WO2017114011A1 - Her-2 gene and/or top2a gene detection probe, preparation method therefor, and test kit - Google Patents

Abstract

Provided are a HER-2 gene and/or TOP2A gene detection probe, and a preparation method therefor, the method comprising the following steps: selecting a BAC clone for the HER-2 gene to be RP11-1021P11, RP11-62N23 or RP11-1044P23 or selecting the BAC clone to be RP11-98J2, RP11-1065L22 or CTD-2327I15, and/or selecting a BAC clone for the TOP2A gene to be RP11-1152A10 or CTD-3217C5 or selecting the BAC clone to be RP11-737D6, CTD-3087O22, RP11-48O10 or CTD-2134K5; obtaining plasmid DNA; labelling. Also provided is a test kit including the HER-2 gene and/or TOP2A gene detection probe. The present invention is accurate and rapid in terms of signal counting rows, and has good results reproducibility.

Description

HER-2 gene and/or TOP2A gene detection probe and preparation method and kit thereof Technical field

The present invention belongs to the field of biotechnology, and particularly relates to a HER-2 (ERBB2) gene and/or TOP2A gene detecting probe, a preparation method thereof and a kit.

Background technique

The HER-2 (ERBB2) gene has gene amplification and protein overexpression in 20%-30% of breast cancer patients. HER-2 is currently recognized as an important prognostic/predictor for breast cancer. In multivariate analysis, HER-2 is an independent prognostic factor for tumor recurrence and overall survival. HER-2 positive breast cancer is invasive, with short disease-free survival and poor prognosis. Trastuzumab (Herceptin) is indicated for breast cancer with HER-2 overexpression and gene amplification. TOP2A gene regulates the supercoiled state of DNA, directly participates in cellular processes, and is a target enzyme of anthracycline antibiotics. Patients with abnormal TOP2A gene have poor prognosis and shortened recurrence-free survival, especially in patients with TOP2A gene deletion. . Gene amplification suggests a recurrence of the tumor, or a long-term decline in efficacy. The state of TOP2A gene has a certain significance in predicting the response of tumor chemotherapy and the effect of drug treatment.

Therefore, it is important to correctly detect and assess the status of HER-2 gene and TOP2A gene in breast cancer.

The HER-2 gene is located in the 17q12 region of chromosome 17, the abnormality of HER-2 is amplified; the TOP2A gene is located in the 17q21 region of chromosome 17, and the abnormality of TOP2A is divided into amplification and deletion, and FISH detection can be used for this. A clear judgment is made in both cases.

Fluorescence in situ hybridization (FISH) is a non-radioactive in situ hybridization technique developed on the basis of the original radioactive in situ hybridization technique in the late 1980s. At present, this technology has been widely used in animal and plant genomic structure research, chromosome fine structure variation analysis, viral infection analysis, human prenatal diagnosis, tumor genetics and genome evolution research in many fields. The basic principle of FISH is to use a known labeled nucleic acid as a probe to heterologously bind to an unknown single-stranded nucleic acid in a material to be tested according to the principle of base complementation to form a hybrid double-stranded nucleic acid which can be detected. Since the DNA molecules are linearly arranged along the longitudinal axis of the chromosome on the chromosome, the probe can be directly hybridized to the chromosome to localize the specific gene on the chromosome. Compared with traditional radiolabeled in situ hybridization, fluorescence in situ hybridization has the characteristics of rapid detection signal, high hybridization specificity and multi-staining, so it has received widespread attention in the field of molecular cytogenetics.

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. , hybrid signal is strong, easy to detect; 2) whole chromosome or chromosomal region-specific probe, which is on a chromosome or a segment on a chromosome Consisting of extremely different nucleotide fragments, which can be obtained from chromosome-specific large fragments cloned into phage and plasmids; 3) specific position probes 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 HER-2 gene and/or the TOP2A gene.

Summary of the invention

One of the objects of the present invention is to provide a HER-2 gene and/or TOP2A gene detecting probe and a preparation method thereof, which can be used for detecting the HER-2 gene and the TOP2A gene state, that is, detecting the HER-2 gene. And the change in the copy number of the TOP2A gene has good specificity.

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

A method for preparing a HER-2 gene and/or a TOP2A gene detection probe, comprising the steps of:

(1) The BAC clone targeting the HER-2 gene is at least one of RP11-1021P11, RP11-62N23, and RP11-1044P23, or the BAC clone is at least one of RP11-98J2, RP11-1065L22, and CTD-2327I15. And/or selecting a BAC clone for the TOP2A gene as at least one of RP11-1152A10, CTD-3217C5, or selecting a BAC clone as at least one of RP11-737D6, CTD-3087O22, RP11-48O10, CTD-2134K5;

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

(3) The plasmid DNA is labeled with fluorescein, and the fluorescein labeled with the plasmid DNA of the same gene is the same, and the color of the fluorescein labeled with the HER-2 gene and the detection probe for the TOP2A gene is different.

In one embodiment, the BAC clones for the TOP2A gene are RP11-1152A10 and CTD-3217C5.

In one embodiment, the BAC clones for the TOP2A gene are RP11-737D6, CTD-3087O22, RP11-48O10, and CTD-2134K5.

In one of the examples, the BAC clones directed against the HER-2 gene are RP11-1021P11, RP11-62N23, and RP11-1044P23.

In one embodiment, the BAC clones for the TOP2A gene are RP11-98J2, RP11-1065L22, and CTD-2327I15.

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 a commercially available nick translation labeling kit and/or a random primer labeling kit, preferably abbott and/or 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 label has a temperature of from 24 ° C to 26 ° C and a marked time of from 4 to 6 hours.

Another object of the present invention is to provide a HER-2 gene and TOP2A gene detecting kit.

The technical solution for achieving this purpose is as follows.

A HER-2 gene and/or TOP2A gene detection kit comprising the above HER-2 gene and/or TOP2A gene detection probe.

In one embodiment, a chromosome 17 discrimination probe (CSP17) probe for internal control is included, the identification probe being different in color from the fluorescein labeled by the HER-2 gene and the TOP2A gene detection 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 uses the FISH (Fluorescence In-Situ Hybridization) method to detect the copy number of the HER-2 gene and the TOP2A gene by screening the optimal HER-2 gene and/or TOP2A gene detection probes and their combinations.

2. The preferred clone of the present invention has good detection specificity and high sensitivity. By adjusting the labeling time and temperature, the length probe is limited to about 500 bp, which improves the hybridization efficiency and reduces the hybridization background.

3. The signal counting line is accurate and fast, and the result is reproducible; through the simultaneous detection and evaluation of HER-2, TOP2A copy number and chromosome 17, it is beneficial to determine the treatment plan and screen more breasts that benefit from targeted drugs. Cancer patients improve patient survival and overall survival.

4. Through the HER-2 gene and TOP2A kit of the present invention, the HER-2 gene and TOP2A state changes are known from the gene level, and various signal types exhibit tumor cell genetic diversity of solid tissues, which can be realized in tumor organisms. The application in the fields of learning and cytogenetics can help comprehensively evaluate various molecular markers, assist breast cancer prognosis and clinical targeted therapy drugs and treatment options.

DRAWINGS

Figure 1A is a schematic illustration of the HER-2 gene detection probe sequence of Example 1.

Fig. 1B is a schematic diagram showing the TOP2A gene detecting probe sequence of Example 1.

Fig. 2 is a graph showing the results of FISH detection of the HER-2 gene and the TOP2A gene detecting probe of human peripheral blood culture cell sheets in Example 1.

3 is a diagram showing the results of FISH detection of breast cancer tissue samples in Example 4, wherein the detection signal type is 2R2G2A, the HER-2 gene is not amplified, and the TOP2A gene is not amplified.

4 is a diagram showing the results of FISH detection of a breast cancer tissue sample in Example 4, wherein the detection signal type is clustered R cluster G2A, and the HER-2 gene and the TOP2A gene are co-amplified.

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 HER-2 Gene and TOP2A Gene Detection Probes

A method for preparing a HER-2 detection probe (GSP HER-2), comprising the following steps:

(1) Cloning screening: Clones containing the target gene HER-2 and the sequences at both ends were selected, as shown in Fig. 1A.

GSP HER-2 consists of two groups, and the following two sets of detection probes are prepared separately.

HER-2 chr17: 37, 856, 254-37, 884, 915, 28, 662 bp

HER-2 probe set 1	BAC	Insert start and end position
First probe	RP11-1021P11	Chr17:37497503...37725641(228Kb)
Second probe	RP11-62N23	Chr17:37725641...37882864(157Kb)
Third probe	RP11-1044P23	Chr17:37871783...38066611(195Kb)
HER-2 probe set 2	BAC	Insert start and end position
Fourth probe	RP11-98J2	Chr17:37572511...37753435 (180Kb)
Fifth probe	RP11-1065L22	Chr17:37758567...37990799(232Kb)
Sixth probe	CTD-2327I15	Chr17:37963139...38082538(119Kb)

The preparation method of the TOP2A detection probe (GSP TOP2A) comprises the following steps:

Cloning Screening: Clones containing the TOP2A gene of interest and the sequences at both ends were selected, as shown in Figure 1B.

GSP TOP2A consists of two groups, as shown in the following table, which were purchased from the Invitrogen RP11BAC and CTD BAC clone libraries. The following two sets of detection probes were separately prepared.

TOP2A chr17: 38, 544, 773-38, 574, 202, 29, 430 bp

TOP2A probe set 2	BAC	Insert start and end position
Third probe	RP11-737D6	Chr17:38257742...38440899(183Kb)
Fourth probe	CTD-3087O22	Chr17:38430841...38564776(134Kb)
Fifth probe	RP11-48O10	Chr17:38564771...38724970(160Kb)
Sixth probe	CTD-2134K5	Chr17:38716958...38834072(117Kb)

(2) Preparation of GSP HER-2 and GSP TOP2A 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, by measuring 260 nm and 280 nm. The absorbance was quantified by plasmid DNA; diluted with auto-sterilized ultrapure water to 200 ng/ul, and packed in a 1.5 ml centrifuge tube, and finally two or three or four kinds of corresponding HER-2 gene or TOP2A gene were obtained. The corresponding combined plasmid DNA was mixed and stored at -20 ° C in a sealed form.

(3) The plasmid DNA mixture was fluorescently labeled by the nick translation method. The fluorescein labeled for each probe of the GSP HER-2 gene was Spectrum-Orange, and the fluorescein labeled for each probe of the GSP TOP2A gene was Spectrum- Green. 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 25 ° C for 5 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 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 precipitate was dissolved in 1 ul of purified water to obtain a GSP TOP2A gene probe, which was stored at -20 ° C in the dark.

(4) GSP HER-2 and GSP TOP2A gene probe validation: hybridization solution prepared using HER-2 probe set 1 + TOP2A probe set 1 and HER-2 probe set 2+ TOP2A probe set 2, respectively The hybridization solution is a reagent to be detected, and probe verification is performed using 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. Figure 2 (experimental results applied to the HER-2 probe set 1 + TOP2A probe set 1) shows the results of FISH hybridization of metaphase chromosomes. In the figure, the red signal shows GSP HER-2, the green signal shows GSP TOP2A, and the cyan signal shows CSP17 (for positioning the chromosome 17 centromere probe, purchased from D17Z1SpectrumAqua Probe, article number: 06J38-017). It can be seen that the HER-2/TOP2A/17 chromosome probe signal is bright, and sensitivity and specificity can be observed on the metaphase chromosome in human peripheral blood cultured cells. 100% can be clearly recorded by using paraffin sample tablets for hybridization detection. Three target copy numbers. The results of other corresponding probe verification experiments are the same as those of the HER-2 probe set 1 + TOP2A probe set 2, and the figures are omitted.

Example 2: Preparation method of HER-2 gene and TOP2A gene detection kit

The HER-2 gene and TOP2A gene detection kit includes two components of HER-2/TOP2A hybridization solution and DAPI counterstaining agent, wherein the HER-2 and TOP2A hybridization liquids comprise the set of GSP TOP2A gene probes described in Example 1. A combination of a needle and a set of GSP HER-2 gene probes. There are two sets of detection probes for the HER-2 and TOP2A genes, respectively. The two groups of the two genes can be combined at will. In this embodiment, the HER-2 gene and TOP2A gene detection kits are of the following four types: HER- 2 (group 1) + TOP2A (group 1) combination, HER-2 (group 2) + TOP2A (group 2) combination, HER-2 (group 1) + TOP2A (group 2) combination, HER-2 ( Group 2) + TOP2A (Group 1) combination, CSP17 probe (Chromosome 17 identification Needle, purchased from D17Z1 SpectrumAqua Probe, item number: 06J38-017), buffer component for hybridization environment (promoting hybridization), closed repetitive sequence of COT Human DNA, etc.). 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:

(1) Preparation of hybridization solution

Ingredients	Every test
A group of GSP HER-2 (100ng/ul)	0.7μl
A group of GSP TOP2A (100ng/ul)	0.6μl
CSP 17 (50ng/ul)	0.2μl
Hybridization buffer	7μl
COT Human DNA (1mg/ml)	0.6μl
H 2 O	Supplement to 10μl

(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 HER-2 gene and TOP2A gene detection kit

1, slide pretreatment

1.1 slides are placed in a 65±5°C incubator for overnight baking;

1.2 Remove the slide, put it into xylene and dewax it for 15 minutes at room temperature;

1.3 Remove the slide, then put it into another cylinder of xylene and continue dewaxing for 15 minutes at room temperature;

1.4 Remove the slide, then place it in absolute ethanol for 10 minutes at room temperature to remove residual xylene;

1.5 Remove the slide, and then put it into 100%, 90%, 70% gradient ethanol room temperature for 3 minutes each time;

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

1.7 Remove the slide and place it in purified water at 100±5 °C for 25 minutes (the slice is placed horizontally in the container with the sample side facing up);

1.8 Remove the slide and let it dry at room temperature;

1.9 Place the slide face up on the shelf, add appropriate amount of pepsin reaction solution in the sample area, and digest for 5 to 15 minutes;

1.10 remove excess liquid and place it in room temperature 2 × SSC for 5 minutes;

1.11 remove the slide and place it in another cylinder at room temperature 2 × SSC for 5 minutes;

1.12 remove the slide, and then put it into room temperature 70%, 90%, 100% gradient ethanol dehydration for 3 minutes each;

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

The reaction time of pepsin needs to be determined by preliminary tests. Samples prepared in the same batch can be pre-tested as described, usually at intervals of 5 minutes. For example, the digestion time is 5 minutes, 10 minutes, and 15 minutes, respectively. After the "slide pretreatment" is completed, the tissue digestion state can be observed under a bright field using a 10× or 20× objective lens; or DAPI counterstaining can be directly performed. The digestion state is judged.

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

2.1 The hybridization solution in the test kit described in Example 2 was taken out from the refrigerator at -20 ± 5 ° C, shaken and mixed, and centrifuged instantaneously;

2.2 Add 10 μl of the hybridization solution to the hybridization area, quickly cover the 18×18mm 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 apparatus, wet the humidity strip of the in situ hybridization instrument, insert the wet strip, cover the top of the hybridization apparatus, 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 heating 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.

The above listed reagents were all prepared in a circular dyeing tank (40 ml each), and up to 5 slices per dyeing tank. For non-room temperature solutions, pre-heat the reagents to the specified temperature before starting the operation. During the washing process, the dyeing tank can be gently shaken at intervals of 2 to 3 minutes to improve the washing effect.

4, the result analysis

The relevant fluorescence and DAPI need to be observed with a suitable filter block. Among them, the red signal indicates GSP HER-2, the green signal indicates GSP TOP2A, and the cyan signal indicates CSP 17.

4.1 Use a suitable filter, look under a 40× objective, and count under a 100× objective;

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

4.3 Sweeping several tumor cell areas, selecting at least 4 areas with good nuclear boundaries, requiring complete nuclear boundary, DAPI staining uniform, no nuclear overlap, CSP17 probe (cyan signal point) signal clear;

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

4.5 Organizational Counting Requirements:

a. Count only tumor tissue (use of HE stained tablets for control observation before FISH detection)

b. Avoid counting in areas with necrotic areas and unclear nuclear boundaries

c. Subjective discrimination is not counted

d. skip the weak signal and the core count without a specific signal or high background

4.6 Go to the 100× objective lens, adjust the focal length, and find all signal points at different levels of the core;

4.7 Count signal points in each core; focus to find all signal points in each core, count two signals in one region, count only one or more FISH signals for each color, no signal or only The core of a color signal is not counted; the total number of cells observed (signal normal and abnormal) is recorded;

4.8 counting method

A total of 40 to 100 tumor cell nucleus GSP TOP2A (red) and CSP17 (green) signals were counted in five clear tumor regions, and the number of GSP TOP2A and CSP 17 signals in individual nuclei was counted.

Example 4: Evaluation of clinical use of HER-2 gene and TOP2A gene detection kit

Using the combination of the HER-2 gene and the TOP2A gene detection probe described in Example 1, the combination of the four detection kits described in Example 2 (HER-2 (probe group 1) + TOP2A (probe group 1), HER -2 (probe group 2) + TOP2A (probe group 2) combination, HER-2 (probe group 1) + TOP2A (probe group 2) combination, HER-2 (probe group 2) + TOP2A (Combination of probe set 1)) For 20 clinical samples (which were confirmed by pathological examination, see the table below), respectively. The detection of the two probe combinations is consistent. Compared with commercially available commercial reagents, the test results are completely consistent, and the specificity and sensitivity of the reagents are high. Figure 3 and Figure 4 show the results of the combined kit detection of HER-2 (probe group 2) + TOP2A (probe group 2), as shown in Figure 3, FISH detection results of paraffin-embedded tissue samples of breast cancer, signal type The expression was 2R2G2A. The results showed that there was no abnormality in the HER-2/TOP2A gene and the chromosome 17 was normal. In Figure 4, the FISH detection results of the breast cancer tissue samples showed that the signal type was clustered R clustered G2～6A, and the result was judged as The HER-2/TOP2A gene was co-amplified, and chromosome 17 was multiplied. The results of the combination of the other two different gene probes were the same, and the figures were omitted.

The combination of HER-2 (probe group 1) + TOP2A (probe group 2), and the combination of HER-2 (probe group 2) + TOP2A (probe group 1) was the same as the results shown in the above table. Due to the length of the article, the specific results are omitted.

In the present invention, the detection of the HER-2 gene and the TOP2A gene can be achieved by using one probe, respectively, but the detection signal can be better by using the combination probe in combination with the probe. 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 BAC clones of the probe set 1 and probe set 2 of the HER-2 gene of the present invention have lengths of 510 Kb and 569 Kb, respectively, and are nucleic acid mixtures containing the HER-2 gene and its both ends. . The total lengths of the BAC clones of the probe sets 1 and 2 of the TOP2A gene of the present invention are: 353 Kb and 576 Kb, respectively, which are nucleic acid mixtures comprising the TOP2A gene and its both ends.

The inventors found in the verification of the probe of the present invention that a longer detection probe does obtain a stronger fluorescent signal, and The same results were obtained in the verification of the 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 (10)

1. A method for preparing a HER-2 gene and/or TOP2A gene detecting probe, comprising the steps of:

   (1) The BAC clone targeting the HER-2 gene is at least one of RP11-1021P11, RP11-62N23, and RP11-1044P23, or the BAC clone is at least one of RP11-98J2, RP11-1065L22, and CTD-2327I15. And/or selecting a BAC clone for the TOP2A gene as at least one of RP11-1152A10, CTD-3217C5, or selecting a BAC clone as at least one of RP11-737D6, CTD-3087O22, RP11-48O10, CTD-2134K5;

   (2) extracting plasmids from BAC clones respectively, obtaining plasmid DNA, and quantifying;

   (3) Labeling the plasmid DNA with fluorescein, the fluorescein labeled with the plasmid DNA of the same gene is the same, and the color of the fluorescein labeled with the HER-2 gene and/or the detection probe for the TOP2A gene is different, that is, Got it.
2. The method for producing a HER-2 gene and/or TOP2A gene detecting probe according to claim 1, wherein the BAC clone for the HER-2 gene is RP11-1021P11, RP11-62N23 and RP11-1044P23, or is BAC. The clones were RP11-98J2, RP11-1065L22 and CTD-2327I15.
3. The method for producing a HER-2 gene and/or TOP2A gene detecting probe according to claim 1, wherein said BAC clone for TOP2A gene is RP11-1152A10 and CTD-3217C5, or said TOP2A gene The BAC clones were RP11-737D6, CTD-3087O22, RP11-48O10, and CTD-2134K5.
4. The method for producing a HER-2 gene and/or TOP2A gene detecting probe according to claim 1, wherein the BAC clones for the HER-2 gene are RP11-1021P11, RP11-62N23 and RP11-1044P23, and for TOP2A The BAC clones of the genes are RP11-1152A10 and CTD-3217C5.
5. The method for preparing a HER-2 gene and/or TOP2A gene detecting probe according to claim 1, wherein the BAC clone for the HER-2 gene is a BAC clone of RP11-98J2, RP11-1065L22 and CTD-2327I15. And the BAC clones for the TOP2A gene are RP11-737D6, CTD-3087O22, RP11-48O10, and CTD-2134K5.
6. The method for preparing a HER-2 gene and/or TOP2A gene detecting probe according to claim 1, wherein the fluorescein is Alexa

   

   FITC, Alexa

   

   Rhodamine, Texas Red, pacific

   

   DEAC.
7. The method for preparing a HER-2 gene and/or TOP2A gene detecting probe according to any one of claims 1 to 6, wherein the step (3) uses a random primer method or a nick translation method to fluorescein label the plasmid DNA. The temperature of the label is 24 ° C - 26 ° C, and the time is 4-6 hours.
8. The HER-2 gene and/or TOP2A gene detecting probe obtained by the production method according to any one of claims 1 to 7.
9. A kit for detecting a HER-2 gene and/or a TOP2A gene, comprising the HER-2 gene and/or TOP2A gene detecting probe according to claim 8.
10. The HER-2 gene and/or TOP2A gene detection kit according to claim 9, further comprising a chromosome 17 identification probe for internal control, the detection probe and the HER-2 gene and the TOP2A gene detection probe The needle-labeled fluorescein is not the same color; it also includes COT Human DNA for blocking the repeat sequence, and DAPI counterstain.

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