CN106367518A - Reagent for detecting gene mutation of Ras related C3 butulinum toxins 1 and application - Google Patents

Abstract

The invention discloses a reagent, a PCR detection method and an application for detecting the Ras-related C3 botulinum toxin 1 (Rac1) gene mutation, and belongs to the technical field of basic biological research and biological detection. Detection reagents include primers, peptide nucleic acid fluorescent probes and wild-type complementary peptide nucleic acid sequences, forward primers such as SEQ ID NO.1 in the sequence listing; reverse primers such as SEQ ID NO.2 in the sequence listing; peptide nucleic acid fluorescent probes For example, SEQ ID NO.3 and NO.4 in the sequence listing; the wild-type complementary peptide nucleic acid sequence is as SEQ ID NO.5 and NO.6 in the sequence listing. The invention can rapidly discover the mutation of the Rac1 gene downstream of the Wnt signaling pathway from the transcriptional level, has the remarkable advantages of strong specificity and high sensitivity, and provides tools for screening anticancer drugs, exploring new targeted drugs, and basic scientific research.

Description

Reagent and application for detecting mutation of Ras-related C3 botulinum toxin 1 gene

technical field

The invention relates to the technical fields of molecular biology and clinical molecular diagnosis, in particular to a reagent and application for detecting the mutation of Ras-related C3 Clostridium botulinum toxin 1 (Rac1) gene.

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.

Rac1 is one of the main members of the rac subfamily of the Rho family of small G proteins. Activated Rac1 is involved in cytoskeleton reorganization, cell migration, apoptosis and gene transcription regulation. In the Wnt signaling pathway, Rac1 is mainly activated through contact with dishevelled-3 (Dvl-3), which leads to the disassembly of the GSK3B-Axin2-APC complex, promotes the entry of β-catenin into the nucleus, and stimulates Tcf-mediated Wnt downstream target genes Transcription, thereby regulating the malignant phenotype of tumor cells, plays an important role in the Wnt signaling pathway. At present, more and more studies have found that Rac1 protein is mutated to varying degrees in many tumors.

At present, the study of the core molecular regulation mechanism of core signaling pathways and the expression levels of some important members in cells has become a key method for the treatment of tumors. In recent years, the research on the Wnt signaling pathway in cancer, as well as the research on the mutation level of Rac1 protein as a factor regulating the activity of Wnt signaling pathway, has become an important problem that needs to be solved urgently in the research and development of tumor drugs. In particular, mutations in the conserved domain of the RAS family of the Rac1 protein play a very important role in the function initiation of the Rac1 protein. For example, the Y40S mutation was detected in renal clear cell carcinoma, and the G142S mutation was detected in head and neck squamous cell carcinoma.

Peptide nucleic acid (peptide nucleic acid, 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 .

This method 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.

Contents of the invention

The purpose of the present invention is to provide a method for detecting the Wnt signaling pathway in view of the difficulty in determining the mutation of the core regulatory molecule Rac1 in the Wnt signaling pathway in the prior art and how to explain the change in the mutation level of the core molecule Rac1 in tumor cells. Rac1 gene mutation detection reagent and its application.

The object of the present invention is to provide a reagent for Rac1 gene mutation detection, including a wild-type PNA sequence for blocking the amplification of the wild-type Rac1 gene, and a reagent for specifically amplifying the Y40S and G142S mutant Rac1 gene sequences. Set of primer pairs and mutant PNA fluorescent probes:

The forward primer for detecting the Rac1 gene Y40S or G142S mutation is as shown in SEQ ID NO.1 in the sequence listing;

The reverse primer for detecting the Rac1 gene Y40S or G142S mutation is as shown in SEQ ID NO.2 in the sequence listing;

The PNA fluorescent probe for detecting the Rac1 gene Y40S mutation is as shown in SEQ ID NO.3 in the sequence listing;

The PNA fluorescent probe for detecting the G142S mutation of the Rac1 gene is shown in SEQ ID NO.4 in the sequence listing;

The specific binding includes the wild-type PNA sequence of 40 codons of the Rac1 gene, such as SEQ ID NO.5 in the sequence listing;

The specific binding includes the wild-type PNA sequence of codon 142 of the Rac1 gene, such as SEQ ID NO.6 in the sequence listing;

The fluorescent reporter group at the 5' end of the PNA fluorescent probe is: FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, and the quenching group at the 3' end is: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.

The detection reagent of the present invention also includes PCR reaction solution, 40 codons and 142 codons mutant type reference product, contains 40 codons and 142 codons wild-type reference product, wherein PCR reaction solution comprises: DEPC water, has 5' → 3'exo-active DNA polymerase, dNTPs, 10×PCR Buffer, RNASIN, M-MLV reverse transcriptase, oligo(dT) and a solution containing Mg ions.

The mutant reference product containing 40 and 142 codons is a recombinant plasmid DNA containing SEQ NO.7;

The wild-type reference product containing 40 and 142 codons is a recombinant plasmid DNA containing SEQ NO.8;

The DNA polymerase with 5'→3' exolytic activity is Taq enzyme;

The final concentration of each component of the PCR reaction solution in the PCR amplification reaction system is: Taq enzyme 0.01U/μL～0.05U/μL, dNTPs 0.2～0.6mM, 10×PCR Buffer 1×, RNASIN 40U/μL～ 60U/μL, M-MLV reverse transcriptase 200U/μL～320U/μL, MgCl ₂ 1.5～5.0mM, solvent DEPC water.

Specifically, 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, the final concentration of the oligo (dT) is 0.05-0.9 μM, and the complementary PNA sequence The final concentration is 0.05-0.9 μM, and the final concentration of the fluorescent probe is 0.05-0.9 μM.

The reaction procedure for real-time fluorescent quantitative PCR with the reagent of the present invention is: reverse transcription at 42°C for 20 minutes; pre-denaturation at 94°C for 2 minutes; denaturation at 95°C for 30s, 58°C for 45s (collecting fluorescence), and 40 cycles.

The principle of the present invention is to construct a peptide nucleic acid PNA sequence complementary to the wild type of the Rac1 gene. When the peptide nucleic acid PNA sequence is complementary to the Rac1 gene, any mutation can cause a mismatch of PNA/DNA, thereby causing the melting temperature to occur. Change. Then design specific peptide nucleic acid PNA probes and primers according to the Rac1 gene mutant type to perform fluorescence quantitative PCR amplification on the Rac1 mutant gene. After a round of PCR amplification, the Rac1 gene mutant type can be distinguished from the wild type open.

Furthermore, the present invention also provides the application of the reagent in the preparation of a detection reagent for detecting cancer cells, the cancer cells being clear cell carcinoma of the kidney and squamous cell carcinoma of the head and neck.

Compared with the prior art, the advantages and positive effects of the present invention are: the Rac1 gene is a gene that plays an important function upstream of the β-catenin protein in the Wnt signaling pathway, and the present invention provides a method for directly detecting the mutation of the Rac1 gene in the Wnt signaling pathway. Reagent, with the help of the detection reagent, the mutation of the Rac1 gene can be quickly detected at the transcription level by the real-time fluorescent quantitative PCR method, and different from the ordinary real-time fluorescent quantitative PCR, the wild-type PNA sequence designed by the present invention can effectively inhibit the wild-type Genome amplification only enriches the mutant gene amplification, which greatly improves the detection specificity. The mutant PNA fluorescent probe we use is more sensitive. The invention provides anticancer drug screening and mechanism research of new targeted drugs. very powerful tool.

Simultaneously, the experimental system of the present invention can be carried out on any real-time fluorescent quantitative PCR instrument, and the above-mentioned primers are placed in eight tubes for real-time fluorescent quantitative PCR detection. The experimental operation is simple, the cost is low, the result is repeatable, and the sensitivity is good. It is an important means to study the mechanism of action of tumor-related drugs and basic scientific research.

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 sample, Y40S mutant type and wild type reference product;

Fig. 3 is the PCR amplification figure of sample, G142S mutant type and wild type reference product;

detailed description

The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The examples provided are only illustrative of the method of the present invention and do not limit the rest of the present disclosure in any way.

The present invention is illustrated by renal clear cell carcinoma cells, but the present invention is not limited to renal clear cell carcinoma cells, and the detection reagent of the present invention can also be applied to squamous cell carcinoma of the head and neck.

Relevant DNA and RNA basic operation in the embodiment all refer to " Molecular Cloning: Laboratory Manual " (Jin Dongyan et al. translate, Science Press, Beijing (1998)) and " Elaborate Molecular Biology Guide " (Yan Zitranslation, Science Press, Beijing (1998)).

In the present invention, Caki-2 (human renal clear cell carcinoma cell line) was purchased from ATCC, USA, RPMI-1640 medium and 10% fetal bovine serum used for culturing cells were purchased from Invitrogen Company, and other reagents were mainly purchased from Treasure Bioengineering (Dalian) Co., Ltd.

[Example 1] Primer probe design

According to the Rac1 mRNA sequence (NCBIReference Sequence: NM_006908.4) reported by the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov), PrimerExpress developed by Applied Biosystems was used Software for Real-Time PCR software designed specific Rac1 primers and probes.

Rac1Y40S:

Rac1-F:5'-CTTGCCTACTGATCAGTTACACAACC-3'; (SEQ NO.1)

Rac1-R:5'-CGGATCGCTTCGTCAAACAC-3'; (SEQ NO.2)

Mutant Rac1(PNA)-P: 5'FAM-CTTTGACAATTCTTCTGCCAATGT-BHQ 3'; (SEQ NO.3)

Wild-type Rac1-PNA: 5'-ACATTGGCAGAATAATTGTCAAAG-3'; (SEQ NO.5)

Target sequence:

GACGGAGCTGTAGGTAAAACTTGCCTACTGATCAGTTACACAACCAATGCATTTCCTGGAGAATATATCCCTACTGTCTTTGACAATTATTCTGCCAATGTTATGGTAGATGGAAAACCGGTGAATCTGGGCTTATGGGATACAGCTGGACAAGAAGATTATGACAGATTACGCCCCCTATCCTATCCGCAAACAGATGTGTTCTTAATTTGCTTTTCCCTTGTGAGTCCTGCATCATTTGAAAATGTCCGTGCAAAGTGGTATCCTGAGGTGCGGCACCACTGTCCCAACACTCCCATCATCCTAGTGGGAACTAAACTTGATCTTAGGGATGATAAAGACACGATCGAGAAACTGAAGGAGAAGAAGCTGACTCCCATCACCTATCCGCAGGGTCTAGCCATGGCTAAGGAGATTGGTGCTGTAAAATACCTGGAGTGCTCGGCGCTCACACAGCGAGGCCTCAAGACAGTGTTTGACGAAGCGATCCGAGCAGTCCTCTGC

Rac1G142S:

Rac1-F: 5'-CTTGCCTACTGATCAGTTACACAACC-3'; (SEQ NO.1)

Rac1-R:5'-CGGATCGCTTCGTCAAACAC-3'; (SEQ NO.2)

Rac1(PNA)-P:5'FAM-TATCCGCAGAGTCTAGCCAT-BHQ 3'; (SEQ NO.4)

Rac1-PNA: 5'-ATGGCTAGACCCTGCGGATA-3'; (SEQ NO.6)

The target sequence is the same as above;

Above: F: forward, forward; Rac1-F indicates the forward primer for detecting nucleic acid.

R: reverse, reverse; Rac1-R indicates a reverse primer for detecting nucleic acid.

P: probe, fluorescent probe; Rac1-P represents a fluorescent probe for detecting 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.

[Example 2] Construction of recombinant plasmids containing DNA fragments of the Rac1 gene mutant type and wild type

1. Human renal clear cell carcinoma Caki-2 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% CO ₂ .

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 % fetal bovine serum DMEM medium, after repeated gentle blowing, 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% CO ₂ incubator.

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

2. 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 12000 rpm 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.

3. Construction of recombinant plasmids

1) performing PCR amplification on the DNA fragments containing the Rac1 gene mutant type and wild type;

The DNA fragment containing Y40S and G142S codon mutants is the sequence shown in SEQ NO.7;

GGAGCTGTAGGTAAAACTTGCCTACTGATCAGTTACACAACCAATGCATTTCCTGGAGAATATATCCCTACTGTCTTTGACAATTCTTCTGCCAATGTTATGGTAGATGGAAAACCGGTGAATCTGGGCTTATGGGATACAGCTGGACAAGAAGATTATGACAGATTACGCCCCCTATCCTATCCGCAAACAGATGTGTTCTTAATTTGCTTTTCCCTTGTGAGTCCTGCATCATTTGAAAATGTCCGTGCAAAGTGGTATCCTGAGGTGCGGCACCACTGTCCCAACACTCCCATCATCCTAGTGGGAACTAAACTTGATCTTAGGGATGATAAAGACACGATCGAGAAACTGAAGGAGAAGAAGCTGACTCCCATCACCTATCCGCAGAGTCTAGCCATGGCTAAGGAGATTGGTGCTGTAAAATACCTGGAGTGCTCGGCGCTCACACAGCGAGGCCTCAAGACAGTGTTTGACGAAGCGATCCGAGCAGTCCTCTGCCCGCCT；(SEQ NO.7)

The wild-type DNA fragment containing codons 40 and 142 is the sequence shown in SEQ NO.8;

GGAGCTGTAGGTAAAACTTGCCTACTGATCAGTTACACAACCAATGCATTTCCTGGAGAATATATCCCTACTGTCTTTGACAATTATTCTGCCAATGTTATGGTAGATGGAAAACCGGTGAATCTGGGCTTATGGGATACAGCTGGACAAGAAGATTATGACAGATTACGCCCCCTATCCTATCCGCAAACAGATGTGTTCTTAATTTGCTTTTCCCTTGTGAGTCCTGCATCATTTGAAAATGTCCGTGCAAAGTGGTATCCTGAGGTGCGGCACCACTGTCCCAACACTCCCATCATCCTAGTGGGAACTAAACTTGATCTTAGGGATGATAAAGACACGATCGAGAAACTGAAGGAGAAGAAGCTGACTCCCATCACCTATCCGCAGGGTCTAGCCATGGCTAAGGAGATTGGTGCTGTAAAATACCTGGAGTGCTCGGCGCTCACACAGCGAGGCCTCAAGACAGTGTTTGACGAAGCGATCCGAGCAGTCCTCTGCCCGCCT；(SEQ NO.8)

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, 2μl; PCR fragment, 6μl; 10xT4buffer, 1μl; T4DNA ligase, 1μl.

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 LB plates containing 100ng/ml antibiotics (100-150μl). 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.

[Example 3] Amplified sample by real-time fluorescent quantitative PCR method

Take 1-5 μg of extracted total RNA and add it to the PCR reaction solution, which includes: sterile water, DNA polymerase with 5'→3' exolytic activity, dNTPs, 10×PCR Buffer, RNASIN, M-MLV reversal Recording enzyme, 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×PCR Buffer, 0.6 μL of RNASIN at a concentration of 40 U/μL, and a concentration of Add 0.6 μL of ₂₀₀ U/μL 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' excision 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

Read the fluorescence value at the end of the third step of the amplification program;

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

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

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

Figure 2 is the PCR amplification diagram of the Y40S mutant type and wild-type reference product described in the examples of the present invention. The upper, middle and lower lines in the figure represent the 40th codon mutant reference product, sample and wild-type reference product of Rac1 respectively. The amplification curve of the product;

Fig. 3 is the PCR amplification diagram of the G142S mutant type and the wild type reference product described in the embodiment of the present invention. The upper, middle and lower lines in the figure respectively represent the 142nd codon mutant reference product, sample and wild type reference product of Rac1. The amplification curve of the product;

The above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still understand the foregoing embodiments. Modifications to the recorded technical solutions, or equivalent replacements for some of the technical features, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed in the present invention.

SEQUENCE LISTING

<110> Hubei University of Technology

<120> A Reagent for Ras-related C3 Clostridium Botulinum Toxin 1 Gene Mutation Detection and Its Application

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 26

<212>DNA

<213> Unknown

<220>

<223> Y40S or G142S mutation forward primer

<400> 1

cttgcctact gatcagttac acaacc 26

<210> 2

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Y40S or G142S mutation reverse primer

<400> 2

cggatcgctt cgtcaaacac 20

<210> 3

<211> 24

<212>DNA

<213> Unknown

<220>

<223> Y40S mutant PNA fluorescent probe

<400> 3

ctttgacaat tcttctgcca atgt 24

<210> 4

<211> 20

<212>DNA

<213> Unknown

<220>

<223> G142S mutant PNA fluorescent probe

<400> 4

tatccgcaga gtctagccat 20

<210> 5

<211> 24

<212>DNA

<213> Unknown

<220>

<223> 40 codon wild-type PNA sequence

<400> 5

acattggcag aataattgtc aaag 24

<210> 6

<211> 20

<212>DNA

<213> Unknown

<220>

<223> 142 codon wild-type PNA sequence

<400> 6

atggctagac cctgcggata 20

<210> 7

<211> 507

<212>DNA

<213> Unknown

<220>

<223> 40, 142 codon mutant reference

<400> 7

ggagctgtag gtaaaacttg cctactgatc agttacacaa ccaatgcatt tcctggagaa 60

tatatcccta ctgtctttga caattcttct gccaatgtta tggtagatgg aaaaccggtg 120

aatctgggct tatgggatac agctggaca gaagattatg acagattacg ccccctatcc 180

tatccgcaaa cagatgtgtt cttaatttgc ttttcccttg tgagtcctgc atcatttgaa 240

aatgtccgtg caaagtggta tcctgaggtg cggcaccact gtcccaacac tcccatcatc 300

ctagtgggaa ctaaacttga tcttagggat gataaagaca cgatcgagaa actgaaggag 360

aagaagctga ctcccatcac ctatccgcag agtctagcca tggctaagga gattggtgct 420

gtaaaatacc tggagtgctc ggcgctcaca cagcgaggcc tcaagacagt gtttgacgaa 480

gcgatccgag cagtcctctg cccgcct 507

<210> 8

<211> 507

<212>DNA

<213> Unknown

<220>

<223> 40, 142 codon wild type reference

<400> 8

ggagctgtag gtaaaacttg cctactgatc agttacacaa ccaatgcatt tcctggagaa 60

tatatcccta ctgtctttga caattattct gccaatgtta tggtagatgg aaaaccggtg 120

aatctgggct tatgggatac agctggaca gaagattatg acagattacg ccccctatcc 180

tatccgcaaa cagatgtgtt cttaatttgc ttttcccttg tgagtcctgc atcatttgaa 240

aatgtccgtg caaagtggta tcctgaggtg cggcaccact gtcccaacac tcccatcatc 300

ctagtgggaa ctaaacttga tcttagggat gataaagaca cgatcgagaa actgaaggag 360

aagaagctga ctcccatcac ctatccgcag ggtctagcca tggctaagga gattggtgct 420

gtaaaatacc tggagtgctc ggcgctcaca cagcgaggcc tcaagacagt gtttgacgaa 480

gcgatccgag cagtcctctg cccgcct 507

Claims (6)

1. A reagent for Ras-related C3 botulinum toxin 1 Rac1 gene mutation detection, characterized in that, comprising a wild-type PNA sequence for blocking wild-type Rac1 gene amplification, for specific amplification of Y40S , a set of primer pairs for the G142S mutant Rac1 gene sequence and the mutant PNA fluorescent probe: The forward primer for detecting the Rac1 gene Y40S or G142S mutation is as shown in SEQ ID NO.1 in the sequence listing; The reverse primer for detecting the Rac1 gene Y40S or G142S mutation is as shown in SEQ ID NO.2 in the sequence listing; The PNA fluorescent probe for detecting the Rac1 gene Y40S mutation is as shown in SEQ ID NO.3 in the sequence listing; The PNA fluorescent probe for detecting the G142S mutation of the Rac1 gene is shown in SEQ ID NO.4 in the sequence listing; The specific binding includes the wild-type PNA sequence of 40 codons of the Rac1 gene, such as SEQ ID NO.5 in the sequence listing; The specific binding includes the wild-type PNA sequence of codon 142 of the Rac1 gene, such as SEQ ID NO.6 in the sequence listing; The fluorescent reporter group at the 5' end of the PNA fluorescent probe is: FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5, and the quenching group at the 3' end is: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL. 2. the reagent that is used for Ras-related C3 botulinum toxin 1 Rac1 gene mutation detection according to claim 1, is characterized in that, described detection reagent also comprises PCR reaction liquid, contains 40 codons and 142 codons Mutant reference product, wild-type reference product containing 40 codons and 142 codons; the PCR reaction solution includes: DEPC water, DNA polymerase with 5'→3' excision activity, dNTPs, 10×PCR Buffer, RNASIN, M-MLV reverse transcriptase, oligo (dT) and a solution containing Mg ions; the mutant reference substance containing 40 and 142 codons is a recombinant plasmid DNA containing SEQ NO.7; the wild containing 40 and 142 codons The type reference product is the recombinant plasmid DNA containing SEQ NO.8. 3 . The reagent for detecting the mutation of Ras-related C3 botulinum toxin 1 Rac1 gene according to claim 2 , wherein the DNA polymerase with 5'→3' exolytic activity is Taq enzyme. 4 . 4. the reagent for Ras-related C3 botulinum toxin 1 Rac1 gene mutation detection according to claim 3, is characterized in that, the final concentration of each component of described PCR reaction solution in PCR amplification reaction system is : Taq enzyme 0.01U/µL～0.05U/µL, dNTPs 0.2～0.6 mM, 10×PCR Buffer 1×, RNASIN 40U/µL～60U/µL, M-MLV reverse transcriptase 200U/µL～320U/µL, MgCl ₂ 1.5~5.0mM, the solvent is DEPC water; the final concentration of the forward primer is 0.05~0.9μM, the final concentration of the reverse primer is 0.05~0.9μM, and the final concentration of the oligo (dT) is 0.05~ 0.9 μM, the final concentration of the complementary PNA sequence is 0.05-0.9 μM, and the final concentration of the fluorescent probe is 0.05-0.9 μM. 5. according to the reagent described in any one of claim 1-4 that is used for Ras related C3 botulinum toxin 1Rac1 gene mutation detection, it is characterized in that, the reaction program that described reagent carries out real-time fluorescent quantitative PCR is: 42 Reverse transcription at °C for 20 min; pre-denaturation at 94°C for 2 min; denaturation at 95°C for 30 s, 58°C for 45 s, and fluorescence was collected at this time for 40 cycles. 6. The application of the reagent according to claim 1 in the preparation of a detection reagent for detecting cancer cells, wherein the cancer cells are clear cell carcinoma of the kidney and squamous cell carcinoma of the head and neck.