CN113667749A - Diagnostic kit for evaluating breast cancer risk by combining four key genes - Google Patents

Diagnostic kit for evaluating breast cancer risk by combining four key genes Download PDF

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CN113667749A
CN113667749A CN202110888350.6A CN202110888350A CN113667749A CN 113667749 A CN113667749 A CN 113667749A CN 202110888350 A CN202110888350 A CN 202110888350A CN 113667749 A CN113667749 A CN 113667749A
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breast cancer
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侯铁英
张喜钦
张玉
陈杰荣
盛梦秋
朱丹丹
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Guangdong General Hospital
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Abstract

The invention relates to the technical field of medicine, in particular to a diagnostic kit for evaluating breast cancer risk by combining four key genes, which comprises a standard substance, primers of four specific genes and internal reference genes, wherein the specific detection genes and the internal reference genes are respectively as follows: DAB2(ID:1601), KDELC1(ID:79070), G6PD (ID:2539), PPP1CB (ID:5500) and β -actin (GeneID: 60); the invention relates to a method for evaluating the morbidity risk of breast cancer, which comprises the steps of analyzing by using methods such as multiple SNP sites or single susceptibility genes and the like at present, having poor practicability, lacking effective sensitivity and specificity and rarely being used for actual detection and application.

Description

Diagnostic kit for evaluating breast cancer risk by combining four key genes
Technical Field
The invention relates to the technical field of medicine, in particular to a diagnostic kit for evaluating breast cancer risk by combining four key genes.
Background
Breast cancer is one of the most common nausea tumors in women worldwide, and the incidence is first in women throughout the year, and is the second leading factor of cancer-related deaths in women, second only to lung cancer. The incidence rate of the breast cancer is more than 7 percent of various malignant tumors of the whole body, the breast cancer has strong metastatic capacity, and the prognosis of general patients is poor. With the increasing environmental and population aging, the incidence of breast cancer tends to rise significantly. Early detection and timely treatment are the key points for preventing and treating the breast cancer.
However, the conventional methods for evaluating the morbidity risk of the breast cancer, such as a multi-purpose SNP (single nucleotide polymorphism) locus or a single susceptibility gene, are used for analyzing, and risk evaluation, have poor practicability and lack of effective sensitivity and specificity, and can be rarely used for actual detection and application, so that combined detection of some genes associated with the breast cancer is found, timeliness and accuracy of the breast cancer risk evaluation can be greatly improved, and therefore, a simple, reliable and rapid high-efficiency detection method for detecting the associated genes can be established to screen high-risk groups susceptible to the breast cancer in time, and preventive measures can be taken as soon as possible according to results, which has important significance for improving early diagnosis and treatment of the breast cancer.
Disclosure of Invention
The invention aims to provide a diagnostic kit for evaluating breast cancer risk by combining four key genes, which solves the problems that the existing common methods for evaluating the morbidity risk of breast cancer, such as multi-purpose SNP loci or single susceptibility genes, have poor practicability, lack of effective sensitivity and specificity and can hardly be used for actual detection and application.
In order to achieve the purpose, the invention provides the following technical scheme: the diagnostic kit for evaluating the breast cancer risk by combining four key genes comprises a standard substance, primers of four specific genes and an internal reference gene, wherein the specific detection genes and the internal reference gene are respectively as follows: DAB2(ID:1601), KDELC1(ID:79070), G6PD (ID:2539), PPP1CB (ID:5500) and beta-actin (GeneID:60), and the primers can specifically amplify the corresponding genes.
Preferably, the partial sequence of the DAB2 gene is as follows:
AGTCCTCAGCTGCCAGCATCTGATTAGAACCATATCTCTCGCCGGGAGTGGCCGCGCGGCTC CGAAGCTCCCGGCCGGCGGCTATTTAAGCGAGGCCCGCCGCATCCGCTGCGCTGTAGCCTGGAGGC TCCGGGCGCGGGGAAGTCATGCTCGCTTCACGGAGGCAATAGCTAGCCGGTGTCTGTGGGAGGTTA TGTTTATTTGAGACTTCTCCATCGGGATCGCCTGGTGTCACCAAGTGTCCACTGGTACTGAGGTTT GCTGCCTGCCTTCTTGCCATGTCTAACGAAGTAGAAACAAGTGCAACCAATGGTCAGCCCGACCAA CAGGCCGCACCAAAAGCACCCTCAAAGAAGGAAAAAAAGAAAGGCCCTGAAAAGACAGATGAATAT CTCTTAGCAAGGTTCAAAGGCGATGGTGTAAAATATAAGGCCAAGCTGATTGGCATTGATGATGTG CCAGATGCAAGAGGGGATAAAATGAGCCAAGACTCTATGATG(SEQ ID NO：1)。
preferably, the KDELC1 gene partial sequence:
AAATGATGCTCCAGTGGCAGGAGCAACTCAAGTTCATCATTGTCCTGAGAGAGAGGAGCAGC GCGGTTCTCGGCCGGGACAGCAGAACGCCAGGGGACCCTCACCTGGGCGCGCCGGGGCACGGGCTT TGATTGTCCTGGGGTCGCGGAGACCCGCGCGCCTGCCCTGCACGCCGGGCGGCAACCTTTGCAGTC GCGTTGGCTGCTGCGATCGGCCGGCGGGTCCCTGCCGAAGGCTCGGCTGCTTCTGTCCACCTCTTA CACTTCTTCATTTATCGGTGGATCATTTCGAGAGTCCGTCTTGTAAATGTTTGGCACTTTGCTACT TTATTGCTTCTTTCTGGCGACAGTTCCAGCACTCGCCGAGACCGGCGGAGAAAGGCAGCTGAGCCC GGAGAAGAGCGAAATATGGGGACCCGGGCTAAAAGCAGACGTCGTCCTTCCCGCCCGCTATTTCTA TATTCAGGCAGTGGATACATCAGGGAATAAATTCACATCTTC(SEQ ID NO：2)。
preferably, the partial sequence of the G6PD gene:
AGAGGCAGGGGCTGGCCTGGGATGCGCGCGCACCTGCCCTCGCCCCGCCCCGCCCGCACGAG GGGTGGTGGCCGAGGCCCCGCCCCGCACGCCTCGCCTGAGGCGGGTCCGCTCAGCCCAGGCGCCCG CCCCCGCCCCCGCCGATTAAATGGGCCGGCGGGGCTCAGCCCCCGGAAACGGTCGTACACTTCGGG GCTGCGAGCGCGGAGGGCGACGACGACGAAGCGCAGACAGCGTCATGGCAGAGCAGGTGGCCCTGA GCCGGACCCAGGTGTGCGGGATCCTGCGGGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGG ATACACACATATTCATCATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCA TCTGGTGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCCGTTCCC GCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCT(SEQ ID NO：3)。
preferably, the PPP1CB gene partial sequence:
GCGGAGCTGGGCGGTGCCGAGGAGGAGGAGGTGGCGGCCTGGGTCTGACGCGGCCCTGTTCG AGGGGGCCTCTCTTGTTTATTTATTTATTTTCCGTGGGTGCCTCCGAGTGTGCGCGCGCTCTCGCT ACCCGGCGGGGAGGGGGTGGGGGGAGGGCCCGGGAAAAGGGGGAGTTGGAGCCGGGGTCGAAACGC CGCGTGACTTGTAGGTGAGAGAACGCCGAGCCGTCGCCGCAGCCTCCGCCGCCGAGAAGCCCTTGT TCCCGCTGCTGGGAAGGAGAGTCTGTGCCGACAAGATGGCGGACGGGGAGCTGAACGTGGACAGCC TCATCACCCGGCTGCTGGAGGTACGAGGATGTCGTCCAGGAAAGATTGTGCAGATGACTGAAGCAG AAGTTCGAGGCTTATGTATCAAGTCTCGGGAGATCTTTCTCAGCCAGCCTATTCTTTTGGAATTGG AAGCACCGCTGAAAATTTGTGGAGATATTCATGGACAATATA(SEQ ID NO：4)。
preferably, the beta-actin gene partial sequence:
gttgctatccaggctgtgctatccctgtacgcctctggccgtaccactggcatcgtgatgga ctccggtgacggggtcacccacactgtgcccatctacgaggggtatgccctcccccatgccatcct gcgtctggacctggctggccgggacctgactgactacctcatgaagatcctcaccgagcgcggcta cagcttcaccaccacggccgagcgggaaatcgtgcgtgacattaaggagaagctgtgctacgtcgc cctggacttcgagcaagagatggccacggctgcttccagctcctccctggagaagagctacgagct gcctgacggccaggtcatcaccattggcaatgagcggttccgctgccctgaggcactcttccagcc ttccttcctgggcatggagtcctgtggcatccacgaaactaccttcaactccatcatgaagtgtga cgtggacatccgcaaagacctgtacgccaacacagtgctgtc(SEQ ID NO：5)。
compared with the prior art, the invention has the following beneficial effects:
at present, the common methods for evaluating the morbidity risk of breast cancer, such as multi-purpose SNP loci or single susceptibility gene, and the like, have poor practicability, lack of effective sensitivity and specificity, and can be rarely used for actual detection and application, the gene combination for detection used by the invention comprises four breast cancer related specific expression genes, meanwhile, the detection result is effectively corrected by using the standard substance and the reference gene, and the morbidity risk of the breast cancer can be effectively predicted through combined detection and analysis, when the ct value of the DAB2 gene is less than that of the standard product among the four detected genes, meanwhile, when the ct values of KDELC1, G6PD and PPP1CB genes are all greater than the ct value of the standard substance, early warning that breast cancer is ill is high risk, and the kit is used for risk assessment, so that the practicability is high, the sensitivity and the specificity can be effectively improved, and the kit is more suitable for actual detection and application.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The diagnostic kit for evaluating the breast cancer risk by combining four key genes comprises a standard substance, primers of four specific genes and an internal reference gene, wherein the specific detection genes and the internal reference gene are respectively as follows: DAB2(ID:1601), KDELC1(ID:79070), G6PD (ID:2539), PPP1CB (ID:5500) and beta-actin (GeneID:60), and the primers can specifically amplify the corresponding genes.
The kit is a box for containing chemical reagents for detecting chemical components, drug residues, virus species and the like. Is generally used by hospitals and pharmaceutical enterprises.
Common elisa kits are:
the special protein detection elisa kit comprises:
such as immunoglobulin, anti-chain O-aso, rheumatoid factor RF, C-reactive protein, microalbumin, beta-2 microglobulin human, ferritin, transferrin, and the like.
Tumor marker detection elisa kit:
such as tumor markers, tissue polypeptide antigens, tumor-associated factors, pancreatic cancer, rectal cancer, cytokeratin fragments, gastrointestinal cancer, ferritin, carbohydrate chain antigens, nerve-specific rare-earth alcoholase, epithelial membrane antigens, breast cancer, human anti-mouse antibodies, prostate, alpha-fetoprotein, liver cancer, methamphetamine, colorectal cancer, lung cancer, occult blood tests of stool and urine, carcinoembryonic antigen, beta-2 microglobulin and the like.
Food safety test elisa kit:
a detection kit for hormone, medicine, mycotoxin, allergen residue and transgenic products in food, and detection products for microorganisms, vitamins and the like. Such as plant virus, bacteria, fungi, plant hormone and transgenic crops, animal and plant disease diagnosis such as pig, cattle, sheep, horse, livestock and poultry and pet detection kit.
Infectious disease detection elisa kit:
such as helicobacter pylori, encephalitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis Heptanthus, Chlamydia, venereal disease, adenovirus, parvovirus B19, pemphigus, varicella-zoster virus, Mycoplasma genitalium, typhoid fever, trachoma, mumps, Mycoplasma hominis, measles, rotavirus, epidemic hemorrhagic fever, gonococcus, Lyme disease, Coxsackie, ureaplasma antidiuresis, Legionella, tuberculosis, collagen, condyloma acuminatum, hepatitis A, poliomyelitis, acute pancreatitis, urinary trypsin, cholera, respiratory syncytial virus, liver fluke, parainfluenza, pneumonia, herpes zoster, infectious mononucleosis, laminin, Brucella, pertussis, diphtheria, echovirus, EB virus, group A streptococcus, and the like.
Dab2 is a phosphoprotein with signal transduction ability, plays an important role in the mitogen signal transduction pathway (MAPK pathway), and plays its role through the interaction with Dab2 binding protein (Dab2IP) and protein phosphorylation, and is involved in the regulation of cell growth.
disable-2(Dab2/DOC-2) is a mitogen-responsive adaptor protein involved in the progression of multiple cellular functions. It participates in many signaling pathways and plays an important role in regulating immune function, protein-protein interactions, cell homeostasis and differentiation, tumorigenesis and organ system inflammation. It contains domains that bind to adaptor proteins containing the NPXY motif and containing the SH3 domain, phosphatidylinositol, integrin, clathrin and myosin VI.
KDELC1 is a member of the endoplasmic reticulum protein family and encodes a protein comprising a lysl-asp-glu-leu or KDEL motif, located at the c-terminus, which prevents secretion of all endoplasmic reticulum resident proteins.
Glucose-6-phosphate dehydrogenase (G6PD) is a widely expressed enzyme that has a housekeeping role in all cells, and is particularly critical to the integrity and function of erythrocytes.
The PPP1CB gene is involved in glycogen metabolism and muscle contraction, and is a myosin light chain phosphatase responsible for transient elevation of calcium ions and regulation of cell mitosis and other processes.
Each part of the kit comprises:
reverse transcription translation system: 4ul of 5X reverse transcription buffer solution, 1ul of 10nm dNTP, 1ul of oligo primer, 1ul of random primer, 1ul of reverse transcriptase, 0.5ul of RNase inhibitor and 12ul of enzyme-free water;
and (3) PCR reaction system: SYBR GREEN 2XPCR mixed reaction buffer 10ul, each primer 1ul, ddH2O 7 ul;
and (3) standard substance: 4 ul. The standard substance is prepared by mixing the following components in proportion: DAB 2: KDELC 1: g6 PD: PPP1 CB: β -actin ═ 1: 2.5: 2.5: 2: 2.
in this example, the partial sequence of the DAB2 gene:
AGTCCTCAGCTGCCAGCATCTGATTAGAACCATATCTCTCGCCGGGAGTGGCCGCGCGGCTC CGAAGCTCCCGGCCGGCGGCTATTTAAGCGAGGCCCGCCGCATCCGCTGCGCTGTAGCCTGGAGGC TCCGGGCGCGGGGAAGTCATGCTCGCTTCACGGAGGCAATAGCTAGCCGGTGTCTGTGGGAGGTTA TGTTTATTTGAGACTTCTCCATCGGGATCGCCTGGTGTCACCAAGTGTCCACTGGTACTGAGGTTT GCTGCCTGCCTTCTTGCCATGTCTAACGAAGTAGAAACAAGTGCAACCAATGGTCAGCCCGACCAA CAGGCCGCACCAAAAGCACCCTCAAAGAAGGAAAAAAAGAAAGGCCCTGAAAAGACAGATGAATAT CTCTTAGCAAGGTTCAAAGGCGATGGTGTAAAATATAAGGCCAAGCTGATTGGCATTGATGATGTG CCAGATGCAAGAGGGGATAAAATGAGCCAAGACTCTATGATG(SEQ ID NO：1)。
in this example, the partial sequence of KDELC1 gene:
AAATGATGCTCCAGTGGCAGGAGCAACTCAAGTTCATCATTGTCCTGAGAGAGAGGAGCAGCGCGG TTCTCGGCCGGGACAGCAGAACGCCAGGGGACCCTCACCTGGGCGCGCCGGGGCACGGGCTTTGAT TGTCCTGGGGTCGCGGAGACCCGCGCGCCTGCCCTGCACGCCGGGCGGCAACCTTTGCAGTCGCGT TGGCTGCTGCGATCGGCCGGCGGGTCCCTGCCGAAGGCTCGGCTGCTTCTGTCCACCTCTTACACT TCTTCATTTATCGGTGGATCATTTCGAGAGTCCGTCTTGTAAATGTTTGGCACTTTGCTACTTTAT TGCTTCTTTCTGGCGACAGTTCCAGCACTCGCCGAGACCGGCGGAGAAAGGCAGCTGAGCCCGGAG AAGAGCGAAATATGGGGACCCGGGCTAAAAGCAGACGTCGTCCTTCCCGCCCGCTATTTCTATATT CAGGCAGTGGATACATCAGGGAATAAATTCACATCTTC(SEQ ID NO：2)。
in this example, the partial sequence of the G6PD gene:
AGAGGCAGGGGCTGGCCTGGGATGCGCGCGCACCTGCCCTCGCCCCGCCCCGCCCGCACGAG GGGTGGTGGCCGAGGCCCCGCCCCGCACGCCTCGCCTGAGGCGGGTCCGCTCAGCCCAGGCGCCCG CCCCCGCCCCCGCCGATTAAATGGGCCGGCGGGGCTCAGCCCCCGGAAACGGTCGTACACTTCGGG GCTGCGAGCGCGGAGGGCGACGACGACGAAGCGCAGACAGCGTCATGGCAGAGCAGGTGGCCCTGA GCCGGACCCAGGTGTGCGGGATCCTGCGGGAAGAGCTTTTCCAGGGCGATGCCTTCCATCAGTCGG ATACACACATATTCATCATCATGGGTGCATCGGGTGACCTGGCCAAGAAGAAGATCTACCCCACCA TCTGGTGGCTGTTCCGGGATGGCCTTCTGCCCGAAAACACCTTCATCGTGGGCTATGCCCGTTCCC GCCTCACAGTGGCTGACATCCGCAAACAGAGTGAGCCCTTCT(SEQ ID NO：3)。
in this example, the PPP1CB gene partial sequence:
GCGGAGCTGGGCGGTGCCGAGGAGGAGGAGGTGGCGGCCTGGGTCTGACGCGGCCCTGTTCG AGGGGGCCTCTCTTGTTTATTTATTTATTTTCCGTGGGTGCCTCCGAGTGTGCGCGCGCTCTCGCT ACCCGGCGGGGAGGGGGTGGGGGGAGGGCCCGGGAAAAGGGGGAGTTGGAGCCGGGGTCGAAACGC CGCGTGACTTGTAGGTGAGAGAACGCCGAGCCGTCGCCGCAGCCTCCGCCGCCGAGAAGCCCTTGT TCCCGCTGCTGGGAAGGAGAGTCTGTGCCGACAAGATGGCGGACGGGGAGCTGAACGTGGACAGCC TCATCACCCGGCTGCTGGAGGTACGAGGATGTCGTCCAGGAAAGATTGTGCAGATGACTGAAGCAG AAGTTCGAGGCTTATGTATCAAGTCTCGGGAGATCTTTCTCAGCCAGCCTATTCTTTTGGAATTGG AAGCACCGCTGAAAATTTGTGGAGATATTCATGGACAATATA(SEQ ID NO：4)。
in this example, the partial sequence of the beta-actin gene:
gttgctatccaggctgtgctatccctgtacgcctctggccgtaccactggcatcgtgatgga ctccggtgacggggtcacccacactgtgcccatctacgaggggtatgccctcccccatgccatcct gcgtctggacctggctggccgggacctgactgactacctcatgaagatcctcaccgagcgcggcta cagcttcaccaccacggccgagcgggaaatcgtgcgtgacattaaggagaagctgtgctacgtcgc cctggacttcgagcaagagatggccacggctgcttccagctcctccctggagaagagctacgagct gcctgacggccaggtcatcaccattggcaatgagcggttccgctgccctgaggcactcttccagcc ttccttcctgggcatggagtcctgtggcatccacgaaactaccttcaactccatcatgaagtgtga cgtggacatccgcaaagacctgtacgccaacacagtgctgtc(SEQ ID NO：5)。
the working principle is as follows: when the diagnostic kit for evaluating breast cancer risk by using the combined gene detection method is used in a blood sample, 1ml of venous blood of a patient is firstly extracted, total RNA is extracted by using a chloroform method, then quantitative analysis is carried out on the RNA, 100ug of the total RNA is used as an initial amount to carry out the next operation, then a reverse transcription reagent provided by the kit is used for carrying out reverse transcription reaction, the system is 20ul, and the reaction conditions are as follows: 42 ℃/60 minutes and 75 ℃/5 minutes, then using a PCR reagent provided by the kit to perform PCR reaction, taking 1ul of the reverse transcription reaction product as a template, and respectively and independently amplifying DAB2, KDELC1, G6PD, PPP1CB and beta-actin, wherein the used primers are respectively as follows: (1) DAB2 forward primer: 5 '-TTCACAGACAGATCTGGAGC-3' (SEQ ID NO: 6), reverse primer: 5 '-TCCTCTTCCACAGGAAGGAT-3' (SEQ ID NO: 7); (2) KDELC1 forward primer: 5 '-GAGACTCGAGCTGGTTAAAC-3' (SEQ ID NO: 8), reverse primer: 5 '-TAAGCTGCTACAGTGCCATC-3' (SEQ ID NO: 9); (3) g6PD forward primer: 5 '-GAAGAGAGTGGGTTTCCAGT-3' (SEQ ID NO: 10), reverse primer: 5 '-ATGTGCAGCTGAGGTCAATG-3' (SEQ ID NO: 11); (4) PPP1CB forward primer: 5 '-AGCTACCCAGTATCCATGCT-3' (SEQ ID NO: 12), reverse primer: 5 '-CGTGAACACACTCATACTTC-3' (SEQ ID NO: 13); (5) beta-actin forward primer: 5 '-TCGTGCGTGACATTAAGGAG-3' (SEQ ID NO: 14), reverse primer: 5 '-AACCGCTCATTGCCAATGGT-3' (SEQ ID NO: 15), simultaneously carrying out PCR reaction of the standard substance, taking 1ul of the standard substance provided in the kit as a template, and respectively and independently amplifying DAB2, KDELC1, G6PD, PPP1CB and beta-actin, wherein the primers are respectively as follows: (1) DAB2 forward primer: 5 '-TTCACAGACAGATCTGGAGC-3' (SEQ ID NO: 16), reverse primer: 5 '-TCCTCTTCCACAGGAAGGAT-3' (SEQ ID NO: 17); (2) KDELC1 forward primer: 5 '-GAGACTCGAGCTGGTTAAAC-3' (SEQ ID NO: 18), reverse primer: 5 '-TAAGCTGCTACAGTGCCATC-3' (SEQ ID NO: 19); (3) g6PD forward primer: 5 '-GAAGAGAGTGGGTTTCCAGT-3' (SEQ ID NO: 20), reverse primer: 5 '-ATGTGCAGCTGAGGTCAATG-3' (SEQ ID NO: 21); (4) PPP1CB forward primer: 5 '-AGCTACCCAGTATCCATGCT-3' (SEQ ID NO: 22), reverse primer: 5 '-CGTGAACACACTCATACTTC-3' (SEQ ID NO: 23); (5) beta-actin forward primer: 5 '-TCGTGCGTGACATTAAGGAG-3' (SEQ ID NO: 24), reverse primer: 5 '-AACCGCTCATTGCCAATGGT-3' (SEQ ID NO: 25), the reaction system for PCR amplification is as follows: SYBR GREEN 2XPCR mixed reaction buffer 10ul, each primer 1ul, ddH2O 7ul, reverse transcription product 1ul or standard 1ul, and PCR reaction conditions are as follows: activating a denaturing enzyme at 94 ℃/5 minutes, denaturing at 94 ℃/30 seconds, annealing at 60 ℃/30 seconds, extending at 72 ℃/30 seconds, performing 40 cycles, using an instrument as a real-time quantitative PCR instrument, and finally performing result analysis, wherein the data of PCR off-line adopts a method for directly reading ct values, firstly correcting through an internal reference gene, comparing the corrected data with a standard product, and warning that the breast cancer is ill at high risk when the ct value of DAB2 gene is less than the ct value of the standard product and the ct values of KDELC1, G6PD and PPP1CB genes are greater than the ct value of the standard product;
when the diagnosis kit for evaluating breast cancer risk by combined gene detection method is used in biopsy tissue, the tissue sample taken out by biopsy firstly uses chloroform method to extract total RNA, RNA is quantitatively analyzed, 100ug of total RNA is used as initial amount to carry out next operation, then reverse transcription reaction is carried out by using reverse transcription reagent provided by the kit, the system is 20ul, and the reaction conditions are as follows: 42 ℃/60 minutes and 75 ℃/5 minutes, then using a PCR reagent provided by the kit to perform PCR reaction, taking 1ul of the reverse transcription reaction product as a template, and respectively and independently amplifying DAB2, KDELC1, G6PD, PPP1CB and beta-actin, wherein the used primers are respectively as follows: (1) DAB2 forward primer: 5 '-TTCACAGACAGATCTGGAGC-3' (SEQ ID NO: 26), reverse primer: 5 '-TCCTCTTCCACAGGAAGGAT-3' (SEQ ID NO: 27); (2) KDELC1 forward primer: 5 '-GAGACTCGAGCTGGTTAAAC-3' (SEQ ID NO: 28), reverse primer: 5 '-TAAGCTGCTACAGTGCCATC-3' (SEQ ID NO: 29); (3) g6PD forward primer: 5 '-GAAGAGAGTGGGTTTCCAGT-3' (SEQ ID NO: 30), reverse primer: 5 '-ATGTGCAGCTGAGGTCAATG-3' (SEQ ID NO: 31); (4) PPP1CB forward primer: 5 '-AGCTACCCAGTATCCATGCT-3' (SEQ ID NO: 32), reverse primer: 5 '-CGTGAACACACTCATACTTC-3' (SEQ ID NO: 33); (5) beta-actin forward primer: 5 '-TCGTGCGTGACATTAAGGAG-3' (SEQ ID NO: 34), reverse primer: 5 '-AACCGCTCATTGCCAATGGT-3' (SEQ ID NO: 35), performing PCR reaction of the standard substance, taking 1ul of the standard substance provided in the kit as a template, and respectively and independently amplifying DAB2, KDELC1, G6PD, PPP1CB and beta-actin, wherein the primers are respectively as follows: (1) DAB2 forward primer: 5 '-TTCACAGACAGATCTGGAGC-3' (SEQ ID NO: 36), reverse primer: 5 '-TCCTCTTCCACAGGAAGGAT-3' (SEQ ID NO: 37); (2) KDELC1 forward primer: 5 '-GAGACTCGAGCTGGTTAAAC-3' (SEQ ID NO: 38), reverse primer: 5 '-TAAGCTGCTACAGTGCCATC-3' (SEQ ID NO: 39); (3) g6PD forward primer: 5 '-GAAGAGAGTGGGTTTCCAGT-3' (SEQ ID NO: 40), reverse primer: 5 '-ATGTGCAGCTGAGGTCAATG-3' (SEQ ID NO: 41); (4) PPP1CB forward primer: 5 '-AGCTACCCAGTATCCATGCT-3' (SEQ ID NO: 42), reverse primer: 5 '-CGTGAACACACTCATACTTC-3' (SEQ ID NO: 6); (5) beta-actin forward primer: 5 '-TCGTGCGTGACATTAAGGAG-3' (SEQ ID NO: 43), reverse primer: 5 '-AACCGCTCATTGCCAATGGT-3' (SEQ ID NO: 44), the reaction system for PCR amplification is as follows: SYBR GREEN 2XPCR mixed reaction buffer 10ul, each primer 1ul, ddH2O 7ul, reverse transcription product 1ul or standard 1ul, and PCR reaction conditions are as follows: activating a denaturing enzyme at 94 ℃/5 minutes, denaturing at 94 ℃/30 seconds, annealing at 60 ℃/30 seconds, extending at 72 ℃/30 seconds, performing 40 cycles, using an instrument as a real-time quantitative PCR instrument, and finally performing result analysis, wherein the CT value is directly read by using PCR off-machine data, firstly correcting through an internal reference gene, comparing the corrected data with a standard product, and warning that the breast cancer is at high risk when the CT value meeting DAB2 gene is smaller than that of the standard product and the CT values of KDELC1, G6PD and PPP1CB genes are larger than that of the standard product.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> Guangdong province people hospital
<120> diagnostic kit for evaluating breast cancer risk by combining four key genes
<141> 2021-08-03
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 500
<212> DNA
<213> Artificial sequence
<400> 1
agtcctcagc tgccagcatc tgattagaac catatctctc gccgggagtg gccgcgcggc 60
tccgaagctc ccggccggcg gctatttaag cgaggcccgc cgcatccgct gcgctgtagc 120
ctggaggctc cgggcgcggg gaagtcatgc tcgcttcacg gaggcaatag ctagccggtg 180
tctgtgggag gttatgttta tttgagactt ctccatcggg atcgcctggt gtcaccaagt 240
gtccactggt actgaggttt gctgcctgcc ttcttgccat gtctaacgaa gtagaaacaa 300
gtgcaaccaa tggtcagccc gaccaacagg ccgcaccaaa agcaccctca aagaaggaaa 360
aaaagaaagg ccctgaaaag acagatgaat atctcttagc aaggttcaaa ggcgatggtg 420
taaaatataa ggccaagctg attggcattg atgatgtgcc agatgcaaga ggggataaaa 480
tgagccaaga ctctatgatg 500
<210> 2
<211> 500
<212> DNA
<213> Artificial sequence
<400> 2
aaatgatgct ccagtggcag gagcaactca agttcatcat tgtcctgaga gagaggagca 60
gcgcggttct cggccgggac agcagaacgc caggggaccc tcacctgggc gcgccggggc 120
acgggctttg attgtcctgg ggtcgcggag acccgcgcgc ctgccctgca cgccgggcgg 180
caacctttgc agtcgcgttg gctgctgcga tcggccggcg ggtccctgcc gaaggctcgg 240
ctgcttctgt ccacctctta cacttcttca tttatcggtg gatcatttcg agagtccgtc 300
ttgtaaatgt ttggcacttt gctactttat tgcttctttc tggcgacagt tccagcactc 360
gccgagaccg gcggagaaag gcagctgagc ccggagaaga gcgaaatatg gggacccggg 420
ctaaaagcag acgtcgtcct tcccgcccgc tatttctata ttcaggcagt ggatacatca 480
gggaataaat tcacatcttc 500
<210> 3
<211> 500
<212> DNA
<213> Artificial sequence
<400> 3
agaggcaggg gctggcctgg gatgcgcgcg cacctgccct cgccccgccc cgcccgcacg 60
aggggtggtg gccgaggccc cgccccgcac gcctcgcctg aggcgggtcc gctcagccca 120
ggcgcccgcc cccgcccccg ccgattaaat gggccggcgg ggctcagccc ccggaaacgg 180
tcgtacactt cggggctgcg agcgcggagg gcgacgacga cgaagcgcag acagcgtcat 240
ggcagagcag gtggccctga gccggaccca ggtgtgcggg atcctgcggg aagagctttt 300
ccagggcgat gccttccatc agtcggatac acacatattc atcatcatgg gtgcatcggg 360
tgacctggcc aagaagaaga tctaccccac catctggtgg ctgttccggg atggccttct 420
gcccgaaaac accttcatcg tgggctatgc ccgttcccgc ctcacagtgg ctgacatccg 480
caaacagagt gagcccttct 500
<210> 4
<211> 500
<212> DNA
<213> Artificial sequence
<400> 4
gcggagctgg gcggtgccga ggaggaggag gtggcggcct gggtctgacg cggccctgtt 60
cgagggggcc tctcttgttt atttatttat tttccgtggg tgcctccgag tgtgcgcgcg 120
ctctcgctac ccggcgggga gggggtgggg ggagggcccg ggaaaagggg gagttggagc 180
cggggtcgaa acgccgcgtg acttgtaggt gagagaacgc cgagccgtcg ccgcagcctc 240
cgccgccgag aagcccttgt tcccgctgct gggaaggaga gtctgtgccg acaagatggc 300
ggacggggag ctgaacgtgg acagcctcat cacccggctg ctggaggtac gaggatgtcg 360
tccaggaaag attgtgcaga tgactgaagc agaagttcga ggcttatgta tcaagtctcg 420
ggagatcttt ctcagccagc ctattctttt ggaattggaa gcaccgctga aaatttgtgg 480
agatattcat ggacaatata 500
<210> 5
<211> 500
<212> DNA
<213> Artificial sequence
<400> 5
gttgctatcc aggctgtgct atccctgtac gcctctggcc gtaccactgg catcgtgatg 60
gactccggtg acggggtcac ccacactgtg cccatctacg aggggtatgc cctcccccat 120
gccatcctgc gtctggacct ggctggccgg gacctgactg actacctcat gaagatcctc 180
accgagcgcg gctacagctt caccaccacg gccgagcggg aaatcgtgcg tgacattaag 240
gagaagctgt gctacgtcgc cctggacttc gagcaagaga tggccacggc tgcttccagc 300
tcctccctgg agaagagcta cgagctgcct gacggccagg tcatcaccat tggcaatgag 360
cggttccgct gccctgaggc actcttccag ccttccttcc tgggcatgga gtcctgtggc 420
atccacgaaa ctaccttcaa ctccatcatg aagtgtgacg tggacatccg caaagacctg 480
tacgccaaca cagtgctgtc 500

Claims (6)

1. The diagnostic kit for evaluating the breast cancer risk by combining four key genes is characterized in that: the kit comprises a standard substance, four primers of specific genes and an internal reference gene, wherein the specific detection genes and the internal reference gene are respectively as follows: DAB2(ID:1601), KDELC1(ID:79070), G6PD (ID:2539), PPP1CB (ID:5500) and beta-actin (GeneID:60), and the primers can specifically amplify the corresponding genes.
2. The diagnostic kit for evaluating the risk of breast cancer according to the combination of four key genes of claim 1, wherein: the partial sequence of the DAB2 gene is as follows:
agtcctcagctgccagcatctgattagaaccatatctctcgccgggagtggccgcgcggctccgaagctcccggccggcggctatttaagcgaggcccgccgcatccgctgcgctgtagcctggaggctccgggcgcggggaagtcatgctcgcttcacggaggcaatagctagccggtgtctgtgggaggttatgtttatttgagacttctccatcgggatcgcctggtgtcaccaagtgtccactggtactgaggtttgctgcctgccttcttgccatgtctaacgaagtagaaacaagtgcaaccaatggtcagcccgaccaacaggccgcaccaaaagcaccctcaaagaaggaaaaaaagaaaggccctgaaaagacagatgaatatctcttagcaaggttcaaaggcgatggtgtaaaatataaggccaagctgattggcattgatgatgtgccagatgcaagaggggataaaatgagccaagactctatgatg(SEQ ID NO：1)。
3. the diagnostic kit for evaluating the risk of breast cancer according to the combination of four key genes of claim 1, wherein: the partial sequence of the KDELC1 gene:
aaatgatgctccagtggcaggagcaactcaagttcatcattgtcctgagagagaggagcagcgcggttctcggccgggacagcagaacgccaggggaccctcacctgggcgcgccggggcacgggctttgattgtcctggggtcgcggagacccgcgcgcctgccctgcacgccgggcggcaacctttgcagtcgcgttggctgctgcgatcggccggcgggtccctgccgaaggctcggctgcttctgtccacctcttacacttcttcatttatcggtggatcatttcgagagtccgtcttgtaaatgtttggcactttgctactttattgcttctttctggcgacagttccagcactcgccgagaccggcggagaaaggcagctgagcccggagaagagcgaaatatggggacccgggctaaaagcagacgtcgtccttcccgcccgctatttctatattcaggcagtggatacatcagggaataaattcacatcttc(SEQ ID NO：2)。
4. the diagnostic kit for evaluating the risk of breast cancer according to the combination of four key genes of claim 1, wherein: the partial sequence of the G6PD gene:
agaggcaggggctggcctgggatgcgcgcgcacctgccctcgccccgccccgcccgcacgaggggtggtggccgaggccccgccccgcacgcctcgcctgaggcgggtccgctcagcccaggcgcccgcccccgcccccgccgattaaatgggccggcggggctcagcccccggaaacggtcgtacacttcggggctgcgagcgcggagggcgacgacgacgaagcgcagacagcgtcatggcagagcaggtggccctgagccggacccaggtgtgcgggatcctgcgggaagagcttttccagggcgatgccttccatcagtcggatacacacatattcatcatcatgggtgcatcgggtgacctggccaagaagaagatctaccccaccatctggtggctgttccgggatggccttctgcccgaaaacaccttcatcgtgggctatgcccgttcccgcctcacagtggctgacatccgcaaacagagtgagcccttct(SEQ ID NO：3)。
5. the diagnostic kit for evaluating the risk of breast cancer according to the combination of four key genes of claim 1, wherein: the PPP1CB gene partial sequence:
gcggagctgggcggtgccgaggaggaggaggtggcggcctgggtctgacgcggccctgttcgagggggcctctcttgtttatttatttattttccgtgggtgcctccgagtgtgcgcgcgctctcgctacccggcggggagggggtggggggagggcccgggaaaagggggagttggagccggggtcgaaacgccgcgtgacttgtaggtgagagaacgccgagccgtcgccgcagcctccgccgccgagaagcccttgttcccgctgctgggaaggagagtctgtgccgacaagatggcggacggggagctgaacgtggacagcctcatcacccggctgctggaggtacgaggatgtcgtccaggaaagattgtgcagatgactgaagcagaagttcgaggcttatgtatcaagtctcgggagatctttctcagccagcctattcttttggaattggaagcaccgctgaaaatttgtggagatattcatggacaatata(SEQ ID NO：4)。
6. the diagnostic kit for evaluating the risk of breast cancer according to the combination of four key genes of claim 1, wherein: the beta-actin gene partial sequence:
gttgctatccaggctgtgctatccctgtacgcctctggccgtaccactggcatcgtgatggactccggtgacggggtcacccacactgtgcccatctacgaggggtatgccctcccccatgccatcctgcgtctggacctggctggccgggacctgactgactacctcatgaagatcctcaccgagcgcggctacagcttcaccaccacggccgagcgggaaatcgtgcgtgacattaaggagaagctgtgctacgtcgccctggacttcgagcaagagatggccacggctgcttccagctcctccctggagaagagctacgagctgcctgacggccaggtcatcaccattggcaatgagcggttccgctgccctgaggcactcttccagccttccttcctgggcatggagtcctgtggcatccacgaaactaccttcaactccatcatgaagtgtgacgtggacatccgcaaagacctgtacgccaacacagtgctgtc(SEQ ID NO：5)。
CN202110888350.6A 2021-08-03 2021-08-03 Diagnostic kit for evaluating breast cancer risk by combining four key genes Pending CN113667749A (en)

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