CN107760785A - Primer sets, kit and method for tumor-targeting drug related gene HER1 and HER2 detection of expression - Google Patents

Primer sets, kit and method for tumor-targeting drug related gene HER1 and HER2 detection of expression Download PDF

Info

Publication number
CN107760785A
CN107760785A CN201711220609.XA CN201711220609A CN107760785A CN 107760785 A CN107760785 A CN 107760785A CN 201711220609 A CN201711220609 A CN 201711220609A CN 107760785 A CN107760785 A CN 107760785A
Authority
CN
China
Prior art keywords
her1
her2
detection
expression
tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201711220609.XA
Other languages
Chinese (zh)
Inventor
不公告发明人
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangdong Great Medical Laboratory Co Ltd
Original Assignee
Guangdong Great Medical Laboratory Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangdong Great Medical Laboratory Co Ltd filed Critical Guangdong Great Medical Laboratory Co Ltd
Priority to CN201711220609.XA priority Critical patent/CN107760785A/en
Publication of CN107760785A publication Critical patent/CN107760785A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pathology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention relates to a kind of primer sets for tumor-targeting drug related gene HER1 and HER2 detection of expression.The primer sets include the specific primer and probe for HER1, HER2 gene and reference gene Actin.The present invention relates to a kind of kit for tumor-targeting drug related gene HER1 and HER2 detection of expression.The kit includes detection HER1 genes, HER2 genes and reference gene Actin specific primer and probe, in addition to PCR buffer, dNTP, HS Taq enzymes and ultra-pure water.The invention further relates to the method for tumor-targeting drug related gene HER1 and HER2 detection of expression.Kit provided by the invention and its detection method have the advantages that simple to operate, detection time is short, detection sensitivity is high and specific good, are particularly suitable for the popularization and application in clinical examination works.

Description

Primer sets for tumor-targeting drug related gene HER1 and HER2 detection of expression, Kit and method
Technical field
The present invention relates to vitro diagnostic techniques field, particularly, is related to one kind and is used for tumor-targeting drug related gene Primer sets, kit and the method for HER1 and HER2 detection of expression.
Background technology
Human epidermal growth factor acceptor (HER) family of tyrosine kinase receptor by erbB-1 (HER1, EGFR), ErbB-2 (HER2), erbB-3 (HER3) and erbB-4 (HER4) four are in the member structurally and functionally with high homology Composition.These acceptors can activate the extracellular ligand of numerous downstream passages, and then regulation includes differentiation, migration, propagation and life The a variety of processes deposited.
HER1(EGFR)：EGFR is a kind of transmembrane glycopeptide polymeric immunoglobulin receptor with tyrosine kinase activity.Swollen in a variety of entities High expression or unconventionality expression in knurl, such as:Non-small cell lung cancer, head and neck cancer, stomach cancer, cancer of the esophagus, the straight cancer of knot, breast cancer, carcinoma of urinary bladder Deng, the propagation, angiogenesis, tumor invasion, transfer and the suppression of Apoptosis with tumour cell are relevant, research show about 30% breast cancer expression EGFR, the prognosis of EGFR positive patients are significantly worse than EGFR patients with negative, and this is in ER feminine genders and without leaching Fawn on and show and must become apparent in the breast cancer of transfer.Therefore EGFR is presently the most main target spot, has multi-medicament to be For this target spot.
EGFR monoclonal antibodies such as Cetuximab (Cetuximab) and Victibix (Panitumumab), can with high special Combined with EGF-R ELISA (EGFR), competitiveness blocks EGF and other ligand bindings, by increasing Cyclin-dependent kinase Factor p27kip expression, inducing cell stop the G1 phases, suppress cell propagation；Increase Bax expression and reduction bcl-2 are expressed and lured Lead apoptosis of tumor cells；TGF- α, the growth factor such as amphiregulin and VEGF, basis are reduced into fiber The angiogenic factors such as Porcine HGF and interleukin-8, suppress Tumor angiogenesis, play a role.One research is built View, EGFR and VEGFR mRNA expression and the KRAS situation in tumor tissues observed, which can be used as, receives western appropriate former times list The prediction biomarker of new chemoradiation therapy Locally Advanced rectal cancer patient based on anti-；Separately there are some researches show EGFR is high Expression is to add Cetuximab to the benefited predictive factor of First-line chemotherapy treatment advanced NSCLC patient's existence, assesses EGFR tables Up to contribute to provide individualized treatment.
HER2：ErbB-2 (human epidermal growth factor receptor-2, HER2 it is) a kind of proto-oncogene encoding proteins, belongs to family tyrosine kinase.HER2 can then suppress ER, PR generation simultaneously, its Protein molecular intracellular region has PTK active, itself contains multiple tyrosine residue phosphorylation sites, by mediating Ras-Raf-Mek- MAPK
And P13K-Akt paths shorten cell propagation, pernicious performance enhancing and anti-apoptotic, so as to cause the hair of tumour It is raw.Clinical data is shown：HER2 high expression in kinds of tumors tissue, and in 20%-30% breast cancer and 18%-27% stomaches Visible HER2 is overexpressed in cancer patient, is that important Prognosis in Breast Cancer judges the factor, HER2 it is high express make tumor cell proliferation, Survival, anti-apoptotic ability increase, migration and infiltration ability increase, often imply that patient's prognosis is bad, and HER1 and Breast cancer patients prognosis positive HER2 is worst.Research simultaneously also confirms that HER2 positive (be overexpressed or expand) breast cancer is suffered from Person shows the effect of more preferable when being treated using Herceptin, Lapatinib equimolecular targeted drug than negative patient.
Indicate that infiltrative type breast cancer need to carry out HER2 genetic tests in National Cancer network (NCCN) breast cancer guide, Stomach cancer guide new content：If metastatic disease be present during diagnosis must confirm to have detected HER2-neu.It is main in clinical practice Instruct Herceptin, handkerchief trastuzumab, Lapatinib, the application of capecitabine and its combined chemotherapy.
At present, in the actual detection method of clinical disease mark, main immunohistochemical technique (chemical staining), stream Formula cell technology, immune radiating method etc., technology popularization is good, observation is directly perceived and relatively conventional, but it is excessively numerous experimentation to be present It is trivial as a result read the shortcomings of subjective impact is larger, it is necessary to reagent type is various, this method is limited to a certain extent clinically Further apply.In recent years, Real Time-PCR are by detection time is short, high detection sensitivity (is immunohistochemical staining More than 10 times of method), and real-time online detection, the initial content of response gene in the tissue can be carried out to PCR, experiment is saved Substantial amounts of detection time, it is thus also avoided that gradual clinically part substitution conventional inspection side the advantages that the generation of carryover contamination Method.Common methods have SYBR GreenI dye methods, double probe hybrid methods and Taqman probe techniques；Wherein SYBR GreenI methods Due to being non-saturable dye, specificity must be sentenced not as double probe hybrid methods and Taqman methods by observing solubility curve Breaking, it is specific；And two probe method hybrid method cost is costly.
In summary, the detection kit that the present invention is combined using TAQMAN probe techniques with real-time fluorescence PCR technology, Suitable for clinical expansion and industrialization.
The content of the invention
It is excessively cumbersome, it is necessary to reagent kind in order to experimentation be present when solving Immunohistochemical Method detection clinical biomarkers thing Class is various, as a result reads the larger technical problem of subjective impact, present invention offer one kind is simple to operate, detection time is short, detection Sensitiveness height and the specific good primer sets for tumor-targeting drug related gene HER1 and HER2 detection of expression, kit And method.
The invention provides a kind of primer sets for tumor-targeting drug related gene HER1 and HER2 detection of expression, bag The specific primer and probe for being directed to HER1 genes, HER2 genes and reference gene Actin respectively are included, it is as follows：
HER1-RF sense primers：
5'-GAGTTGATCATCGAATTCTCCAAAA-3'(SEQ ID NO.1)；
HER1-RR anti-sense primers：
5'-GCATTCTTTCATCCCCCTGAA-3'(SEQ ID NO.2)；
HER1-RP probes：
5'FAM-CCCGAGACCCCCAGCGCTACC-TRAMA-3'(SEQ ID NO.3)；
HER2-RF sense primers：
5'-CACCTACAACACAGACACGTTTGA-3'(SEQ ID NO.4)；
HER2-RR anti-sense primers：
5'-TCCCACGTCCGTAGAAAGGTA-3'(SEQ ID NO.5)；
HER2-RP probes：
5'FAM-CCGGTATACATTCGGCGCCAGC-TRAMA-3'(SEQ ID NO.6)；
Actin-RF sense primers：
5'-CCGACAGGATGCAGAAGGAG-3'(SEQ ID NO.7)；
Actin-RR anti-sense primers：
5'-AGGATGGAGCCGCCGAT-3'(SEQ ID NO.8)；
Actin-RP probes：
5'FAM-ATCAAGATCATTGCTCCTCCTGAGCGC-TRAMA-3'(SEQ ID NO.9)。
Present invention also offers a kind of kit for tumor-targeting drug related gene HER1 and HER2 detection of expression, Including PCR reaction solutions, the specificity that the PCR reaction solutions include detection HER1 genes, HER2 genes and reference gene Actin is drawn Thing and probe, it is as follows：
HER1-RF sense primers：
5'-GAGTTGATCATCGAATTCTCCAAAA-3'；
HER1-RR anti-sense primers：
5'-GCATTCTTTCATCCCCCTGAA-3'；
HER1-RP probes：
5'FAM-CCCGAGACCCCCAGCGCTACC-TRAMA-3'；
HER2-RF sense primers：
5'-CACCTACAACACAGACACGTTTGA-3'；
HER2-RR anti-sense primers：
5'-TCCCACGTCCGTAGAAAGGTA-3'；
HER2-RP probes：
5'FAM-CCGGTATACATTCGGCGCCAGC-TRAMA-3'；
Actin-RF sense primers：
5'-CCGACAGGATGCAGAAGGAG-3'；
Actin-RR anti-sense primers：
5'-AGGATGGAGCCGCCGAT-3'；
Actin-RP probes：
5'FAM-ATCAAGATCATTGCTCCTCCTGAGCGC-TRAMA-3'；
The PCR reaction solutions also include PCR buffer, dNTP, HS Taq enzymes and ultra-pure water.
In a kind of preferred embodiment of the kit provided by the invention, in addition to positive reference substance, negative control Product and blank control product.
In a kind of preferred embodiment of the kit provided by the invention, the positive reference substance is respectively to contain HER1 With the solution of HER2 genes；The negative controls are the solution without HER1 and HER2 genes；The blank control product are super Pure water.
Present invention also offers a kind of method for tumor-targeting drug related gene HER1 and HER2 detection of expression, bag Include following steps：
Step 1：Sample extracting mRNA to be detected is taken, sample mRNA progress reverse transcriptions are obtained into cDNA, mould is used as using cDNA Plate；
Step 2：Above-mentioned kit is provided, takes out appropriate PCR reaction solutions, by the PCR reaction solutions and the appropriate mould Plate mixes；
Step 3：Real-time fluorescence quantitative PCR is carried out using above-mentioned specific primer and probe, records Ct values, and respectively Standard curve is built using HER1 and HER2 standard genes as positive control, the substrate that sample is calculated according to the Ct values of sample is dense Degree；The concentration of substrate of normal sample and lesion sample is calculated on standard of comparison curve, is finally judged in sample to be detected MRNA expressions.
In a kind of preferred embodiment of methods described provided by the invention, the reaction bar of the real-time fluorescence quantitative PCR Part is：95 DEG C of pre-degeneration 5min；95 DEG C of 15sec, 60 DEG C of 30sec, fluorescence signal is caught, circulated 40 times.
Compared to prior art, provided by the present invention for tumor-targeting drug related gene HER1 and HER2 detection of expression Primer sets, the beneficial effect of kit and method is：
First, the primer and TAQMAN-MGB fluorescence probe high by designing specificity, it is reconfigured to easy to use, detection knot The reliable kit of fruit, design scientific and reasonable PCR reaction systems so that the present invention have simple to operate, detection speed it is fast, The advantages that detection sensitivity is high and specific good, is particularly suitable for the popularization and application in clinical examination works；
2nd, it is reference gene by increasing Actin genes in design of primers and kit, is had based on the reference gene The advantages of mRNA expression is stable, it is possible to prevente effectively from the generation of false negative, and preferably destination gene expression amount can also be carried out Correction, so as to improve the sensitivity of the inventive method and repeatability so that testing result is more accurately and reliably；
3rd, the above-mentioned advantage having based on the present invention, it can cause the present invention that clinically there is good economy and society Can benefit, the particularly personalized medicine to clinical tumor targeted drug (such as Cetuximab, Herceptin, Lapatinib) Have and know meaning well, important reference frame can be provided for clinical precisely medication, reduce the unreasonable feelings used of medicine Shape, the medical treatment cost of the whole society is reduced, save society doctor and defend resource.
Brief description of the drawings
The PCR amplification curve diagrams of Actin mRNA when Fig. 1 is clinical same pattern detection；
The PCR amplification curve diagrams of HER 1mRNA when Fig. 2 is clinical same pattern detection；
The PCR amplification curve diagrams of Actin mRNA when Fig. 3 is clinical another pattern detection；
The PCR amplification curve diagrams of HER 2mRNA when Fig. 4 is clinical another pattern detection.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with the accompanying drawings and embodiment, it is right The present invention is described in further detail.It should be appreciated that specific embodiment described herein is only to explain the present invention, and It is not used in the restriction present invention.
Embodiment 1：The preparation of kit
First, the design and synthesis of primer and probe
For HER1 genes, HER2 genes and reference gene Actin, (sequence is referring to the mankind disclosed in ncbi database respectively Whole genome sequence), using Primer Premier 3.0 and Methyl Primer Express v1.0 softwares, separately design Specific primer and probe, probe are marked using 5'-FAM and 3'-TRAMAMGB.
Specific primer and probe sequence, it is as shown in the table：
2nd, reference substance selects
Positive reference substance is respectively the solution of the gene containing HER1 and HER2；Negative controls are without HER1 and HER2 genes Solution；Blank control product are ultra-pure water.
3rd, PCR reaction solutions form
Including the PCR reaction solutions containing above-mentioned specific primer and probe, the PCR reaction solutions include detection HER1 bases The specific primer and probe of cause, HER2 genes and reference gene Actin；Also include PCR buffer, dNTP, HS Taq enzymes and Ultra-pure water.
Wherein, it is (precious biological (big to be purchased from the precious biology in Dalian for PCR buffer, dNTP, HS Taq enzyme and ultra-pure water Even) Engineering Co., Ltd)；The ultra-pure water is that (Nuclease-Free Water) uses DEPC (Diethyl Pyrocarbonate, pyrocarbonic acid diethyl ester) the treated and ultra-pure water through autoclave sterilization；HS Taq enzymes (TaKaRa Taq Hot Start Version) it is to be directed to general T aq archaeal dna polymerase high sensitivities, the situation of non-specific band is also easy to produce, specially The thermophilic archaeal dna polymerase product of high specific that door is developed.
4th, PCR reaction systems configure
Embodiment 2：Clinical tumor targeted drug (such as Cetuximab, Herceptin, drawing are carried out using mentioned reagent box Pa replaces Buddhist nun) the horizontal detection of personalized medicine related gene expression method
First, biological specimen
Biomaterial of the present invention is all from Guangdong and attains medical test institute greatly.For during the 1-12 months in 2016 wide Attain 100 remaining tissue samples that medical test is detected greatly in east.
2nd, sample extracting mRNA to be detected is taken, sample mRNA progress reverse transcriptions are obtained into cDNA, template is used as using cDNA：
Sample mRNA extracts kits are QIAGEN miRNeasy FFPE Kit kit (paraffin-embedded tissue samples RNA is extracted) (Kai Jie Bioisystech Co., Ltd (Shanghai)) and QIAGEN RNeasy Mini Kit kit (flesh tissue samples This RNA is extracted) (Kai Jie Bioisystech Co., Ltd (Shanghai))；
Sample mRNA Reverse Transcriptase kits are that (treasured is raw for Takara-PrimeScriptTM RT reagent Kit kits Thing engineering (Dalian) Co., Ltd).
1) take and receive qualified clinical detection sample (the normal structure sample and tumor tissues sample each one of same patient Part)；
2) reagent used required for extraction mRNA is taken out from mRNA extracts kits, normal structure sample is extracted and swells The mRNA of tumor tissue sample；
3) the mRNA samples of extraction need to carry out reverse transcription operation in 24 hours, otherwise need to be placed in -80 DEG C of preservations；
4) reagent used required for reverse transcription mRNA is taken out from mRNA Reverse Transcriptase kits, by extraction in step 3) MRNA reverse transcriptions are cDNA, and are used as template using cDNA.
The 3rd, the kit of the present invention is provided, take out appropriate PCR reaction solutions, by the PCR reaction solutions and the appropriate template Mix；
1) fluorescence quantitative PCR reaction solution, Taq enzyme, primer, probe are taken out from kit, room temperature is melted and vibrates mixing Afterwards, 2000rpm centrifuges 10sec；
It is determined that detection reaction tube number is n (standard items+negative control of the pipe various concentrations gradient of n=sample numbers+4) by following Single detection reaction system prepares the dosage that meter calculates reaction system (40ul) each reagent, adds a proper volume centrifuge tube, fills Divide and mix, be distributed into after 2000rpm centrifugations 10sec in each PCR reaction tubes, 38 μ l/ pipes；Pay attention to avoiding producing bubble, cover Specimen Treatment Chamber is transferred to after lid.
Single detection reaction system is with tabulation：
2) sample rna reverse transcription product is taken out, puts thaw at RT, 2000rpm centrifugations 10sec；Divide in PCR reaction tubes Not Jia Ru in normal sample RNA reverse transcription products, lesion sample rna reverse transcription product and kit various concentrations gradient standard The μ l of product 2；2ul sterilizing ultra-pure waters are added in negative control pipe；Sample should be directly added into reaction solution, avoid sample adhesion in pipe On wall, low-speed centrifugal 10sec after lid fully mixes is covered tightly, is transferred to amplification region.
3) PCR reaction tubes are sequenced and is put into ABI7500 quantitative real time PCR Instruments (U.S. AppliedBiosystems public affairs Department)；PCR response parameters are set；
Cycling condition is set：
Reaction system is 40 μ l.Instrument sense channel selects：Fluorescence signal collection：Fam fluoresceins；Collect temperature：60℃. (should strictly ensure that the normal sample of same patient is consistent with the amplification condition of lesion sample)
4th, PCR result judgements：
The interpretation of result software of quantitative real time PCR Instrument is opened, imports the experimental data of preservation；Using spot fitting method, enter Row interpretation of result, build the standard curve of standard items；Carry out the determination of interpretation of result base line：Take what 3-10 or 3-15 were circulated Fluorescence signal.Threshold value can adjust according to instrument noise tolerance limit.Threshold value setting principle is negative right just above normal with threshold line It is defined according to the peak of product amplification curve；Missing inspection strong positive sample is avoided, baseline should be set to 3-10 circulation fluorescence letter first Number observation analysis Ct values, if without Ct values ＜ 16.0 sample, baseline is changed and is set to the fluorescence signal of 3-15 circulation and enters Row interpretation of result.
Quality control standard：The testing result of negative control should be negative；The Ct values of blank control should be equal to 40.0；Otherwise, this It is invalid that secondary experiment is considered as.The coefficient correlation of standard curve is more than 0.99；Otherwise, this time experiment is considered as invalid.
As a result judge：
The standard curve built according to standard items, and calculate according to the Ct values of sample the concentration of substrate of sample.
The concentration of substrate of normal sample and lesion sample is calculated on standard of comparison curve, it can be determined that patient's pathological tissues MRNA expressions in sample.
Comparative result HER1, HER2mRNA expression
Normal sample concentration is less than lesion concentration of specimens Pathological tissues mRNA is high level expression
Normal sample concentration is higher than lesion concentration of specimens Pathological tissues mRNA is low expression level
Clinical meaning：
Clinical diagnosis finds that HER1 (EGFR) is high, and expression patient's prognosis is poor, while its height expression is that addition western appropriate former times is single Resist the benefited predictive factor of First-line chemotherapy treatment advanced NSCLC patient's existence；HER2 high expression makes tumor cell proliferation, deposited The ability increase of living, anti-apoptotic ability increase, migration and infiltration, often imply that patient's prognosis is bad, while the high tables of HER2 The cancer patient reached is shown more preferably when being treated using Herceptin, Lapatinib equimolecular targeted drug than negative patient The effect of.
Based on the clinical sample mRNA expression testing results of the present invention, referring to Fig. 1-Fig. 3, wherein Fig. 1-2 is clinic The PCR amplification curve diagrams of Actin mRNA during same pattern detection and HER1mRNA PCR amplification curve diagrams；Fig. 3-4 is to face Actin mRNA PCR amplification curve diagrams and HER2mRNA PCR amplification curve diagrams during another pattern detection of bed.
Primer sets, kit provided by the present invention for tumor-targeting drug related gene HER1 and HER2 detection of expression And the beneficial effect of method is：
First, the primer and TAQMAN-MGB fluorescence probe high by designing specificity, it is reconfigured to easy to use, detection knot The reliable kit of fruit, design scientific and reasonable PCR reaction systems so that the present invention have simple to operate, detection speed it is fast, The advantages that detection sensitivity is high and specific good, is particularly suitable for the popularization and application in clinical examination works；
2nd, it is reference gene by increasing Actin genes in design of primers and kit, is had based on the reference gene The advantages of mRNA expression is stable, it is possible to prevente effectively from the generation of false negative, and preferably destination gene expression amount can also be carried out Correction, so as to improve the sensitivity of the inventive method and repeatability so that testing result is more accurately and reliably；
3rd, the above-mentioned advantage having based on the present invention, it can cause the present invention that clinically there is good economy and society Can benefit, the particularly personalized medicine to clinical tumor targeted drug (such as Cetuximab, Herceptin, Lapatinib) Have and know meaning well, important reference frame can be provided for clinical precisely medication, reduce the unreasonable feelings used of medicine Shape, the medical treatment cost of the whole society is reduced, save society doctor and defend resource.
Embodiments of the invention are the foregoing is only, are not intended to limit the scope of the invention, it is every to utilize this hair The equivalent flow conversion that bright description is made, or other related technical fields are directly or indirectly used in, similarly wrap Include in the scope of patent protection of the present invention.
Sequence table
<110>Attain Co., Ltd of medical test institute greatly in Guangdong
<120>Primer sets, kit and method for tumor-targeting drug related gene HER1 and HER2 detection of expression
<130> 2017
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gagttgatca tcgaattctc caaaa 25
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gcattctttc atccccctga a 21
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
cccgagaccc ccagcgctac c 21
<210> 4
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
cacctacaac acagacacgt ttga 24
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
tcccacgtcc gtagaaaggt a 21
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
ccggtataca ttcggcgcca gc 22
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
ccgacaggat gcagaaggag 20
<210> 8
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
aggatggagc cgccgat 17
<210> 9
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
atcaagatca ttgctcctcc tgagcgc 27

Claims (6)

  1. A kind of 1. primer sets for tumor-targeting drug related gene HER1 and HER2 detection of expression, it is characterised in that including The specific primer and probe of HER1 genes, HER2 genes and reference gene Actin are directed to respectively, it is as follows：
    HER1-RF sense primers：
    5'-GAGTTGATCATCGAATTCTCCAAAA-3'；
    HER1-RR anti-sense primers：
    5'-GCATTCTTTCATCCCCCTGAA-3'；
    HER1-RP probes：
    5'FAM-CCCGAGACCCCCAGCGCTACC-TRAMA-3'；
    HER2-RF sense primers：
    5'-CACCTACAACACAGACACGTTTGA-3'；
    HER2-RR anti-sense primers：
    5'-TCCCACGTCCGTAGAAAGGTA-3'；
    HER2-RP probes：
    5'FAM-CCGGTATACATTCGGCGCCAGC-TRAMA-3'；
    Actin-RF sense primers：
    5'-CCGACAGGATGCAGAAGGAG-3'；
    Actin-RR anti-sense primers：
    5'-AGGATGGAGCCGCCGAT-3'；
    Actin-RP probes：
    5'FAM-ATCAAGATCATTGCTCCTCCTGAGCGC-TRAMA-3'。
  2. A kind of 2. kit for tumor-targeting drug related gene HER1 and HER2 detection of expression, it is characterised in that including PCR reaction solutions, the PCR reaction solutions include detection HER1 genes, HER2 genes and reference gene Actin specific primer and Probe, it is as follows：
    HER1-RF sense primers：
    5'-GAGTTGATCATCGAATTCTCCAAAA-3'；
    HER1-RR anti-sense primers：
    5'-GCATTCTTTCATCCCCCTGAA-3'；
    HER1-RP probes：
    5'FAM-CCCGAGACCCCCAGCGCTACC-TRAMA-3'；
    HER2-RF sense primers：
    5'-CACCTACAACACAGACACGTTTGA-3'；
    HER2-RR anti-sense primers：
    5'-TCCCACGTCCGTAGAAAGGTA-3'；
    HER2-RP probes：
    5'FAM-CCGGTATACATTCGGCGCCAGC-TRAMA-3'；
    Actin-RF sense primers：
    5'-CCGACAGGATGCAGAAGGAG-3'；
    Actin-RR anti-sense primers：
    5'-AGGATGGAGCCGCCGAT-3'；
    Actin-RP probes：
    5'FAM-ATCAAGATCATTGCTCCTCCTGAGCGC-TRAMA-3'；
    The PCR reaction solutions also include PCR buffer, dNTP, HS Taq enzymes and ultra-pure water.
  3. 3. the kit according to claim 2 for tumor-targeting drug related gene HER1 and HER2 detection of expression, Characterized in that, also include positive reference substance, negative controls and blank control product.
  4. 4. the kit according to claim 3 for tumor-targeting drug related gene HER1 and HER2 detection of expression, Characterized in that, the positive reference substance is respectively the solution of the gene containing HER1 and HER2；The negative controls be without The solution of HER1 and HER2 genes；The blank control product are ultra-pure water.
  5. A kind of 5. method for tumor-targeting drug related gene HER1 and HER2 detection of expression, it is characterised in that including such as Lower step：
    Step 1：Sample extracting mRNA to be detected is taken, sample mRNA progress reverse transcriptions are obtained into cDNA, template is used as using cDNA；
    Step 2：Kit as claimed in claim 4 is provided, takes out appropriate PCR reaction solutions, by the PCR reaction solutions with fitting The template is measured to mix；
    Step 3：Real-time fluorescence quantitative PCR is carried out using the specific primer described in claim 1 and probe, records Ct values, and Standard curve is built using HER1 and HER2 standard genes as positive control respectively, the bottom of sample is calculated according to the Ct values of sample Thing concentration；The concentration of substrate of normal sample and lesion sample is calculated on standard of comparison curve, is finally judged in sample to be detected MRNA expressions.
  6. 6. the method according to claim 5 for tumor-targeting drug related gene HER1 and HER2 detection of expression, its It is characterised by, the reaction condition of the real-time fluorescence quantitative PCR is：95 DEG C of pre-degeneration 5min；95 DEG C of 15sec, 60 DEG C of 30sec, Fluorescence signal is caught, is circulated 40 times.
CN201711220609.XA 2017-11-29 2017-11-29 Primer sets, kit and method for tumor-targeting drug related gene HER1 and HER2 detection of expression Pending CN107760785A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201711220609.XA CN107760785A (en) 2017-11-29 2017-11-29 Primer sets, kit and method for tumor-targeting drug related gene HER1 and HER2 detection of expression

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201711220609.XA CN107760785A (en) 2017-11-29 2017-11-29 Primer sets, kit and method for tumor-targeting drug related gene HER1 and HER2 detection of expression

Publications (1)

Publication Number Publication Date
CN107760785A true CN107760785A (en) 2018-03-06

Family

ID=61275760

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201711220609.XA Pending CN107760785A (en) 2017-11-29 2017-11-29 Primer sets, kit and method for tumor-targeting drug related gene HER1 and HER2 detection of expression

Country Status (1)

Country Link
CN (1) CN107760785A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100151463A1 (en) * 2008-09-05 2010-06-17 Baehner Frederick L Method for Determining the Likelihood of Response to HER2 Inhibitors
CN103882139A (en) * 2014-04-04 2014-06-25 上海赛安生物医药科技有限公司 Multi-gene detection kit for selection of chemotherapy regimens
CN106011239A (en) * 2016-05-19 2016-10-12 北京安必奇生物科技有限公司 Kit for amplifying Her2 (Human epidermal growth factor receptor-2) gene in human peripheral blood circulating tumor DNA (Deoxyribonucleic Acid)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100151463A1 (en) * 2008-09-05 2010-06-17 Baehner Frederick L Method for Determining the Likelihood of Response to HER2 Inhibitors
CN103882139A (en) * 2014-04-04 2014-06-25 上海赛安生物医药科技有限公司 Multi-gene detection kit for selection of chemotherapy regimens
CN106011239A (en) * 2016-05-19 2016-10-12 北京安必奇生物科技有限公司 Kit for amplifying Her2 (Human epidermal growth factor receptor-2) gene in human peripheral blood circulating tumor DNA (Deoxyribonucleic Acid)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ANGELOS KOUTRAS等: "Evaluation of the prognostic significance of HER family mRNA expression in high‑risk early breast cancer: a Hellenic Cooperative Oncology Group (HeCOG) validation study", 《J TRANSL MED》 *

Similar Documents

Publication Publication Date Title
CN105087769A (en) Probe and kit for detecting HER-2 (human epidermal growth factor receptor-2) gene amplification
CN107619865B (en) Target for predicting sensitivity of small cell lung cancer patient to chemotherapeutic drugs and application
CN103074430B (en) Special primer, kit and method for testing miRNA-155 in bladder cancer urine
CN107586842A (en) A kind of biomarker for clear cell carcinoma of kidney diagnosis and treatment
CN110408703A (en) Colorectal cancer miRNA marker and its application
CN107475386B (en) Long-chain non-coding RNA marker for diagnosis and treatment osteosarcoma
CN107760786A (en) Primer sets, kit and method for the detection of tumor chemotherapeutic drug related gene TYMS expressions
CN110317878A (en) A kind of long-chain non-coding RNA and its application for bladder cancer diagnosis and treatment monitoring
CN110257514A (en) A kind of new cancer of the esophagus blood miRNA marker and its application
CN107760785A (en) Primer sets, kit and method for tumor-targeting drug related gene HER1 and HER2 detection of expression
CN105985961B (en) Inhibit siRNA and its application of EGFR gene expression
CN107858431A (en) Primer sets, kit and method for cancer target medicine phases correlation gene VEGFR1 and VEGFR2 detection of expression
CN109136373A (en) It is a kind of for early diagnosing the lncRNA detection kit and its application of lung cancer metastasis
CN107868818A (en) Primer sets, kit and method for the detection of tyrosine kinase inhibitor related gene IGF1R expressions
CN107868826A (en) Primer sets, kit and method for the detection of Imatinib related gene C kit expressions
CN106916887A (en) Application of the ERH genes in carcinoma of urinary bladder diagnosis and treatment product is prepared
CN106521022A (en) Application of SET gene in preparing product for diagnosing and/or treating gastric cancer
CN104774916B (en) Biomarker combination used for detection of chemotherapy curative effect and/or prognosis of metastatic colorectal cancer and application thereof
CN109266736A (en) Molecular marked compound and its application for osteoporosis early screening
CN109280706A (en) A kind of circular rna hsa-circ-0102618 and its specificity amplification primer and application
CN109055556A (en) A kind of lncRNA detection kit and its application for diagnosing transfer
CN108949981A (en) Utilize the kit of real-time fluorescence quantitative PCR detection VEGFR2 gene relative expression quantity
CN110777206B (en) Application of LOC90024RNA and product for diagnosis, prognosis evaluation and tumor treatment
CN107365859A (en) Molecular markers of the LncRNA as diagnosis and treatment osteosarcoma
CN110628909B (en) miRNA molecule miR-4500 related to endometrial cancer and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20180306

RJ01 Rejection of invention patent application after publication