WO2017114005A1 - Terc gene and/or myc gene detection probe, preparation method therefor, and reagent kit - Google Patents

Abstract

A TERC gene and MYC gene detection probe, and a preparation method therefor. The method comprises the following steps: selecting at least one of CTD-3214J12, RP11-816J6 and RP11-778I3 as a BAC clone for a TERC gene, or selecting at least one of RP11-3K16, CTD-2582E13 and RP11-886J24 as a BAC clone; and/or selecting CTD-2511O8 as a BAC clone for an MYC gene, or selecting at least one of RP11-440N18 and CTD-2205H22 as the BAC clone; obtaining a plasmid DNA; performing labeling. Also provided is a reagent kit comprising the TERC gene and MYC gene detection probe.

Description

TERC gene and/or MYC 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 TERC (ERBB2) gene and/or MYC gene detecting probe, a preparation method thereof and a kit.

Background technique

Cervical cancer is a common malignant tumor in gynecology. From normal cervix, cervical intraepithelial neoplasia to cervical cancer is a gradual development process, its occurrence and development are closely related to the abnormal expression or deletion of various genes and proteins, which is a complex multi-step process. During the treatment of cervical cancer, the therapeutic effect on cervical cancer is related to the pathological grade. CIN and early cervical cancer (including carcinoma in situ and early invasive carcinoma) have very good therapeutic effects, and the treatment measures are appropriate. survive. Therefore, early diagnosis and accurate grading of cervical cancer is very important in the prevention and treatment of cervical cancer. Human telomerase RNA component (TERC) is a major component of human telomerase. Mutation, activation and amplification of TERC gene will lead to enhanced telomerase activity. The protooncogene MYC (v-myc avian myelocytomatosis viral oncogene homolog, MYC) is an important member of the MYC family and encodes a phosphorylated protein in the nucleus. The MYC gene is a major regulatory gene of cell proliferation, and its enhanced expression can initiate a non-controlled proliferation process of cells, thereby promoting the occurrence of most human tumors. The study found that compared with normal cervical and low-grade cervical precancerous lesions (CIN1), the amplification frequency of TERC gene and MYC gene is enhanced with the progression of cervical lesions, suggesting that detection of TERC/MYC gene status can assist clinical differentiation. Low-grade cervical precancerous lesions improve the sensitivity and specificity of cytology and HPV screening for cervical lesions.

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 letter Strong, easy to detect; 2) Whole-chromosomal or chromosomal region-specific probes consisting of extremely different nucleotide segments on a chromosome or a segment of a chromosome, which can be cloned into phage and plasmid-specific chromosomes Large fragment acquisition; 3) 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 detection kits with high sensitivity and specificity for FISH detection of TERC gene and/or MYC gene.

Summary of the invention

One of the objects of the present invention is to provide a TERC gene and/or MYC gene detecting probe and a preparation method thereof, which can be used for detecting the state of the TERC gene and the MYC gene, that is, detecting a copy of the TERC gene and the MYC gene. The number changes and has good specificity.

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

A method for preparing a TERC gene and/or a MYC gene detection probe, comprising the steps of:

(1) selecting a BAC clone for the TERC gene as at least one of CTD-3214J12, RP11-816J6, and RP11-778I3, or selecting a BAC clone as at least one of RP11-3K16, CTD-2582E13, and RP11-886J24; / or select the BAC clone for the MYC gene as CTD-2511O8, or select the BAC clone as at least one of RP11-440N18 and CTD-2205H22;

(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 fluoresin labeled with the TERC gene and the detection probe for the MYC gene is different.

In one embodiment, the BAC clones for the TERC gene are CTD-3214J12, RP11-816J6, and RP11-778I3.

In one embodiment, the BAC clones for the TERC gene are RP11-3K16, CTD-2582E13, and RP11-886J24.

In one embodiment, the BAC clone for the MYC gene is CTD-2511O8.

In one of the examples, the BAC clones directed against the MYC gene are RP11-440N18 and CTD-2205H22.

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 14 ° C to 18 ° C and a labeling time of from 10 to 14 hours.

Another object of the present invention is to provide a TERC gene and MYC gene detecting kit.

The technical solution for achieving this purpose is as follows.

A TERC gene and/or MYC gene detection kit comprising the above TERC gene and/or MYC gene detection probe.

In one embodiment, a chromosome 7 discrimination probe (CSP 7) probe for internal control is included, the identification probe being different in color from the fluorescein labeled by the TERC gene and the MYC 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 detects the TERC/MYC gene amplification by FISH (Fluorescence In-Situ Hybridization) by screening the optimal MYC gene amplification detection probe and its combination, and the signal counting is accurate and rapid, and The reproducibility of the results is good; supplementing the shortage of detection reagents in the clinic depends on the import, which is beneficial to the subsequent application of the detection probe in clinical research, early diagnosis, grading diagnosis and guiding treatment of cervical cancer.

(2) Preferably, the cloning detection specificity of the present invention is good, and the TERC/MYC gene amplification detection kit according to the present invention understands the state change of the MYC gene from the gene level, and changes the abnormal signal pattern and the number of abnormal signal cells. Judging pathological grades is diagnostic in cytological height and low-grade lesions.

DRAWINGS

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

Figure 1B is a schematic illustration of the MYC gene detection probe sequence of Example 1.

2 is a FISH detection result of a human peripheral blood culture cell sheet TERC gene and a MYC gene detection probe in Example 1. Figure.

3 is a FISH detection result of a cervical exfoliated cell sample in Example 4, wherein the detection signal type is 2R2G2A, and the detection signal type is 2R2G2A, and the detection result is: the TERC gene is not amplified, and the MYC gene is not expanded. increase.

4 is a FISH detection result of the cervical tissue sample in Example 4, wherein the detection signal type is 2R2G2A, and the detection result is that the TERC gene is not amplified, and the MYC gene is not amplified.

Fig. 5 is a graph showing the results of FISH detection of cervical tissue samples in Example 4, wherein the detection signal type is 3R3G2A, and the detection results are: TERC gene amplification, MYC gene amplification.

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 TERC gene and MYC gene detection probe

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

(1) A clone comprising the TERC of the gene of interest and the sequences at both ends was selected, as shown in Fig. 1A. The GSP TERC comprises two sets of probes, the first set of probes comprising a first probe, a second probe and a third probe, as shown in the following table, 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.

First group:

Group one	BAC	Insert start and end position
First probe	CTD-3214J12	(chr3: 169152582..169377736) 225Kb

Second probe	RP11-816J6	(chr3: 169447253..169494853) 48Kb
Third probe	RP11-778I3	(chr3:169492904..169717073)224Kb

Group two	BAC	Insert start and end position
Fourth probe	RP11-3K16	(chr3: 169249515..169449349) 200Kb
Fifth probe	CTD-2582E13	(chr3: 169408931..169606612) 198Kb
Sixth probe	RP11-886J24	(chr3: 169580072..169746683 167Kb

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

Clones containing the MYC of the gene of interest and the sequences at both ends were selected as shown in Figure 1B. The GSP MYC includes two sets of probes:

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

The second set of probes included a second probe and a third probe, as detailed below, which were purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.

The following two sets of detection probes were separately prepared.

First group:

Group one	BAC	Insert start and end position
First probe	CTD-2511O8	Chr8:128654266...128867152(213Kb)

Second Group:

Group two	BAC	Insert start and end position
Second probe	RP11-440N18	Chr8:128596756...128777986(181Kb)
Third probe	CTD-2205H22	Chr8:128740195...128887949(148Kb)

(2) Preparation of GSP TERC and GSP MYC 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 determined. Quantification of plasmid DNA; diluted with auto-sterilized ultrapure water to 200 ng / ul, divided into 1.5 ml centrifuge tubes, and finally obtained one or two or four corresponding combinations of TERC gene or MYC gene DNA was mixed and stored at -20 °C.

(3) Fluorescent labeling of the plasmid DNA mixture by nick translation, each probe for the GSP TERC gene The labeled fluorescein is Spectrum-Orange, and the fluorescein labeled for each probe of the GSP MYC gene is 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 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 a band of between 50 bp and 500 bp was required.

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 GSP TERC and GSP MYC and gene probes, respectively, and stored at -20 ° C in the dark.

(4) GSP TERC and GSP MYC gene probe verification: hybridization solution prepared by using one probe of TERC group + probe of MYC group, and hybridization solution prepared by two probes of TERC group and two probes of MYC group as reagents to be detected The probe was verified using human normal dividing 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 a probe in the TERC group + probe in the MYC group) shows the results of FISH hybridization of metaphase chromosomes. The red signal shows GSP TERC, the green signal shows GSP MYC, and the cyan signal CSP 7 (Chromosome 7 probe, purchased from D7Z1SpectrumAqua Probe, article number: 06J54-007). It can be seen that the TERC/MYC/7 chromosome probe signal is bright, and sensitivity and specificity can be observed on the metaphase chromosome in human peripheral blood cultured cell sheets. 100% sex; using a paraffin sample for hybridization detection, three target copy numbers can be clearly recorded. The results of other corresponding probe verification experiments are the same as those of the TERC group one probe + MYC group one probe, and the figures are omitted.

Example 2: Preparation method of TERC gene and MYC gene detection kit

The TERC gene and MYC gene detection kit includes two components of a TERC/MYC hybridization solution and a DAPI counterstain, wherein the TERC and MYC hybridization solution comprises the set of GSP MYC gene probes described in Example 1 and a set of GSP TERCs. A combination of gene probes. The TERC and MYC genes have two sets of detection probes respectively, and the two groups of the two genes can be combined at will. In this embodiment, the TERC gene and the MYC gene detection kit are four, respectively: TERC (group one) + Combination of MYC (Group 1), combination of TERC (Group 2) + MYC (Group 2), combination of TERC (Group 1) + MYC (Group 2), TERC (Group 2) + MYC (Group 1), CSP7 (Chromosome 7 probe, available from D7Z1 Spectrum Aqua Probe, Cat. No. 06J54-007), buffer component for hybridization environment (promoting hybridization), COT Human DNA with closed repeats, 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

(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 TERC gene and MYC 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 and place it in room temperature xylene (or environmental dewaxing agent) for 10 minutes;

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

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

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 sterile purified water for 3 minutes at room temperature, and use the lint-free paper towel to absorb excess water;

1.7 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 time when the sound starts to sound);

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

1.9 remove the slide and place it in room temperature 2 × SSC for 3 minutes;

1.10 remove the slide and let it dry at room temperature;

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

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

1.13 remove the slide, and then put it into another cylinder at room temperature 2 × SSC for 5 minutes;

1.14 Take out the slides, and then put them into 70%, 90%, 100% gradient ethanol at room temperature for 2 minutes each time;

1.15 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 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.

4, the result analysis

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

4.1 Use a suitable filter, look under a 10× 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 should be 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 glance at several cell regions, requiring complete cell boundaries, 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;

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

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

4.6.1 normal signal: 2R2G2A;

4.6.2 Abnormal signal: >2R2G2A (TERC amplification), 2G>2G2A (MYC amplification).

4.7 setting 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: Clinical evaluation of TERC gene and MYC gene detection kit

Using the TERC gene and MYC gene detection probe combination described in Example 1, the four detection kits described in Example 2 (TERC (Group 1) + MYC (Group 1) combination, TERC (Group 2) + MYC (Group) 2) combination, TERC (group 1) + MYC (group 2) combination, TERC (group 2) + MYC (group 1) combination) for 20 clinical samples (sample type is formalin fixed paraffin embedded) Tissue samples and cervical exfoliated cell samples were diagnosed by pathological examination, as shown in the table below, and tested separately. 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 and Figure 5 show the combined kit detection results of TERC (Group 1) + MYC (Group 1). As shown in Figure 3 and Figure 4, the detection signal type is 2R2G2A, and TERC/MYC is not amplified; As shown in Fig. 5, the detection signal type is 3R3G2A, and the TERC/MYC gene is amplified. The detection results of the combinations of the other three different gene probes are the same, and the figures are omitted.

The combination of the combination of TERC (group 1) + MYC (group 2), TERC (group 2) + MYC (group 1) is the same as the results shown in the above table, and the specific results are omitted for reasons of space.

In the present invention, the detection of the TERC gene and the MYC gene by using one probe can also achieve the corresponding detection, but the use of the combined probe can be better than the combination of the probes, especially in the present embodiment. In the example, the signal of TERC (group 1) + MYC (group 1) and TERC (group 2) + MYC (group 2) combination detection is the best. 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 detection probes of Groups 1 and 2 for the TERC gene of the present invention have lengths of 564 Kb and 497 Kb, respectively, and are nucleic acid mixtures comprising the TERC gene and its both ends. The total lengths of the BAC clones of the detection probes of Groups 1 and 2 for the MYC gene of the present invention are: 213 Kb and 291 Kb, respectively, which are nucleic acid mixtures comprising the MYC gene and its both ends.

The inventors found in the verification of the probe of the present invention that the longer detection probe did obtain a stronger fluorescent signal, and the same result was obtained in the verification of the clinical sample. 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. different.

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 TERC gene and/or a MYC gene detecting probe, comprising the steps of:

   (1) selecting a BAC clone for the TERC gene as at least one of CTD-3214J12, RP11-816J6, and RP11-778I3, or selecting a BAC clone as at least one of RP11-3K16, CTD-2582E13, and RP11-886J24; / or select the BAC clone for the MYC gene as CTD-2511O8, or select the BAC clone as at least one of RP11-440N18 and CTD-2205H22;

   (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 fluoresin labeled with the TERC gene and the detection probe for the MYC gene is different.
2. The method for preparing a TERC gene and/or MYC gene detecting probe according to claim 1, wherein the BAC clone for the TERC gene is CTD-3214J12, RP11-816J6 and RP11-778I3, or the BAC clone is RP11- 3K16, CTD-2582E13 and RP11-886J24.
3. The method for producing a TERC gene and/or MYC gene detecting probe according to claim 1, wherein the BAC clone for the MYC gene is CTD-2511O8, or the BAC clone for the MYC gene is RP11-440N18 And CTD-2205H22.
4. The method for producing a TERC gene and/or MYC gene detecting probe according to claim 1, wherein the BAC clones for the TERC gene are RP11-3K16, CTD-2582E13 and RP11-886J24, and the MYC gene The BAC clones were RP11-440N18 and CTD-2205H22.
5. The method for preparing a TERC gene and/or MYC gene detecting probe according to claim 1, wherein the BAC clone for the TERC gene is a BAC clone of CTD-3214J12, RP11-816J6 and RP11-778I3, and a MYC gene. The BAC clone was CTD-2511O8.
6. The method for preparing a TERC gene and/or MYC 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 TERC gene and/or MYC gene detecting probe according to any one of claims 1 to 6, wherein the step (3) adopts a random primer method or a nick translation method to perform fluorescein labeling on the plasmid DNA. The temperature of the label is 14 ° C - 18 ° C and the time of labeling is 10-14 hours.
8. The TERC gene and/or MYC gene detecting probe obtained by the production method according to any one of claims 1 to 7.
9. A TERC gene and/or MYC gene detecting kit comprising the TERC gene of claim 8 and a MYC gene detecting probe.
10. The TERC gene and/or MYC gene detection kit according to claim 9, further comprising a chromosome 7 discrimination probe for internal control, the identification probe and the fluorescent label of the TERC gene and the MYC gene detection probe The colors of the pigments are different; there are also COT Human DNA for blocking repeats, and DAPI counterstains.

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