CN107881224A - A kind of kit and its detection method of detection HER3 mutation - Google Patents

A kind of kit and its detection method of detection HER3 mutation Download PDF

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CN107881224A
CN107881224A CN201711176268.0A CN201711176268A CN107881224A CN 107881224 A CN107881224 A CN 107881224A CN 201711176268 A CN201711176268 A CN 201711176268A CN 107881224 A CN107881224 A CN 107881224A
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nucleotide sequence
primer
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王洁
王瑜
张亚飞
吴叶君
申爱丽
吴子侠
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Qiagen (suzhou) Translational Medicine Co Ltd
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Qiagen (suzhou) Translational Medicine Co Ltd
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Abstract

The present invention, which provides a kind of kit of detection HER3 mutation and its detection method, the kit, mainly includes 9 pairs of PCR primers and 9 sequencing primers.The kit of the present invention can accurately analyze the gene frequency of target gene specific locus mutation and quantitative analysis mixed cell population, have high sensitivity, high specificity, take the advantages of short, detection method fast accurate is easy to operate.

Description

A kind of kit and its detection method of detection HER3 mutation
Technical field
The present invention relates to biology field, more particularly to a kind of kit of detection HER3 mutation and its detection side Method.
Background technology
Human epidermal growth factor acceptor (Human epidermal-growth-factor receptor, HER) family Including four EGFR (erbB1/HER1), HER2, (erbB2/neu), HER3 (erbB3) and HER4 (erbB4) family members, category In I receptors family tyrosine kinase (Receptor tyrosine kinase, RTKs).HER after ligand binding with activating, shape Into homodimer or heterodimer, adjust MAPK paths or activate the signal transmission that PI3K/AKT paths participate in cell, Signal is transmitted by kinase cascade into the cell, it is final to adjust the growth and division of cell, and participate in the propagation of tumour cell, infiltration And angiogenesis etc..The clinical pathologic characteristic of numerous tumours such as HER family proteins and breast cancer, lung cancer and melanoma and prognosis It is closely related.
HER3 is also known as erbB3, and its mRNA is the glycosylated albumen of 6.2Kb, molecular weight 160kd, containing 1342 amino acid Residue.HER3 kinase regulatories area can be with phosphatidyl-inositol 3-kinase (phosphoinositide3-kinase, PI3K) comprising 6 The sites that combine of subunit P85, so HER3 turns into the major activator of PI3K and protein kinase B signal transduction pathway, So as to processes such as modulate tumor cell growth, apoptosis, infiltration, transfers.There is research to show that HER3 exists in various tumor tissues It is overexpressed, it is overexpressed the up-regulation that may be to transcribe (mRNA) or post-transcriptional level (albumen synthesis) and caused, in clinical tumor Play biological action is receptor protein.Normal tissue cell in addition to hematopoietic tissue has the expression of HER3 albumen, shows HER3 plays very important physiological action in normal cell differentiation and proliferation.
It is reported that there are 11% generation HER3 mutation in colon cancer and stomach cancer, there is 11.8% generation HER3 in gallbladder cancer Mutation, the HER3 that non-small cell lung cancer, oophoroma and glioma also have different proportion are mutated.HER3 gene mutations change carefully The non-ligand-dependent mode of born of the same parents' epithelium, cell normally differentiation and propagation are have impact on, so as to cause canceration.Experiment in vivo shows anti- Body class and micromolecular inhibitor can effectively suppress to be mutated the carcinogenesis of HER3 regulations and the process of cancer.Although it there is no at present Listed for the medicine of HER3 target spots, but increasing businesses and institutions participate in the research and development of HER3 medicines, and some, which have been enter into, faces The bed stage.And it is the premise and basis for instructing HER3 targeted drugs to use to detect HER3 mutation.
At present, the detection method of HER3 mutation is mainly direct sequencing, and PCR sequencing PCR prepares to sequencing result solution from sample Read, efficiency is low, and time-consuming, needs 2 days or so time；And direct sequencing detection sensitivity is low, in the case of mutation rate is relatively low, The method of direct Sequencing is almost difficult to its accurate detection, can cause substantial amounts of missing inspection and the generation of false negative.Therefore, directly The technologies such as sequencing are far from the demand for reaching its practical application.
Therefore, research and develop the detection kit of efficient and sensible and method detect survival rate of the HER3 mutation for raising patient, Extend life cycle, avoid excessive chemotherapy, improve life quality and have great significance.
The content of the invention
In view of the shortcomings of the prior art and actual demand, the present invention provide a kind of kit that detection HER3 is mutated and its Detection method, the kit can realize easy, quick detection HER3 gene mutations, and the kit uses pyrosequencing Method, the method for the pyrosequencing has that specificity is good, and detection method is easy to be quick, can provide specific mutant proportion The advantages that, the product for detecting tumor sample HER3 gene mutations using pyrosequencing techniques technically possesses competitive advantage.
To use following technical scheme up to this purpose, the present invention：
On the one hand, the present invention provides a kind of kit of detection HER3 mutation；
The kit includes amplimer and sequencing primer, the nucleotide sequence such as SEQ ID of the amplimer Shown in NO.1-SEQ ID NO.18, the nucleotide sequence of the sequencing primer is as shown in SEQ ID NO.19-27；
The nucleotide sequence of the amplimer is specifically as follows：
SEQ ID NO.1:TATGTCCTCGTGGCCATGAAT；
SEQ ID NO.2:AACATGACGAAGATGGCAAACT；
SEQ ID NO.3:AGACCATCTGTGCTCCTCAGTG；
SEQ ID NO.4:GCCACATCCCAATTTCAGAAC；
SEQ ID NO.5:TTGGGATGTGGCCTTTGA；
SEQ ID NO.6:ACTGATACTTGGTGTGGGGATTG；
SEQ ID NO.7:AACCCAATCCCCACACCAAG；
SEQ ID NO.8:ATCATTGTTCCTTCCCCTCAGAC；
SEQ ID NO.9:GTGGACTCGAGCAACATTGATG；
SEQ ID NO.10:GCTGGGGTCTAAGGAGGGAAG；
SEQ ID NO.11:CAAGATCCCTGCCCTGGAC；
SEQ ID NO.12:GGGCAAACTCTCTGCCACT；
SEQ ID NO.13:TATTTGCCTCTGGGTTCTCTGCT；
SEQ ID NO.14:GGCTTCTCTCACCTTGGCAATTT；
SEQ ID NO.15:GGAACATGGTATGGTGCATAGAA；
SEQ ID NO.16:ATCAGGAGGCAGCAGGTCAG；
SEQ ID NO.17:CAGTTTGGGAGTTGATGACCTTCG；
SEQ ID NO.18:CACACTTGACCATCACCATGTAG。
The nucleotide sequence of the sequencing primer is specifically as follows：
SEQ ID NO.19:TCCCATCGTAGACCTG；
SEQ ID NO.20:CCTGAGCAGCCCCCG；
SEQ ID NO.21:TTAGCTTGTTGTAGACAAGA；
SEQ ID NO.22:CCCACACCAAGTATCA；
SEQ ID NO.23:ATTGATGGATTTGTGAAC；
SEQ ID NO.24:CTGTGATCTCCCGTACT；
SEQ ID NO.25:GGGGCACTGGGGCCA；
SEQ ID NO.26:CGAAACGTGCTACTCAAG；
SEQ ID NO.27:TGCAGATCTGGGGCTGT。
The kit of the present invention uses pyrosequencing techniques, completes the mutation detecting analysis to HER3 amplified productions.It is burnt Phosphoric acid sequencing technologies (Pyrosequencing) are a kind of sequencing technologies being sequenced in synthesis, by each dNTP on primer Polymerization is coupled with first order fluorescence reaction, by detecting release and the intensity of fluorescence, the real time measure DNA sequence dna and Accurate Analysis sequence Change.Its general principle is to cascade chemiluminescence reaction by the enzyme in the same reaction system of 4 kinds of enzymatics, surveyed in each round In sequence reaction, a kind of dNTP is only added, if the dNTP matches with template, polymerase can is incorporated into primer strand and released The pyrophosphoric acid group (PPi) of equimolar number is released, PPi can be eventually converted into visible light signal, and be converted into one by Pyrogram Individual peak value, the height of each peak value are directly proportional to the nucleotide number mixed in reaction；Then a kind of dNTP under adding, continue The synthesis of DNA；Finally by the situation for analyzing peak value, reach the real time measure DNA sequence dna and Accurate Analysis sequence variation Purpose.Pyrosequencing techniques are simple to operate, and result is accurately and reliably.
In the present invention, the kit can be exactly to HER3's by using above-mentioned amplimer and sequencing primer 9 higher sites of the frequency of mutation are detected, so as to realize the detection of tumour.
Preferably, the kit also includes the combination of glycerine and/or glycine betaine, preferably glycerine and glycine betaine.
Because pyrosequencing requires that PCR primer can not be long, design of primers region is caused to be limited, to be easy to detect, and It is required that this 9 pairs of primers expand under identical conditions, this PCR amplification high to specificity brings challenge；It is and non-specific Amplification, have harmful effect to follow-up sequencing sensitivity.The present invention expands body in order to improve primer specific amplification, in PCR Glycerine and glycine betaine are with the addition of in system.Fluorescent quantitative PCR solubility curve result is shown, after adding glycerine and glycine betaine, amplification Specificity greatly improves, and eliminates the interference that non-specific amplification product is reacted follow-up pyrosequencing, improves pyrophosphoric acid The sensitivity of sequencing.
Preferably, the mass fraction of the glycerine is 2-4%, such as can be 2%, 3% or 4%, preferably 3%.
Preferably, the molar concentration of the glycine betaine is 0.25-0.5M, for example, can be 0.25M, 0.3M, 0.35M, 0.4M or 0.5M, preferably 0.4M.
Preferably, the kit also includes the conventional reagent of pyrosequencing, including：Ago-Gel pearl, absorption are slow Appointing in fliud flushing, annealing buffer, Sequenase, sequencing substrate, 10 × washing lotion, denaturing liquid, dATP а S, dCTP, dGTP or dTTP Anticipate a kind of or at least two combinations.
PCR conventional reagents include：1 × Ex Taq Buffer (Mg2+Plus), 0.2mM dNTPMixture, 1 × Fluorescent dye Evagreen, 0.75U/ μ L Ex Taq HS.
Preferably, the kit includes the routine of amplimer, sequencing primer, glycerine, glycine betaine and pyrosequencing Reagent.
The nucleotide sequence of the amplimer as shown in SEQ ID NO.1-SEQ ID NO.18, the sequencing primer Nucleotide sequence is as shown in SEQ ID NO.19-SEQ ID NO.27.
The mass fraction of the glycerine is 2%-4%, and the molar concentration of the glycine betaine is 0.25M-0.5M.
The kit also includes the conventional reagent of pyrosequencing, including：Ago-Gel pearl, adsorption-buffering liquid, move back Fiery buffer solution, Sequenase, sequencing substrate, 10 × washing lotion, denaturing liquid, in dATP а S, dCTP, dGTP or dTTP any one or At least two combination.
Second aspect, the present invention provide a kind of method for the kit detection HER3 mutation applied as described in relation to the first aspect, Comprise the following steps：
(1) tumor tissues sample genomic dna is extracted；
(2) performing PCR amplification is entered to 9 sites of the HER3 genes of genomic DNA；
(3) pyrosequencing is carried out to PCR primer, detects the mutation in 9 sites.
Due to HER3 genes larger mutational site related to tumour, i.e., the higher site of the frequency of mutation includes in tumour 9 sites of V104M, A232V, P262H, G284R, T355A, T389K, Q809R, S846I and E928G, present invention is generally directed to This 9 sites of HER3 genes 104,232,262,284,355,389,809,846 and 928 carry out pyrosequencing detection.
Preferably, 9 sites of step (2) PCR amplifications be 104 sites, 232 sites, 262 sites, 284 sites, 355 sites, 389 sites, 809 sites, 846 sites and 928 sites.
Preferably, the primer of step (2) the PCR amplifications includes：
The nucleotide sequence such as SEQ ID NO.1 and SEQ ID NO.2 institutes for the PCR primer that the 104 site amplification uses Show, the nucleotide sequence shown in the SEQ ID NO.1 and SEQ ID NO.2 is as follows：
SEQ ID NO.1 (sense primer):TATGTCCTCGTGGCCATGAAT；
SEQ ID NO.2 (anti-sense primer):AACATGACGAAGATGGCAAACT；
The nucleotide sequence such as SEQ ID NO.3 and SEQ ID NO.4 institutes for the PCR primer that the 232 site amplification uses Show, the nucleotide sequence shown in the SEQ ID NO.3 and SEQ ID NO.4 is as follows：
SEQ ID NO.3 (sense primer):AGACCATCTGTGCTCCTCAGTG；
SEQ ID NO.4 (anti-sense primer):GCCACATCCCAATTTCAGAAC；
The nucleotide sequence such as SEQ ID NO.5 and SEQ ID NO.6 institutes for the PCR primer that the 262 site amplification uses Show, the nucleotide sequence shown in the SEQ ID NO.5 and SEQ ID NO.6 is as follows：
SEQ ID NO.5 (sense primer):TTGGGATGTGGCCTTTGA；
SEQ ID NO.6 (anti-sense primer):ACTGATACTTGGTGTGGGGATTG；
The nucleotide sequence such as SEQ ID NO.7 and SEQ ID NO.8 institutes for the PCR primer that the 284 site amplification uses Show, the nucleotide sequence shown in the SEQ ID NO.7 and SEQ ID NO.8 is as follows：
SEQ ID NO.7 (sense primer):AACCCAATCCCCACACCAAG；
SEQ ID NO.8 (anti-sense primer):ATCATTGTTCCTTCCCCTCAGAC；
The nucleotide sequence such as SEQ ID NO.9 and SEQ ID NO.10 institutes for the PCR primer that the 355 site amplification uses Show, the nucleotide sequence shown in the SEQ ID NO.9 and SEQ ID NO.10 is as follows：
SEQ ID NO.9 (sense primer):GTGGACTCGAGCAACATTGATG；
SEQ ID NO.10 (anti-sense primer):GCTGGGGTCTAAGGAGGGAAG；
The nucleotide sequence such as SEQ ID NO.11 and SEQ ID NO.12 for the PCR primer that the 389 site amplification uses Shown, the nucleotide sequence shown in the SEQ ID NO.11 and SEQ ID NO.12 is as follows：
SEQ ID NO.11 (sense primer):CAAGATCCCTGCCCTGGAC；
SEQ ID NO.12 (anti-sense primer):GGGCAAACTCTCTGCCACT；
The nucleotide sequence such as SEQ ID NO.13 and SEQ ID NO.14 for the PCR primer that the 809 site amplification uses Shown, the nucleotide sequence shown in the SEQ ID NO.13 and SEQ ID NO.14 is as follows：
SEQ ID NO.13 (sense primer):TATTTGCCTCTGGGTTCTCTGCT；
SEQ ID NO.14 (anti-sense primer):GGCTTCTCTCACCTTGGCAATTT；
The nucleotide sequence such as SEQ ID NO.15 and SEQ ID NO.16 for the PCR primer that the 846 site amplification uses Shown, the nucleotide sequence shown in the SEQ ID NO.15 and SEQ ID NO.16 is as follows：
SEQ ID NO.15 (sense primer):GGAACATGGTATGGTGCATAGAA；
SEQ ID NO.16 (anti-sense primer):ATCAGGAGGCAGCAGGTCAG；
The nucleotide sequence such as SEQ ID NO.17 and SEQ ID NO.18 for the PCR primer that the 928 site amplification uses Shown, the nucleotide sequence shown in the SEQ ID NO.17 and SEQ ID NO.18 is as follows：
SEQ ID NO.17 (sense primer):CAGTTTGGGAGTTGATGACCTTCG；
SEQ ID NO.18 (anti-sense primer):CACACTTGACCATCACCATGTAG.
Preferably, final concentration of 0.1-0.3 μM of the PCR primer of step (2) the PCR amplifications, such as can be 0.1 μ M, 0.2 μM or 0.3 μM, preferably 0.2 μM.
Preferably, the program of step (2) described amplified reaction is specific as follows：
(1 ') carries out preheating 3 minutes at 98 DEG C；
(2 ') keep being denatured for 10 seconds at 98 DEG C, keep being annealed for 30 seconds at 65 DEG C, keep entering for 20 seconds at 72 DEG C Row amplification and extension, circulate the process 45 times；
(3 ') melting curve is carried out.
Preferably, the sequencing primer of the pyrosequencing described in step (3) includes：
The nucleotide sequence of the sequencing primer in 104 sites is as shown in SEQ ID NO.19：
SEQ ID NO.19:TCCCATCGTAGACCTG；
The nucleotide sequence of the sequencing primer in 232 sites is as shown in SEQ ID NO.20：
SEQ ID NO.20:CCTGAGCAGCCCCCG；
The nucleotide sequence of the sequencing primer in 262 sites is as shown in SEQ ID NO.21：
SEQ ID NO.21:TTAGCTTGTTGTAGACAAGA；
The nucleotide sequence of the sequencing primer in 284 sites is as shown in SEQ ID NO.22：
SEQ ID NO.22:CCCACACCAAGTATCA；
The nucleotide sequence of the sequencing primer in 355 sites is as shown in SEQ ID NO.23：
SEQ ID NO.23:ATTGATGGATTTGTGAAC；
The nucleotide sequence of the sequencing primer in 389 sites is as shown in SEQ ID NO.24：
SEQ ID NO.24:CTGTGATCTCCCGTACT；
The nucleotide sequence of the sequencing primer in 809 sites is as shown in SEQ ID NO.25：
SEQ ID NO.25:GGGGCACTGGGGCCA；
The nucleotide sequence of the sequencing primer in 846 sites is as shown in SEQ ID NO.26：
SEQ ID NO.26:CGAAACGTGCTACTCAAG；
The nucleotide sequence of the sequencing primer in 928 sites is as shown in SEQ ID NO.27：
SEQ ID NO.27:TGCAGATCTGGGGCTGT。
Preferably, final concentration of 0.7-0.9 μM of the primer of step (3) described sequencing, for example, can be 0.7 μM, 0.8 μM or 0.9 μM, preferably 0.8 μM.
Preferably, step (3) described pyrosequencing uses AQ patterns.
Preferably, the nucleotides Loading sequence of step (3) described pyrosequencing is as follows：
V104M nucleotides Loading sequence is SEQ ID NO.28:CGTCTCGCACGACTGCG；
A232V nucleotides Loading sequence is SEQ ID NO.29:CAGCACAC；
P262H nucleotides Loading sequence is SEQ ID NO.30:CTGCTGTG；
G284R nucleotides Loading sequence is SEQ ID NO.31:CGTATGTACGAGT；
T355A nucleotides Loading sequence is SEQ ID NO.32:GTGTCAGCAG；
T389K nucleotides Loading sequence is SEQ ID NO.33:CGTCGAGA；
Q809R nucleotides Loading sequence is SEQ ID NO.34:TCAGCTGC；
S846I nucleotides Loading sequence is SEQ ID NO.35:CTCACGAGTCAG；
E928G nucleotides Loading sequence is SEQ ID NO.36:CGCACTGCCTCCTCTCTAGC.
In the present invention, the V104M refers to that the V in 104 sites is mutated into M, and the A232V refers to the A mutation of 232 Into V, the P262H refers to that the P of 262 is mutated into H, and the G284R refers to that the G of 284 is mutated into R, and the T355A refers to Be that the T of 355 is mutated into A, the T389K refers to that the T of 389 is mutated into K, and the Q809R refers to that the Q of 809 dashes forward It is changed into R, the S846I refers to that the S of 846 sports I, and the E928G refers to that the E of 928 is mutated into G.
Compared with prior art, the present invention has the advantages that：
(1) kit provided by the invention can carry out HER3 mutation with the genomic DNA of tumor tissues sample extraction Detection, it is accurate to analyze the gene frequency of target gene specific locus mutation and quantitative analysis mixed cell population, and provide standard True base mutation ratio, there is the advantages of specificity is good, flux is high, cost is low, time-consuming short, be applicable to aid in HER3 targetings The guidance of medication and examination of curative effect.
(2) detection method fast accurate provided by the invention, it is easy to operate.
Brief description of the drawings
Fig. 1 (A) is by 1 in the present invention to HER3 gene V104M site mutation type plasmids and wild plasmid:1 hybrid guided mode Intend the pyrosequencing result of 50% sudden change sample；Fig. 1 (B) is to HER3 Gene A 232V site mutation type plasmids in the present invention 1 is pressed with wild plasmid:The pyrosequencing result of the sudden change sample of 1 hybrid analog-digital simulation 50%；Fig. 1 (C) be in the present invention with pair HER3 gene P262H site mutation type plasmids and wild plasmid press 1:The pyrosequencing of the sudden change sample of 1 hybrid analog-digital simulation 50% As a result；Fig. 1 (D) is by 1 in the present invention to HER3 gene G284R site mutation type plasmids and wild plasmid:1 hybrid analog-digital simulation The pyrosequencing result of 50% sudden change sample；Fig. 1 (E) be in the present invention to HER3 gene T355A site mutation type plasmids and Wild plasmid presses 1:The pyrosequencing result of the sudden change sample of 1 hybrid analog-digital simulation 50%；Fig. 1 (F) is to HER3 bases in the present invention Because T389K site mutation type plasmids and wild plasmid press 1:The pyrosequencing result of the sudden change sample of 1 hybrid analog-digital simulation 50%；Figure 1 (G) be in the present invention HER3 genes Q809R site mutation type plasmids and wild plasmid by 1:1 hybrid analog-digital simulation 50% is mutated sample This pyrosequencing result；Fig. 1 (H) be in the present invention HER3 genes S846I site mutation type plasmids and wild plasmid by 1:The pyrosequencing result of the sudden change sample of 1 hybrid analog-digital simulation 50%；Fig. 1 (I) is HER3 genes E928G site mutations in the present invention Type plasmid and wild plasmid press 1:The pyrosequencing result of the sudden change sample of 1 hybrid analog-digital simulation 50%；
Fig. 2 (A) is the pyrosequencing result in clinical sample HER3 gene V104M sites in the present invention；Fig. 2 (B) is this The pyrosequencing result in clinical sample HER3 Gene A 232V sites in invention；Fig. 2 (C) is clinical sample HER3 in the present invention The pyrosequencing result in gene P262H sites；Fig. 2 (D) is the burnt phosphorus in clinical sample HER3 gene G284R sites in the present invention Sour sequencing result；Fig. 2 (E) is the pyrosequencing result in clinical sample HER3 gene T355A sites in the present invention；Fig. 2 (F) is The pyrosequencing result in clinical sample HER3 gene T389K sites in the present invention；Fig. 2 (G) is clinical sample in the present invention The pyrosequencing result in HER3 gene Q809R sites；Fig. 2 (H) is clinical sample HER3 gene S846I sites in the present invention Pyrosequencing result；Fig. 2 (I) is the pyrosequencing result in clinical sample HER3 gene E928G sites in the present invention.
Embodiment
Further to illustrate the technological means and its effect of the invention taken, below in conjunction with accompanying drawing and by specific real Mode is applied to further illustrate technical scheme, but the present invention is not limited in scope of embodiments.Following embodiments In test method, be conventional method unless otherwise specified.Test material used in following embodiments, such as without special Illustrate, be to be commercially available from routine biochemistry reagent suppliers.
The assembling of the kit of embodiment 1
The kit includes amplimer, sequencing primer, glycerine, glycine betaine and conventional reagent, specific as follows：
Amplimer：
SEQ ID NO.1:TATGTCCTCGTGGCCATGAAT；
SEQ ID NO.2:AACATGACGAAGATGGCAAACT；
SEQ ID NO.3:AGACCATCTGTGCTCCTCAGTG；
SEQ ID NO.4:GCCACATCCCAATTTCAGAAC；
SEQ ID NO.5:TTGGGATGTGGCCTTTGA；
SEQ ID NO.6:ACTGATACTTGGTGTGGGGATTG；
SEQ ID NO.7:AACCCAATCCCCACACCAAG；
SEQ ID NO.8:ATCATTGTTCCTTCCCCTCAGAC；
SEQ ID NO.9:GTGGACTCGAGCAACATTGATG；
SEQ ID NO.10:GCTGGGGTCTAAGGAGGGAAG；
SEQ ID NO.11:CAAGATCCCTGCCCTGGAC；
SEQ ID NO.12:GGGCAAACTCTCTGCCACT；
SEQ ID NO.13:TATTTGCCTCTGGGTTCTCTGCT；
SEQ ID NO.14:GGCTTCTCTCACCTTGGCAATTT；
SEQ ID NO.15:GGAACATGGTATGGTGCATAGAA；
SEQ ID NO.16:ATCAGGAGGCAGCAGGTCAG；
SEQ ID NO.17:CAGTTTGGGAGTTGATGACCTTCG；
SEQ ID NO.18:CACACTTGACCATCACCATGTAG；
Sequencing primer：
SEQ ID NO.19:TCCCATCGTAGACCTG；
SEQ ID NO.20:CCTGAGCAGCCCCCG；
SEQ ID NO.21:TTAGCTTGTTGTAGACAAGA；
SEQ ID NO.22:CCCACACCAAGTATCA；
SEQ ID NO.23:ATTGATGGATTTGTGAAC；
SEQ ID NO.24:CTGTGATCTCCCGTACT；
SEQ ID NO.25:GGGGCACTGGGGCCA；
SEQ ID NO.26:CGAAACGTGCTACTCAAG；
SEQ ID NO.27:TGCAGATCTGGGGCTGT；
Wherein, the mass fraction of glycerine is 3%, and the molar concentration of glycine betaine is 0.4M；
Conventional reagent：
PCR conventional reagents include：1 × Ex Taq Buffer (Mg2+Plus), 0.2m Μ dNTP Mixture, 1 × Fluorescent dye Evagreen, 0.75U/ μ L Ex Taq HS.
Pyrosequencing conventional reagent includes：Ago-Gel pearl, adsorption-buffering liquid, annealing buffer, Sequenase, sequencing Substrate, 10 × washing lotion, denaturing liquid, dATPaS, dCTP, dGTP and dTTP.
Embodiment 2
First extract tumor tissues sample genomic dna, then using patient's tumor tissues sample genomic dna extract as Template, in 30 μ L reaction systems, to HER3 genes the 104th, 232,262,284,355,389,809,846 in genomic DNA And 928 totally 9 sites enter performing PCR amplification, the optimal concentration of PCR primer be 0.2 μM；
The nucleotide sequence of the amplimer is as shown in SEQ ID NO.1-SEQ ID NO.18；
The nucleotide sequence such as SEQ ID NO.1 and SEQ ID NO.2 institutes for the PCR primer that the 104 site amplification uses Show, the nucleotide sequence shown in the SEQ ID NO.1 and SEQ ID NO.2 is as follows：
SEQ ID NO.1 (sense primer):TATGTCCTCGTGGCCATGAAT
SEQ ID NO.2 (anti-sense primer):AACATGACGAAGATGGCAAACT；
The nucleotide sequence such as SEQ ID NO.3 and SEQ ID NO.4 institutes for the PCR primer that the 232 site amplification uses Show, the nucleotide sequence shown in the SEQ ID NO.3 and SEQ ID NO.4 is as follows：
SEQ ID NO.3 (sense primer):AGACCATCTGTGCTCCTCAGTG
SEQ ID NO.4 (anti-sense primer):GCCACATCCCAATTTCAGAAC；
The nucleotide sequence such as SEQ ID NO.5 and SEQ ID NO.6 institutes for the PCR primer that the 262 site amplification uses Show, the nucleotide sequence shown in the SEQ ID NO.5 and SEQ ID NO.6 is as follows：
SEQ ID NO.5 (sense primer):TTGGGATGTGGCCTTTGA
SEQ ID NO.6 (anti-sense primer):ACTGATACTTGGTGTGGGGATTG；
The nucleotide sequence such as SEQ ID NO.7 and SEQ ID NO.8 institutes for the PCR primer that the 284 site amplification uses Show, the nucleotide sequence shown in the SEQ ID NO.7 and SEQ ID NO.8 is as follows：
SEQ ID NO.7 (sense primer):AACCCAATCCCCACACCAAG
SEQ ID NO.8 (anti-sense primer):ATCATTGTTCCTTCCCCTCAGAC；
The nucleotide sequence such as SEQ ID NO.9 and SEQ ID NO.10 institutes for the PCR primer that the 355 site amplification uses Show, the nucleotide sequence shown in the SEQ ID NO.9 and SEQ ID NO.10 is as follows：
SEQ ID NO.9 (sense primer):GTGGACTCGAGCAACATTGATG
SEQ ID NO.10 (anti-sense primer):GCTGGGGTCTAAGGAGGGAAG；
The nucleotide sequence such as SEQ ID NO.11 and SEQ ID NO.12 for the PCR primer that the 389 site amplification uses Shown, the nucleotide sequence shown in the SEQ ID NO.11 and SEQ ID NO.12 is as follows：
SEQ ID NO.11 (sense primer):CAAGATCCCTGCCCTGGAC；
SEQ ID NO.12 (anti-sense primer):GGGCAAACTCTCTGCCACT；
The nucleotide sequence such as SEQ ID NO.13 and SEQ ID NO.14 for the PCR primer that the 809 site amplification uses Shown, the nucleotide sequence shown in the SEQ ID NO.13 and SEQ ID NO.14 is as follows：
SEQ ID NO.13 (sense primer):TATTTGCCTCTGGGTTCTCTGCT；
SEQ ID NO.14 (anti-sense primer):GGCTTCTCTCACCTTGGCAATTT；
The nucleotide sequence such as SEQ ID NO.15 and SEQ ID NO.16 for the PCR primer that the 846 site amplification uses Shown, the nucleotide sequence shown in the SEQ ID NO.15 and SEQ ID NO.16 is as follows：
SEQ ID NO.15 (sense primer):GGAACATGGTATGGTGCATAGAA；
SEQ ID NO.16 (anti-sense primer):ATCAGGAGGCAGCAGGTCAG；
The nucleotide sequence such as SEQ ID NO.17 and SEQ ID NO.18 for the PCR primer that the 928 site amplification uses Shown, the nucleotide sequence shown in the SEQ ID NO.17 and SEQ ID NO.18 is as follows：
SEQ ID NO.17 (sense primer):CAGTTTGGGAGTTGATGACCTTCG；
SEQ ID NO.18 (anti-sense primer):CACACTTGACCATCACCATGTAG.
PCR courses of reaction are as follows：Preheating 3 minutes is first carried out at 98 DEG C；Keep being denatured for 10 seconds at 98 DEG C, 65 DEG C Lower to keep being annealed, keeping being expanded and being extended for 20 seconds at 72 DEG C for 30 seconds, signal collection passage is " Green " and circulation should Process 45 times；Then kept for 5 minutes at 72 DEG C；Finally carry out solubility curve.
The Ct values that are shown by fluorescent PCR amplification instrument and solubility curve check expanding effect, detect the glimmering of reaction system Light signal strength, cycle-index Ct values required during the threshold value of setting and solubility curve and positive quality control are reached with fluorescence signal Contrast situation as criterion, Ct values be less than 38 and solubility curve it is consistent with positive quality control be it is qualified, can be used for next Walk pyrosequencing；Ct values are less than 38 but solubility curve as non-specific amplification inconsistent with positive quality control or Ct are more than or equal to 38 To be unqualified, next step pyrosequencing is not used to.
With SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ The PCR primer of sequence carries biotin mark shown in ID NO11, SEQ ID NO.13, SEQ ID NO.15 and SEQ ID NO.17 Note, the primer with biotin labeling is during pyrosequencing to fixed purification of single stranded DNA.
The pyrosequencing of embodiment 3
Experiment material：
Pyrosequencing instrument and matched reagent (adsorption-buffering liquid, denaturation buffer, annealing buffer, lavation buffer solution, Enzyme, substrate and 4 kinds of dNTPs) purchase and purchased in QIAGEN companies, the coated Agarose microbead of streptavidin in GE companies.
Pyrosequencing detecting step：
(1) PCR primer is fixed：In the unions of PCR eight add be separately added into 29 μ L ultra-pure waters, 40 μ L adsorption-buffering liquid and The 1 coated Ago-Gel pearl of μ L Streptavidins, 10 μ L biotin labeling pcr amplification product is added, by the unions of PCR eight It is placed on normal temperature on turbula shaker to vibrate 10 minutes, PCR primer is fixed on Ago-Gel pearl；
(2) preparation of sequencing primer：Sequencing primer is diluted to 0.8 μM using annealing buffer.Add what 25 μ l had diluted Sequencing primer is into each reacting hole of the orifice plates of PyroMark Q24 24 to be used；
(3) purifying of single-stranded template：Vavuum pump is opened, filter probe is moved on in the townhouse pipes of PCR eight, capture has combined The microballon of PCR primer, retain 15 seconds, taking-up checks whether that most of microballon has all been attracted on vacuum filter probe, then will Vacuum filter probe is put into the slight oscillatory 5-10 seconds in 70% ethanol, then moves on to the suction 10-15 seconds in denaturation buffer, makes DNA is denatured the single-stranded DNA templates to be purified, and finally to the 5-10 seconds are cleaned in lavation buffer solution, washs loose list Chain DNA, so as to obtain the single stranded DNA as sequencing template；
(4) primer hybridization：The sequencing primer that 25 μ L steps (2) prepare first is added in the orifice plate of pyrosequencing 24, then Vacuum filter probe is taken out from PCR plate, vavuum pump is closed, moves into pyrosequencing microwell plate, jiggle, will purify Single-stranded DNA templates respectively with SEQIDNO.19, SEQIDNO.20, SEQIDNO.21, SEQIDNO.22, The sequencing primer of sequence shown in SEQIDNO.23, SEQIDNO.24, SEQIDNO.25, SEQIDNO.26, SEQIDNO.27 is abundant Mix, be denatured at 80 DEG C 1.5 minutes, then room temperature warm bath 10-15 minutes, primer is hybridized with template annealing；
The sequencing primer of pyrosequencing includes：
The nucleotide sequence of the sequencing primer in 104 sites is as shown in SEQ ID NO.19：
SEQ ID NO.19:TCCCATCGTAGACCTG；
The nucleotide sequence of the sequencing primer in 232 sites is as shown in SEQ ID NO.20：
SEQ ID NO.20:CCTGAGCAGCCCCCG；
The nucleotide sequence of the sequencing primer in 262 sites is as shown in SEQ ID NO.21：
SEQ ID NO.21:TTAGCTTGTTGTAGACAAGA；
The nucleotide sequence of the sequencing primer in 284 sites is as shown in SEQ ID NO.22：
SEQ ID NO.22:CCCACACCAAGTATCA；
The nucleotide sequence of the sequencing primer in 355 sites is as shown in SEQ ID NO.23：
SEQ ID NO.23:ATTGATGGATTTGTGAAC；
The nucleotide sequence of the sequencing primer in 389 sites is as shown in SEQ ID NO.24：
SEQ ID NO.24:CTGTGATCTCCCGTACT；
The nucleotide sequence of the sequencing primer in 809 sites is as shown in SEQ ID NO.25：
SEQ ID NO.25:GGGGCACTGGGGCCA；
The nucleotide sequence of the sequencing primer in 846 sites is as shown in SEQ ID NO.26：
SEQ ID NO.26:CGAAACGTGCTACTCAAG；
The nucleotide sequence of the sequencing primer in 928 sites is as shown in SEQ ID NO.27：
SEQ ID NO.27:TGCAGATCTGGGGCTGT。
(5) pyrosequencing：Using the PyroMark Q24 sequenators and kit produced by QIAGEN companies, exist successively Enzyme, substrate and 4 kinds of dNTPs are added in agent bin, from AQ detection patterns, nucleotides is added by following Loading sequence and carries out instead Should：
V104M nucleotides Loading sequence is SEQ ID NO.28:CGTCTCGCACGACTGCG；
A232V nucleotides Loading sequence is SEQ ID NO.29:CAGCACAC；
P262H nucleotides Loading sequence is SEQ ID NO.30:CTGCTGTG；
G284R nucleotides Loading sequence is SEQ ID NO.31:CGTATGTACGAGT；
T355A nucleotides Loading sequence is SEQ ID NO.32:GTGTCAGCAG；
T389K nucleotides Loading sequence is SEQ ID NO.33:CGTCGAGA；
Q809R nucleotides Loading sequence is SEQ ID NO.34:TCAGCTGC；
S846I nucleotides Loading sequence is SEQ ID NO.35:CTCACGAGTCAG；
E928G nucleotides Loading sequence is SEQ ID NO.36:CGCACTGCCTCCTCTCTAGC.
Product after sample-adding every time is detected by CCD optical systems, to obtain special detection peak, according to acquisition Accurate DNA sequence dna information can be read in peak value figure, as a result as shown in Fig. 1 (A-I) and Fig. 2 (A-I).
Fig. 1 (A) is the pyrosequencing knot to known V104M site mutations ratio for 50% master sample with AQ patterns Fruit, detection mutant proportion are G：T=48%：52%；
Pyrosequencing knots of the Fig. 1 (B) with AQ patterns to known A232V site mutations ratio for 50% master sample Fruit, detection mutant proportion are A：G=50%：50%；
Pyrosequencing knots of the Fig. 1 (C) with AQ patterns to known P262H site mutations ratio for 50% master sample Fruit, detection mutant proportion are G：T=58%：42%；
Pyrosequencing knots of the Fig. 1 (D) with AQ patterns to known G284R site mutations ratio for 50% master sample Fruit, detection mutant proportion are C：G=55%：45%；
Pyrosequencing knots of the Fig. 1 (E) with AQ patterns to known T355A site mutations ratio for 50% master sample Fruit, detection mutant proportion are A：G=51%：49%；
Pyrosequencing knots of the Fig. 1 (F) with AQ patterns to known T389K site mutations ratio for 50% master sample Fruit, detection mutant proportion are G：T=59%：41%；
Pyrosequencing knots of the Fig. 1 (G) with AQ patterns to known Q809R site mutations ratio for 50% master sample Fruit, detection mutant proportion are A：G=54%：46%；
Pyrosequencing knots of the Fig. 1 (H) with AQ patterns to known S846I site mutations ratio for 50% master sample Fruit, detection mutant proportion are G：T=50%：50%；
Pyrosequencing knots of the Fig. 1 (I) with AQ patterns to known E928G site mutations ratio for 50% master sample Fruit, detection mutant proportion are C：T=51%：49%；
The mutant proportion numerical value that pyrosequencing result provides it can be seen from Fig. 1 (A)-Fig. 1 (I) and known standard sample This mutant proportion goodness of fit is high, illustrates the method for the present invention accurately and reliably.
Fig. 2 (A)-Fig. 2 (I) is the and of clinical sample HER3 genes 104,232,262,284,355,389,809,846 respectively The pyrosequencing result figure in 928 9 sites.
Clinical sample detects a certain proportion of mutation it can be seen from Fig. 2 (A)-Fig. 2 (I).
In summary, according to multiple assay compared with HER3 gene master sample pyrosequencing results, sequencing Primer can accurately detect the mutation in above-mentioned 9 sites of HER3 genes, it can thus be appreciated that kit provided by the invention and method Can enter performing PCR amplification and pyrosequencing detection to HER3 gene purpose fragments, it is accurate analyze target gene specific locus mutation and The gene frequency of quantitative analysis mixed cell population, and the base mutation ratio of accurate corresponding site can be provided, With specificity it is good, flux is high, cost is low and it is time-consuming short the advantages of, be applicable to aid in HER3 targeting medication guidance and curative effect Detection, detection method fast accurate is easy to operate, and laboratory foundation is provided for clinical rational drug use.
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implemented.Art Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosing.
Sequence table
<110>Kai Jie（Suzhou）Translational medicine studies Co., Ltd
<120>A kind of kit and its detection method of detection HER3 mutation
<130> 2017
<141> 2017-11-22
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial synthesized ()
<400> 1
tatgtcctcg tggccatgaa t 21
<210> 2
<211> 22
<212> DNA
<213>Artificial synthesized ()
<400> 2
aacatgacga agatggcaaa ct 22
<210> 3
<211> 22
<212> DNA
<213>Artificial synthesized ()
<400> 3
agaccatctg tgctcctcag tg 22
<210> 4
<211> 21
<212> DNA
<213>Artificial synthesized ()
<400> 4
gccacatccc aatttcagaa c 21
<210> 5
<211> 18
<212> DNA
<213>Artificial synthesized ()
<400> 5
ttgggatgtg gcctttga 18
<210> 6
<211> 23
<212> DNA
<213>Artificial synthesized ()
<400> 6
actgatactt ggtgtgggga ttg 23
<210> 7
<211> 20
<212> DNA
<213>Artificial synthesized ()
<400> 7
aacccaatcc ccacaccaag 20
<210> 8
<211> 23
<212> DNA
<213>Artificial synthesized ()
<400> 8
atcattgttc cttcccctca gac 23
<210> 9
<211> 22
<212> DNA
<213>Artificial synthesized ()
<400> 9
gtggactcga gcaacattga tg 22
<210> 10
<211> 21
<212> DNA
<213>Artificial synthesized ()
<400> 10
gctggggtct aaggagggaa g 21
<210> 11
<211> 19
<212> DNA
<213>Artificial synthesized ()
<400> 11
caagatccct gccctggac 19
<210> 12
<211> 18
<212> DNA
<213>Artificial synthesized ()
<400> 12
ggcaaactct ctgccact 18
<210> 13
<211> 23
<212> DNA
<213>Artificial synthesized ()
<400> 13
tatttgcctc tgggttctct gct 23
<210> 14
<211> 23
<212> DNA
<213>Artificial synthesized ()
<400> 14
ggcttctctc accttggcaa ttt 23
<210> 15
<211> 23
<212> DNA
<213>Artificial synthesized ()
<400> 15
ggaacatggt atggtgcata gaa 23
<210> 16
<211> 20
<212> DNA
<213>Artificial synthesized ()
<400> 16
atcaggaggc agcaggtcag 20
<210> 17
<211> 24
<212> DNA
<213>Artificial synthesized ()
<400> 17
cagtttggga gttgatgacc ttcg 24
<210> 18
<211> 23
<212> DNA
<213>Artificial synthesized ()
<400> 18
cacacttgac catcaccatg tag 23
<210> 19
<211> 16
<212> DNA
<213>Artificial synthesized ()
<400> 19
tcccatcgta gacctg 16
<210> 20
<211> 15
<212> DNA
<213>Artificial synthesized ()
<400> 20
cctgagcagc ccccg 15
<210> 21
<211> 20
<212> DNA
<213>Artificial synthesized ()
<400> 21
ttagcttgtt gtagacaaga 20
<210> 22
<211> 16
<212> DNA
<213>Artificial synthesized ()
<400> 22
cccacaccaa gtatca 16
<210> 23
<211> 18
<212> DNA
<213>Artificial synthesized ()
<400> 23
attgatggat ttgtgaac 18
<210> 24
<211> 17
<212> DNA
<213>Artificial synthesized ()
<400> 24
ctgtgatctc ccgtact 17
<210> 25
<211> 15
<212> DNA
<213>Artificial synthesized ()
<400> 25
ggggcactgg ggcca 15
<210> 26
<211> 18
<212> DNA
<213>Artificial synthesized ()
<400> 26
cgaaacgtgc tactcaag 18
<210> 27
<211> 17
<212> DNA
<213>Artificial synthesized ()
<400> 27
tgcagatctg gggctgt 17
<210> 28
<211> 17
<212> DNA
<213>Artificial synthesized ()
<400> 28
cgtctcgcac gactgcg 17
<210> 29
<211> 8
<212> DNA
<213>Artificial synthesized ()
<400> 29
cagcacac 8
<210> 30
<211> 8
<212> DNA
<213>Artificial synthesized ()
<400> 30
ctgctgtg 8
<210> 31
<211> 13
<212> DNA
<213>Artificial synthesized ()
<400> 31
cgtatgtacg agt 13
<210> 32
<211> 10
<212> DNA
<213>Artificial synthesized ()
<400> 32
gtgtcagcag 10
<210> 33
<211> 8
<212> DNA
<213>Artificial synthesized ()
<400> 33
cgtcgaga 8
<210> 34
<211> 8
<212> DNA
<213>Artificial synthesized ()
<400> 34
tcagctgc 8
<210> 35
<211> 12
<212> DNA
<213>Artificial synthesized ()
<400> 35
ctcacgagtc ag 12
<210> 36
<211> 20
<212> DNA
<213>Artificial synthesized ()
<400> 36
cgcactgcct cctctctagc 20

Claims (10)

1. a kind of kit of detection HER3 mutation, it is characterised in that the kit includes amplimer and sequencing primer, institute The nucleotide sequence of amplimer is stated as shown in SEQ ID NO.1-SEQ ID NO.18, the nucleotide sequence of the sequencing primer As shown in SEQ ID NO.19-SEQ ID NO.27.
2. kit according to claim 1, it is characterised in that the kit also includes glycerine and/or glycine betaine, excellent Elect the combination of glycerine and glycine betaine as；
Preferably, the mass fraction of the glycerine is 2%-4%, preferably 3%；
Preferably, the molar concentration of the glycine betaine is 0.25M-0.5M, preferably 0.4M；
Preferably, the kit also includes the conventional reagent of pyrosequencing；
Preferably, the conventional reagent includes：Ago-Gel pearl, adsorption-buffering liquid, annealing buffer, Sequenase, sequencing bottom In thing, 10 × washing lotion, denaturing liquid, dATP а S, dCTP, dGTP or dTTP any one or at least two combination.
3. kit according to claim 1 or 2, it is characterised in that the kit includes amplimer, sequencing is drawn Thing, glycerine, the conventional reagent of glycine betaine and pyrosequencing；
The nucleotide sequence of the amplimer is as shown in SEQ ID NO.1-SEQ ID NO.18, the nucleosides of the sequencing primer Acid sequence is as shown in SEQ ID NO.19-SEQ ID NO.27；
The mass fraction of the glycerine is 2%-4%, and the molar concentration of the glycine betaine is 0.25M-0.5M；
The conventional reagent includes：Ago-Gel pearl, adsorption-buffering liquid, annealing buffer, Sequenase, sequencing substrate, 10 × In washing lotion, denaturing liquid, dATPaS, dCTP, dGTP or dTTP any one or at least two combination.
A kind of 4. method for the kit detection HER3 mutation applied as any one of claim 1-3, it is characterised in that Comprise the following steps：
(1) tumor tissues sample genomic dna is extracted；
(2) performing PCR amplification is entered to 9 sites of the HER3 genes of genomic DNA；
(3) pyrosequencing is carried out to PCR primer, detects the mutation in 9 sites.
5. according to the method for claim 4, it is characterised in that 9 sites of step (2) the PCR amplifications include 104 Point, 232 sites, 262 sites, 284 sites, 355 sites, 389 sites, 809 sites, 846 sites and 928 sites.
6. according to the method for claim 5, it is characterised in that the primer of step (2) the PCR amplifications includes：
The nucleotide sequence for the PCR primer that the 104 site amplification uses is as shown in SEQ ID NO.1 and SEQ ID NO.2；
The nucleotide sequence for the PCR primer that the 232 site amplification uses is as shown in SEQ ID NO.3 and SEQ ID NO.4；
The nucleotide sequence for the PCR primer that the 262 site amplification uses is as shown in SEQ ID NO.5 and SEQ ID NO.6；
The nucleotide sequence for the PCR primer that the 284 site amplification uses is as shown in SEQ ID NO.7 and SEQ ID NO.8；
The nucleotide sequence for the PCR primer that the 355 site amplification uses is as shown in SEQ ID NO.9 and SEQ ID NO.10；
The nucleotide sequence for the PCR primer that the 389 site amplification uses is as shown in SEQ ID NO.11 and SEQ ID NO.12；
The nucleotide sequence for the PCR primer that the 809 site amplification uses is as shown in SEQ ID NO.13 and SEQ ID NO.14；
The nucleotide sequence for the PCR primer that the 846 site amplification uses is as shown in SEQ ID NO.15 and SEQ ID NO.16；
The nucleotide sequence for the PCR primer that the 928 site amplification uses is as shown in SEQ ID NO.17 and SEQ ID NO.18；
Preferably, final concentration of 0.1-0.3 μM, preferably 0.2 μM of the PCR primer of step (2) the PCR amplifications.
7. according to the method any one of claim 4-6, it is characterised in that the program of step (2) described amplified reaction It is specific as follows：
(1 ') carries out preheating 3 minutes at 98 DEG C；
(2 ') keep being denatured for 10 seconds at 98 DEG C, keep being annealed for 30 seconds at 65 DEG C, keep being expanded for 20 seconds at 72 DEG C Increase and extend, circulate the process 45 times；
(3 ') melting curve is carried out.
8. according to the method any one of claim 4-7, it is characterised in that pyrosequencing described in step (3) Sequencing primer includes：
The nucleotide sequence of the sequencing primer in 104 site is as shown in SEQ ID NO.19；
The nucleotide sequence of the sequencing primer in 232 site is as shown in SEQ ID NO.20；
The nucleotide sequence of the sequencing primer in 262 site is as shown in SEQ ID NO.21；
The nucleotide sequence of the sequencing primer in 284 site is as shown in SEQ ID NO.22；
The nucleotide sequence of the sequencing primer in 355 site is as shown in SEQ ID NO.23；
The nucleotide sequence of the sequencing primer in 389 site is as shown in SEQ ID NO.24；
The nucleotide sequence of the sequencing primer in 809 site is as shown in SEQ ID NO.25；
The nucleotide sequence of the sequencing primer in 846 site is as shown in SEQ ID NO.26；
The nucleotide sequence of the sequencing primer in 928 site is as shown in SEQ ID NO.27；
Preferably, final concentration of 0.7-0.9 μM of the primer of step (3) described sequencing, preferably 0.8 μM.
9. according to the method any one of claim 4-8, it is characterised in that step (3) pyrosequencing uses AQ patterns；
Preferably, the nucleotides Loading sequence of step (3) described pyrosequencing is as follows：
V104M nucleotides Loading sequence is SEQ ID NO.28；
A232V nucleotides Loading sequence is SEQ ID NO.29；
P262H nucleotides Loading sequence is SEQ ID NO.30；
G284R nucleotides Loading sequence is SEQ ID NO.31；
T355A nucleotides Loading sequence is SEQ ID NO.32；
T389K nucleotides Loading sequence is SEQ ID NO.33；
Q809R nucleotides Loading sequence is SEQ ID NO.34；
S846I nucleotides Loading sequence is SEQ ID NO.35；
E928G nucleotides Loading sequence is SEQ ID NO.36.
10. a kind of detection kit as any one of claim 1-3 is used for medicine and/or the examination for preparing detection tumour Agent.
CN201711176268.0A 2017-11-22 2017-11-22 A kind of kit and its detection method of detection HER3 mutation Pending CN107881224A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103834747A (en) * 2014-03-16 2014-06-04 山东国际旅行卫生保健中心 Method for detecting pathogenicity of influenza A (H1N1) virus based on pyrosequencing

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103834747A (en) * 2014-03-16 2014-06-04 山东国际旅行卫生保健中心 Method for detecting pathogenicity of influenza A (H1N1) virus based on pyrosequencing

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIJAY S. JAISWAL ET AL: "Oncogenic ERBB3 Mutations in Human Cancers", 《CELL》 *
李靖轩等: "基于焦磷酸测序技术的基因突变检测在精准医疗中的应用研究进展", 《药学进展》 *

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