are interchanged.

[0008] The second aspect of the present invention provides use of the above-mentioned FISH probe combination in the preparation of a reagent for detecting extrachromosomal MYCL gene amplification.

[0009] The third aspect of the present invention provides a kit for detecting extrachromosomal MYCL gene amplification, wherein the kit comprises the aforementioned FISH probe combination.

[0010] Compared with the prior art, the present invention has the following beneficial technical effects: 1) This invention introduces the MYCL amplification probe for clinical diagnosis for the first time. The amplification probe is superior to the break probe in terms of detection intuitiveness, quantitative analysis, clinical guidance value and ease of operation. In practical applications, it is more suitable for detecting genes related to targeted therapy with known amplification, greatly reducing the detection cost; 2) The MYCL staining in the FISH images of SCLC of the present invention has good specificity and sensitivity and can be used to judge the MYCL gene amplification of clinical samples; 3) This study has conducted MYCL probe testing including positive and negative controls on more than 100 cases and found that about 10% (10 / 104) of the patients had MYCL gene amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 The MYCL probe internal reference gene map is selected in the embodiment of the present invention; Figure 2 For positive pathological sample results; Figure 3 Negative pathology sample results; Figure 4 This is the staining result of NCI-H889 human small cell lung cancer cell line; Figure 5 These are the results of peripheral blood culture cell testing. DETAILED DESCRIPTION

[0012] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0013] The main sources of raw materials and equipment for the following embodiments, comparative examples and test designs of the present invention are as follows:

[0014] The samples described in the following examples are 104 SCLC pathological samples from the Department of Thoracic Surgery, Peking University People's Hospital, and 10 of them were known to be positive for MYCL gene amplification after genome sequencing.

[0015] Example 1 Preparation of MYCL DNA FISH probe The MYCL gene located in the chromosome 1p34 segment (genomic coordinates: chr1:39,895,428-39,901,917, reference version: GRCh38 / hg38, the same below) and the corresponding bacterial artificial chromosomes (BAC clones) on both sides were searched through http: / / genome.ucsc.edu / , and two clones that can cover the MYCL gene were selected. The two clones should have as high an overlap as possible, and the MYCL gene should be located in the middle of the genomic DNA fragments covered by the two clones. Finally, two BAC clones, RP11-318B17 (chr1:39,799,550-39,957,743) and RP11-204L3 (chr1:39,817,299-40,003,043), were selected, with a fragment length of 203kb.

[0016] The probe OR14I1 (1q44) on the opposite arm of chromosome 1 was selected as the internal reference, corresponding to two BAC clones, RP11-378I20 (chr1: 248,538,648-248,714,123) and RP11-469B24 (chr1: 248,553,488-248,655,656). The genomic location and length of the fluorescence in situ hybridization MYCL counting probe and its internal reference probe are shown in Figure 2. Figure 1 As shown. The corresponding BAC clones were purchased from Invitrogen, and the plasmids were extracted after culturing the BAC clones. Using the nick translation method, the two BAC clone fragments corresponding to MYCL (RP11-318B17 and RP11-204L3) were labeled with TRITC-labeled dUTP as red fluorescence, and the two BAC clone fragments corresponding to the internal reference gene OR14I1 (1q44) (RP11-378I20 and RP11-469B24) were labeled with Fluorescein-labeled dUTP as green fluorescence. The labeled probes were mixed with Human Cot-1 DNA, hybridization buffer, and purified water in proportion to form a probe hybridization solution, and stored frozen at -20 degrees in the dark.

[0017] Example 2 Accuracy detection of MYCL DNA FISH probe 3-micron-thick small cell lung cancer tissue pathological sections (from the Department of Thoracic Surgery, Peking University People's Hospital, and all patients have signed informed consent) were placed in a 65±1 degree incubator for overnight baking. After taking out, soak in xylene for 30 minutes, and then soak in anhydrous ethanol for 10 minutes. Then, rehydrate in 100%, 85%, 70% gradient ethanol and purified water for 3 minutes each, and then boil in purified water at 100±5 degrees for 20 minutes. After taking out and drying, add 100 microliters of pepsin reaction solution to the sample area and digest for 5 minutes. Quickly put in 2 Wash in SSC for 5 minutes, and finally dehydrate in 70%, 85%, and 100% ethanol at room temperature for 3 minutes each, then take out and dry. Add 10 microliters of hybridization solution until dry. 22mm cover glass, turn the sample slide over so that the sample faces down, quickly cover it, and gently press the cover glass to evenly distribute the hybridization solution; seal the slide along the edge of the cover glass with sealing glue, completely covering the edge where the cover glass and the slide are in contact; place the slide on a hot plate at 85±1 degrees and denature for 5 minutes; quickly put the slide in a water bath at 37±1 degrees to preheat; remove the sealing glue and cover glass, and place the slide in a 2 Wash in SSC solution for 10 min; then transfer to 0.1% NP-40 / 2 Wash in SSC solution for 5 minutes; then dehydrate in 70% ethanol at room temperature for 3 minutes. After taking out the slide and drying it in the dark, add 10 microliters of DAPI counterstain to the dry 22 On a 22 mm cover glass, invert the sample slide so that the target area of ​​the cover glass and the slide are in contact. After inversion, press lightly to avoid generating bubbles and store in a dark place until observation.

[0018] Result determination method: refer to the commonly used dual-color counting probe usage standards and use Olympus B 51 fluorescence microscope DAPI / FITC / TexasRed three-color filter excitation, 100 times objective lens to observe interphase cells, and identify fluorescence signals. Use the fluorescence in situ hybridization software provided by Guangzhou Anping Company to analyze the image and evaluate the probe hybridization of the entire slice. The entire core area of ​​each slice must have at least 100 non-overlapping cell nuclei and clear fluorescence signals to determine that the fluorescence in situ hybridization detection is effective.

[0019] Result determination content: 1) Scan the entire slide under a 40× objective lens. A satisfactory specimen should have hybridization signals in more than 75% of the cancer cell nuclei. Observe whether there is heterogeneity.

[0020] 2) Find a clear tumor area under a 100× objective lens, observe and count the fluorescent signals of tumor cell nuclei, count MYCL (red) and OR14I1 (green) signals in 50 nuclei, record the total number of MYCL and OR14I1 signals and the average copy number, and then calculate the ratio of the average copy number of MYCL to OR14I1.

[0021] 1. If MYCL / OR14I1 < 2.0, MYCL is interpreted as negative (no amplification); 2. If MYCL / OR14I1 ≥ 2.0, MYCL is interpreted as positive (amplification).

[0022] In this example, a total of 104 SCLC pathological samples from the Department of Thoracic Surgery of Peking University People's Hospital were tested. The method of this example can accurately distinguish and confirm 10 MYCL positive cases and 94 MYCL negative cases. Figure 2-3 As shown, the red and green fluorescence intensities are both 2+, which meets the interpretation criteria. The hybridization result of positive pathological samples is shown in ( Figure 2 ), positive pathological samples mainly showed positive signal results of more red and 2 green, while negative pathological samples showed 2 red and 2 green ( Figure 3 ).

[0023] Example 3 Sensitivity Detection of MYCL DNA FISH Probe 1. Plasmid extraction: Use a commercially available plasmid extraction kit (QIAfilter Plasmid Maxi Kit, QIAGEN) to extract plasmids from the corresponding BAC clones of MYCL and OR14I1 according to the kit instructions to obtain plasmid DNA, and quantify the DNA using Nanodrop 2000.

[0024] 2. Fluorescent labeling of plasmid DNA: 1) Use the nick translation method to fluorescently label the BAC cloned plasmid DNA, where MYCL corresponds to the cloned plasmid DNA labeled with TRITC-labeled dUTP fluorescein; OR14I1 corresponds to the cloned plasmid DNA labeled with Fluorescein-labeled dUTP. Prepare the PCR reaction system on ice under strict light-proof conditions. The probe labeling reaction system is as follows:

[0025] 2) After the system is prepared, shake and mix, centrifuge, mark at 25℃ for 2 hours, and incubate at 80℃ for 10 minutes to inactivate the enzyme.

[0026] 3. After labeling the product, use sodium acetate to precipitate the nucleic acid and obtain the labeled probe after high-speed centrifugation. The purification steps are as follows: 1) Prepare the purified solution by mixing 3M sodium acetate and anhydrous ethanol in a ratio of 1:25; 2) Ethanol precipitate and concentrate the labeled product. Add the labeled product to a 1.5 mL centrifuge tube containing the purified solution at a volume ratio of 5:13. Vortex to mix and centrifuge. Place the mixture in a -80°C ultra-low temperature refrigerator for 30 to 60 minutes. Centrifuge at 13,000 rpm for 2 minutes to precipitate the probe, discard the supernatant, and dry in a dark place.

[0027] 3) Add 200 μL of 70% ethanol to gently rinse the precipitate, centrifuge briefly to fully remove the ethanol, and dry at 45°C for 3 minutes.

[0028] 4) Finally, dissolve in 2 μL purified water to obtain the labeled MYCL (red) and OR14I1 (green) probe materials, and store in a dark place.

[0029] In order to evaluate the performance of the probe prepared in Example 1, the probe in Example 1 was used to test the NCI-H889 human small cell lung cancer cell line, and 50 metaphase or interphase cells were analyzed to analyze the fluorescence brightness of the hybridization signal, the hybridization efficiency, whether the hybridization position was correct, etc. The results are shown in Figure 4 : It can be seen that the corresponding MYCL (1p34) and OR14I1 (1q44) chromosome loci should be marked with red and green fluorescence, respectively, the signal is bright and there is no cross-hybridization between chromosome loci, indicating that the hybridization efficiency of this example is 100% (50 / 50).

[0030] Example 4 MYCL (1p34) probe hybridization efficiency detection Cell droplet processing steps:

Instruments and materials

[0031] Cell preservation solution: 2 g polyethylene glycol 1500 (Sigma P5402) dissolved in 100 ml 50% ethanol; Hypotonic solution (0.075 mol / L KCl): Accurately weigh 5.59 g potassium chloride, add 500 ml pure water, and after fully dissolving, adjust the volume to 1 liter; Fixative: methanol: glacial acetic acid in a volume ratio of 3:1, prepared in a chemical fume hood and mixed thoroughly; <Preparation> Anti-slide slides (Boster, Wuhan), pipette, microscope.

[0032] <Ethanol aging of slides> Anhydrous ethanol; flat plate heater (Shanghai Broadcom BHW-05A), 24×50mm cover glass (Shanghai Biotech), gauze, square cover (length×width ≈ 9cm×13cm, can process 4 slides at the same time), and tweezers.

[0033] <Slide pretreatment> 20×PBS (Shanghai Bioengineering); 70%, 85%, 100% gradient ethanol; round glass staining jar (Shanghai Bioengineering); constant temperature water bath (37±1℃); Pepsin solution: add 400 μl 1 mol / L HCl to 40 ml water, place in a 37±1℃ water bath, add 20 μl 10% pepsin (Shanghai Biotech) before use, mix well, and replace after one day of use; 4% paraformaldehyde: Add 500 μl 1 mol / L NaOH to 900 ml deionized water and heat to 65-70°C; add 40 g paraformaldehyde (Shanghai Shenggong) while stirring, and continue stirring until dissolved; cool to room temperature and add 100 ml 10× PBS; adjust pH to 7.3 with HCl; filter and sterilize, and store at 2-8°C away from light; 1% paraformaldehyde (water: 26ml + 20xPBS: 2ml + 4% paraformaldehyde: 10ml + 1M MgCl2: 2ml).

[0034] <Simultaneous denaturation of samples and probes> 20×20 mm coverslip (Shanghai Biotech), tweezers, rubber cement, plate heater, hybridization box, constant temperature water bath (37±1°C) <Post-hybridization washing and counterstaining> 20×SSC (Shanghai Bio-Tech); tweezers; round glass staining jar; constant temperature water bath (37±1℃); 70%, 85%, 100% gradient ethanol; 24×24mm cover glass; Wash solution I (50% formamide / 2×SSC): 16 ml pure water + 4 ml 20×SSC + 20 ml formamide, total volume 40 ml; Wash solution II (2×SSC): 36 ml pure water + 4 ml 20×SSC, total volume 40 ml; Wash solution III (2×SSC / 0.1% NP-40): 35.96 ml pure water + 4 ml 20×SSC + 40 μl NP-40, total volume 40 ml.

[0035] Wash solution IV (0.4×SSC / 0.3% NP-40): 39.08 ml pure water + 800 μl 20×SSC + 120 μl NP-40, total volume 40 ml.

[0036]

Test method

[0037] 1.2 Take 1 ml of cells and add 10 ml of 0.075 M KCl, mix by pipetting, and let stand for 3 min.

[0038] 1.3 Incubate in a 37±1℃ water bath for 30 minutes in hypotonic condition.

[0039] 1.4 Add 1 ml of fixative solution, mix well by pipetting, and pre-fix at room temperature for 10 minutes.

[0040] 1.5 Mix by pipetting and centrifuge at 2000 rpm for 5 min.

[0041] 1.6 Remove the supernatant, add 5ml-10ml of fixative solution to the precipitate, mix well by pipetting, and let stand at room temperature for 10 minutes.

[0042] 1.7 Centrifuge at 2000 rpm for 5 min and remove the supernatant.

[0043] 1.8 Repeat the above washing steps until the cell pellet is clean (this step does not require standing at room temperature for 10 minutes).

[0044] 2. Production 2.1 Take a clean slide; 2.2 After resuspending the cells, take 3μl, 10μl and 30μl of the suspension and drop them on different positions on the slide, making sure not to overlap; 2.3 Dry at room temperature; 2.4 Observe the cell density of the three areas under a phase contrast microscope using a 20× objective lens, and record the amount of cell suspension added when there is no obvious overlap of cells and the number of cells is above 100 to 200; a) If the cell density and number are appropriate, proceed to step 2.5; b) If the area where 30 μl of suspension was dripped still has too few cells, take another 30 μl of suspension and drip it onto the area again, let it dry, and observe; c) If there are still too many cells in the area where 3 μl of the suspension was added, dilute the cell suspension with fresh fixative and repeat steps 2.1 to 2.4. 2.5 Take another clean slide, divide it into two areas, left and right, and add appropriate amount of cell suspension to each area; 2.6 Observe under a phase contrast microscope. If there are too many cell fragments, pretreatment is required and appropriate hybridization areas should be selected. Note: At least one more slide is required for each case. The cell drop slide can be placed in a sealed container with anhydrous ethanol and can be stored at -20±5℃ for one year. The remaining cell suspension can be stored at 2-8℃ for one month so that it can be re-slided when necessary.

[0045] 3. Ethanol aging of slides 3.1 Heat the hot plate to 95±1℃; 3.2 Take out a 24×50 mm cover glass (preserved in anhydrous ethanol), wipe it dry and set aside; 3.3 Take a piece of gauze folded into a square with 4 to 8 layers and soak it in anhydrous ethanol; 3.4 Add 160 μl of anhydrous ethanol to each hybridization area of ​​the slide; 3.5 Gently cover with a 24×50 mm cover glass and press lightly; 3.6 Place the slide on a hot plate preheated at 95±1℃, cover it with gauze soaked in anhydrous ethanol (to completely cover the slide), and then cover it with a pre-prepared square cover (to completely cover the slide), and time for 2 minutes; 3.7 Remove the lid and gauze from the hot stage, take out the slide, gently remove the coverslip, and continue with the pretreatment steps.

[0046] Note: Alternatively, you can age the slides by leaving them at room temperature overnight or baking them at 56°C for 1 hour, then continuing with the pretreatment steps.

[0047] 4. Slide pretreatment 4.1 Place the slide in 1× PBS at 37±1℃ and incubate for 5 minutes; 4.2 Remove the slide and place it in a 37±1℃ pepsin solution for digestion for 7 to 15 minutes (the enzyme potency can be determined by a preliminary test); 4.3 Take out the slide and wash it in 1×PBS at room temperature for 3 minutes; 4.4 Take out the slide and fix it in 1% paraformaldehyde / PBS at room temperature for 10 minutes; 4.5 Take out the slide and wash it in 1×PBS at room temperature for 3 minutes; 4.6 Take out the slide and wash it in 1×PBS at room temperature for 3 minutes; 4.7 Take out the slides and dehydrate them in 70%, 85%, and 100% graded ethanol for 3 minutes each; then take out the slides and dry them at room temperature.

[0048] Note: If the sample is fresh, you can also replace 4.1-4.8 with the following steps: a: Soak the glass slide in 2×SSC (PH7.0) solution at room temperature for 2 minutes; b: Dehydrate by soaking in 70%, 85%, and 100% ethanol at room temperature for 2 minutes; then take out the slides and dry them at room temperature.

[0049] 5. Simultaneous denaturation of samples and probes 5.1 Take out the hybridization solution, leave it at room temperature, and centrifuge it at low speed; 5.2 Pipette 10 μl of hybridization solution onto a dry 20x20 mm cover glass, flip the sample slide so that the sample faces downward, quickly cover it, and gently press the cover glass to evenly distribute the hybridization solution after inversion to avoid bubbles, and mark it; 5.3 Use rubber glue to seal the slide along the edge of the cover glass, completely covering the edge where the cover glass meets the slide; 5.4 Place the slide on a hot plate at 78±1℃ and denature for 2 minutes and 30 seconds (Note: the temperature must be confirmed to be accurate before use); 5.5 Quickly place the slide into the preheated hybridization box, protect from light, and incubate at 37±1℃ overnight (about 16 hours); 5.6 Continue with post-hybridization wash step.

[0050] 6. Post-hybridization washing and counterstaining 6.1 30 minutes before washing, place the prepared wash solution I, wash solution II, and wash solution III in a 37±1℃ water bath and measure to ensure that the temperature is appropriate; 6.2 Take out the hybridization box that has been incubated overnight and carefully remove the rubber glue and coverslip; 6.3 Quickly place the slides into a preheated staining jar containing wash solution I and wash at 37±1℃ for 15 minutes; 6.4 Take out the slide and put it into the staining jar containing washing solution II and wash it at 37±1℃ for 15 minutes; 6.5 Take out the slide and put it into the staining jar containing washing solution III and wash it at 37±1℃ for 5 minutes; 6.6 Take out the slides and dehydrate them in 70%, 85%, and 100% graded ethanol at room temperature for 3 minutes each; 6.7 Take out the slide, place it vertically on a paper towel, and dry it in a dark place; 6.8 Add 10 μl of DAPI counterstain onto a dry 24x24 mm coverslip, invert the sample slide so that the coverslip contacts the target area on the slide, press gently after inversion to avoid creating bubbles, and store in a dark place until observation.

[0051] Note: If the sample is fresh, you can also use the following steps instead of 6.3-6.5: a: Place the slide in wash solution IV (0.4×SSC / 0.3% NP-40) at 72°C for 2 minutes; b: Place in wash buffer III (2×SSC / 0.1% NP-40) for 30 seconds at room temperature; then proceed to steps 6.6-6.8.

[0052] Note: The above reagents are prepared in round staining jars (40 ml for each reagent), and each jar can hold up to 5 slices. Non-room temperature solutions require preheating of the reagents to the specified temperature before the operation. During the washing process, the staining jar can be gently shaken every 2 to 3 minutes to improve the washing effect.

[0053] 7. Results Analysis Correlated fluorescence and DAPI should be observed using appropriate filter cubes.

[0054] 7.1 Use appropriate filters, search under 40× objective lens, and count under 100× objective lens; 7.2 Adjust the focal length appropriately and have a clear concept of signal and background. The signal points are located inside the cells. When there are fluorescent signal points outside the cells, it is important to distinguish them from the signal points inside the cells. It is best to avoid counting in this area. 7.3 Scan several cell areas, requiring complete nuclear boundaries, uniform DAPI staining, no nuclear overlap, and clear green and red signal points; skip counting nuclei with weak signals, no specific signals, or high background; do not count nuclei that require subjective identification; 7.4 Start the analysis from the upper left corner of the selected area, scan from left to right, and observe multiple fields of view; switch to the 100× objective lens, adjust the focus, and find all signal points at different levels of the nucleus; count the signal points in each nucleus; focus to find all signal points in each nucleus, count two signals in one area, and only count those with 1 or more FISH signals in each color. Do not count nuclei with no signal or only one color signal; record the total number of cells observed (normal and abnormal signal numbers); 7.7 Set the negative threshold according to the relevant probe instructions and perform counting and result determination.

[0055]

Test results

[0056] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A FISH probe combination for detecting extrachromosomal MYCL gene amplification, the FISH probe combination consisting of BAC clone fragments RP11-318B17, RP11-204L3, RP11-378I20 and RP11-469B24, wherein the two BAC clone fragments RP11-318B17 and RP11-204L3 corresponding to MYCL are labeled with the same fluorescence of any color; RP11-378I20 and RP11-469B24 are labeled with the same fluorescent marker but with a different color from RP11-318B17 and RP11-204L3.

2. The FISH probe combination according to claim 1, characterized in that: RP11-318B17 and RP11-204L3 are both labeled with red fluorescence, and RP11-378I20 and RP11-469B24 are both labeled with green fluorescence; or the labeled fluorescence colors can be interchanged.

3. Use of the FISH probe combination according to claim 1 or 2 in the preparation of a reagent for detecting extrachromosomal MYCL gene amplification.

4. A kit for detecting extrachromosomal MYCL gene amplification, the kit comprising the FISH probe combination according to claim 1 or 2.

Images