CN112646895A - Primer, probe, kit, detection method and application for detecting gene expression level - Google Patents

Abstract

The application discloses a primer, a probe, a kit, a detection method and application for detecting gene expression level, wherein the oncogene comprises at least one of C-MYC, C-FOS, K-RAS, RB, P21 and P53, and correspondingly, the sequence of the primer and the sequence of the probe are also disclosed. By adopting the technical scheme, the corresponding primers and probes can be set according to requirements, quantitative detection of at least one oncogene is realized, the problem that the oncogene can only be detected one by one in detection can be solved, and the improvement of the detection efficiency and the reduction of the detection cost are facilitated.

Description

Primer, probe, kit, detection method and application for detecting gene expression level

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a primer, a probe, a kit, a detection method and application for detecting mRNA expression levels of C-MYC, C-FOS, K-RAS, RB, P21 and P53 genes, which are used for detecting the expression of the genes in cells.

Background

The generation and development of tumors are a complex biological process which is carried out step by step, and intracellular genes can be inserted near a strong promoter through translocation and generate mutation in a plurality of copies, losses, base changes and the like in a chromosome. The stem cells cultured in vitro have the potential of differentiation, and if the genes related to the cell cycle and the differentiation in the cells are mutated, the expression level of the genes can be obviously changed, so that various vital activities of the cells are influenced, and even the cells are developed into cancer cells. The expression level of these genes can be detected and can reflect or indirectly reflect the mutation condition. Activation of intracellular proto-oncogenes and inactivation of cancer suppressor genes are essential causes of tumorigenesis. The influence of the expression levels and mutation status of proto-oncogenes and tumor suppressor genes on cell physiology is a dynamic process. Among protooncogenes, 3 genes of C-MYC, C-FOS and K-RAS are relatively important; of the cancer suppressor genes, 3 genes, RB, P21 and P53, are relatively important.

In the prior art, the detection of oncogene expression is usually carried out on a single gene, the detection of different oncogenes can be carried out one by one, the detection time is long, the consumption of detection consumables is large, and the efficient detection is difficult for a precious sample with a small sample amount.

Disclosure of Invention

In order to solve the technical problems, the application discloses a primer, a probe, a kit, a detection method and application for detecting mRNA expression levels of C-MYC, C-FOS, K-RAS, RB, P21 and P53 genes, which can solve the problem that oncogene detection can only be performed one by one in the prior art, and are beneficial to improvement of detection efficiency and reduction of detection cost.

In order to solve the technical problems, the application discloses a primer and a probe for detecting the expression level of 6 oncogenes, wherein the oncogenes comprise at least one of C-MYC, C-FOS, K-RAS, RB, P21 and P53, and the sequence of the primer and the sequence of the probe comprise:

further, the application also discloses a kit comprising the primer and the probe, wherein the kit comprises:

a cell lysis reagent;

a cDNA synthesis reagent;

qPCR quantitative detection reagent.

Wherein, the cell lysis reagent comprises lysis solution, RNase inhibitor and DNase; the cDNA synthesis reagent comprises 2 x reverse transcription reaction liquid.

The qPCR quantitative detection reagent comprises 2 xProbe qPCR Mix, a primer Probe mixed solution 1 and a primer Probe mixed solution 2.

Wherein the primer probe mixture 1 comprises: primers and probes of an internal reference gene; primers and probes for detecting the C-MYC gene; primers and probes for detecting the C-FOS gene; the primer probe mixed solution 2 comprises: primers and probes for detecting the K-RAS gene; primers and probes for detecting the RB gene; primers and probes for detecting the P21 gene; primers and probes for detecting the P53 gene.

Wherein the internal reference gene is ATCB gene, and the primer sequence comprises SEQ ID NO.19 and SEQ ID NO. 20; the probe sequence includes SEQ ID NO 21.

Further, the application also discloses a method for detecting the expression level of the oncogene by using the kit, wherein the method comprises the following steps:

cracking the target cells by using a cell cracking reagent to obtain RNA;

reverse transcribing the RNA using a cDNA synthesis reagent to obtain cDNA;

mixing the cDNA, the 2 xProbe qPCR Mix and the primer Probe mixture 1 and/or the primer Probe mixture 2 to obtain a sample to be detected;

and (3) placing the sample to be detected into a quantitative fluorescence PCR detector for detection according to a preset program, and obtaining the CT value of each gene.

Wherein the preset program comprises: 95 ℃ for 1min, 95 ℃ for 15s × 40 cycles, 45 ℃ for 40s × 40 cycles.

Wherein the method comprises the following steps: the quantitative fluorescence PCR detector can detect at least 4 kinds of fluorescence: FAM, HEX, ROX and CY 5.

Furthermore, the application also discloses application of the kit in detecting the expression quantity of the C-MYC, C-FOS, K-RAS, RB, P21 and P53 genes.

Different from the prior art, the primers and the probes for detecting the mRNA expression levels of the C-MYC, C-FOS, K-RAS, RB, P21 and P53 genes disclosed by the application can be set as required, so that the quantitative detection of at least one oncogene can be realized, the problem that the oncogene can be detected only one by one can be solved, and the improvement of the detection efficiency and the reduction of the detection cost are facilitated.

Drawings

FIG. 1 is a schematic flow chart showing a method for detecting an oncogene expression level using the kit according to an embodiment of the present invention.

FIG. 2 is a graph showing the results of one embodiment of the present invention for calculating the difference in expression of a target gene between different samples.

FIG. 3 is a graph showing the results of cell changes in one embodiment of the present application during the experiment in which MSC cells are placed at 2-8 ℃.

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

The present invention will be described in further detail with reference to the following examples, but the present invention is not limited thereto. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without any creative effort belong to the protection scope of the present invention; modifications and equivalents may be made to the disclosed embodiments without departing from the spirit and scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers.

A protooncogene refers to a gene present in the genome of a normal cell of an organism. Normally in a low or non-expression state and exert important physiological functions. Under certain conditions, such as viral infection, chemical carcinogen or radiation, proto-oncogenes can be abnormally activated and converted into oncogenes, which can promote cell proliferation, prevent apoptosis and inhibit the escape cycle of tumors, thereby inducing cells to cancerate by uncontrolled growth of cells. Of these, 3 genes, C-MYC, C-FOS and K-RAS, are relatively important. C-MYC is a translocatable gene, an adjustable gene regulated by a plurality of substances, a gene which can lead cells to be infinitely proliferated, obtain an immortalization function and promote cell division, and is related to the occurrence and development of a plurality of tumors. C-FOS: it is involved in the normal differentiation, growth, learning and memory of cells, and is closely related to the expression of FOS protein in cortex, hippocampus, limbic system, dorsal hippocampus and striatum in brain. The K-RAS protein has a molecular switching action and plays an important role in many signal paths. Studies have shown that about 30% of human malignancies are associated with RAS gene mutations, and that the RAS mutated product can be always in an activated state. At present, K-RAS gene mutation status detection has become a routine test item for individualized targeted drug therapy in the United states, Europe and Asia. Inappropriate expression or mutation can directly interfere with cell cycle operation, or induce cell degeneration, or induce apoptosis at different stages of the cell cycle.

Cancer suppressor genes are a class of genes that inhibit cell proliferation and thus potentially inhibit carcinogenesis. Normally, cancer suppressor genes monitor cell state by inhibiting cell cycle, promoting cell senescence, or promoting apoptosis, and prevent abnormal cell proliferation. Of these, 3 genes RB, P21 and P53 are relatively important. RB is an important tumor suppressor gene and is mutated in many different carcinomas. The protein product of this gene is a transcription factor that controls the expression of important genes that drive cells into the process of division. P21, an important member of the cyclin-dependent kinase inhibitor family. Not only is closely related to the tumor inhibition, but also can coordinate the relationship among the cell cycle, DNA replication and repair by inhibiting the activity of cyclin-dependent kinase CDKs complex, thereby closely connecting the tumor inhibition with the cell cycle control process. P53, an important anticancer gene, whose wild type causes apoptosis of cancer cells to prevent canceration, also has the function of helping cell gene repair defects. Mutants of P53 increased the chance of canceration.

The application discloses a primer and a probe for detecting expression levels of multiple oncogenes, wherein the oncogenes comprise at least one of C-MYC, C-FOS, K-RAS, RB, P21 and P53, and correspondingly, the nucleotide sequence of the C-MYC mRNA is shown as SEQ ID NO.22 (the design of the primer is referred to a Homo sapiens (Homo sapiens) sequence in a sequence table), the nucleotide sequence of the C-FOS mRNA is shown as SEQ ID NO.23, the nucleotide sequence of the K-RAS mRNA is shown as SEQ ID NO.24, the nucleotide sequence of the RB mRNA is shown as SEQ ID NO.25, the nucleotide sequence of the P21 mRNA is shown as SEQ ID NO.26, and the nucleotide sequence of the P53 mRNA is shown as SEQ ID NO. 27. Wherein the sequences of the primer and the probe comprise:

in the embodiment, corresponding primers and probes can be set as required, so that quantitative detection of at least one oncogene is realized, the problem that detection of the oncogene can be only performed one by one can be solved, and the improvement of detection efficiency and the reduction of detection cost are facilitated.

In order to solve the above problems, the present application also discloses a kit comprising the above primer and probe, wherein the kit comprises: a cell lysis reagent; a cDNA synthesis reagent; qPCR quantitative detection reagent. The cell lysis reagent comprises lysis solution, an RNase inhibitor and a DNase and is used for lysing target cells to obtain RNA; the cDNA synthesis reagent comprises 2 x reverse transcription reaction liquid and is used for carrying out reverse transcription on the obtained RNA to obtain cDNA. The qPCR quantitative detection reagent comprises 2 xProbe qPCR Mix, a primer Probe mixed solution 1 and a primer Probe mixed solution 2. The kit is convenient to use and high in sensitivity.

Further, the primer probe mixture 1 comprises: primers and probes of an internal reference gene; primers and probes for detecting the C-MYC gene; primers and probes for detecting the C-FOS gene; the primer probe mixed solution 2 comprises: primers and probes for detecting the K-RAS gene; primers and probes for detecting the RB gene; primers and probes for detecting the P21 gene; primers and probes for detecting the P53 gene. Wherein the internal reference gene is ATCB gene and is used for detecting the nucleotide sequence of the internal reference gene ACTB mRNA shown as SEQ ID NO.28, and the primer sequence comprises SEQ ID NO.19 and SEQ ID NO. 20; the probe sequence includes SEQ ID NO 21. Of course, the kit further includes a container or a box for containing or carrying the above components, and the details are not repeated herein.

In order to solve the above problems, the present application also discloses a method for detecting an expression level of an oncogene using the kit, which comprises, in general, lysing a cell of interest (e.g., MES cell) to release RNA, reverse transcribing the RNA in the lysate into cDNA, performing real-time fluorescent quantitative PCR amplification on the C-MYC, C-FOS, K-RAS, RB, P21, and P53 genes and the reference gene in the cDNA using quantitative polymerase chain reaction using C-MYC, C-FOS, K-RAS, RB, P21, and P53 gene-specific primers and primers specific to the reference gene, respectively, and calculating relative expression levels of the C-MYC, C-FOS, K-RAS, RB, P21, and P53 genes based on the detection result of the amplification product. Of course, other RNA extraction kits, cDNA synthesis kits, and qPCR reagents may also be selected according to sample requirements. Specifically, referring to fig. 1, the method includes:

and S100, cracking the target cells by using a cell cracking reagent to obtain RNA.

In step S100, the process of obtaining RNA by lysing cells is mainly performed by purchasing an optimized kit according to actual conditions, and the specific operation method can be referred to the instruction of the related product, and thus is not described herein again.

And step S200, carrying out reverse transcription on the RNA by using a cDNA synthesis reagent to obtain cDNA.

In step S200, cDNA synthesis may be performed in a one-step process or a two-step process, and the following description will be made with reference to specific embodiments.

The one-step method for synthesizing cDNA is mainly aimed at pure cells which are separated, such as in vitro pure cultured cells and white blood cells separated from whole blood. The collected cell suspension was centrifuged at × 500g for 3min, and the liquid fraction was aspirated off using a 200 μ L pipette tip. If the sample cells are in the culture medium, the cells are washed once with 500. mu.L of physiological saline. If not immediately detected, stored at-80 ℃.

Example 1, the synthesis using the one-step method comprises: the method comprises the following steps: cell lysis: using SuperPrep II Cell lysine&RT Kit for qPCR Kit (purchased from Toyo Boseki Biotech Co., Ltd., cat # SCQ-401). Determining the number of cells n, the number of cells in the cell sample is generally optimally 2X 10⁴Not more than 7X 10⁴. A cell lysis mixture was prepared as "29.35 Xnxn.1. 1. mu.L lysate, 0.5 X0 nx1.1. mu.L RNase inhibitor, and 0.15 Xnxn.1.1. mu.L DNase". Adding 30 mu L of cell lysis mixed solution into each cell sample, standing and incubating at room temperature for 5min to obtain cell lysis solution; step two: synthesis of cDNA: a reverse transcription mixture was prepared as "12 XnX 1.1. mu.L of enzyme-free sterile water, 4 XnX 1.1. mu.L of 2X reverse transcription reaction solution". mu.L of cell lysate was added per 16. mu.L of reverse transcription mix. The reverse transcription procedure was performed in a PCR instrument (15 min at 37 ℃, 5min at 50 ℃, 5min at 98 ℃). Reverse transcription to obtain cDNA, and storing at-20 deg.c.

Example 2, a two-step method for synthesizing cDNA, comprising the steps of: the method comprises the following steps: and (3) RNA extraction: an appropriate RNA extraction method, such as RNeasy series kit (manufactured by QIAGEN), is selected depending on the type of specimen, and reverse transcription is immediately performed. The step is operated according to the reagent instruction or an optimized conventional RNA extraction scheme is adopted, so the operation is not specifically explained; step two: synthesis of cDNA: using the First Strand cDNA Synthesis Kit ReverTra Ace-Kit (purchased from Toyo Boseki Biotech Co., Ltd., cat # FSK-101): a reverse transcription system was prepared by completing the system to 20. mu.L with "4. mu.L of 5 Xreverse transcription buffer, 2. mu.L of dNTP (10nM), 1. mu.L of RNase inhibitor, 1. mu.L of Random Primer, 1. mu.L of ReverTra Ace, 0.1 to 1. mu.g of sample RNA, and enzyme-free sterile water" per sample. The reverse transcription procedure was performed in a PCR instrument (30 ℃ 10min, 42 ℃ 20min, 99 ℃ 5 min). Reverse transcription to obtain cDNA, and storing at-20 deg.c.

And step S300, mixing the cDNA, the 2 xProbe qPCR Mix and the primer Probe mixture 1 and/or the primer Probe mixture 2 to obtain a sample to be detected.

In the step S300, a method of primer probe mixture for qPCR includes: primers and probes were designed to quantitatively detect ACTB, C-MYC, C-FOS, K-RAS, RB, P21, and P53 mRNA as described in example 3 below:

the ACTB mRNA quantitative detection primers are shown as SEQ ID NO.19 and SEQ ID NO.20, and the probes are as follows: CY 5-5'-cctgcgtctggacctggctggc-3' -BHQ2(SEQ ID NO. 21);

the primers for quantitative detection of C-MYC mRNA are shown as SEQ ID NO.1 and SEQ ID NO.2, and the probe is: ROX-5'-aacaaccgaaaatgcaccagccccagg-3' -BHQ2(SEQ ID NO. 3);

the C-FOS mRNA quantitative detection primers are shown as SEQ ID NO.4 and SEQ ID NO.5, and the probe is: HEX-5'-ccgagattgccaacctgctg-3' -BHQ1(SEQ ID NO. 6);

the K-RAS mRNA quantitative detection primers are shown as SEQ ID NO.7 and SEQ ID NO.8, and the probes are: FAM-5'-attgagtgcctaccagatgcca-3' -BHQ1(SEQ ID NO. 9);

RB mRNA quantitative detection primers are shown as SEQ ID NO.10 and SEQ ID NO.11, and probes are as follows: HEX-5'-atcagttggtccttctcggtcc-3' -BHQ1(SEQ ID NO. 12);

the P21 mRNA quantitative detection primer is shown as SEQ ID NO.13 and SEQ ID NO.14, and the probe is: CY 5-5'-tcatgctggtctgccgccgtt-3' -BHQ2(SEQ ID NO. 15);

the P53 mRNA quantitative detection primer is shown as SEQ ID NO.16 and SEQ ID NO.17, and the probe is: ROX-5'-ACAGTCAGAGCCAACCTCAGG-3' -BHQ2(SEQ ID NO. 18).

After dissolving the primers and the probes to 50 μ M by using TE Buffer, mixing the upstream and downstream primers of each gene according to a ratio of 1:1 to form 7 primer mixed solutions (which are corresponding PrimerMix in the following table), and preparing a primer-probe mixed solution 1 and a primer-probe mixed solution 2 according to the formulas in the following tables 1 and 2.

TABLE 1 primer Probe Mixed solution 1 formulation

TABLE 2 primer Probe Mixed solution 2 formulation

Composition (I)	Final concentration/nM
		K-RAS Primer Mix	200
RB Primer Mix	100
		P21 Primer Mix	250
P53 Primer Mix	100
		K-RAS Probe(SEQ ID NO.9)	100
RB Probe(SEQ ID NO.12)	100
		P21 Probe(SEQ ID NO.15)	150
P53 Probe(SEQ ID NO.18)	175

After obtaining the primer Probe mixture 1 and the primer Probe mixture 2, mixing the obtained primer Probe mixture with the corresponding cDNA and 2 × Probe qPCR Mix, specifically, the sample to be tested (i.e. qPCR system) includes: upstream and downstream primers, probes, cDNA and 2 XProbe qPCR Mix of corresponding genes.

And S400, placing the sample to be detected into a quantitative fluorescence PCR detector for detection according to a preset program, and obtaining the CT value of each gene.

In step S400, the quantitative detection may be a single-fold fluorescence detection or a multiple-fold fluorescence detection. In one embodiment, a single fluorescence detection and a multiple fluorescence detection are respectively configured for detecting a sample to be detected, specifically referring to example 4, the steps include:

the method comprises the following steps: preparing qPCR mixed solution of a single fluorescence method: a Probe qPCR Mix kit (available from Toyo Boseki Biotech Co., Ltd., product No. QPS-101) was used. qPCR system for each gene (30 μ L) was prepared: upstream and downstream primers and probes (final concentrations same as in example 3), 2 × Probe qPCR Mix, 5% cDNA (v/v).

Step two: preparing qPCR mixed solution of a multiple fluorescence method: a Probe qPCR Mix kit (available from Toyo Boseki Biotech Co., Ltd., product No. QPS-101) was used. Only 2 qPCR systems (30 μ L) were prepared for each cDNA specimen, as follows: primer Probe Mix 1 or 2, 2 XProbe qPCR Mix, 5% cDNA (v/v) of example 3.

Step three: fluorescence quantification: placing the solution prepared in the first step or the second step into a fluorescence quantitative PCR (instrument needs to have the detection capability of 4 types of fluorescence, namely FAM, HEX, ROX and CY 5), and performing a fluorescence quantitative program (95 ℃ for 1min, 95 ℃ for 15s × 40 cycles, and 45 ℃ for 40s × 40 cycles). Thus, CT values of the respective genes were obtained. Among them, the level of the threshold line affects the CT value of the underexpressed gene, and the setting thereof is different depending on the quantitative fluorescence PCR used, but it is not recommended to exceed 1.7% of the maximum fluorescence value.

TABLE 3 measurement of data on expression of 6 oncogenes in MSC cells by single fluorescence and multiplex fluorescence

As can be seen from the above Table 3, the single fluorescence and the multiple fluorescence can achieve good experimental effect, and the experimental results have small difference and can be flexibly applied according to the requirements. The method and the kit for detecting the expression quantity of the oncogene, which are provided by the application, realize the measurement of the expression quantity of the oncogene and can effectively obtain the real condition of the expression quantity of the oncogene;

in the above table 3, the expression level of the target gene (one of C-MYC, C-FOS, K-RAS, RB, P21 and P53) of a single specimen relative to the reference gene ACTB can be calculated by the method of delta CT: 2^-ΔCT＝2^{- (CT (Gene of interest) -CT (ACTB))}。2^-ΔCTThat is, the expression level of the target gene relative to the reference gene. By using 2^-ΔΔCTThe method is rigorous enough to truly reflect the relative gene expression level. The expression level data can be compared with each other between different batches of experiments. Is suitable for being popularized in the pathology department of a hospital, and is promoted to become a conventional detection item which can be developed in the hospital.

The following tests were performed on the performance of the kits of the present application, in particular;

example 5: the method for detecting the expression difference of 6 oncogenes among MSC cells, Hela cells and HEK 293 and analyzing the difference of the expression levels of the 6 oncogenes among different sample cells comprises the following steps:

the method comprises the following steps: obtaining CT values: cDNA solutions of MSC cells, Hela cells and HEK 293 cells were obtained according to the method of example 1, and CT values of each gene were obtained according to the multiplex fluorescence fractions of example 4 as a sample for analysis; step two: and (3) data analysis: through 2^-ΔΔCTThe expression difference of the target gene among different samples is calculated in the following way: 2^-ΔΔCT＝2^{- (Delta CT (specimen of interest) -Delta CT (control specimen))}。

The results of the detection of 3 types of cells in a certain batch are shown in FIG. 2, and MSC cells are used as reference cells in FIG. 2 (the expression level of all genes in the reference cells is set to 1). As can be seen from fig. 2: the P21 gene of Hela cell is much lower than that of MSC cell, while the K-RAS, RB, P21 and P53 gene of HEK 293 cell is higher than that of MSC cell and Hela cell. This result demonstrates that the method can be used for cell-like comparison, for example: and comparing the expression difference of the cancer genes of the cancer tissues and the tissues beside the cancer.

Example 6: the method for detecting the expression difference between MSC cells with 6 oncogenes placed at 2-8 ℃ for different time and analyzing the difference between the expression levels of the 6 oncogenes in different processed sample cells comprises the following steps:

the method comprises the following steps: freshly harvested MSC cells at 2X 10⁴Storing each part at 4 deg.C, taking out at 0, 1, 3, 6, 10 days, centrifuging at x 500g for 3min, sucking off liquid part with 200 μ L suction head, and storing at-80 deg.C. The same batch was run when all samples were taken: step two: a cDNA solution of cells was obtained according to the method of example 1, and CT values of each gene were obtained according to the multiplex fluorescence part of example 4 as a sample for analysis; step three: and (3) data analysis: the calculation was performed in the same manner as in example 5.

FIG. 3 shows the detection results of experiments performed by placing a certain batch of MSC cells at 2-8 ℃, and the results prove that the oncogenes of the cells are expressed and reduced in different degrees after being placed for more than 1 day, and the cell quality tends to be reduced. Meanwhile, the experimental method is proved to be useful in representing the difference between different sample cells to be treated.

In addition, the application also discloses application of the kit in detecting expression levels of C-MYC, C-FOS, K-RAS, RB, P21 and P53 genes. The kit is used for detecting the oncogenes, at least one of the 6 oncogenes can be selected and detected according to requirements, the detection efficiency is high, the detection process is flexible, and different use requirements can be better met.

In conclusion, the method and the kit for detecting the expression level of the oncogene, provided by the invention, can realize the measurement of the expression level of the oncogene, can effectively obtain the real situation of the expression level of the gene, can carry out multiple detection compared with the common qPCR, improves the flux exponentially, has high accuracy in batch (CV is less than 1.5 percent), and is suitable for the detection of a large amount of samples. In the field of cell production/treatment, such as example 5, it is necessary to evaluate whether the expression of each oncogene is stable between different generations of cells, and therefore, it is necessary to ensure that a problem is quickly and effectively found before a cancer tendency (abnormal expression of each oncogene in a cell) occurs in a cell. In the scientific research field, such as example 6, the method and kit for detecting the expression level of the oncogene provided by the present invention often evaluate the difference between samples processed by different experiments, and the difference often involves the change of cell traits, such as canceration tendency or premature senility. The method can adopt single-kind cell individual, batch-to-batch comparison or comparison between different kinds of cells according to actual requirements. By using 2^-ΔΔCTThe method is rigorous enough to truly reflect the relative gene expression level. Currently, IVD (in vitro diagnosis) product mode becomes the choice of more tumor gene detection companies. At present, most of the kit products aim at a certain specific gene, belong to the field of small Pannel gene detection, are suitable for popularization in the pathology department of hospitals, and are promoted to become a conventional detection project which can be developed in hospitals. Oncogene expression of current cell preparationsThere is no established standard for testing in each hospital and each medical unit.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

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gaagagccgg gcgagcagag ctgcgctgcg ggcgtcctgg gaagggagat ccggagcgaa 120

tagggggctt cgcctctggc ccagccctcc cgctgatccc ccagccagcg gtccgcaacc 180

cttgccgcat ccacgaaact ttgcccatag cagcgggcgg gcactttgca ctggaactta 240

caacacccga gcaaggacgc gactctcccg acgcggggag gctattctgc ccatttgggg 300

acacttcccc gccgctgcca ggacccgctt ctctgaaagg ctctccttgc agctgcttag 360

acgctggatt tttttcgggt agtggaaaac cagcagcctc ccgcgacgat gcccctcaac 420

gttagcttca ccaacaggaa ctatgacctc gactacgact cggtgcagcc gtatttctac 480

tgcgacgagg aggagaactt ctaccagcag cagcagcaga gcgagctgca gcccccggcg 540

cccagcgagg atatctggaa gaaattcgag ctgctgccca ccccgcccct gtcccctagc 600

cgccgctccg ggctctgctc gccctcctac gttgcggtca cacccttctc ccttcgggga 660

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atcaaaaaca tcatcatcca ggactgtatg tggagcggct tctcggccgc cgccaagctc 840

gtctcagaga agctggcctc ctaccaggct gcgcgcaaag acagcggcag cccgaacccc 900

gcccgcggcc acagcgtctg ctccacctcc agcttgtacc tgcaggatct gagcgccgcc 960

gcctcagagt gcatcgaccc ctcggtggtc ttcccctacc ctctcaacga cagcagctcg 1020

cccaagtcct gcgcctcgca agactccagc gccttctctc cgtcctcgga ttctctgctc 1080

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ccgcccacca ccagcagcga ctctgaggag gaacaagaag atgaggaaga aatcgatgtt 1200

gtttctgtgg aaaagaggca ggctcctggc aaaaggtcag agtctggatc accttctgct 1260

ggaggccaca gcaaacctcc tcacagccca ctggtcctca agaggtgcca cgtctccaca 1320

catcagcaca actacgcagc gcctccctcc actcggaagg actatcctgc tgccaagagg 1380

gtcaagttgg acagtgtcag agtcctgaga cagatcagca acaaccgaaa atgcaccagc 1440

cccaggtcct cggacaccga ggagaatgtc aagaggcgaa cacacaacgt cttggagcgc 1500

cagaggagga acgagctaaa acggagcttt tttgccctgc gtgaccagat cccggagttg 1560

gaaaacaatg aaaaggcccc caaggtagtt atccttaaaa aagccacagc atacatcctg 1620

tccgtccaag cagaggagca aaagctcatt tctgaagagg acttgttgcg gaaacgacga 1680

gaacagttga aacacaaact tgaacagcta cggaactctt gtgcgtaagg aaaagtaagg 1740

aaaacgattc cttctaacag aaatgtcctg agcaatcacc tatgaacttg tttcaaatgc 1800

atgatcaaat gcaacctcac aaccttggct gagtcttgag actgaaagat ttagccataa 1860

tgtaaactgc ctcaaattgg actttgggca taaaagaact tttttatgct taccatcttt 1920

tttttttctt taacagattt gtatttaaga attgttttta aaaaatttta agatttacac 1980

aatgtttctc tgtaaatatt gccattaaat gtaaataact ttaataaaac gtttatagca 2040

gttacacaga atttcaatcc tagtatatag tacctagtat tataggtact ataaacccta 2100

atttttttta tttaagtaca ttttgctttt taaagttgat ttttttctat tgtttttaga 2160

aaaaataaaa taactggcaa atatatcatt gagccaaatc ttaagttgtg aatgttttgt 2220

ttcgtttctt ccccctccca accaccacca tccctgtttg ttttcatcaa ttgccccttc 2280

agagggtggt cttaagaaag gcaagagttt tcctctgttg aaatgggtct gggggcctta 2340

aggtctttaa gttcttggag gttctaagat gcttcctgga gactatgata acagccagag 2400

ttgacagtta gaaggaatgg cagaaggcag gtgagaaggt gagaggtagg caaaggagat 2460

acaagaggtc aaaggtagca gttaagtaca caaagaggca taaggactgg ggagttggga 2520

ggaaggtgag gaagaaactc ctgttacttt agttaaccag tgccagtccc ctgctcactc 2580

caaacccagg aattctgccc agttgatggg gacacggtgg gaaccagctt ctgctgcctt 2640

cacaaccagg cgccagtcct gtccatgggt tatctcgcaa accccagagg atctctggga 2700

ggaatgctac tattaaccct atttcacaaa caaggaaata gaagagctca aagaggttat 2760

gtaacttatc tgtagccacg cagataatac aaagcagcaa tctggaccca ttctgttcaa 2820

aacacttaac ccttcgctat catgccttgg ttcatctggg tctaatgtgc tgagatcaag 2880

aaggtttagg acctaatgga cagactcaag tcataacaat gctaagctct atttgtgtcc 2940

caagcactcc taagcatttt atccctaact ctacatcaac cccatgaagg agatactgtt 3000

gatttcccca tattagaagt agagagggaa gctgaggcac acaaagactc atccacatgc 3060

ccaagattca ctgataggga aaagtggaag cgagatttga acccaggctg tttactccta 3120

acctgtccaa gccacctctc agacgacggt aggaatcagc tggctgcttg tgagtacagg 3180

agttacagtc cagtgggtta tgttttttaa gtctcaacat ctaagcctgg tcaggcatca 3240

gttccccttt ttttgtgatt tattttgttt ttattttgtt gttcattgtt taatttttcc 3300

ttttacaatg agaaggtcac catcttgact cctaccttag ccatttgttg aatcagactc 3360

atgacggctc ctgggaagaa gccagttcag atcataaaat aaaacatatt tattctttgt 3420

catgggagtc attattttag aaactacaaa ctctccttgc ttccatcctt ttttacatac 3480

tcatgacaca tgctcatcct gagtccttga aaaggtattt ttgaacatgt gtattaatta 3540

taagcctctg aaaacctatg gcccaaacca gaaatgatgt tgattatata ggtaaatgaa 3600

ggatgctatt gctgttctaa ttacctcatt gtctcagtct caaagtaggt cttcagctcc 3660

ctgtactttg ggattttaat ctaccaccac ccataaatca ataaataatt actttctttg 3720

a 3721

<210> 23

<211> 2104

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 23

aaccgcatct gcagcgagca tctgagaagc caagactgag ccggcggccg cggcgcagcg 60

aacgagcagt gaccgtgctc ctacccagct ctgctccaca gcgcccacct gtctccgccc 120

ctcggcccct cgcccggctt tgcctaaccg ccacgatgat gttctcgggc ttcaacgcag 180

actacgaggc gtcatcctcc cgctgcagca gcgcgtcccc ggccggggat agcctctctt 240

actaccactc acccgcagac tccttctcca gcatgggctc gcctgtcaac gcgcaggact 300

tctgcacgga cctggccgtc tccagtgcca acttcattcc cacggtcact gccatctcga 360

ccagtccgga cctgcagtgg ctggtgcagc ccgccctcgt ctcctccgtg gccccatcgc 420

agaccagagc ccctcaccct ttcggagtcc ccgccccctc cgctggggct tactccaggg 480

ctggcgttgt gaagaccatg acaggaggcc gagcgcagag cattggcagg aggggcaagg 540

tggaacagtt atctccagaa gaagaagaga aaaggagaat ccgaagggaa aggaataaga 600

tggctgcagc caaatgccgc aaccggagga gggagctgac tgatacactc caagcggaga 660

cagaccaact agaagatgag aagtctgctt tgcagaccga gattgccaac ctgctgaagg 720

agaaggaaaa actagagttc atcctggcag ctcaccgacc tgcctgcaag atccctgatg 780

acctgggctt cccagaagag atgtctgtgg cttcccttga tctgactggg ggcctgccag 840

aggttgccac cccggagtct gaggaggcct tcaccctgcc tctcctcaat gaccctgagc 900

ccaagccctc agtggaacct gtcaagagca tcagcagcat ggagctgaag accgagccct 960

ttgatgactt cctgttccca gcatcatcca ggcccagtgg ctctgagaca gcccgctccg 1020

tgccagacat ggacctatct gggtccttct atgcagcaga ctgggagcct ctgcacagtg 1080

gctccctggg gatggggccc atggccacag agctggagcc cctgtgcact ccggtggtca 1140

cctgtactcc cagctgcact gcttacacgt cttccttcgt cttcacctac cccgaggctg 1200

actccttccc cagctgtgca gctgcccacc gcaagggcag cagcagcaat gagccttcct 1260

ctgactcgct cagctcaccc acgctgctgg ccctgtgagg gggcagggaa ggggaggcag 1320

ccggcaccca caagtgccac tgcccgagct ggtgcattac agagaggaga aacacatctt 1380

ccctagaggg ttcctgtaga cctagggagg accttatctg tgcgtgaaac acaccaggct 1440

gtgggcctca aggacttgaa agcatccatg tgtggactca agtccttacc tcttccggag 1500

atgtagcaaa acgcatggag tgtgtattgt tcccagtgac acttcagaga gctggtagtt 1560

agtagcatgt tgagccaggc ctgggtctgt gtctcttttc tctttctcct tagtcttctc 1620

atagcattaa ctaatctatt gggttcatta ttggaattaa cctggtgctg gatattttca 1680

aattgtatct agtgcagctg attttaacaa taactactgt gttcctggca atagtgtgtt 1740

ctgattagaa atgaccaata ttatactaag aaaagatacg actttatttt ctggtagata 1800

gaaataaata gctatatcca tgtactgtag tttttcttca acatcaatgt tcattgtaat 1860

gttactgatc atgcattgtt gaggtggtct gaatgttctg acattaacag ttttccatga 1920

aaacgtttta ttgtgttttt aatttattta ttaagatgga ttctcagata tttatatttt 1980

tattttattt ttttctacct tgaggtcttt tgacatgtgg aaagtgaatt tgaatgaaaa 2040

atttaagcat tgtttgctta ttgttccaag acattgtcaa taaaagcatt taagttgaat 2100

gcga 2104

<210> 24

<211> 5430

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 24

ctaggcggcg gccgcggcgg cggaggcagc agcggcggcg gcagtggcgg cggcgaaggt 60

ggcggcggct cggccagtac tcccggcccc cgccatttcg gactgggagc gagcgcggcg 120

caggcactga aggcggcggc ggggccagag gctcagcggc tcccaggtgc gggagagagg 180

cctgctgaaa atgactgaat ataaacttgt ggtagttgga gctggtggcg taggcaagag 240

tgccttgacg atacagctaa ttcagaatca ttttgtggac gaatatgatc caacaataga 300

ggattcctac aggaagcaag tagtaattga tggagaaacc tgtctcttgg atattctcga 360

cacagcaggt caagaggagt acagtgcaat gagggaccag tacatgagga ctggggaggg 420

ctttctttgt gtatttgcca taaataatac taaatcattt gaagatattc accattatag 480

agaacaaatt aaaagagtta aggactctga agatgtacct atggtcctag taggaaataa 540

atgtgatttg ccttctagaa cagtagacac aaaacaggct caggacttag caagaagtta 600

tggaattcct tttattgaaa catcagcaaa gacaagacag agagtggagg atgcttttta 660

tacattggtg agagagatcc gacaatacag attgaaaaaa atcagcaaag aagaaaagac 720

tcctggctgt gtgaaaatta aaaaatgcat tataatgtaa tctgggtgtt gatgatgcct 780

tctatacatt agttcgagaa attcgaaaac ataaagaaaa gatgagcaaa gatggtaaaa 840

agaagaaaaa gaagtcaaag acaaagtgtg taattatgta aatacaattt gtactttttt 900

cttaaggcat actagtacaa gtggtaattt ttgtacatta cactaaatta ttagcatttg 960

ttttagcatt acctaatttt tttcctgctc catgcagact gttagctttt accttaaatg 1020

cttattttaa aatgacagtg gaagtttttt tttcctctaa gtgccagtat tcccagagtt 1080

ttggtttttg aactagcaat gcctgtgaaa aagaaactga atacctaaga tttctgtctt 1140

ggggcttttg gtgcatgcag ttgattactt cttatttttc ttaccaattg tgaatgttgg 1200

tgtgaaacaa attaatgaag cttttgaatc atccctattc tgtgttttat ctagtcacat 1260

aaatggatta attactaatt tcagttgaga ccttctaatt ggtttttact gaaacattga 1320

gggaacacaa atttatgggc ttcctgatga tgattcttct aggcatcatg tcctatagtt 1380

tgtcatccct gatgaatgta aagttacact gttcacaaag gttttgtctc ctttccactg 1440

ctattagtca tggtcactct ccccaaaata ttatattttt tctataaaaa gaaaaaaatg 1500

gaaaaaaatt acaaggcaat ggaaactatt ataaggccat ttccttttca cattagataa 1560

attactataa agactcctaa tagcttttcc tgttaaggca gacccagtat gaaatgggga 1620

ttattatagc aaccattttg gggctatatt tacatgctac taaattttta taataattga 1680

aaagatttta acaagtataa aaaattctca taggaattaa atgtagtctc cctgtgtcag 1740

actgctcttt catagtataa ctttaaatct tttcttcaac ttgagtcttt gaagatagtt 1800

ttaattctgc ttgtgacatt aaaagattat ttgggccagt tatagcttat taggtgttga 1860

agagaccaag gttgcaaggc caggccctgt gtgaaccttt gagctttcat agagagtttc 1920

acagcatgga ctgtgtcccc acggtcatcc agtgttgtca tgcattggtt agtcaaaatg 1980

gggagggact agggcagttt ggatagctca acaagataca atctcactct gtggtggtcc 2040

tgctgacaaa tcaagagcat tgcttttgtt tcttaagaaa acaaactctt ttttaaaaat 2100

tacttttaaa tattaactca aaagttgaga ttttggggtg gtggtgtgcc aagacattaa 2160

tttttttttt aaacaatgaa gtgaaaaagt tttacaatct ctaggtttgg ctagttctct 2220

taacactggt taaattaaca ttgcataaac acttttcaag tctgatccat atttaataat 2280

gctttaaaat aaaaataaaa acaatccttt tgataaattt aaaatgttac ttattttaaa 2340

ataaatgaag tgagatggca tggtgaggtg aaagtatcac tggactagga agaaggtgac 2400

ttaggttcta gataggtgtc ttttaggact ctgattttga ggacatcact tactatccat 2460

ttcttcatgt taaaagaagt catctcaaac tcttagtttt ttttttttac aactatgtaa 2520

tttatattcc atttacataa ggatacactt atttgtcaag ctcagcacaa tctgtaaatt 2580

tttaacctat gttacaccat cttcagtgcc agtcttgggc aaaattgtgc aagaggtgaa 2640

gtttatattt gaatatccat tctcgtttta ggactcttct tccatattag tgtcatcttg 2700

cctccctacc ttccacatgc cccatgactt gatgcagttt taatacttgt aattccccta 2760

accataagat ttactgctgc tgtggatatc tccatgaagt tttcccactg agtcacatca 2820

gaaatgccct acatcttatt tcctcagggc tcaagagaat ctgacagata ccataaaggg 2880

atttgaccta atcactaatt ttcaggtggt ggctgatgct ttgaacatct ctttgctgcc 2940

caatccatta gcgacagtag gatttttcaa acctggtatg aatagacaga accctatcca 3000

gtggaaggag aatttaataa agatagtgct gaaagaattc cttaggtaat ctataactag 3060

gactactcct ggtaacagta atacattcca ttgttttagt aaccagaaat cttcatgcaa 3120

tgaaaaatac tttaattcat gaagcttact tttttttttt ggtgtcagag tctcgctctt 3180

gtcacccagg ctggaatgca gtggcgccat ctcagctcac tgcaacctcc atctcccagg 3240

ttcaagcgat tctcgtgcct cggcctcctg agtagctggg attacaggcg tgtgccacta 3300

cactcaacta atttttgtat ttttaggaga gacggggttt caccctgttg gccaggctgg 3360

tctcgaactc ctgacctcaa gtgattcacc caccttggcc tcataaacct gttttgcaga 3420

actcatttat tcagcaaata tttattgagt gcctaccaga tgccagtcac cacacaaggc 3480

actgggtata tggtatcccc aaacaagaga cataatcccg gtccttaggt agtgctagtg 3540

tggtctgtaa tatcttacta aggcctttgg tatacgaccc agagataaca cgatgcgtat 3600

tttagttttg caaagaaggg gtttggtctc tgtgccagct ctataattgt tttgctacga 3660

ttccactgaa actcttcgat caagctactt tatgtaaatc acttcattgt tttaaaggaa 3720

taaacttgat tatattgttt ttttatttgg cataactgtg attcttttag gacaattact 3780

gtacacatta aggtgtatgt cagatattca tattgaccca aatgtgtaat attccagttt 3840

tctctgcata agtaattaaa atatacttaa aaattaatag ttttatctgg gtacaaataa 3900

acaggtgcct gaactagttc acagacaagg aaacttctat gtaaaaatca ctatgatttc 3960

tgaattgcta tgtgaaacta cagatctttg gaacactgtt taggtagggt gttaagactt 4020

acacagtacc tcgtttctac acagagaaag aaatggccat acttcaggaa ctgcagtgct 4080

tatgagggga tatttaggcc tcttgaattt ttgatgtaga tgggcatttt tttaaggtag 4140

tggttaatta cctttatgtg aactttgaat ggtttaacaa aagatttgtt tttgtagaga 4200

ttttaaaggg ggagaattct agaaataaat gttacctaat tattacagcc ttaaagacaa 4260

aaatccttgt tgaagttttt ttaaaaaaag ctaaattaca tagacttagg cattaacatg 4320

tttgtggaag aatatagcag acgtatattg tatcatttga gtgaatgttc ccaagtaggc 4380

attctaggct ctatttaact gagtcacact gcataggaat ttagaaccta acttttatag 4440

gttatcaaaa ctgttgtcac cattgcacaa ttttgtccta atatatacat agaaactttg 4500

tggggcatgt taagttacag tttgcacaag ttcatctcat ttgtattcca ttgatttttt 4560

ttttcttcta aacatttttt cttcaaacag tatataactt tttttagggg attttttttt 4620

agacagcaaa aactatctga agatttccat ttgtcaaaaa gtaatgattt cttgataatt 4680

gtgtagtaat gttttttaga acccagcagt taccttaaag ctgaatttat atttagtaac 4740

ttctgtgtta atactggata gcatgaattc tgcattgaga aactgaatag ctgtcataaa 4800

atgaaacttt ctttctaaag aaagatactc acatgagttc ttgaagaata gtcataacta 4860

gattaagatc tgtgttttag tttaatagtt tgaagtgcct gtttgggata atgataggta 4920

atttagatga atttagggga aaaaaaagtt atctgcagat atgttgaggg cccatctctc 4980

cccccacacc cccacagagc taactgggtt acagtgtttt atccgaaagt ttccaattcc 5040

actgtcttgt gttttcatgt tgaaaatact tttgcatttt tcctttgagt gccaatttct 5100

tactagtact atttcttaat gtaacatgtt tacctggaat gtattttaac tatttttgta 5160

tagtgtaaac tgaaacatgc acattttgta cattgtgctt tcttttgtgg gacatatgca 5220

gtgtgatcca gttgttttcc atcatttggt tgcgctgacc taggaatgtt ggtcatatca 5280

aacattaaaa atgaccactc ttttaattga aattaacttt taaatgttta taggagtatg 5340

tgctgtgaag tgatctaaaa tttgtaatat ttttgtcatg aactgtacta ctcctaatta 5400

ttgtaatgta ataaaaatag ttacagtgac 5430

<210> 25

<211> 4772

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 25

gctcagttgc cgggcggggg agggcgcgtc cggtttttct caggggacgt tgaaattatt 60

tttgtaacgg gagtcgggag aggacggggc gtgccccgac gtgcgcgcgc gtcgtcctcc 120

ccggcgctcc tccacagctc gctggctccc gccgcggaaa ggcgtcatgc cgcccaaaac 180

cccccgaaaa acggccgcca ccgccgccgc tgccgccgcg gaacccccgg caccgccgcc 240

gccgccccct cctgaggagg acccagagca ggacagcggc ccggaggacc tgcctctcgt 300

caggcttgag tttgaagaaa cagaagaacc tgattttact gcattatgtc agaaattaaa 360

gataccagat catgtcagag agagagcttg gttaacttgg gagaaagttt catctgtgga 420

tggagtattg ggaggttata ttcaaaagaa aaaggaactg tggggaatct gtatctttat 480

tgcagcagtt gacctagatg agatgtcgtt cacttttact gagctacaga aaaacataga 540

aatcagtgtc cataaattct ttaacttact aaaagaaatt gataccagta ccaaagttga 600

taatgctatg tcaagactgt tgaagaagta tgatgtattg tttgcactct tcagcaaatt 660

ggaaaggaca tgtgaactta tatatttgac acaacccagc agttcgatat ctactgaaat 720

aaattctgca ttggtgctaa aagtttcttg gatcacattt ttattagcta aaggggaagt 780

attacaaatg gaagatgatc tggtgatttc atttcagtta atgctatgtg tccttgacta 840

ttttattaaa ctctcacctc ccatgttgct caaagaacca tataaaacag ctgttatacc 900

cattaatggt tcacctcgaa cacccaggcg aggtcagaac aggagtgcac ggatagcaaa 960

acaactagaa aatgatacaa gaattattga agttctctgt aaagaacatg aatgtaatat 1020

agatgaggtg aaaaatgttt atttcaaaaa ttttatacct tttatgaatt ctcttggact 1080

tgtaacatct aatggacttc cagaggttga aaatctttct aaacgatacg aagaaattta 1140

tcttaaaaat aaagatctag atgcaagatt atttttggat catgataaaa ctcttcagac 1200

tgattctata gacagttttg aaacacagag aacaccacga aaaagtaacc ttgatgaaga 1260

ggtgaatgta attcctccac acactccagt taggactgtt atgaacacta tccaacaatt 1320

aatgatgatt ttaaattcag caagtgatca accttcagaa aatctgattt cctattttaa 1380

caactgcaca gtgaatccaa aagaaagtat actgaaaaga gtgaaggata taggatacat 1440

ctttaaagag aaatttgcta aagctgtggg acagggttgt gtcgaaattg gatcacagcg 1500

atacaaactt ggagttcgct tgtattaccg agtaatggaa tccatgctta aatcagaaga 1560

agaacgatta tccattcaaa attttagcaa acttctgaat gacaacattt ttcatatgtc 1620

tttattggcg tgcgctcttg aggttgtaat ggccacatat agcagaagta catctcagaa 1680

tcttgattct ggaacagatt tgtctttccc atggattctg aatgtgctta atttaaaagc 1740

ctttgatttt tacaaagtga tcgaaagttt tatcaaagca gaaggcaact tgacaagaga 1800

aatgataaaa catttagaac gatgtgaaca tcgaatcatg gaatcccttg catggctctc 1860

agattcacct ttatttgatc ttattaaaca atcaaaggac cgagaaggac caactgatca 1920

ccttgaatct gcttgtcctc ttaatcttcc tctccagaat aatcacactg cagcagatat 1980

gtatctttct cctgtaagat ctccaaagaa aaaaggttca actacgcgtg taaattctac 2040

tgcaaatgca gagacacaag caacctcagc cttccagacc cagaagccat tgaaatctac 2100

ctctctttca ctgttttata aaaaagtgta tcggctagcc tatctccggc taaatacact 2160

ttgtgaacgc cttctgtctg agcacccaga attagaacat atcatctgga cccttttcca 2220

gcacaccctg cagaatgagt atgaactcat gagagacagg catttggacc aaattatgat 2280

gtgttccatg tatggcatat gcaaagtgaa gaatatagac cttaaattca aaatcattgt 2340

aacagcatac aaggatcttc ctcatgctgt tcaggagaca ttcaaacgtg ttttgatcaa 2400

agaagaggag tatgattcta ttatagtatt ctataactcg gtcttcatgc agagactgaa 2460

aacaaatatt ttgcagtatg cttccaccag gccccctacc ttgtcaccaa tacctcacat 2520

tcctcgaagc ccttacaagt ttcctagttc acccttacgg attcctggag ggaacatcta 2580

tatttcaccc ctgaagagtc catataaaat ttcagaaggt ctgccaacac caacaaaaat 2640

gactccaaga tcaagaatct tagtatcaat tggtgaatca ttcgggactt ctgagaagtt 2700

ccagaaaata aatcagatgg tatgtaacag cgaccgtgtg ctcaaaagaa gtgctgaagg 2760

aagcaaccct cctaaaccac tgaaaaaact acgctttgat attgaaggat cagatgaagc 2820

agatggaagt aaacatctcc caggagagtc caaatttcag cagaaactgg cagaaatgac 2880

ttctactcga acacgaatgc aaaagcagaa aatgaatgat agcatggata cctcaaacaa 2940

ggaagagaaa tgaggatctc aggaccttgg tggacactgt gtacacctct ggattcattg 3000

tctctcacag atgtgactgt ataactttcc caggttctgt ttatggccac atttaatatc 3060

ttcagctctt tttgtggata taaaatgtgc agatgcaatt gtttgggtga ttcctaagcc 3120

acttgaaatg ttagtcattg ttatttatac aagattgaaa atcttgtgta aatcctgcca 3180

tttaaaaagt tgtagcagat tgtttcctct tccaaagtaa aattgctgtg ctttatggat 3240

agtaagaatg gccctagagt gggagtcctg ataacccagg cctgtctgac tactttgcct 3300

tcttttgtag catataggtg atgtttgctc ttgtttttat taatttatat gtatattttt 3360

ttaatttaac atgaacaccc ttagaaaatg tgtcctatct atcttccaaa tgcaatttga 3420

ttgactgccc attcaccaaa attatcctga actcttctgc aaaaatggat attattagaa 3480

attagaaaaa aattactaat tttacacatt agattttatt ttactattgg aatctgatat 3540

actgtgtgct tgttttataa aattttgctt ttaattaaat aaaagctgga agcaaagtat 3600

aaccatatga tactatcata ctactgaaac agatttcata cctcagaatg taaaagaact 3660

tactgattat tttcttcatc caacttatgt ttttaaatga ggattattga tagtactctt 3720

ggtttttata ccattcagat cactgaattt ataaagtacc catctagtac ttgaaaaagt 3780

aaagtgttct gccagatctt aggtatagag gaccctaaca cagtatatcc caagtgcact 3840

ttctaatgtt tctgggtcct gaagaattaa gatacaaatt aattttactc cataaacaga 3900

ctgttaatta taggagcctt aatttttttt tcatagagat ttgtctaatt gcatctcaaa 3960

attattctgc cctccttaat ttgggaaggt ttgtgttttc tctggaatgg tacatgtctt 4020

ccatgtatct tttgaactgg caattgtcta tttatctttt atttttttaa gtcagtatgg 4080

tctaacactg gcatgttcaa agccacatta tttctagtcc aaaattacaa gtaatcaagg 4140

gtcattatgg gttaggcatt aatgtttcta tctgattttg tgcaaaagct tcaaattaaa 4200

acagctgcat tagaaaaaga ggcgcttctc ccctccccta cacctaaagg tgtatttaaa 4260

ctatcttgtg tgattaactt atttagagat gctgtaactt aaaatagggg atatttaagg 4320

tagcttcagc tagcttttag gaaaatcact ttgtctaact cagaattatt tttaaaaaga 4380

aatctggtct tgttagaaaa caaaatttta ttttgtgctc atttaagttt caaacttact 4440

attttgacag ttattttgat aacaatgaca ctagaaaact tgactccatt tcatcattgt 4500

ttctgcatga atatcataca aatcagttag tttttaggtc aagggcttac tatttctggg 4560

tcttttgcta ctaagttcac attagaatta gtgccagaat tttaggaact tcagagatcg 4620

tgtattgaga tttcttaaat aatgcttcag atattattgc tttattgctt ttttgtattg 4680

gttaaaactg tacatttaaa attgctatgt tactattttc tacaattaat agtttgtcta 4740

ttttaaaata aattagttgt taagagtctt aa 4772

<210> 26

<211> 2378

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 26

atgttgagct ctggcataga agaggctggt ggctattttg tccttgggct gcctgttttc 60

aggtgaggaa ggggatggta ggagacagga gacctctaaa gaccccagaa ataaaggatg 120

acaagcagag agccccgggc aggaggcaaa agtcctgtgt tccaactata gtcatttctt 180

tgctgcatga tctgagttag gtcaccagac ttctctgagc cccagtttcc ccagcagtgt 240

atacgggcta tgtggggagt attcaggaga cagacaactc actcgtcaaa tcctcccctt 300

cctggccaac aaagctgctg caaccacagg gatttcttct gttcaggcgc catgtcagaa 360

ccggctgggg atgtccgtca gaacccatgc ggcagcaagg cctgccgccg cctcttcggc 420

ccagtggaca gcgagcagct gagccgcgac tgtgatgcgc taatggcggg ctgcatccag 480

gaggcccgtg agcgatggaa cttcgacttt gtcaccgaga caccactgga gggtgacttc 540

gcctgggagc gtgtgcgggg ccttggcctg cccaagctct accttcccac ggggccccgg 600

cgaggccggg atgagttggg aggaggcagg cggcctggca cctcacctgc tctgctgcag 660

gggacagcag aggaagacca tgtggacctg tcactgtctt gtacccttgt gcctcgctca 720

ggggagcagg ctgaagggtc cccaggtgga cctggagact ctcagggtcg aaaacggcgg 780

cagaccagca tgacagattt ctaccactcc aaacgccggc tgatcttctc caagaggaag 840

ccctaatccg cccacaggaa gcctgcagtc ctggaagcgc gagggcctca aaggcccgct 900

ctacatcttc tgccttagtc tcagtttgtg tgtcttaatt attatttgtg ttttaattta 960

aacacctcct catgtacata ccctggccgc cccctgcccc ccagcctctg gcattagaat 1020

tatttaaaca aaaactaggc ggttgaatga gaggttccta agagtgctgg gcatttttat 1080

tttatgaaat actatttaaa gcctcctcat cccgtgttct ccttttcctc tctcccggag 1140

gttgggtggg ccggcttcat gccagctact tcctcctccc cacttgtccg ctgggtggta 1200

ccctctggag gggtgtggct ccttcccatc gctgtcacag gcggttatga aattcacccc 1260

ctttcctgga cactcagacc tgaattcttt ttcatttgag aagtaaacag atggcacttt 1320

gaaggggcct caccgagtgg gggcatcatc aaaaactttg gagtcccctc acctcctcta 1380

aggttgggca gggtgaccct gaagtgagca cagcctaggg ctgagctggg gacctggtac 1440

cctcctggct cttgataccc ccctctgtct tgtgaaggca gggggaaggt ggggtcctgg 1500

agcagaccac cccgcctgcc ctcatggccc ctctgacctg cactggggag cccgtctcag 1560

tgttgagcct tttccctctt tggctcccct gtaccttttg aggagcccca gctacccttt 1620

ttctccagct gggctctgca attcccctct gctgctgtcc ctcccccttg tcctttccct 1680

tcagtaccct ctcagctcca ggtggctctg aggtgcctgt cccaccccca cccccagctc 1740

aatggactgg aaggggaagg gacacacaag aagaagggca ccctagttct acctcaggca 1800

gctcaagcag cgaccgcccc ctcctctagc tgtgggggtg agggtcccat gtggtggcac 1860

aggccccctt gagtggggtt atctctgtgt taggggtata tgatggggga gtagatcttt 1920

ctaggaggga gacactggcc cctcaaatcg tccagcgacc ttcctcatcc accccatccc 1980

tccccagttc attgcacttt gattagcagc ggaacaagga gtcagacatt ttaagatggt 2040

ggcagtagag gctatggaca gggcatgcca cgtgggctca tatggggctg ggagtagttg 2100

tctttcctgg cactaacgtt gagcccctgg aggcactgaa gtgcttagtg tacttggagt 2160

attggggtct gaccccaaac accttccagc tcctgtaaca tactggcctg gactgttttc 2220

tctcggctcc ccatgtgtcc tggttcccgt ttctccacct agactgtaaa cctctcgagg 2280

gcagggacca caccctgtac tgttctgtgt ctttcacagc tcctcccaca atgctgaata 2340

tacagcaggt gctcaataaa tgattcttag tgacttta 2378

<210> 27

<211> 2588

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 27

gatgggattg gggttttccc ctcccatgtg ctcaagactg gcgctaaaag ttttgagctt 60

ctcaaaagtc tagagccacc gtccagggag caggtagctg ctgggctccg gggacacttt 120

gcgttcgggc tgggagcgtg ctttccacga cggtgacacg cttccctgga ttggccagac 180

tgccttccgg gtcactgcca tggaggagcc gcagtcagat cctagcgtcg agccccctct 240

gagtcaggaa acattttcag acctatggaa actacttcct gaaaacaacg ttctgtcccc 300

cttgccgtcc caagcaatgg atgatttgat gctgtccccg gacgatattg aacaatggtt 360

cactgaagac ccaggtccag atgaagctcc cagaatgcca gaggctgctc cccccgtggc 420

ccctgcacca gcagctccta caccggcggc ccctgcacca gccccctcct ggcccctgtc 480

atcttctgtc ccttcccaga aaacctacca gggcagctac ggtttccgtc tgggcttctt 540

gcattctggg acagccaagt ctgtgacttg cacgtactcc cctgccctca acaagatgtt 600

ttgccaactg gccaagacct gccctgtgca gctgtgggtt gattccacac ccccgcccgg 660

cacccgcgtc cgcgccatgg ccatctacaa gcagtcacag cacatgacgg aggttgtgag 720

gcgctgcccc caccatgagc gctgctcaga tagcgatggt ctggcccctc ctcagcatct 780

tatccgagtg gaaggaaatt tgcgtgtgga gtatttggat gacagaaaca cttttcgaca 840

tagtgtggtg gtgccctatg agccgcctga ggttggctct gactgtacca ccatccacta 900

caactacatg tgtaacagtt cctgcatggg cggcatgaac cggaggccca tcctcaccat 960

catcacactg gaagactcca gtggtaatct actgggacgg aacagctttg aggtgcgtgt 1020

ttgtgcctgt cctgggagag accggcgcac agaggaagag aatctccgca agaaagggga 1080

gcctcaccac gagctgcccc cagggagcac taagcgagca ctgcccaaca acaccagctc 1140

ctctccccag ccaaagaaga aaccactgga tggagaatat ttcacccttc agatccgtgg 1200

gcgtgagcgc ttcgagatgt tccgagagct gaatgaggcc ttggaactca aggatgccca 1260

ggctgggaag gagccagggg ggagcagggc tcactccagc cacctgaagt ccaaaaaggg 1320

tcagtctacc tcccgccata aaaaactcat gttcaagaca gaagggcctg actcagactg 1380

acattctcca cttcttgttc cccactgaca gcctcccacc cccatctctc cctcccctgc 1440

cattttgggt tttgggtctt tgaacccttg cttgcaatag gtgtgcgtca gaagcaccca 1500

ggacttccat ttgctttgtc ccggggctcc actgaacaag ttggcctgca ctggtgtttt 1560

gttgtgggga ggaggatggg gagtaggaca taccagctta gattttaagg tttttactgt 1620

gagggatgtt tgggagatgt aagaaatgtt cttgcagtta agggttagtt tacaatcagc 1680

cacattctag gtaggggccc acttcaccgt actaaccagg gaagctgtcc ctcactgttg 1740

aattttctct aacttcaagg cccatatctg tgaaatgctg gcatttgcac ctacctcaca 1800

gagtgcattg tgagggttaa tgaaataatg tacatctggc cttgaaacca ccttttatta 1860

catggggtct agaacttgac ccccttgagg gtgcttgttc cctctccctg ttggtcggtg 1920

ggttggtagt ttctacagtt gggcagctgg ttaggtagag ggagttgtca agtctctgct 1980

ggcccagcca aaccctgtct gacaacctct tggtgaacct tagtacctaa aaggaaatct 2040

caccccatcc cacaccctgg aggatttcat ctcttgtata tgatgatctg gatccaccaa 2100

gacttgtttt atgctcaggg tcaatttctt ttttcttttt tttttttttt tttctttttc 2160

tttgagactg ggtctcgctt tgttgcccag gctggagtgg agtggcgtga tcttggctta 2220

ctgcagcctt tgcctccccg gctcgagcag tcctgcctca gcctccggag tagctgggac 2280

cacaggttca tgccaccatg gccagccaac ttttgcatgt tttgtagaga tggggtctca 2340

cagtgttgcc caggctggtc tcaaactcct gggctcaggc gatccacctg tctcagcctc 2400

ccagagtgct gggattacaa ttgtgagcca ccacgtccag ctggaagggt caacatcttt 2460

tacattctgc aagcacatct gcattttcac cccacccttc ccctccttct ccctttttat 2520

atcccatttt tatatcgatc tcttatttta caataaaact ttgctgccac ctgtgtgtct 2580

gaggggtg 2588

<210> 28

<211> 1812

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 28

accgccgaga ccgcgtccgc cccgcgagca cagagcctcg cctttgccga tccgccgccc 60

gtccacaccc gccgccagct caccatggat gatgatatcg ccgcgctcgt cgtcgacaac 120

ggctccggca tgtgcaaggc cggcttcgcg ggcgacgatg ccccccgggc cgtcttcccc 180

tccatcgtgg ggcgccccag gcaccagggc gtgatggtgg gcatgggtca gaaggattcc 240

tatgtgggcg acgaggccca gagcaagaga ggcatcctca ccctgaagta ccccatcgag 300

cacggcatcg tcaccaactg ggacgacatg gagaaaatct ggcaccacac cttctacaat 360

gagctgcgtg tggctcccga ggagcacccc gtgctgctga ccgaggcccc cctgaacccc 420

aaggccaacc gcgagaagat gacccagatc atgtttgaga ccttcaacac cccagccatg 480

tacgttgcta tccaggctgt gctatccctg tacgcctctg gccgtaccac tggcatcgtg 540

atggactccg gtgacggggt cacccacact gtgcccatct acgaggggta tgccctcccc 600

catgccatcc tgcgtctgga cctggctggc cgggacctga ctgactacct catgaagatc 660

ctcaccgagc gcggctacag cttcaccacc acggccgagc gggaaatcgt gcgtgacatt 720

aaggagaagc tgtgctacgt cgccctggac ttcgagcaag agatggccac ggctgcttcc 780

agctcctccc tggagaagag ctacgagctg cctgacggcc aggtcatcac cattggcaat 840

gagcggttcc gctgccctga ggcactcttc cagccttcct tcctgggcat ggagtcctgt 900

ggcatccacg aaactacctt caactccatc atgaagtgtg acgtggacat ccgcaaagac 960

ctgtacgcca acacagtgct gtctggcggc accaccatgt accctggcat tgccgacagg 1020

atgcagaagg agatcactgc cctggcaccc agcacaatga agatcaagat cattgctcct 1080

cctgagcgca agtactccgt gtggatcggc ggctccatcc tggcctcgct gtccaccttc 1140

cagcagatgt ggatcagcaa gcaggagtat gacgagtccg gcccctccat cgtccaccgc 1200

aaatgcttct aggcggacta tgacttagtt gcgttacacc ctttcttgac aaaacctaac 1260

ttgcgcagaa aacaagatga gattggcatg gctttatttg ttttttttgt tttgttttgg 1320

tttttttttt ttttttggct tgactcagga tttaaaaact ggaacggtga aggtgacagc 1380

agtcggttgg agcgagcatc ccccaaagtt cacaatgtgg ccgaggactt tgattgcaca 1440

ttgttgtttt tttaatagtc attccaaata tgagatgcgt tgttacagga agtcccttgc 1500

catcctaaaa gccaccccac ttctctctaa ggagaatggc ccagtcctct cccaagtcca 1560

cacaggggag gtgatagcat tgctttcgtg taaattatgt aatgcaaaat ttttttaatc 1620

ttcgccttaa tactttttta ttttgtttta ttttgaatga tgagccttcg tgccccccct 1680

tccccctttt ttgtccccca acttgagatg tatgaaggct tttggtctcc ctgggagtgg 1740

gtggaggcag ccagggctta cctgtacact gacttgagac cagttgaata aaagtgcaca 1800

ccttaaaaat ga 1812

Claims (10)

1. A primer and a probe for detecting the expression level of a plurality of oncogenes, wherein the oncogenes comprise at least one of C-MYC, C-FOS, K-RAS, RB, P21 and P53, and the internal reference is ACTB, and wherein the sequence of the primer and the sequence of the probe comprise:

2. a kit comprising the primer and probe of claim 1, wherein the kit comprises:

a cell lysis reagent;

a cDNA synthesis reagent;

qPCR quantitative detection reagent.

3. The kit of claim 2, wherein the cell lysis reagent comprises a lysis solution, an rnase inhibitor, and a dnase; the cDNA synthesis reagent comprises 2 x reverse transcription reaction liquid.

4. The kit according to claim 2, wherein the qPCR quantitative detection reagent comprises 2 XProbe qPCR Mix, primer Probe mixture 1 and primer Probe mixture 2.

5. The kit of claim 4, wherein the primer probe mixture 1 comprises: primers and probes of an internal reference gene; primers and probes for detecting the C-MYC gene; primers and probes for detecting the C-FOS gene; the primer probe mixed solution 2 comprises: primers and probes for detecting the K-RAS gene; primers and probes for detecting the RB gene; primers and probes for detecting the P21 gene; primers and probes for detecting the P53 gene.

6. The kit of claim 5, wherein the reference gene is ATCB gene, and the primer sequences comprise SEQ ID NO 19 and SEQ ID NO 20; the probe sequence includes SEQ ID NO 21.

7. A method for detecting an oncogene expression level using the kit according to any one of claims 2 to 6, comprising:

cracking the target cells by using the cell cracking reagent to obtain RNA;

reverse transcribing the RNA using the cDNA synthesis reagent to obtain cDNA;

mixing the cDNA, the 2 xProbe qPCR Mix and the primer Probe mixture 1 and/or the primer Probe mixture 2 to obtain a sample to be detected;

and (3) placing the sample to be detected into a quantitative fluorescence PCR detector for detection according to a preset program, and obtaining the CT value of each gene.

8. The method of claim 7, wherein the method comprises: the quantitative fluorescent PCR presetting program comprises the following steps: 95 ℃ for 1min, 95 ℃ for 15s × 40 cycles, 45 ℃ for 40s × 40 cycles.

9. The method of claim 7, wherein the method comprises: the quantitative fluorescence PCR detector can detect at least 54 fluorescence of FAM, HEX, ROX and CY.

10. Use of the kit of any one of claims 2 to 6 for detecting expression levels of C-MYC, C-FOS, K-RAS, RB, P21 and P53 genes.

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