CN113293213A - Primer probe for detecting breast cancer recurrence transfer gene HER2 amplification and application thereof - Google Patents
Primer probe for detecting breast cancer recurrence transfer gene HER2 amplification and application thereof Download PDFInfo
- Publication number
- CN113293213A CN113293213A CN202110674748.XA CN202110674748A CN113293213A CN 113293213 A CN113293213 A CN 113293213A CN 202110674748 A CN202110674748 A CN 202110674748A CN 113293213 A CN113293213 A CN 113293213A
- Authority
- CN
- China
- Prior art keywords
- gene
- seq
- sequence
- probe
- her2
- 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
Links
- 239000000523 sample Substances 0.000 title claims abstract description 44
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 title claims abstract description 23
- 230000003321 amplification Effects 0.000 title claims abstract description 18
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 18
- 206010006187 Breast cancer Diseases 0.000 title claims abstract description 17
- 208000026310 Breast neoplasm Diseases 0.000 title claims abstract description 17
- 238000012546 transfer Methods 0.000 title claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 56
- 238000007847 digital PCR Methods 0.000 claims abstract description 17
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 8
- 101100101343 Homo sapiens EFTUD2 gene Proteins 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 3
- 108091026890 Coding region Proteins 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 101150057053 fzd2 gene Proteins 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 18
- 108700020302 erbB-2 Genes Proteins 0.000 abstract description 18
- 101150054472 HER2 gene Proteins 0.000 abstract description 17
- 230000004544 DNA amplification Effects 0.000 abstract description 7
- 238000000034 method Methods 0.000 abstract description 7
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 description 28
- 108020004414 DNA Proteins 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 11
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 9
- 238000010586 diagram Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 206010027476 Metastases Diseases 0.000 description 5
- 230000009401 metastasis Effects 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 229960000575 trastuzumab Drugs 0.000 description 4
- 101000608799 Homo sapiens 116 kDa U5 small nuclear ribonucleoprotein component Proteins 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 102000048651 human EFTUD2 Human genes 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 238000004393 prognosis Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 101150021140 EFTUD2 gene Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 208000017891 HER2 positive breast carcinoma Diseases 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000011528 liquid biopsy Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000005580 one pot reaction Methods 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 101000851181 Homo sapiens Epidermal growth factor receptor Proteins 0.000 description 1
- 239000002136 L01XE07 - Lapatinib Substances 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000011254 conventional chemotherapy Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229940022353 herceptin Drugs 0.000 description 1
- 102000051957 human ERBB2 Human genes 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- BCFGMOOMADDAQU-UHFFFAOYSA-N lapatinib Chemical compound O1C(CNCCS(=O)(=O)C)=CC=C1C1=CC=C(N=CN=C2NC=3C=C(Cl)C(OCC=4C=C(F)C=CC=4)=CC=3)C2=C1 BCFGMOOMADDAQU-UHFFFAOYSA-N 0.000 description 1
- 229960004891 lapatinib Drugs 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 1
- 229960002087 pertuzumab Drugs 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003239 susceptibility assay Methods 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
Images
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
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q2600/118—Prognosis of disease development
-
- 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
Abstract
The invention provides a primer probe for detecting the amplification of a breast cancer recurrence transfer gene HER2 and application thereof, wherein the sequences of the primer probe are respectively an upstream primer sequence FZD2-F of a target gene: 15 '-GTGTGGCAGTTCAGCCCTAT-3' of SEQ ID No, a downstream primer sequence FZD2-R of a target gene: 25 '-AATGTGTGCCACGAAACTGC-3' of SEQ ID No, and a target gene probe sequence FZD 2-P: 35 '-FAM-GGCTGATGGCTCGCGCTGGGCA-BHQ 1-3' of SEQ ID No, and an upstream primer sequence EFTUD2-F of an internal reference gene: SEQ ID No. 45 '-CTCAAAGTGCGGGGACTGAT-3', reference gene downstream primer sequence EFTUD 2-R: 55 '-GGCATCAGGGTGACTCCAAA-3' of SEQ ID No, reference gene probe sequence EFTUD 2-P: SEQ ID No. 65 '-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ 1-3'. The primer probe is used for a digital PCR kit, and the kit can quickly, specifically and sensitively detect HER2 gene amplification. The kit has simple one-step amplification method and low cost, is very suitable for the gene copy number variation condition in various samples such as tumor tissues, circulating tumor cells, circulating tumor DNA and the like, and is suitable for large-scale popularization and application.
Description
Technical Field
The invention belongs to the technical field of tumor molecule detection, and particularly relates to a primer probe for detecting breast cancer recurrence metastasis gene HER2 amplification and application thereof.
Background
The breast cancer is the most common malignant tumor of women, the incidence rate of the tumor of high-lying women is the first, the first cause of death of female residents is occupied, and huge mental and economic burden is caused to families and society. The therapeutic effect of the targeted drug and the prognosis caused by the same are greatly different among patients with different tumors. Therefore, the dynamic detection of the related gene mutation of the tumor patient can accurately guide the clinical selection of the appropriate targeted drug.
HER2(ERBB2) is a member of the human Epidermal Growth Factor Receptor (EGFR) family and is a protooncogene. Over 30% of human tumors have HER-2 gene amplification/overexpression widely, and HER2 gene amplification/overexpression is not only related to the occurrence and development of tumors, but also is an important clinical treatment monitoring and prognosis index and an important target for tumor targeted treatment. HER2 gene amplification is present in about 15-30% of breast cancer patients. For HER2 positive tumors, high expression of the HER2 gene can result in overexpression of the HER2 protein, thereby stimulating the cancer cells to grow crazy, increase invasiveness, and relapse and metastasis relatively quickly. Therefore, HER2 positive tumors are often of higher malignancy, and the conventional chemotherapy treatment has poor effect and poor prognosis. Currently, there are targeted therapeutic schemes for HER2 positive breast cancer, and targeted therapeutic drugs include trastuzumab, pertuzumab, lapatinib, and the like. In the case of trastuzumab (herceptin), which was also the most successful at the earliest use, clinical studies have shown that trastuzumab can significantly prolong PFS (progression free survival) and OS (overall survival) in HER2 positive breast cancer patients. Trastuzumab combined with chemotherapy can reduce the recurrence risk of tumors by 52% and the death risk of patients by 33%. The HER2 gene amplification detection is listed as a breast cancer targeted therapy molecular marker recommended by NCCN at present.
The traditional HER2 gene detection methods include Immunohistochemistry (IHC) detection of the expression level of HER2 protein, in situ hybridization detection of the HER2 gene amplification level and the like. These methods rely on tumor tissue, are complicated in material selection, and cannot achieve dynamic detection. Digital PCR is the third generation PCR technology that has been developed in the last decade, and the main principle is to place a single DNA molecule in an independent reaction chamber, where each reaction chamber either contains no nucleic acid target molecule to be detected or at least one nucleic acid target molecule to be detected, and perform PCR amplification on it, and detect a specific target sequence using TaqMan chemical reagents and dye-labeled probes. After PCR amplification, an analyzer detects each microdroplet one by one, the microdroplet with a fluorescent signal is judged as '1', the microdroplet without the fluorescent signal is judged as '0', and finally the copy number concentration of the target molecule to be detected is given according to the Poisson distribution principle and the proportion of positive microdroplets. The result of digital PCR quantification does not depend on Ct value any more and directly gives the initial copy number concentration of the target sequence, has the advantages of accuracy, hypersensitiveness, insusceptibility to PCR inhibitor and the like, can detect low-abundance tumor DNA variation, reduces false negative results, and is particularly suitable for the field of liquid biopsy (such as circulating tumor cell CTC or circulating tumor DNA). Therefore, the development of related digital PCR technology for amplification detection of the breast cancer recurrence transfer gene HER2 is beneficial to dynamic disease course tracking and drug prognosis monitoring, greatly reduces health damage to patients caused by untimely or excessive medication and economic pressure and burden on individuals, families and national health care systems, and has great benefits for economic, social and scientific and technological development in our market.
Disclosure of Invention
The invention aims to provide a primer probe for detecting the amplification of a breast cancer recurrence transfer gene HER2 based on a digital PCR technology and application thereof, and in order to realize the purpose of the invention, the following technical scheme is adopted:
a primer probe for detecting the amplification of a breast cancer recurrence transfer gene HER2 based on a digital PCR technology is composed of a primer probe with a nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 6, and the sequence is as follows:
the sequence of the target gene upstream primer HER 2-F: 5'-GTGTGGCAGTTCAGCCCTAT-3' SEQ ID NO: 1;
the downstream primer sequence of the target gene HER 2-R: 5'-AATGTGTGCCACGAAACTGC-3' SEQ ID NO: 2;
target gene probe sequence HER 2-P: 5 '-FAM-GGCTGATGGCTCGCGCTGGGCA-BHQ 1-3' SEQ ID NO 3;
the upstream primer sequence of the internal reference gene EFTUD 2-F: 5'-CTCAAAGTGCGGGGACTGAT-3' SEQ ID NO 4;
the downstream primer sequence EFTUD2-R of the internal reference gene: 5'-GGCATCAGGGTGACTCCAAA-3' SEQ ID NO: 5;
reference gene probe sequence EFTUD 2-P: 5 '-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ 1-3' SEQ ID NO 6.
Preferably, the target gene primer probe is designed according to a gene coding region of a human gene HER2, the internal reference gene primer probe is designed according to a human EFTUD2 gene region, the sizes of amplified target fragments are 102bp and 109bp respectively, and an amplification sequence (102bp) is as follows:
5’-GTGTGGCAGTTCAGCCCTATGCCCCATGGCTGATGGCTCGCGCTGGGCAGGTATGCAGGGCTGACGTAGTGCCTTTGTGGCAGCAGTTTCGTGGCACACATT-3’。
the amplified sequence (109bp) was:
5’-CTCAAAGTGCGGGGACTGATTGTTAGCTCTAAAAGCTCTGGCACTATAGATAGTGATAGCCTGCTTCCTGGGAATGTGCTGCTAGAAGATTTGGAGTCACCCTGATGCC-3’。
preferably, the digital PCR primer probe is applied to the preparation of an amplification detection kit for detecting the breast cancer recurrence metastasis gene HER 2.
The invention also provides a digital PCR kit for detecting HER2 gene amplification, which comprises a primer probe with a nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 6.
The use method of the kit comprises the following steps:
(1) preparing a digital PCR reaction solution; the formulation of 20. mu.l of digital PCR reaction solution is: 4 mul of one-step reaction buffer solution, 2 mul of enzyme mixed solution, 1.2 mul of 10 mul of HER2-F, HER2-R, EFTUD2-F and EFTUD2-R primers respectively, 0.6 mul of 10 mul of MHER2-P and EFTUD2-P respectively, and 8 mul of DNA template of a sample to be detected;
(2) preparing microdroplets, transferring the microdroplets into a PCR plate, and carrying out amplification in a PCR instrument; reaction procedure: 15min at 45 ℃; 10min at 95 ℃; followed by 35 cycles, each cycle comprising 95 ℃ for 15s, 55 ℃ for 20s, 72 ℃ for 20 s; setting a temperature rise and fall speed of 2 ℃/sec for each step;
(3) and (3) putting the PCR plate subjected to the PCR amplification into a microdroplet analyzer, detecting microdroplets, analyzing data and displaying a detection result.
The method for judging the detection result of the kit comprises the following steps: (1) positive control: the copy number of the target gene/the copy number of the internal reference gene is more than 2.2; (2) negative control: the copy number of the target gene/the copy number of the internal reference gene is less than 1.8; and (3) judging the result of the sample to be detected: (1) positive: the copy number of the target gene/the copy number of the internal reference gene is more than 2.2; (2) negative: the copy number of the target gene/the copy number of the internal reference gene is less than 1.8; (3) suspected positive: 1.8< copy number of target gene/copy number of internal reference gene < 2.2.
The invention designs a pair of specific primers and probes according to the HER2 gene region, and designs a pair of primers and probes by taking the human EFTUD2 gene region as an internal reference, establishes a detection method based on the digital PCR technology, and has the advantages that:
(1) high sensitivity: the detection sensitivity reaches 10 ten thousand normal DNA molecules, one HER2 amplified molecule is detected, and the kit is suitable for liquid biopsy samples (such as circulating tumor cells CTC or circulating tumor DNA).
(2) High specificity: the specific primer probe designed aiming at the HER2 gene region can specifically detect the copy number change of the HER2 gene, and does not interfere with other homologous genes.
(3) The interference of complex samples on the experimental results can be eliminated: is not influenced by PCR inhibitors, can avoid PCR false negative caused by sample complexity, and is suitable for body fluid samples.
Drawings
FIG. 1 is a schematic diagram of an experimental result of the effectiveness of the primer provided by the present invention, which is a detection result of HER2 target genes, wherein the positions of the holes in the diagram are 3 HER2 gene copy number variation positive samples and 3 HER2 gene copy number variation negative samples in sequence from left to right, and channel 1 represents a FAM channel;
FIG. 2 is a schematic diagram showing the experimental results of the effectiveness of the primers provided by the present invention, which is the detection results of reference genes in human EFTUD2, wherein the positions of the holes in the diagram are 3 HER2 gene copy number variation positive samples and 3 HER2 gene copy number variation negative samples in sequence from left to right, and channel 2 represents a HEX channel;
FIG. 3 is a schematic diagram of the sensitivity test results of the primers provided by the present invention, which is the detection result of HER2 target gene, the holes G01-A02 are the detection results of the critical concentration positive sample, the holes B02-D02 are the detection results of no template control, and channel 1 represents the FAM channel;
FIG. 4 is a schematic diagram of the sensitivity test results of the primers provided by the present invention, which is the detection results of reference genes in human EFTUD2, wherein the positions G01-A02 are the detection results of positive samples with critical concentration, the positions B02-D02 are the detection results of no template control, and channel 2 represents the HEX channel.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation. The technical scheme of the invention is a conventional scheme in the field if not specifically stated, and the used reagents or raw materials are purchased from commercial sources or published if not specifically stated.
Example 1 design of specific primers and probes for detecting amplification of the breast cancer recurrence metastasis gene HER2
The design of specific primers and probes suitable for ddPCR is carried out by taking human HER2 as a target gene and human EFTUD2 as an internal reference gene, wherein the sequences of the primers and the probes are as follows:
the sequence of the target gene upstream primer HER 2-F: 5'-GTGTGGCAGTTCAGCCCTAT-3' SEQ ID NO: 1;
the downstream primer sequence of the target gene HER 2-R: 5'-AATGTGTGCCACGAAACTGC-3' SEQ ID NO: 2;
target gene probe sequence HER 2-P: 5 '-FAM-GGCTGATGGCTCGCGCTGGGCA-BHQ 1-3' SEQ ID NO 3;
the upstream primer sequence of the internal reference gene EFTUD 2-F: 5'-CTCAAAGTGCGGGGACTGAT-3' SEQ ID NO 4;
the downstream primer sequence EFTUD2-R of the internal reference gene: 5'-GGCATCAGGGTGACTCCAAA-3' SEQ ID NO: 5;
reference gene probe sequence EFTUD 2-P: 5 '-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ 1-3' SEQ ID NO 6;
example 2 detection of amplification of the breast cancer recurrence metastasis Gene HER2 Using digital PCR detection primers and probes
1. Plasma free DNA extraction
Nucleic acid extraction of the Plasma sample was carried out using a NucleoSpin Plasma XS kit (cat # 740901.50) manufactured by MNG. Mu.l of plasma was taken, nucleic acid extraction was performed according to the extraction kit instructions, and finally resuspended with 20. mu.l of eluent.
2. Digital PCR amplification reaction
(1) Preparing a digital PCR reaction solution; the formulation of 20. mu.l of digital PCR reaction solution is: 4 mul of one-step reaction buffer solution, 2 mul of enzyme mixed solution, 1.2 mul of 10 mul of HER2-F, HER2-R, EFTUD2-F and EFTUD2-R primers respectively, 0.6 mul of 10 mul of MHER2-P and EFTUD2-P respectively, and 8 mul of DNA template of a sample to be detected;
(2) on the production chip of the droplet PCR, 20. mu.L of the droplet PCR reaction system of step 1) was added, and 40. mu.L of the droplet-producing oil was added to carry out the droplet-producing step, and after completion, the reaction system formed into droplets was transferred to a 96-well plate and sealed.
(3) Putting the 96-well plate after the membrane sealing into a PCR instrument for amplification; reaction procedure: 15min at 45 ℃; 10min at 95 ℃; followed by 35 cycles, each cycle comprising 95 ℃ for 15s, 55 ℃ for 20s, 72 ℃ for 20 s; setting a temperature rise and fall speed of 2 ℃/sec for each step;
(4) the PCR plate with completed PCR amplification is put into a microdroplet analyzer, microdroplets are detected, data are analyzed, and the detection result is displayed (FIG. 1-FIG. 2).
(5) The determination method of the detection result comprises the following steps: (1) positive control: the copy number of the target gene/the copy number of the internal reference gene is more than 2.2; (2) negative control: the copy number of the target gene/the copy number of the internal reference gene is less than 1.8; and (3) judging the result of the sample to be detected: (1) positive: the copy number of the target gene/the copy number of the internal reference gene is more than 2.2; (2) negative: the copy number of the target gene/the copy number of the internal reference gene is less than 1.8; (3) suspected positive: 1.8< copy number of target gene/copy number of internal reference gene < 2.2.
TABLE 1 detection results of HER2 gene and EFTUD2 gene in different samples
EXAMPLE 3 digital PCR primer Probe sensitivity assay for Breast cancer samples
The experiment was performed using a gradient dilution of breast cancer positive samples. The detection was carried out by the detection method given in example 2. The results show that the ontology can detect the number of copies that differ from the no template control at 0.001% template amount (FIGS. 3-4).
TABLE 2 Positive sample HER2 gene and EFTUD2 gene test results under critical concentration
Sequence listing
<110> Shenzhen Huaxinkang Gene science and technology Limited
<120> primer probe for detecting breast cancer recurrence transfer gene HER2 amplification and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 1
gtgtggcagt tcagccctat 20
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 2
aatgtgtgcc acgaaactgc 20
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 3
ggctgatggc tcgcgctggg ca 22
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 4
ctcaaagtgc ggggactgat 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 5
ggcatcaggg tgactccaaa 20
<210> 6
<211> 26
<212> DNA
<213> Artificial sequence ()
<400> 6
agcctgcttc ctgggaatgt gctgct 26
Claims (3)
1. A primer probe for detecting the amplification of a breast cancer recurrence transfer gene HER2 is characterized in that the sequences of the primer probe are respectively:
the upstream primer sequence of the target gene FZD 2-F: 15 '-GTGTGGCAGTTCAGCCCTAT-3' of SEQ ID No,
The downstream primer sequence of the target gene FZD 2-R: SEQ ID Nos 25 '-AATGTGTGCCACGAAACTGC-3',
the sequence of the target gene probe FZD 2-P: 35 '-FAM-GGCTGATGGCTCGCGCTGGGCA-BHQ 1-3' of SEQ ID No,
The upstream primer sequence of the internal reference gene EFTUD 2-F: SEQ ID Nos 45 '-CTCAAAGTGCGGGGACTGAT-3',
the downstream primer sequence EFTUD2-R of the internal reference gene: SEQ ID Nos 55 '-GGCATCAGGGTGACTCCAAA-3',
reference gene probe sequence EFTUD 2-P: SEQ ID No. 65 '-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ 1-3'.
2. The primer probe of claim 1, wherein the primer probe sequences SEQ ID NO 1-SEQ ID NO 3 are designed according to the coding region of human HER2 gene, the amplified target fragment is 102bp, SEQ ID NO 4-SEQ ID NO 6 are designed according to the region of human EFTUD2 gene, and the amplified target fragment is 109 bp.
3. Use of the primer probe of claims 1-2 in the preparation of a digital PCR kit for detecting amplification of FZD2 gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110674748.XA CN113293213A (en) | 2021-06-17 | 2021-06-17 | Primer probe for detecting breast cancer recurrence transfer gene HER2 amplification and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110674748.XA CN113293213A (en) | 2021-06-17 | 2021-06-17 | Primer probe for detecting breast cancer recurrence transfer gene HER2 amplification and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113293213A true CN113293213A (en) | 2021-08-24 |
Family
ID=77328685
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110674748.XA Pending CN113293213A (en) | 2021-06-17 | 2021-06-17 | Primer probe for detecting breast cancer recurrence transfer gene HER2 amplification and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113293213A (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130034546A1 (en) * | 2010-01-19 | 2013-02-07 | Verinata Health, Inc. | Analyzing Copy Number Variation in the Detection of Cancer |
| US20150139946A1 (en) * | 2013-10-19 | 2015-05-21 | Trovagene, Inc. | Detecting mutations in disease over time |
| CA2941161A1 (en) * | 2014-03-13 | 2015-09-17 | F.Hoffmann-La Roche Ag | Methods and compositions for modulating estrogen receptor mutants |
| CN105986028A (en) * | 2015-03-23 | 2016-10-05 | 上海宝藤生物医药科技股份有限公司 | Method for detecting HER 2 positivity of breast cancer through ddPCR technology |
| CN106591438A (en) * | 2016-11-28 | 2017-04-26 | 华东医药（杭州）基因科技有限公司 | Nucleic acid combination for detecting Her2 gene, kit and application |
| CN108504742A (en) * | 2018-06-25 | 2018-09-07 | 北京爱普拜生物技术有限公司 | A kind of kit and method detecting HER-2 gene copy number variations based on digital pcr technology |
| CN110616251A (en) * | 2019-09-29 | 2019-12-27 | 中国计量科学研究院 | Digital PCR method and detection kit for detecting breast cancer HER2 copy number variation |
| CN110951843A (en) * | 2019-12-12 | 2020-04-03 | 苏州绘真医学检验有限公司 | Kit and method for detecting HER2 copy number variation of CTCs based on ddPCR |
| CN210340904U (en) * | 2019-08-19 | 2020-04-17 | 艾普拜生物科技(苏州)有限公司 | Kit for detecting HER-2 gene copy number variation based on digital PCR technology |
| CN111307561A (en) * | 2020-02-27 | 2020-06-19 | 菁良基因科技(深圳)有限公司 | Paraffin embedding reference substance for gene detection and preparation method and application thereof |
-
2021
- 2021-06-17 CN CN202110674748.XA patent/CN113293213A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130034546A1 (en) * | 2010-01-19 | 2013-02-07 | Verinata Health, Inc. | Analyzing Copy Number Variation in the Detection of Cancer |
| US20150139946A1 (en) * | 2013-10-19 | 2015-05-21 | Trovagene, Inc. | Detecting mutations in disease over time |
| CA2941161A1 (en) * | 2014-03-13 | 2015-09-17 | F.Hoffmann-La Roche Ag | Methods and compositions for modulating estrogen receptor mutants |
| CN105986028A (en) * | 2015-03-23 | 2016-10-05 | 上海宝藤生物医药科技股份有限公司 | Method for detecting HER 2 positivity of breast cancer through ddPCR technology |
| CN106591438A (en) * | 2016-11-28 | 2017-04-26 | 华东医药（杭州）基因科技有限公司 | Nucleic acid combination for detecting Her2 gene, kit and application |
| CN108504742A (en) * | 2018-06-25 | 2018-09-07 | 北京爱普拜生物技术有限公司 | A kind of kit and method detecting HER-2 gene copy number variations based on digital pcr technology |
| CN210340904U (en) * | 2019-08-19 | 2020-04-17 | 艾普拜生物科技(苏州)有限公司 | Kit for detecting HER-2 gene copy number variation based on digital PCR technology |
| CN110616251A (en) * | 2019-09-29 | 2019-12-27 | 中国计量科学研究院 | Digital PCR method and detection kit for detecting breast cancer HER2 copy number variation |
| CN110951843A (en) * | 2019-12-12 | 2020-04-03 | 苏州绘真医学检验有限公司 | Kit and method for detecting HER2 copy number variation of CTCs based on ddPCR |
| CN111307561A (en) * | 2020-02-27 | 2020-06-19 | 菁良基因科技(深圳)有限公司 | Paraffin embedding reference substance for gene detection and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| NADIA HOWLADER等: ""US incidence of breast cancer subtypes defined by joint hormone receptor and HER2 status"", 《FULL TEXT LINKS》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2018086263A1 (en) | Real-time fluorescent quantitative pcr detection method, and standard sample and detection kit thereof | |
| CN104762408B (en) | Detect the kit and its detection method of EGFR genetic mutation | |
| CN108949990A (en) | A kind of kit and method detecting EGFR genetic mutation | |
| CN104774962A (en) | Kit for detecting BRAF gene mutation and detecting method thereof | |
| CN105112522A (en) | Detection primer combination and kit for HER2 (human epidermal growth factor receptor 2) gene amplification | |
| CN104789677A (en) | Circulating tumor DNA EGFR detecting technology and reagent kit thereof | |
| CN106755297A (en) | One group is based on primer sets that ARMS fluorescence quantitative PCR detection EGFR genes T790M is mutated and preparation method thereof | |
| CN104774963A (en) | Kit for detecting PIK3CA gene mutation and detecting method thereof | |
| CN111349702A (en) | Marker for predicting prognosis of cancer patient and application thereof | |
| CN110257516A (en) | For developing molecular marker and the application of diagnosing gastric cancer product | |
| CN110093423A (en) | Purposes of the gene marker in adenocarcinoma of lung diagnosis and treatment | |
| CN109371022A (en) | A kind of circular rna hsa_circKPNA2_002 and its specificity amplification primer and application | |
| CN106498029A (en) | The method for improving the diagnosis efficiency of the T790M mutation of EGFR | |
| CN112852967A (en) | Kit for quantitatively detecting ALK, RET and ROS1 fusion genes based on ddPCR | |
| CN108624693A (en) | Applications of the miR-577 in preparing diagnosis of nephropathy marker | |
| CN109022583A (en) | Hsa_circ_0021977 is preparing the application on Diagnosis of Breast cancer product | |
| CN112553337A (en) | Method for detecting ERBB2 gene amplification based on digital PCR technology and detection kit thereof | |
| CN113293213A (en) | Primer probe for detecting breast cancer recurrence transfer gene HER2 amplification and application thereof | |
| CN109666746A (en) | For detecting primer, probe and the kit and its detection method of the mutation of mankind's ROS1 Gene Fusion | |
| CN107760787A (en) | A kind of breast cancer molecular parting real-time fluorescence multiple PCR primer and kit and application | |
| CN106811537A (en) | One kind detection epidermal growth factor receptor gene T790M low frequencies mutant primer and its application | |
| CN108823310B (en) | Complete set of reagent for detecting DPEP1 gene expression level and application thereof | |
| CN107904309B (en) | It is a kind of for diagnose indication three negative type breast cancers Bone tumours gene diagnosis kit | |
| CN106434968B (en) | Application of the CCDC168 as marker in detection cancer of pancreas reagent | |
| CN102242209B (en) | Quantitative detection kit based on multiple genes for assisting diagnosis of patients with multiple myeloma |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210824 |