WO2017114003A1 - Alk gene and eml4 gene detection probe, preparation method therefor, and reagent kit - Google Patents

Abstract

Disclosed in the present invention are an ALK gene detection probe, and an ALK gene and EML4 gene detection probe, and a preparation method therefor. The method comprises the following steps: selecting at least one of RP11-62B19, RP11-100C1 and CTD-2245E6 as a BAC clone for an ALK gene, and selecting at least one of CTD-2544I11, RP11-684O3 and RP11-134O13 as a BAC clone; selecting at least one of CTD-2358L8 and CTD-2547J15 as a BAC clone for an EML4 gene; obtaining a plasmid DNA; and performing labeling. Also disclosed in the present invention is a reagent kit comprising the ALK gene and EML4 gene detection probe.

Description

ALK gene and EML4 gene detection probe and preparation method and kit thereof Technical field

The present invention belongs to the field of biotechnology, and particularly relates to an ALK gene and EML4 gene detecting probe, a preparation method thereof and a kit.

Background technique

In 2014, the annual tumor registration report indicated that lung cancer ranked first among the national incidence of malignant tumors (35.23/100,000, 19.59%) and mortality (27.93/100,000, 24.87%). The occurrence of lung cancer is considered to be a multi-step process that is closely related to the amplification/activation of protooncogenes and/or the inactivation of tumor suppressor genes, and provides targets for patients as more and more cancer-driven factors are discovered. The opportunity to treat. The study found that 64% of patients with lung adenocarcinoma detected cancer-driven factors, of which 8% were ALK rearrangements. ALK gene fusion is not a favorable prognostic factor for advanced NSCLC, but crizotinib improves survival in ALK-positive patients. Previous studies have shown that the EML4-ALK gene fusion mutation rate is 2% to 7%. A study reported at the 2011 American Society of Clinical Oncology (ASCO) annual meeting showed that the 1-year and 2-year survival rates of ALK-positive patients treated with the EML4-ALK inhibitor crizotinib were 74% and 54%, respectively, and received crizotinib II. The survival rate of ALK positive patients with third-line treatment was significantly higher than that of ALK positive control group and ALK negative control group. The ALK mutation in lung cancer is mainly caused by the rearrangement of ALK gene and other gene fusion. EML4-ALK (echinoderma microtubule-associated protein-like 4-anaplastic lymphoma kinase) fusion gene mutation is the main type, accounting for about 5% of all NSCLC. Other genes that are fused to the ALK gene in lung cancer include TFG, KIF5B, and the like. The EML4-ALK fusion gene can be found in a variety of tumors, such as anaplastic large cell lymphoma, inflammatory myofibroblastoma, neuroblastoma, and NSCLC, which are caused by short arm insertion of chromosome 2. The ALK fusion gene forms an intricate signal transduction network, which affects cell proliferation, differentiation and apoptosis through the activation and transmission of downstream substrate molecules and the mutual crossing and overlapping of transduction pathways. The EML4-ALK fusion gene binds the kinase regions of two EML4-ALK molecules through the extracellular helical domain of the fusion partner to form a stable dimer, which activates downstream MAPK, PI3K/AKT, JAK/STAT3 by autophosphorylation. Wait for the pathway, causing the cells to metastasize.

ALK gene rearrangement was first discovered and named in anaplastic lymphoma, and was reported in lung cancer in 2007. Now, as a member of the tyrosine kinase signaling pathway, ALK gene abnormalities may be found in more cancerous species, and the indications for targeted drugs will continue to expand.

Therefore, research kits for this gene and for clinical sample testing are extremely beneficial for screening targeted drug benefit populations.

The probes used for hybridization can be roughly classified into three categories: 1) chromosome-specific repeat probes, such as alpha satellites, satellite class III probes, which often have a hybrid target of more than 1 Mb, do not contain scattered repeats, and bind tightly to the target. Strong hybridization signal, easy to detect; 2) whole chromosome or chromosomal region-specific probe consisting of a very different nucleotide fragment on a chromosome or a segment of a chromosome, which can be cloned into phage and plasmid A chromosome-specific large fragment is obtained; 3) a specific position probe consisting of one or several cloned sequences.

The fluorescein labeling of the probe can be performed by direct and indirect labeling. The indirect labeling is a biotin-labeled DNA probe, which is detected by fluorescein avidin or streptavidin after hybridization, and the avidin-biotin-fluorescein complex can also be used to fluoresce signals. Amplification is performed so that a fragment of about 500 bp can be detected. The direct labeling method is to directly bind fluorescein to the probe nucleotide or the pentose phosphate backbone, or to incorporate fluorescein nucleoside triphosphate in the nick translation labeling probe. The direct labeling method has simple steps in detection and is convenient for clinical use.

At present, there is a lack of a highly specific detection kit for the FISH detection of the ALK/EML4 gene.

Summary of the invention

One of the objects of the present invention is to provide an ALK gene detecting probe and a preparation method thereof, and the prepared probe can be used for detecting ALK gene rearrangement and identification, and has good specificity.

The technical solution for achieving the above object is as follows.

A method for preparing an ALK gene detection probe, comprising the steps of:

(1) The BAC clones targeting the ALK gene were selected as at least one of RP11-62B19, RP11-100C1 and CTD-2245E6 as a set of probes, and the BAC clones were selected as CTD-2544I11, RP11-684O3 and RP11-134O13. At least one as another set of probes;

(2) separately extracting a plasmid from the clone to obtain a plasmid DNA, and quantifying;

(3) Labeling the plasmid DNA with fluorescein, the color of the fluorescein of the same group of probes is the same, the color of the fluorescein of different sets of probes is different, that is, the BAC clones are RP11-62B19, RP11-100C1 and CTD-2245E6. The probe is different from the color of the fluorescein derived from the probes of the BAC clones CTD-2544I11, RP11-684O3 and RP11-134O13.

Another aspect of the present invention provides an ALK gene and EML4 gene detecting probe and a preparation method thereof, and the prepared probe can be used for detecting the ALK gene and the EML4 gene state, that is, for EML4/ALK gene fusion identification, which is excellent. Specificity.

A method for preparing an ALK gene and an EML4 gene detection probe, comprising the steps of:

(1) The BAC clones targeting the ALK gene were selected as at least one of RP11-62B19, RP11-100C1 and CTD-2245E6 as a set of probes, and the BAC clones were selected as CTD-2544I11, RP11-684O3 and RP11-134O13. At least one of the other set of probes; and the BAC clone selected for the EML4 gene is at least one of CTD-2358L8 and CTD-2547J15;

(2) separately extracting a plasmid from the clone to obtain a plasmid DNA, and quantifying;

(3) Labeling the plasmid DNA with fluorescein, the color of the fluorescein labeled with the ALK gene and the detection probe for the EML4 gene is different, and for the ALK gene, the color of the fluorescein of the same group of probes is the same, different groups The color of the fluorescein of the needle is different, that is, the probe derived from BAC clones as RP11-62B19, RP11-100C1 and CTD-2245E6 and the probe derived from BAC clones as CTD-2544I11, RP11-684O3 and RP11-134O13. The color of fluorescein is not the same, that is.

In one embodiment, the ALK rearrangement probe comprises two groups, the first group of BAC clones being RP11-62B19, RP11-100C1 and CTD-2245E6, respectively; the second group of BAC clones being CTD-2544I11, RP11- 684O3 and RP11-134O13.

In one embodiment, the EML4 probe comprises a set and the third set of BAC clones are CTD-2358L8 and CTD-2547J15.

In one embodiment, the labeled fluorescein selects a fluorescent dye known in the art, preferably fluorescein is Alexa

FITC, Alexa

Rhodamine, Texas Red, pacific

DEAC.

In one embodiment, the labeling of the gene probe can be performed by labeling the corresponding fluorescein to the double-stranded nucleic acid by methods in the prior art, including but not limited to: random primer method, nick translation, etc., marker gene probe The needle can be used with a commercially available nick translation labeling kit and a random primer labeling kit, preferably abbott and Roche's Nick Translation Kit. In the step (3) of the present invention, the plasmid DNA is preferably subjected to fluorescein labeling by a random primer method or a nick translation method.

In one embodiment, the temperature of the label is between 15 ° C and 18 ° C and the time of labeling is between 8 and 12 hours.

Another object of the present invention is to provide an ALK gene and EML4 gene detecting kit.

The technical solution for achieving this purpose is as follows.

A kit for detecting ALK gene and EML4 gene, comprising the above ALK gene and EML4 gene detecting probe.

In one embodiment, there is also a COT Human DNA for blocking the repeat sequence, and a DAPI counterstain.

The invention has the following beneficial effects:

(1) The present invention can detect unknown rearrangement associated with ALK or directly identify EML4/ALK rearrangement by screening for optimal EML4/ALK gene rearrangement detection probes and combinations thereof.

(2) Using the method of the present invention to detect ALK gene rearrangement, the signal counting is accurate and rapid, and the result is reproducible; supplementing the shortage of detection reagents in the clinic depends on the import, which is beneficial to the subsequent detection probe. It is used in clinical research for molecular typing and targeted therapy of lung cancer.

DRAWINGS

Fig. 1A is a schematic diagram showing the sequence of the ALK gene detecting probe in Example 1.

Fig. 1B is a schematic diagram showing the probe sequence of the EML4 gene detection in Example 1.

Fig. 2A is a graph showing the results of FISH detection of the human peripheral blood culture cell sheet ALK gene detecting probe in Example 1.

Fig. 2A is a graph showing the results of FISH detection of the human peripheral blood culture cell sheet ALK gene and EML4 gene detection probe in Example 1.

Fig. 3A is a diagram showing the results of FISH detection of the ALK gene of the lung cancer tissue sample in Example 4, wherein the detection signal type is 2F (RG adjacent or coincident), and the ALK gene is not rearranged.

FIG. 3B is a diagram showing the results of FISH detection of the ALK gene and the EML4 gene of the lung cancer tissue sample in Example 4, wherein the detection signal type is 2F2A, the ALK gene is not rearranged, and no EML4/ALK rearrangement occurs.

4A is a diagram showing the results of FISH detection of the ALK gene of the lung cancer tissue sample in Example 4, wherein the detection signal type is 1F1G1R, and the F signal is broken compared with the normal signal type 2F, and the detection result is: ALK gene rearrangement.

4B is a diagram showing the results of FISH detection of the ALK gene and the EML4 gene in the lung cancer tissue sample of Example 4, wherein the detection signal type is 1F1A1G1RA, and the F signal is broken and the RA signal fusion occurs simultaneously with the normal signal type 2F2A. For: ALK gene is broken and the rearrangement type is EML4/ALK.

detailed description

In order to facilitate the understanding of the present invention, the present invention will be described more fully hereinafter. The invention can be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the understanding of the present disclosure will be more fully understood.

Experimental methods in which the specific conditions are not indicated in the following examples are generally carried out according to the conditions described in conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. The various common chemical reagents used in the examples are commercially available products.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning meaning The terms used in the description of the present invention are for the purpose of describing the specific embodiments and are not intended to limit the invention. The term "and/or" used in the present invention includes any and all combinations of one or more of the associated listed items.

Example 1 Preparation of ALK Gene and EML4 Gene Detection Probes

The preparation method of the ALK detection probe of the present embodiment comprises the following steps:

(1) A clone comprising the target gene ALK and the sequences at both ends was selected, as shown in Fig. 1A. GSP ALK includes two sets of probes:

The first set of probes included a first probe, a second probe, and a third probe, as shown in the following table, which was purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.

The second set of probes included a fourth probe, a fifth probe, and a sixth probe, as specifically listed below, which were purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.

The following two sets of detection probes were separately prepared.

Probe set 1 - marked red R, total length of BAC: 462 Kb.

Probe Set 2 - Mark Green G, total length of BAC: 513 Kb.

Probe set 1 (red)	BAC	Insert start and end position
First probe	RP11-62B19	Chr2:29354267..29528536
Second probe	RP11-100C1	Chr2:29210476..29383355
Third probe	CTD-2245E6	Chr2:29066898..29207778

Probe set 2 (green)	BAC	Insert start and end position
Fourth probe	CTD-2544I11	Chr2:29923580..30121092
Fifth probe	RP11-684O3	Chr2:30116506..30315853
Sixth probe	RP11-134O13	Chr2:30282641..30436938

The preparation method of the EML4 detection probe of the present embodiment comprises the following steps:

Clones containing the near-ALK end sequence of the EML4 gene of interest were picked as shown in Figure 2B. GSP EML4 includes a set of probes including a first probe and a second probe, as shown in the following table, which was purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.

Probe Set 3 - Label Cyan A.

BAC total length: 261Kb

Probe Set 3 (cyan)	BAC	Insert start and end position
First probe	CTD-2358L8	Chr2:42305395..42410427

Second probe

CTD-2547J15

Chr2:42149101..42340641

(2) Preparation of GSP ALK and GSP EML4 gene detection probes: Ultra-low copy plasmid DNA extraction was performed on different BAC clones using Qiagen's Plasmid Maxi Kit according to the method described in the specification, and the absorbance at 260 nm and 280 nm was measured. The plasmid DNA was quantified; diluted with auto-sterilized ultrapure water to 200 ng/ul, and packed in a 1.5 ml centrifuge tube, and finally the plasmid DNA corresponding to three or two corresponding combinations of the ALK gene or the EML4 gene was mixed. Store at -20 ° C.

(3) Fluorescent labeling of the plasmid DNA mixture by nick translation method, group 2 probe-labeled fluorescein for GSP ALK gene is green-dUTP, group 3 probe-labeled fluorescein Aqua-dUTP, for GSP EML4 gene The probe-labeled fluorescein is red-dUTP. Using abbott's Nick Translation Kit, the PCR reaction system was prepared on ice under strict light conditions as follows.

After mixing, mix and shake, mark at 16 ° C for 12 hours, then incubate at 80 ° C for 10 minutes to inactivate the enzyme. 5 ul of 2% agarose gel was used for electrophoresis, and there were diffused bands at around 50-500 bp.

The labeled product was subjected to ethanol precipitation and concentration, and sodium acetate and absolute ethanol were sequentially added to a 1.5 ml centrifuge tube in the following manner, and protected from light and ice:

Labeled product 45ul

Sodium acetate (3mol/L) 5ul

Anhydrous ethanol 125ul

After mixing, put in a -70 ° C refrigerator for at least 2 hours, centrifuge at 13000 rpm for 30 minutes at 4 ° C, carefully remove the supernatant, do not stir the precipitate, add 1 ml of 70% ethanol, centrifuge at 13000 rpm for 15 minutes at 4 ° C, carefully go Clear, do not stir the sediment, dry away from light. The pellet was dissolved in 1 ul of purified water to obtain GSP ALK and GSP EML4 gene probes, and stored at -20 ° C in the dark.

(4) GSP ALK and GSP EML4 gene probe validation: hybridization solution prepared using ALK probe set 1 + ALK probe set 2+ EML4 probe set 3, respectively, and ALK probe set 1 + ALK probe set 2 preparation The hybridization solution is subjected to probe verification on human normal division metaphase lymphocyte droplets (detection method is referred to in Example 3). The cultured cells contain metaphase or interphase chromosomal DNA. When fluorescent in situ hybridization, the chromosomal DNA appears as a morphologically recognizable chromosome or nucleus. FISH hybridization of metaphase chromosomes as shown in Fig. 2A (experimental results applied to probe set 1 + probe set 2) and Fig. 2B (experimental results applied to probe set 1 + probe set 2+ probe set 3) Results map. Fig. 2A shows the upper metaphase chromosome of human peripheral blood cultured cells. The figure shows that the ALK gene (2p23) segment of chromosome 2 shows a red-green fusion signal (yellow), the other segments have no hybridization signal point, and the ALK probe group 1 and probe Group 2 has excellent specificity. Fig. 2B shows the upper metaphase chromosome of human peripheral blood culture cells. The red-green fusion signal of 2p23 segment (yellow), the cyan signal of 2p21 segment, the hybridization signal point of other segments, ALK probe group 1, ALK probe group are shown in the figure. 2 and EML4 probe set 3 have superior specificity.

Example 2: Kit for ALK gene detection, ALK gene and EML4 gene detection

This embodiment provides two kits, namely an ALK gene detection kit, and an ALK gene and EML4 gene detection kit.

The ALK gene rearrangement detection kit includes two components of a hybridization solution and a DAPI counterstain, wherein the hybridization solution comprises the GSP ALK two-set probe (two-color) described in Example 1, and is used in a hybrid environment (promoting hybridization). Buffer components, closed repeats of COT Human DNA, and the like. DAPI counterstaining agent is mainly used for counterstaining of cells after hybridization, in which DAPI binds to DNA, so that the nucleus shows blue fluorescence, and the counterstaining agent containing p-phenylenediamine can maintain fluorescence stability.

The specific formula is as follows:

Hybrid solution preparation

The ALK gene and EML4 gene detection kit includes two components of ALK/EML4 hybridization solution and DAPI counterstaining agent, wherein the ALK and EML4 hybridization solution comprises the set of GSP EML4 gene probes described in Example 1 and two sets of GSP ALK. A combination of gene probes. The ALK gene has two sets of detection probes, and the EML4 gene has a set of detection probes. In this embodiment, the ALK gene and the EML4 gene detection kit are: ALK (group 1+group 2)+EML4 (group 3) Combinations, buffer components for hybrid environment (promoting hybridization), CDM Human DNA such as closed repeats, DAPI counterstains are mainly used for counterstaining of cells after hybridization, in which DAPI binds to DNA, allowing the nucleus to show Blue fluorescence, a counterstain containing p-phenylenediamine, maintains fluorescence stability.

The specific formula is as follows:

(1) Preparation of hybridization solution

(2) DAPI counterstain preparation

10 mg of p-phenylenediamine was dissolved in 1 ml of PBS, the pH was adjusted to 9.0, 9 ml of glycerol was added, and the mixture was repeatedly shaken and stored at -20 ° C. 2.5 μl of DAPI solution (0.1 mg/ml) was dissolved in 1 ml of anti-fade solution, and shaken and shaken repeatedly in the dark, and stored at -20 °C in the dark.

(3) Finished product assembly

Component name	Specification/10test	Quantity
Hybrid solution	100μl / tube	1 tube
DAPI counterstain	100μl / tube	1 tube
Instruction manual		1 serving

Example 3: Detection method of ALK gene and EML4 gene detection kit

1, slide pretreatment

1. Slide the slide into a 65±5°C incubator for overnight;

2. Remove the slide and place it in room temperature xylene (or environmental dewaxing agent) for 10 minutes;

3. Remove the slide and place it in another cylinder of room temperature xylene (or environmental dewaxing agent) for 10 minutes;

4. Remove the slide and place it in absolute ethanol for 10 minutes at room temperature to remove residual xylene (or environmental dewaxing agent);

5. Remove the slide and place it in 100%, 90%, 70% gradient ethanol at room temperature for 3 minutes each time;

6. Remove the slide, then put it in sterile purified water for 3 minutes at room temperature, and use the lint-free paper towel to absorb excess water;

7. Remove the slide, then put the slide into the 1×EDTA repair solution, and pressurize the autoclave for 2 minutes (the pressure cooker has gas release, and the timing starts to sound);

8. After cooling to room temperature, remove the slide and rinse off the tap water;

9. Remove the slide and place it in room temperature 2 x SSC for 3 minutes;

10. Remove the slide and let it dry at room temperature;

11. Place the slide face up on the shelf and add the appropriate amount of pepsin solution to the sample area. Digest at room temperature for 5 to 15 minutes;

12. Remove excess liquid and place it in room temperature 2 x SSC for 5 minutes;

13. Remove the slide and place it in another tank at room temperature 2 × SSC for 5 minutes;

14. Remove the slides, and then place them in a 70%, 90%, 100% gradient ethanol at room temperature for 2 minutes each;

15. Remove the slide and allow to dry at room temperature.

2. Simultaneous denaturation of samples and probes (light protection operation)

2.1 Remove the hybridization solution from the -20±5°C refrigerator, shake and mix, and centrifuge instantaneously;

2.2 Add 10 μl of the hybridization solution to the hybridization area, quickly cover the 22×22mm coverslip, and gently press to make the hybridization solution evenly distributed to avoid air bubbles;

2.3 Use rubber rubber to seal the edge of the cover glass to completely cover the contact between the cover glass and the slide;

2.4 Place the slide into the hybridization instrument, wet the humidity strip of the in situ hybridization instrument, insert the wet strip, cover the top of the hybridization instrument, set the “Denat&Hyb” program, denature at 85 ° C for 5 minutes, and hybridize at 37 ° C for 10 to 18 hours; If there is no hybrid instrument, you can use alternative instruments, such as constant temperature hot stage for denaturation, electric oven / or water bath for hybridization, pay attention to accurate temperature and maintain hybrid humidity.

3. Washing and counterstaining after hybridization (protection from light)

3.1 30 minutes before washing, the prepared lotion I, lotion II, placed in a water bath of 37 ± 1 ° C, measured to ensure that the temperature is appropriate;

3.2 Turn off the power of the hybridization instrument, take out the slide, gently remove the rubber glue, and remove the cover slip. (If the cover slip is difficult to remove, it can be shaken in the wash solution I to facilitate its falling off;

3.3 slides were placed in 37 ± 1 ° C lotion I (2 × SSC) for 10 minutes;

3.4 remove the slide, and then put it into 37 ± 1 ° C lotion II (0.1% NP-40/2 × SSC) for 5 minutes;

3.5 Remove the slides and let them stand in 70% ethanol for 3 minutes at room temperature;

3.6 Remove the slide and naturally dry the slide in the dark;

3.7 Room temperature, add 10μl DAPI counterstain to 22×22mm coverslip, the target area of the slide is facing down, put it on the cover slip, gently press to avoid air bubbles, store in the dark, to be observed.

4, the result analysis

The relevant fluorescence and DAPI need to be observed with a suitable filter block.

1. Use a suitable filter, look under a 10× objective, and count under a 100× objective;

2. Adjust the appropriate focal length, have a clear concept of signal and background; the signal point should be located in the cell; when there is a fluorescent signal point outside the cell, pay attention to distinguish it from the intracellular signal point, it is best to avoid the area for counting ;

3. Scan several cell regions, requiring complete cell borders, no overlap, uniform DAPI staining, clear green and red signals, skip cell counts and cell counts without specific signals or high background; cells that require subjective discrimination are not counted;

4. Start the analysis from the upper left corner of the selection area, scan from left to right, and observe multiple fields of view;

5. Go to the 100× objective, adjust the focus, and find all the signal points at different levels of the core;

6. Count the signal points in each cell; focus to find all the signal points in each cell, count two or three signals in one region, and count only one or more FISH signals in each color. Cells with no signal or only a single color signal were not counted; the total number of cells observed (normal and abnormal signal number) was recorded.

6.1 normal signal: 2F (ALK rearrangement test kit) or 2F2A (EML4/ALK rearrangement test kit);

6.2 Abnormal signal: 1R1G1F (ALK rearrangement detection kit)

1F1A1G1RA (EML4/ALK Rearrangement Detection Kit).

7. Set the negative threshold

More than 10 samples were randomly selected as negative control tablets. Each negative control panel randomly counts the complete 100 cells, counts the number and percentage of abnormal signal cells in each sample, the average and standard deviation of the statistical percentage, and the negative threshold is set to the mean + 3 standard deviation .

Example 4: Evaluation of clinical use of ALK gene and EML4 gene detection kit

Using the ALK gene and EML4 gene detection probe combination described in Example 1, the two detection kits described in Example 2, one is a combination of (ALK (Group 1) + ALK (Group 2) + EML4 (Group 3) (hereinafter referred to as EML4/ALK rearrangement test kit), and another kit for combination of ALK (group 1) + ALK (group 2) (hereinafter referred to as ALK rearrangement detection kit), for 20 parts Clinical samples (which were confirmed by pathological examination, the specimens were formalin-fixed paraffin-embedded tissue samples, see the table below), and were tested separately. The experiments of the two kits were repeated three times, and the test results were repeated. The same, the detection consistency is good. Compared with commercially available commercial reagents, the test results are completely consistent, and the specificity and sensitivity of the reagents are high. 3A, FIG. 3B, FIG. 4A, and FIG. 4B are the results of the detection of the two sets of probe combinations of the two genes, wherein FIG. 3A is a diagram of the FISH detection result of the lung tissue sample of the ALK rearrangement detection kit, wherein the detection signal type For 2F (RG adjacent or coincident), the ALK gene is not rearranged. Fig. 3B is a diagram showing the results of FISH detection of lung cancer tissue samples by the EML4/ALK rearrangement detection kit, wherein the detection signal type is 2F2A, the ALK gene is not rearranged, and no EML4/ALK rearrangement occurs. FIG. 3B is a diagram showing the results of FISH detection of lung cancer tissue samples in the examples, wherein the detection signal type is 2F2A, the ALK gene is not rearranged, and no EML4/ALK rearrangement occurs. Fig. 4A is a diagram showing the results of FISH detection of lung cancer tissue samples by ALK rearrangement detection kit, wherein the detection signal type is 1F1G1R, and the F signal is broken compared with the normal signal type 2F, and the detection result is: ALK gene rearrangement. Figure 4B is a diagram showing the results of FISH detection of lung cancer tissue samples by EML4/ALK rearrangement detection kit. The detection signal type is 1F1A1G1RA. Compared with the normal signal type 2F2A, F signal rupture occurs and RA signal fusion occurs at the same time. The detection result is : ALK gene is broken and the rearrangement type is EML4/ALK.

In the present invention, for the detection kits of the EML4 gene and the ALK gene, gene rearrangement and fusion type detection are performed using three sets of probes, respectively. However, in combination with the use of probes, it is better to use a combination of cloned probes to detect signals. Theoretically, the longer the length of the probe, the brighter the fluorescence signal obtained during actual detection, but because more gene sequences may be involved, the complexity of the resulting signal is increased, and the difficulty of detection is also enhanced. The length of the probe group 3 of the EML4 gene, the probe set 1 of the ALK gene, and the probe set of the probe set 2 of the ALK gene of the present invention are 261 Kb, 462 Kb, and 513 Kb, respectively.

The ALK rearrangement partner has many genes, and EML4/ALK rearrangement is one of them. The ALK gene rearrangement detection probe (including the ALK two-group probe) can detect ALK rearrangement, and breakage means that rearrangement occurs, but the rearrangement type cannot be judged. The EML4/ALK rearrangement detection probe (including the ALK two-set probe and the EML4 set of probes) can detect ALK rearrangement and simultaneously determine whether EML4/ALK rearrangement has occurred.

The inventors found in the verification of the probe of the present invention that the longer detection probe is indeed stronger. Fluorescent signals, and the same results were obtained in the validation of clinical samples. Therefore, in the design of the fluorescent probe, the signal brightness can be increased by appropriately extending the length of the fluorescent probe, but how to use it in combination, there are certain technical difficulties, and a good detection result is to be achieved, in addition to the experience in design. It is also necessary to verify the difference in signal type through clinical sample verification.

From the above experimental results, it is known that after molecular markers are detected for these samples, the samples can be molecularly classified according to the detection results, and used for clinical treatment plan formulation, drug selection and efficacy judgment according to the significance of the detection indicators.

The technical features of the embodiments may be combined in any combination. For the sake of brevity of description, all possible combinations of the technical features in the above embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, It should be considered as the scope of this manual.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (12)

1. A method for preparing an ALK gene detecting probe, comprising the steps of:

   (1) The BAC clones targeting the ALK gene were selected as at least one of RP11-62B19, RP11-100C1 and CTD-2245E6 as a set of probes, and the BAC clones were selected as CTD-2544I11, RP11-684O3 and RP11-134O13. At least one as another set of probes;

   (2) separately extracting a plasmid from the clone to obtain a plasmid DNA, and quantifying;

   (3) Labeling the plasmid DNA with fluorescein, the color of the fluorescein of the same set of probes is the same, and the color of the fluorescein of the different sets of probes is different.
2. The method for preparing an ALK gene detecting probe according to claim 1, wherein the BAC clones are RP11-62B19, RP11-100C1 and CTD-2245E6, and the BAC clones are CTD-2544I11, RP11- 684O3 and RP11-134O13.
3. The ALK gene detecting probe obtained by the production method according to any one of claims 1 or 2.
4. An ALK gene detecting kit comprising the ALK gene detecting probe according to claim 3.
5. A method for preparing an ALK gene and an EML4 gene detecting probe, comprising the steps of:

   (1) selecting a BAC clone for the ALK gene as at least one of RP11-62B19, RP11-100C1, and CTD-2245E6, and selecting a BAC clone as at least one of CTD-2544I11, RP11-684O3, and RP11-134O13; The BAC clone selected for the EML4 gene is at least one of CTD-2358L8 and CTD-2547J15;

   (2) separately extracting a plasmid from the clone to obtain a plasmid DNA, and quantifying;

   (3) Labeling the plasmid DNA with fluorescein, the color of the fluorescein labeled with the ALK gene and the detection probe for the EML4 gene is different, and for the ALK gene, the color of the fluorescein of the same group of detection probes is the same, different groups The color of the fluorescein of the detection probe is not the same.
6. The method for preparing an ALK gene and an EML4 gene detecting probe according to claim 5, wherein the ALK gene detecting probe has two groups, wherein one group of BAC clones is RP11-62B19, RP11-100C1 and CTD- 2245E6; another group of BAC clones is CTD-2544I11, RP11-684O3 and RP11-134O13.
7. The method for producing an ALK gene and EML4 gene detecting probe according to claim 5, wherein the BAC clones for the EML4 gene detecting probe are CTD-2358L8 and CTD-2547J15.
8. The method for preparing an ALK gene and an EML4 gene detecting probe according to any one of claims 5 to 7, wherein the fluorescein is Alexa

   

   FITC, Alexa

   

   Rhodamine, Texas Red, pacific

   

   DEAC.
9. The method for preparing an ALK gene and an EML4 gene detecting probe according to any one of claims 5 to 7, wherein the step (3) performs fluorescein labeling on the plasmid DNA by a random primer method or a nick translation method, the label The temperature is between 15 ° C and 18 ° C and the labeling time is 8-12 hours.
10. The ALK gene and EML4 gene detecting probe obtained by the production method according to any one of claims 5-9.
11. A kit for detecting an ALK gene and an EML4 gene, comprising the ALK gene and the EML4 gene detecting probe according to claim 10.
12. The ALK gene and EML4 gene detecting kit according to claim 11, further comprising a COT Human DNA for blocking a repeat sequence, and a DAPI counterstaining agent.

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