CN105039522A - Detection reagent, PCR detection method and application of FoxM1 gene mutation in Wnt signaling pathway based on peptide nucleic acid probe - Google Patents

Abstract

The invention discloses a detection reagent for FoxM1 gene mutation in a Wnt signal channel based on a peptide nucleic acid probe, a PCR detection method and application, wherein the detection reagent comprises primers and a peptide nucleic acid fluorescent probe, the primers comprise forward and reverse primers, and the forward primers are represented by SEQ ID NO.1, NO.3, NO.5 and NO.7 in a sequence table; the reverse primer is shown as SEQ ID NO.2, NO.4, NO.6 and NO.8 in the sequence table; the acid fluorescent probe is shown as SEQ ID NO.9, NO.10, NO.11 and NO.12 in the sequence table; the wild complementary peptide has the nucleic acid sequence shown as SEQ ID No.13, NO.14, NO.15 and NO.16 in the sequence list. The detection method is a real-time fluorescent quantitative PCR method based on peptide nucleic acid PNA as a fluorescent probe. The invention provides a tool for screening anti-cancer drugs, discussing new targeted drugs, basic scientific research and the like.

Description

Detection reagent, PCR detection method and application of FoxM1 gene mutation in Wnt signaling pathway based on peptide nucleic acid probe

technical field

The invention belongs to the technical field of molecular biology biological nucleic acid detection, and in particular relates to a detection reagent, PCR detection method and application of a FoxM1 gene mutation in the Wnt signaling pathway based on a peptide nucleic acid probe.

Background technique

The Wnt signaling pathway widely exists in invertebrates and vertebrates, and is a highly conserved signaling pathway in the evolution of species. Wnt signaling plays a vital role in the early development of animal embryos, organ formation, tissue regeneration and other physiological processes. If the key protein in this signaling pathway is mutated or abnormally expressed, leading to abnormal activation of the signal, it may induce cancer. The Wnt signaling pathway includes the classic Wnt signaling pathway and the non-canonical Wnt signaling pathway. In the classic pathway, the Wnt-β-catenin signaling pathway, the Wnt factor inhibits intracellular free cells by activating the Frizzle/LRP5/6 synergistic receptors on the cell membrane. The phosphorylation and degradation of β-catenin protein, the nuclear translocation of β-catenin protein will occur after the level of β-catenin protein in the cytoplasm increases, resulting in the increase of β-catenin protein in the nucleus, and the β-catenin protein in the nucleus can Together with Pygo2, Bcl-9 and FoxM1 proteins, they form a complex with the TCF/LEF-1 transcription factor family and activate the transcriptional activation of downstream target genes of the Wnt signaling pathway.

FoxM1 protein is one of the important downstream members of β-catenin in the Wnt signaling pathway. At present, more and more studies have found that, in addition to Pygo2 protein, FoxM1 protein is also highly expressed in many tumors.

At present, the study of the core molecular regulation mechanism of the core signaling pathway, as well as the expression level of some important members in cells, has become a key method for the treatment of tumors. The study of the expression levels of important members of the signaling pathway has become an urgent problem to be solved in the research and development of tumor drugs, but at the same time, there are more and more gene mutations in many tumor cells, which play a very important role in the function of the FoxM1 protein , For example, S55C mutation and R256G mutation were detected in lung adenocarcinoma cells, P271Q mutation was detected in skin melanoma cells, and L318F mutation was detected in bladder urothelial cancer cells.

Peptide nucleic acid (peptidenucleicacids, PNA) is a class of DNA analogues that replace the sugar-phosphate backbone with a polypeptide backbone. It is a third-generation antisense reagent that is designed and constructed by computer and finally artificially synthesized on the basis of the first-generation and second-generation antisense reagents. It is a brand-new DNA analog, that is, neutral peptide chain amide The 2-aminoethylglycine bond replaces the pentose phosphodiester bond backbone in DNA, and the rest is the same as DNA. PNA can recognize and bind DNA or RNA sequences through Watson-Crick base pairing to form a stable double helix structure. Because PNA has no negative charge, there is no electrostatic repulsion between DNA and RNA, so the stability and specificity of the combination are greatly improved; unlike the hybridization between DNA or DNA and RNA, the hybridization between PNA and DNA or RNA is almost Not affected by the salt concentration of the hybridization system, the ability to hybridize with DNA or RNA molecules is far superior to that of DNA/DNA or DNA/RNA, which is characterized by high hybridization stability, excellent specific sequence recognition ability, and is not hydrolyzed by nucleases and proteases .

Contents of the invention

The purpose of the present invention is to address the above-mentioned status quo, and aims to provide a method that can determine the mutation of the core signal molecule FoxM1 in the Wnt signaling pathway, can explain the change of the core molecule FoxM1 in tumor cells, and has good repeatability and sensitivity based on peptides. The detection reagent, PCR detection method and application of the FoxM1 gene mutation in the Wnt signaling pathway of the nucleic acid probe.

The way to achieve the purpose of the present invention is that the detection reagent for FoxM1 gene mutation in the Wnt signaling pathway based on peptide nucleic acid probes includes primers and probes, the primers include forward primers and reverse primers, and the probes are peptide nucleic acid probe;

The forward primer for detecting the S55C mutation of the FoxM1 gene is shown as SEQ ID NO.1 in the sequence listing;

The reverse primer for detecting the S55C mutation of the FoxM1 gene is shown in SEQ ID NO.2 in the sequence listing;

The forward primer for detecting the R256G mutation of the FoxM1 gene is shown as SEQ ID NO.3 in the sequence listing;

The reverse primer for detecting the R256G mutation of the FoxM1 gene is shown as SEQ ID NO.4 in the sequence listing;

The forward primer for detecting the P271Q mutation of the FoxM1 gene is shown as SEQ ID NO.5 in the sequence listing;

The reverse primer for detecting the P271Q mutation of the FoxM1 gene is shown in SEQ ID NO.6 in the sequence listing;

The forward primer for detecting the L318F mutation of the FoxM1 gene is shown in SEQ ID NO.7 in the sequence listing;

The reverse primer for detecting the L318F mutation of the FoxM1 gene is shown in SEQ ID NO.8 in the sequence listing;

The PNA fluorescent probe for detecting the S55C mutation of the FoxM1 gene is shown in SEQ ID NO.9 in the sequence listing;

The PNA fluorescent probe for detecting the R256G mutation of the FoxM1 gene is shown in SEQ ID NO.10 in the sequence listing;

The PNA fluorescent probe for detecting the P271Q mutation of the FoxM1 gene is shown in SEQ ID NO.11 in the sequence listing.

The PNA fluorescent probe for detecting the L318F mutation of the FoxM1 gene is shown in SEQ ID NO.12 in the sequence listing;

The 5' end and 3' end of the peptide nucleic acid fluorescent probe are respectively modified with a fluorescent reporter group and a quencher group, and the fluorescent reporter groups modified at the 5' end are: FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, the quenching group to modify the 3' end is: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.

A method for detecting the FoxM1 gene mutation in the Wnt signaling pathway based on a peptide nucleic acid probe. The detection method is a real-time fluorescent quantitative PCR method based on a peptide nucleic acid as a fluorescent probe;

The reaction system of the real-time fluorescent quantitative PCR is: forward primer, reverse primer, DEPC water, DNA polymerase with 5'→3' exolytic activity, dNTPs, 10×PCRBuffer, RNASIN, M-MLV reverse transcriptase , oligo(dT) and a solution containing Mg ions;

The DNA polymerase with 5'→3' excision activity is Taq enzyme.

The application of the detection reagent for FoxM1 gene mutation in the Wnt signaling pathway based on the peptide nucleic acid probe is used to prepare the detection reagent for detecting tumor cells and normal cells, and the cancer cells are lung adenocarcinoma cells, skin melanoma cells or bladder urinary tract skin cells.

The present invention uses PNA oligomers with specific sequences as probes. Since PNA is an artificially synthesized nucleic acid analog, and is an achiral, uncharged molecule, it avoids hybridization instability caused by mutual charge repulsion when oligonucleotides bind to their target genes, and the combination is not easy. Affected by the ionic strength of the hybridization solution, it shows a strong hybridization advantage and greatly improves the detection sensitivity.

Compared with the prior art, the advantages and positive effects of the present invention are: the FoxM1 gene is a gene that plays an important function in the downstream of the β-catenin protein in the newly discovered Wnt signaling pathway, and the present invention provides a method for directly detecting FoxM1 in the Wnt signaling pathway. Reagents for gene mutation detection, by means of the detection reagents, the mutation of the FoxM1 gene can be quickly detected at the transcription level by real-time fluorescent quantitative PCR. Unlike ordinary real-time fluorescent quantitative PCR, the peptide nucleic acid PNA fluorescent probe we use More sensitive, the invention provides a very powerful tool for the screening of anticancer drugs and the mechanism research of new targeted drugs.

Description of drawings

Fig. 1 is the peptide nucleic acid mass spectrogram described in the embodiment of the present invention;

Fig. 2 is the PCR amplification figure of S55C mutant type and wild type reference product described in the embodiment of the present invention;

Fig. 3 is the PCR amplification figure of R256G mutant type and wild type reference product described in the embodiment of the present invention;

Fig. 4 is the PCR amplification figure of P271Q mutant type and wild type reference product described in the embodiment of the present invention;

Fig. 5 is a PCR amplification diagram of the L318F mutant and wild-type reference products described in the examples of the present invention.

Detailed ways

The detection reagent of Pygo2 gene mutation in the Wnt signaling pathway based on peptide nucleic acid probe, described primer comprises forward primer and reverse primer, and described probe is peptide nucleic acid probe, is characterized in that:

The forward primer for detecting the R46S mutation of the Pygo2 gene is shown as SEQ ID NO.1 in the sequence listing;

The reverse primer for detecting the R46S mutation of the Pygo2 gene is shown as SEQ ID NO.2 in the sequence listing;

The forward primer for detecting P98H mutation of Pygo2 gene is as SEQ ID NO.3 in the sequence listing;

The reverse primer for detecting P98H mutation of Pygo2 gene is shown as SEQ ID NO.4 in the sequence listing;

The forward primer for detecting the R334Q mutation of the Pygo2 gene is shown as SEQ ID NO.5 in the sequence listing;

The reverse primer for detecting the R334Q mutation of the Pygo2 gene is shown in SEQ ID NO.6 in the sequence listing;

The forward primer for detecting the R356P mutation of the Pygo2 gene is shown as SEQ ID NO.7 in the sequence listing;

The reverse primer for detecting the Pygo2 gene R356P mutation is shown in SEQ ID NO.8 in the sequence listing;

The PNA fluorescent probe for detecting the R46S mutation of the Pygo2 gene is shown in SEQ ID NO.9 in the sequence listing;

The PNA fluorescent probe for detecting the P98H mutation of the Pygo2 gene is shown in SEQ ID NO.10 in the sequence listing;

The PNA fluorescent probe for detecting the R334Q mutation of the Pygo2 gene is shown in SEQ ID NO.11 in the sequence listing;

The PNA fluorescent probe for detecting the R356P mutation of the Pygo2 gene is shown in SEQ ID NO.12 in the sequence listing.

The 5' end and 3' end of the peptide nucleic acid fluorescent probe are respectively modified with a fluorescent reporter group and a quencher group, and the fluorescent reporter groups modified at the 5' end are: FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, the quenching group to modify the 3' end is: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.

The detection reagent also contains a contrast reagent, which is a peptide nucleic acid sequence complementary to the wild type of the FoxM1 gene,

The specific binding includes the wild-type PNA sequence of FoxM1 gene 55 codon, such as SEQ ID NO.13 in the sequence listing;

The specific binding includes the wild-type PNA sequence of FoxM1 gene 256 codon, such as SEQ ID NO.14 in the sequence listing;

The specific binding includes the wild-type PNA sequence of FoxM1 gene 271 codon, such as SEQ ID NO.15 in the sequence listing;

The specific binding includes the wild-type PNA sequence of codon 318 of FoxM1 gene, such as SEQ ID NO.16 in the sequence listing.

The mass spectrogram of the peptide nucleic acid used in the present invention is shown in FIG. 1 .

A method for detecting the FoxM1 gene mutation in the Wnt signaling pathway based on a peptide nucleic acid probe, the detection method is a PCR detection method, and the PCR detection method is a real-time fluorescent quantitative PCR method based on a peptide nucleic acid as a fluorescent probe;

The reaction system of the real-time fluorescent quantitative PCR is: forward primer, reverse primer, DEPC water, DNA polymerase with 5'→3' exolytic activity, dNTPs, 10×PCRBuffer, RNASIN, M-MLV reverse transcriptase , oligo(dT) and a solution containing Mg ions.

The DNA polymerase with 5'→3' excision activity is Taq enzyme.

The detection method of the FoxM1 gene mutation in the Wnt signaling pathway based on the peptide nucleic acid probe, the detection steps are as follows:

1) Design primers and PNA probes for detecting these sites for the mutations of codons 55, 256, 271, and 318 of the FoxM1 gene;

2) Designing 4 segments of PNA sequences complementary to the wild type of these sites for the sequences of codons 55, 256, 271, and 318 of the FoxM1 gene;

3) Construct plasmids containing FoxM1 gene mutant and wild type, and calculate the copy number to make a reference product;

4) extract the mRNA in the cells to be tested;

5) Use the above-mentioned FoxM1 gene mutation detection reagent to perform fluorescent quantitative PCR detection on reference products and samples, and determine whether codons 55, 256, 271, and 318 of the FoxM1 gene are mutated.

The final concentration of the Taq enzyme in the PCR reaction solution is 0.01U/μL～0.05U/μL, the final concentration of the dNTPs is 0.2～0.6mM, the final concentration of the 10×PCRBuffer is 1×, the The final concentration of RNASIN is 40U/μL～60U/μL, the final concentration of the M-MLV reverse transcriptase is 200U/μL～ _320U /μL, the final concentration of MgCl is 1.5～5.0mM, and the solvent is DEPC water , the final concentration of the forward primer is 0.05-0.9 μM, the final concentration of the reverse primer is 0.05-0.9 μM, and the final concentration of the fluorescent probe is 0.05-0.9 μM.

The reaction program of the real-time fluorescent quantitative PCR is: reverse transcription at 42°C for 20 minutes; pre-denaturation at 94°C for 2 minutes; denaturation at 95°C for 30 seconds; 58°C for 45 seconds; 40 cycles.

The experimental system of the present invention can be carried out on any real-time fluorescent quantitative PCR instrument, and its PCR reaction instrument is Applied Biosystems 7500 fluorescent quantitative PCR instrument. The above primers are placed in eight tubes for real-time fluorescence quantitative PCR detection. The experiment is simple, low in cost, reproducible in results, and good in sensitivity. It is an important means for studying the mechanism of action of tumor-related drugs and basic scientific research.

The detection reagent for Pygo2 gene mutation in the Wnt signaling pathway based on the peptide nucleic acid probe is used to prepare the detection reagent for detecting tumor cells and normal cells, and the cancer cells are lung adenocarcinoma cells, skin melanoma cells or bladder urothelial cells.

The invention is exemplified by pancreatic cancer cells.

In the present invention, PNAC-1 (human pancreatic cancer PNAC-1 cell line) cell line was purchased from ATCC in the United States, RPMI-1640 medium and 10% fetal bovine serum used for culturing cells were purchased from Yingjun Company, and other reagents were mainly purchased from Treasure Bioengineering (Dalian) Co., Ltd.

Utilizing the reagent of the present invention to detect intracellular Wnt signaling pathway Pygo2 gene mutation comprises the following specific steps:

According to the hFoxM1 mRNA sequence (NCBI Reference Sequence: NM_202002.2) reported by the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov), use the PrimerExpressSoftwareforReal-TimePCR software developed by AppliedBiosystems to design specific FoxM1 primers and probes.

FoxM1S55C:

FoxM1-F: 5'-ACATCAGAGGAGGAACCTAAGAGAT-3';

FoxM1-R:5'-GGGTGGTTAATAATCTTGATCCCA-3';

FoxM1(PNA)-P:5'FAM-TGGCAGAGTCCAACTGTTGCAAGT-BHQ3';

FoxM1-PNA: TGGCAGAGTCCAACTCTTGCAAGT

Target sequence:

CAAGTGAAACATCAGAGGAGGAACCTAAGAGATCCCCTGCCCAACAGGAGTCTAATCAAGCAGAGGCCTCCAAGGAAGTGGCAGAGTCCAACTCTTGCAAGTTTCCAGCTGGGATCAATTAACCACCCCACCATGCCC

FoxM1R256G:

FoxM1-F:5'-AATTCGCCATCAACAGCACTG-3';

FoxM1-R:5'-GGTCCTCAATCCACGTATAGATGTC-3';

FoxM1(PNA)-P:5'FAM-AGGAAGGGCATGACTTTGAA-BHQ3';

FoxM1-PNA: AGGAAGCGCATGACTTTGAA

Target sequence:

ATGGCCATGATACAATTCGCCATCAACAGCACTGAGAGGAAGCGCATGACTTTGAAAGACATCTATACGTGGATTGAGGACCACTTT

FoxM1P271Q:

FoxM1-F:5'-GACATCTATACGTGGATTGAGGACC-3';

FoxM1-R:5'-GAGACCTTGCCATTGGCAGA-3';

FoxM1(PNA)-P:5'FAM-TTTCAATACTTTTAAGCACATTGCC-BHQ3';

FoxM1-PNA: TTTCCCTACTTTTAAGCACATTGCC

Target sequence:

GAAAGACATCTATACGTGGATTGAGGACCACTTTCCCTACTTTAAGCACATTGCCAAGCCAGGCTGGAAGAACTCCATCCGCCACAACCTTTCCCTGCACGACATGTTTGTCCGGGAGACGTCTGCCAATGGCAAGGTCTCCTT

FoxM1L318F:

FoxM1-F:5'-TCTGCCAATGGCAAGGTCTC-3';

FoxM1-R:5'-TCGGTCGTTTCTGCTGTGATT-3';

FoxM1(PNA)-P:5'FAM-CCAACCGCTACTTCACATTGGA-BHQ3';

FoxM1-PNA: CCAACCGCTACTTGACATTGGA

Target sequence:

GGAGACGTCTGCCAATGGCAAGGTCTCCTTCTGGACCATTCACCCAGTGCCAACCGCTACTTGACATTGGACCAGGTGTTTAAGCCACTGGACCCAGGGTCTCCACAAATTGCCCGAGCACTTGGAATCACAGCAGAAACGACCGAATCCAG

Above: F: forward, forward; HW-F indicates the forward primer used to detect hookworm nucleic acid.

R: reverse, reverse; HW-R indicates the reverse primer used to detect hookworm nucleic acid.

P: probe, fluorescent probe; HW-P represents a fluorescent probe for detecting hookworm nucleic acid, and the fluorescent probe is a TaqMan fluorescent probe.

In the embodiment of the present invention, the fluorescent reporter group at the 5' end of the modified fluorescent probe can be: FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5; the quencher group at the 3' end of the modified fluorescent probe It can be: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL, the fluorescent reporter group and quencher group will not affect the amplification of fluorescent quantitative PCR, only need to be selected according to the fluorescent reporter group and quencher group of the probe The model of instrument used sets the range of detectable fluorescent signals. Fluorescent probe fluorescent reporter groups provided by the embodiments of the present invention: FAM, HEX, TET and FAM have an excitation wavelength of 470-650nm and an acceptance wavelength of 500-700nm; quenching groups: Eclipse, TAMRA, BHQ1. The purification methods after primer synthesis can be: HAP, PAGE and HPLC purification methods.

2. Human lung adenocarcinoma Calu-3 cell line culture and passage

1) Cell culture

All cell lines were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at 37°C and 5% CO2.

2) Cell passage

First, use a sterilized pipette to suck out the culture medium in the cell culture dish, add PBS buffer to wash twice, slowly add an appropriate amount of trypsin to the cells, wait until the cells become round, adjust the angle and the cells can move, then add 3ml of 10 DMEM medium with % fetal bovine serum, after repeatedly blowing gently, observe the cell morphology and count under the microscope, pass appropriate amount of cells to other sterilized culture dishes according to the content of cells in the culture dish, add 5ml of DMEM medium Then put into 5% CO2 incubator.

Cell counting formula (unit/ml): (total number of cells in 4 grids)×10 ⁴ ×dilution factor/4

3. Extraction of total RNA

1) Pour off the culture medium, and after washing with PBS, pour 1ml Trizol directly into the culture flask ( ⁵ ×106 cells/ml), and repeatedly pump evenly;

2) Add 0.2ml of chloroform (1/5 of the total volume of Trizol) to the centrifuge tube containing the lysate, shake and mix for 30 seconds, and let stand at room temperature for 5 minutes;

3) Centrifuge at 12000 rpm at 4°C for 15 minutes to separate the phases into three layers. Upper layer: RNA (about 60% of Trizol); middle: DNA; lower layer: protein (phenol-chloroform);

4) Aspirate the supernatant carefully and transfer to another EP tube. The volume of supernatant produced from 1ml of lysate is about 0.4-0.6ml. The organic phase and intermediate layer contain DNA and protein, avoid touching;

5) Add about 0.5ml of isopropanol to the supernatant, shake and mix for 30 seconds. Stand at room temperature for 10 minutes;

6) Centrifuge at 12000rpm at 4°C for 10 minutes;

7) RNA pellet will form on the side of the centrifuge bottom. Carefully aspirate the supernatant and avoid aspiration of the RNA pellet;

8) Add 1ml pre-cooled 75% ethanol (1ml Trizol, at least 1ml ethanol to wash DNA) into the centrifuge tube, shake and mix for 30 seconds to shake the precipitate, and centrifuge at room temperature at 12000rpm for 1-2 minutes. Aspirate and discard the supernatant as much as possible to prevent the loss of RNA pellet. Repeat the above washing steps once. In 75% ethanol, store RNA at 4°C for at least 1 week, and at -20°C for at least 1 year;

9) If the fluidity is low at room temperature, invert the centrifuge tube on the filter paper to dry the RNA, but not completely dry (5-10 minutes). Dissolve the precipitate with 15 μL of DEPC water, and incubate at 55-60°C for 10-15 minutes.

10) RNA purity test

Add 2 μL of RNA solution dropwise to an ultramicro spectrophotometer (model: P330-311), and read the OD260/OD280 ratio in the instrument.

4. Construction of recombinant plasmids

1) performing PCR amplification on DNA fragments containing FoxM1 gene mutant type and wild type;

2) The PCR product is subjected to double digestion;

The vector and the PCR product were subjected to double enzyme digestion under the following conditions (both reaction systems were 30ul, 37°C, enzyme digestion for 2 hours);

3) After electrophoresis, the double-digestion product was recovered by tapping the gel (operate according to the kit instructions);

4) Ligate the digested product with the plasmid vector;

The above-mentioned double digestion products were purified (the vector digestion products were recovered by cutting gel, and the purification steps of the PCR fragments after digestion were the same as the purification steps of the above PCR products), and ligated overnight at 16°C under the action of T4 DNA ligase. The connection system is as follows: vector, 2ul; PCR fragment, 6ul; 10xT4buffer, 1ul; T4DNAligase, 1ul.

5) Transform Escherichia coli competent;

Take 5 μl of the above ligation solution and transform it into DH5α chemically competent cells prepared in advance, bathe in ice for 30 minutes, heat shock at 42°C for 2 minutes, place on ice for 5 minutes, add 1ml of LB culture solution, shake at 37°C for 45 minutes, centrifuge at 5000rpm, 1-5min ( Do not centrifuge for too long, so as not to be too solid), and finally evenly spread on the LB plate (100-150ul) containing 100ng/ml antibiotic. Plates were incubated upside down at 37°C overnight. Pick the positive clones and transfer them to another LB plate containing 100ng/ml antibiotics, number them, and culture them upside down at 37°C overnight.

6) QIAGEN kit extracts the plasmid (performed according to the instructions) to make a reference product.

5. Real-time fluorescence quantitative PCR

Take 1-5 μg of extracted total RNA and add it to PCR reaction solution, which includes: sterile water, DNA polymerase with 5'→3' exolytic activity, dNTPs, 10×PCRBuffer, RNASIN, M-MLV reverse transcriptase , oligo(dT) and a solution containing Mg ions. Among them, 0.3 μL of DNA polymerase with 5’→3’ exolytic activity at a concentration of 5 U/μL, 2 μL of dNTPs at a concentration of 10 mmol/L, 5 μL of 10×PCRBuffer, 0.6 μL of RNASIN at a concentration of 40 U/μL, and a concentration of 200 U/L Add 0.6 μL of M-MLV reverse transcriptase, ₅ μL of 25 mmol/L MgCl solution, and add sterile water to a volume of 50 μL. Wherein, the DNA polymerase with 5'→3' exolytic activity can be Taq enzyme.

PCR amplification: put each reaction tube into the reaction tank of the fluorescent quantitative PCR instrument, set the type of labeled fluorescent group, sample name and type, and select the Taqman fluorescent probe to be used (the fluorescent reporter group of this product is FAM, HEX , TAT, and the fluorescence quenching group is Eclipse), define the sample well, and perform PCR amplification according to the amplification program provided in the table below:

Table 1 is the PCR amplification reaction amplification program

Fluorescence readings were taken at the end of the third step of the amplification program.

The PCR amplification diagrams of the S55C mutant and wild-type reference products described in the examples of the present invention are shown in Figure 2, and the upper, middle and lower lines in the figure represent the 55th codon of FoxM1 mutant reference product, sample and wild-type reference. The amplification curve of the product. The PCR amplification diagrams of the R256G mutant type and wild-type reference product are shown in Figure 3; the upper, middle and lower lines in the figure represent the amplification curves of the FoxM1 codon 256 mutant reference product, sample and wild-type reference product . The PCR amplification diagrams of the P271Q mutant and wild-type reference products are shown in Figure 4; the upper, middle and lower lines in the figure represent the amplification curves of the FoxM1 codon 271 mutant reference product, sample and wild-type reference product . The PCR amplification diagrams of the L318F mutant type and wild-type reference product are shown in Figure 5. The upper, middle and lower lines in the figure represent the amplification curves of the FoxM1 codon 318 mutant reference product, sample and wild-type reference product. . It can be seen from the figure that the 55th and 256th codons in the lung adenocarcinoma Calu-3 cell line are mutant types, while the 271st and 318th codons are wild type.

6. Data analysis and judgment:

At the same time, select the mutant-type and wild-type reference wells corresponding to the detected sample and the sample detection site, and compare the PCR amplification curves of the three holes (CT _A represents the CT value of the sample well, CT _W represents the wild-type CT value, and CT _M represents the CT value of the wild-type mutant CT value):

When CT _W < CT _A ≤ CT _M , it indicates that there is a mutation in the sample;

When CT _W =CT _A , it indicates that the sample is wild type.

Claims (7)

1. the detection reagent of FoxM1 gene mutation in the Wnt signaling pathway based on peptide nucleic acid probe, comprises primer and probe, and described primer comprises forward primer and reverse primer, and described probe is peptide nucleic acid probe, is characterized in that : The forward primer for detecting the S55C mutation of the FoxM1 gene is shown as SEQ ID NO.1 in the sequence listing; The reverse primer for detecting the S55C mutation of the FoxM1 gene is shown in SEQ ID NO.2 in the sequence listing; The forward primer for detecting the R256G mutation of the FoxM1 gene is shown as SEQ ID NO.3 in the sequence listing; The reverse primer for detecting the R256G mutation of the FoxM1 gene is shown as SEQ ID NO.4 in the sequence listing; The forward primer for detecting the P271Q mutation of the FoxM1 gene is shown as SEQ ID NO.5 in the sequence listing; The reverse primer for detecting the P271Q mutation of the FoxM1 gene is shown in SEQ ID NO.6 in the sequence listing; The forward primer for detecting the L318F mutation of the FoxM1 gene is shown in SEQ ID NO.7 in the sequence listing; The reverse primer for detecting the L318F mutation of the FoxM1 gene is shown in SEQ ID NO.8 in the sequence listing; The PNA fluorescent probe for detecting the S55C mutation of the FoxM1 gene is shown in SEQ ID NO.9 in the sequence listing; The PNA fluorescent probe for detecting the R256G mutation of the FoxM1 gene is shown in SEQ ID NO.10 in the sequence listing; The PNA fluorescent probe for detecting the P271Q mutation of the FoxM1 gene is shown in SEQ ID NO.11 in the sequence listing. The PNA fluorescent probe for detecting the L318F mutation of the FoxM1 gene is shown in SEQ ID NO.12 in the sequence listing; The 5' end and 3' end of the peptide nucleic acid fluorescent probe are respectively modified with a fluorescent reporter group and a quencher group, and the fluorescent reporter groups modified at the 5' end are: FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, the quenching group to modify the 3' end is: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL. 2. the detection reagent of FoxM1 gene mutation in the Wnt signaling pathway based on peptide nucleic acid probe according to claim 1, it is characterized in that: detection reagent also contains contrast reagent, and contrast reagent is the peptide nucleic acid sequence complementary with FoxM1 gene wild type , The specific binding includes the wild-type PNA sequence of FoxM1 gene 55 codon, such as SEQ ID NO.13 in the sequence listing; The specific binding includes the wild-type PNA sequence of FoxM1 gene 256 codon, such as SEQ ID NO.14 in the sequence listing; The specific binding includes the wild-type PNA sequence of FoxM1 gene 271 codon, such as SEQ ID NO.15 in the sequence listing; The specific binding includes the wild-type PNA sequence of codon 318 of FoxM1 gene, such as SEQ ID NO.16 in the sequence listing. 3. the method for detecting with the detection reagent of FoxM1 gene mutation in the Wnt signaling pathway based on peptide nucleic acid probe claimed in claim 1, it is characterized in that: detection method is PCR detection method, and described PCR detection method is based on peptide nucleic acid A real-time fluorescent quantitative PCR method for fluorescent probes; The reaction system of the real-time fluorescent quantitative PCR is: forward primer, reverse primer, DEPC water, DNA polymerase with 5'→3' exolytic activity, dNTPs, 10×PCRBuffer, RNASIN, M-MLV reverse transcriptase , oligo(dT) and a solution containing Mg ions; The DNA polymerase with 5'→3' excision activity is Taq enzyme. 4. the detection method of FoxM1 gene mutation in the Wnt signaling pathway based on peptide nucleic acid probe according to claim 3, is characterized in that: detection step is as follows: 1) Design primers and PNA probes for detecting these sites for the mutations of codons 55, 256, 271, and 318 of the FoxM1 gene; 2) Designing 4 segments of PNA sequences complementary to the wild type of these sites for the sequences of codons 55, 256, 271, and 318 of the FoxM1 gene; 3) Construct plasmids containing FoxM1 gene mutant and wild type, and calculate the copy number to make a reference product; 4) extract the mRNA in the cells to be tested; 5) Use the above-mentioned FoxM1 gene mutation detection reagent to perform fluorescent quantitative PCR detection on reference products and samples, and determine whether codons 55, 256, 271, and 318 of the FoxM1 gene are mutated. 5. The detection method of FoxM1 gene mutation in the Wnt signaling pathway based on peptide nucleic acid probe according to claim 3, characterized in that: the final concentration of Taq enzyme in the PCR reaction solution is 0.01U/μL～0.05 U/μL, the final concentration of the dNTPs is 0.2～0.6mM, the final concentration of the 10×PCRBuffer is 1×, the final concentration of the RNASIN is 40U/μL～60U/μL, the M-MLV reverse transcription The final concentration of the enzyme is 200U/μL～320U/μL, the final concentration of the _MgCl2 is 1.5～5.0mM, the solvent is DEPC water, the final concentration of the forward primer is 0.05～0.9μM, the reverse primer The final concentration of the fluorescent probe is 0.05-0.9 μM, and the final concentration of the fluorescent probe is 0.05-0.9 μM. 6. The method for detecting FoxM1 gene mutations in the Wnt signaling pathway based on peptide nucleic acid probes according to claim 3, characterized in that: the reaction program of the real-time fluorescent quantitative PCR is: reverse transcription at 42°C for 20min; Denaturation, 2min; 95°C denaturation, 30s; 58°C, 45s; 40 cycles. 7. the application of the detection reagent of FoxM1 gene mutation in the Wnt signaling pathway based on peptide nucleic acid probe of claim 1, it is characterized in that: be used to prepare the detection reagent of detecting tumor cell and normal cell, described cancer cell is lung Adenocarcinoma cells, skin melanoma cells, or bladder urothelial cells.