CN111549134B - Liver cancer early detection kit based on polygenic mutation - Google Patents
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Abstract
The invention discloses a liver cancer early detection kit based on polygenic mutation, and belongs to the technical field of gene detection. The invention discloses a multi-gene mutation-based liver cancer early detection kit, which develops a multi-gene mutation detection liver cancer screening kit based on a fluorescence PCR reaction technology and is used for assisting clinical early screening of liver cancer; the detection is convenient and the sensitivity is high.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a liver cancer early detection kit based on polygenic mutation.
Background
Liver cancer is one of the common malignant tumors in China. 85% of the worldwide liver cancer incidences are concentrated in resource-poor or developing countries and regions. China is a main high incidence area of liver cancer, and the liver cancer is hidden, short in course of disease and poor in prognosis, so that the health of the public is seriously influenced, and the serious burden is caused to the social and economic development of China. About 38.3 thousands of people die of liver cancer each year in China, accounting for 51% of the death cases of liver cancer worldwide, and the severe situation brings heavy burden to the society and medical treatment of China. Most patients lack the consciousness of regular examination because they have insufficient disease importance when they have no obvious symptoms. In addition, the patients with liver diseases with low or medium income do not want to bear the hospital detection cost, or do not want to perform regular examination due to the reasons of difficult medical attendance and the like, and often miss the optimal time for early diagnosis, so that the liver cancer is mostly discovered in the late stage, only 30% of patients have the chance of surgical resection, and the metastasis and recurrence rate within 5 years after the resection is as high as 60% -70%. Therefore, early screening and diagnosis are especially important for effective treatment of liver cancer and improvement of prognosis.
In recent years, there has been much research evidence that the accumulation of normal mutations in human genes induces the risk of cancer, leading to conditions such as oncogene activation and inactivation and loss of cancer suppressor genes. RAS gene as intracellular signal conducting protein proto-oncogene belongs to membrane combined GTP/GDP related binding protein, is an important oncogene in EGFR signal pathway, plays an important role in regulating and controlling related signal pathways such as cell proliferation and angiogenesis, and can be used as a molecular switch to participate in the processes of transmitting related signals such as extracellular proliferation, growth and differentiation to cells. Research shows that the common characteristic genes of the RAS gene family and the human tumorigenesis related are KRAS, HRAS and NRAS, and can cause RAS abnormal activation under the condition that RAS gene mutation occurs, stimulate the continuous growth and differentiation of cells and initiate tumors. Research shows that the KRAS gene of a liver cancer patient has obvious mutation, and the KRAS gene mutation rate of the liver cancer patient with extrahepatic metastasis is obviously increased.
P53 has a wild type (P53-wt) and a mutant type (P53-mt). p53-wt is an important tumor suppressor gene in humans, which normally functions to regulate DNA repair, synthesis and apoptosis in the cell cycle, inhibit the formation of tumor cells, and act as a "molecular policeman". When the p53 gene is lost or mutated, the original function is changed to cause the out-of-control of cell growth and the transcription is p53-mt. p53-mt not only causes loss of 53 cancer suppressing activity, but also has an activity of promoting malignant transformation. Mutations in the p53 gene can be detected in almost half or more malignant tumors.
BCL10 is an apoptosis regulator containing a caspase recruitment domain, and is found from chromosomal translocation in mucosa-associated lymphoid tissue lymphomas. Mutation or overexpression of the Bcl10 gene is closely related to the occurrence and development of lymphoma. In various solid tumors such as ovarian cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, cervical carcinoma, breast cancer and renal clear cell carcinoma, bcl10 is found to be widely involved in invasion and infiltration of cancer cells and promote the growth of the cancer cells.
CTNNB1 (β -catenin 1) is a gene encoding β catenin, and the protein encoded by CTNNB1 gene is a complex protein that constitutes an adhesive junction and maintains epithelial cell layers by regulating cell growth and adhesion between cells. One report in Japan shows that the CTNNB1 gene exon 3 mutation is an important molecular mechanism for causing the inactivation of the Wnt \ beta catenin pathway, is highly related to the occurrence of liver cancer, and has the mutation rate of 35 percent.
The AT-rich domain 1A (ARIDlA) gene is an important member of the SWI/SNF chromosome remodeling complex, is located on human chromosome 1 1p36.11, and the encoded protein thereof consists of 2285 amino acid residues and has a relative molecular weight of about 240X 10 3 . Studies have demonstrated that aberrant chromatin remodeling leading to gene expression can cause a variety of diseases. It has also been reported that ARIDlA gene mutation causes the decrease of protein expression level, and causes various malignant tumors such as ovarian cancer, endometrial cancer, gastric cancer, liver cancer, bladder cancer, etc.
Telomerase reverse transcriptase (TERT) is a key determinant of telomerase activity. The TERT gene is located on chromosome 5p15.33, and normal somatic cells carry a 2-fold copy of the TERT gene. TERT expression plays an important role in cell immortalization and tumor progression, and increased TERT replication leads to tumorigenesis. In recent years, researches show that TERT promoter mutation plays an important role in activating telomerase in various tumors, and the TERT promoter mutation can up-regulate telomerase activity to influence the occurrence and development of tumors.
At present, the liver cancer is often diagnosed clinically by combining imaging technology with a method of detecting a serologic biomarker. The imaging technology detection is usually Computed Tomography (CT), magnetic Resonance Imaging (MRI) and ultrasonic examination (US), and the method has an unsatisfactory display effect on <1cm of liver cancer focus and liver cirrhosis complicated with liver cancer. The method for detecting the serological marker is based on the detection and identification of liver function index, tumor marker Alpha Fetoprotein (AFP) and the like. The most effective diagnostic evidence among these is the amount of AFP expression. However, the sensitivity of AFP for screening and diagnosing liver cancer is only 39-64%, and the effective rate of early diagnosis is only 9-32%, so that the popularization and application of AFP in the early screening and diagnosis of liver cancer are limited.
Therefore, it is an urgent need to solve the problem of providing a kit for early detection of liver cancer based on multiple gene mutations.
Disclosure of Invention
In view of the above, the invention provides a multi-gene mutation-based liver cancer early detection kit, which is based on fluorescence PCR reaction to perform multi-gene mutation detection, and has the advantages of simple detection and high sensitivity.
In order to achieve the purpose, the invention adopts the following technical scheme:
a liver cancer early detection kit based on polygenic mutation comprises a primer and a probe;
the primer sequences are as follows:
KRAS-F：5’-CATAGAGCTTGACATGGTCG-3’；SEQ ID NO.1；
KRAS-R：5’-ACAAGAGGTTATGAGCACCT-3’；SEQ ID NO.2；
P53-F：5’-AGCAGAATAGCATGAACCTC-3’；SEQ ID NO.3；
P53-R：5’-CCGACAGGATATGAGGTCGC-3’；SEQ ID NO.4；
BCL10-136F：5’-GGACCAGGAAGAAGCGCCATCTCC-3’；SEQ ID NO.5；
BCL10-136R：5’-GATCCTCCTGCTGTGGCTCGGACT-3’；SEQ ID NO.6；
CTNNB1-110F：5’-CTGAGGAAGAGGATGTGGATAC-3’；SEQ ID NO.7；
CTNNB1-110R：5’-AGGACTTGGGAGGTATCCACAT-3’；SEQ ID NO.8；
ARID1A-5548F：5’-GGATTGGTGGGGAGA-3’；SEQ ID NO.9；
ARID1A-5548R：5’-CCTCTGCTGTTGTCACAT-3’；SEQ ID NO.10；
ARID1A-6420F：5’-ATTCTGGCCACACCCTCT-3’；SEQ ID NO.11；
ARID1A-6420R：5’-CCAGGTTGGCCAGCAGTACC-3’；SEQ ID NO.12；
TERT-2460F：5’-CCTCACCTCACCCACACA-3’；SEQ ID NO.13；
TERT-2460R：5’-AGGGCCTCGTCTTCTACAGG-3’；SEQ ID NO.14；
internal standard GAPDH-F:5 'AGGCCACTTGTTTGAGTTATTG-3'; SEQ ID No.15;
internal standard GAPDH-R:5 'AACAGCATGTGCAAAATGAGGGGTAG-3'; SEQ ID No.16;
the probe sequence is as follows:
KRAS-P：5’-GATCTTTCACATTCACCAAATA-3’；SEQ ID NO.17；
P53-P：5’-ACATCTAGTCACCGAACATC-3’；SEQ ID NO.18；
BCL10-P：5’-AAAGGGCTGGAAAATTGTTA-3’；SEQ ID NO.19；
CTNNB1-P：5’-CTGAGTGGTAAAGGCAATCCTGAGG-3’；SEQ ID NO.20；
ARID1A-5548P：5’-CCAGACCCACTTCGAGAGCAAG-3’；SEQ ID NO.21；
ARID1A-6420P：5’-TGGTGCGCTTCCTCAGTGACCG-3’；SEQ ID NO.22；
TERT-2460P：5’-GAGTATGGCTGCGTGGTGAA-3’；SEQ ID NO.23；
internal standard GAPDH-P:5 'ATGCATCTGGCTCCGAGTTCCTGAATGC-3'; SEQ ID NO.24.
Further, the kit for detecting the early liver cancer based on polygenic mutation is used for detecting based on fluorescent quantitative PCR;
the reaction system of the fluorescent quantitative PCR is as follows: 10 mul of PCR Buffer, 1 mul of upstream primer, 1 mul of downstream primer, 1 mul of internal standard probe, 1 mul of Taq enzyme, 5 mul of DNA template, H 2 O5. Mu.l, 25. Mu.l in total. The internal standard primer and the gene primer are added separately, and the total amount of the internal standard upstream and downstream primers is 1 mu l.
The reaction tube was placed in a fluorescent PCR detector with the following cycle parameters set:
the fluorescence signals were collected as FAM, VIC, ROX, CY5 and the data were collected at 58 ℃.
When the sample is detected, dividing 6 genes into two tubes for detection, wherein each tube comprises 3 probes corresponding to the genes and an internal standard probe; the 3 gene probes and the internal standard probe are respectively marked with FAM, VIC, ROX and CY5 fluorophores, and the marked fluorophores are not specifically limited as long as the marked fluorophores are different, and other fluorophores besides the enumerated fluorophores in the invention can be marked. The two mutation sites ARID1A-5548 and ARID1A-6420 of the gene ARID1A are marked with the same fluorophore, and further distinction is not needed.
According to the technical scheme, compared with the prior art, the invention discloses and provides a liver cancer early detection kit based on polygenic mutation, and develops a liver cancer screening kit based on polygenic mutation detection of a fluorescence PCR reaction technology, which is used for assisting clinical early screening of liver cancer; the detection is convenient and the sensitivity is high.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 shows the results of detecting mutations in P53, BCL10, and KRAS genes in a sample according to the present invention;
wherein, 1, internal standard; 2,53; 3,BCL10;4,KRAS;
FIG. 2 is a drawing showing the results of detecting CTNNB1, ARID1A, TERT gene mutation in a sample according to the present invention;
wherein, 1, internal standard; 2,CTNNB1;3,ARID1A;4, TERT.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in 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.
EXAMPLE 1 sample testing
(1) Extraction of blood sample DNA: extracting blood of a patient, and extracting DNA in the blood of the patient by using an extraction kit of the TAKARA gene; the operation with the fully automatic analyzer was as follows: (1) 100ml of whole blood containing an anticoagulant was taken and put into a 1.5ml EP tube, and then 100ml of PBS buffer solution was added. Then adding Buffer GB (180 ml), proteinase K (20 ml) and 10mg/ml ribonuclease A (10 ml) into an EP tube, fully blowing and uniformly mixing, and then placing in a water bath at 56 ℃ for 10min constant-temperature water bath. Adding 200ml of 100% ethanol into the lysate, fully blowing and beating the lysate to uniformly mix the lysate and the lysate; (2) placing the adsorption column on a collecting tube, sucking the solution into the adsorption column, performing operation at 12000 r/m for 2 min, and removing the filtrate; (3) absorbing 500ml of Buffer WA, adding into an adsorption column, performing 12000 r/m centrifugation for 2 min, and removing the filtrate; (4) after centrifugation at 2000 rpm for 2 minutes, the DNA was eluted.
(2) Mutation detection was performed in two tubes, 3 genes per tube.
(3) Detecting KRAS, P53, BCL10, CTNNB1, ARID1A and TERT gene mutation based on fluorescent quantitative PCR:
the reaction system of the fluorescent quantitative PCR is as follows: 10 mul of PCR Buffer, 1 mul of upstream primer, 1 mul of downstream primer, 1 mul of internal standard probe, 1 mul of Taq enzyme, 5 mul of DNA template, H 2 O5. Mu.l, 25. Mu.l in total. Wherein probes of P53, BCL10, KRAS and internal standard respectively mark FAM, VIC, ROX and CY5 fluorescent groups; probes for CTNNB1, ARID1A, TERT and internal standard label FAM, VIC, ROX, CY5 fluorophores respectively.
The reaction tube was placed in a fluorescent PCR detector with the following cycle parameters set:
(4) Analysis of results
And (4) judging the standard: and (3) judging whether the sample has gene mutation such as KRAS gene, P53 and the like, and if more than 3 of the gene mutations are detected, the risk of the tested person suffering from liver cancer is high, and further examination is required to confirm whether the tested person suffers from liver cancer.
The results are shown in FIGS. 1-2, and the results in FIGS. 1-2 show that only the KRAS and CTNNB1 genes in the DNA of the blood sample are mutated; the tested patients have less risk of liver cancer.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Zhejiang university college of medicine affiliated with first hospital, zhejiang Mule Biotech Co., ltd
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Claims (2)
1. A liver cancer early detection kit based on polygenic mutation is characterized by comprising a primer and a probe;
the primer sequences are as follows:
KRAS-F：5’-CATAGAGCTTGACATGGTCG-3’；SEQ ID NO.1；
KRAS-R：5’-ACAAGAGGTTATGAGCACCT-3’；SEQ ID NO.2；
P53-F：5’-AGCAGAATAGCATGAACCTC-3’；SEQ ID NO.3；
P53-R：5’-CCGACAGGATATGAGGTCGC-3’；SEQ ID NO.4；
BCL10-136F：5’-GGACCAGGAAGAAGCGCCATCTCC-3’；SEQ ID NO.5；
BCL10-136R：5’-GATCCTCCTGCTGTGGCTCGGACT-3’；SEQ ID NO.6；
CTNNB1-110F：5’-CTGAGGAAGAGGATGTGGATAC-3’；SEQ ID NO.7；
CTNNB1-110R：5’-AGGACTTGGGAGGTATCCACAT-3’；SEQ ID NO.8；
ARID1A-5548F：5’-GGATTGGTGGGGAGA-3’；SEQ ID NO.9；
ARID1A-5548R：5’-CCTCTGCTGTTGTCACAT-3’；SEQ ID NO.10；
ARID1A-6420F：5’-ATTCTGGCCACACCCTCT-3’；SEQ ID NO.11；
ARID1A-6420R：5’-CCAGGTTGGCCAGCAGTACC-3’；SEQ ID NO.12；
TERT-2460F：5’-CCTCACCTCACCCACACA-3’；SEQ ID NO.13；
TERT-2460R：5’-AGGGCCTCGTCTTCTACAGG-3’；SEQ ID NO.14；
internal standard GAPDH-F:5 'AGGCCACTTGTTTGAGTTATTG-3'; SEQ ID No.15;
internal standard GAPDH-R:5 'AACAGCATGTGCAAAATGAGGGGTAG-3'; SEQ ID No.16;
the probe sequence is as follows:
KRAS-P：5’-GATCTTTCACATTCACCAAATA-3’；SEQ ID NO.17；
P53-P：5’-ACATCTAGTCACCGAACATC-3’；SEQ ID NO.18；
BCL10-P：5’-AAAGGGCTGGAAAATTGTTA-3’；SEQ ID NO.19；
CTNNB1-P：5’-CTGAGTGGTAAAGGCAATCCTGAGG-3’；SEQ ID NO.20；
ARID1A-5548P：5’-CCAGACCCACTTCGAGAGCAAG-3’；SEQ ID NO.21；
ARID1A-6420P：5’-TGGTGCGCTTCCTCAGTGACCG-3’；SEQ ID NO.22；
TERT-2460P：5’-GAGTATGGCTGCGTGGTGAA-3’；SEQ ID NO.23；
internal standard GAPDH-P:5 'ATGCATCTGTGCTCCGAGTTCCTGAATGC-3'; SEQ ID NO.24.
2. The kit for the early detection of liver cancer based on polygenic mutations according to claim 1, wherein the detection is based on fluorescent quantitative PCR;
the reaction system of the fluorescent quantitative PCR is as follows: 10 mul of PCR Buffer, 1 mul of upstream primer, 1 mul of downstream primer, 1 mul of internal standard probe, 1 mul of Taq enzyme, 5 mul of DNA template, H 2 O5. Mu.l, 25. Mu.l in total;
the reaction tube was placed in a fluorescent PCR detector with the following cycle parameters set:
the fluorescence signals were collected as FAM, VIC, ROX, CY5 and data were collected at 58 ℃.
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Citations (3)
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|---|---|---|---|---|
| CN104805207A (en) * | 2015-04-29 | 2015-07-29 | 苏州工业园区为真生物医药科技有限公司 | Kit for detecting kras gene mutation, and detection method of kit |
| CN107304441A (en) * | 2016-04-20 | 2017-10-31 | 上海星昕生物科技有限公司 | A kind of Hepatocarcinoma screening method detected based on polygenes |
| CN108753979A (en) * | 2018-07-09 | 2018-11-06 | 安徽达健医学科技有限公司 | A kind of kit and its application method for liver cancer early screening |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805207A (en) * | 2015-04-29 | 2015-07-29 | 苏州工业园区为真生物医药科技有限公司 | Kit for detecting kras gene mutation, and detection method of kit |
| CN107304441A (en) * | 2016-04-20 | 2017-10-31 | 上海星昕生物科技有限公司 | A kind of Hepatocarcinoma screening method detected based on polygenes |
| CN108753979A (en) * | 2018-07-09 | 2018-11-06 | 安徽达健医学科技有限公司 | A kind of kit and its application method for liver cancer early screening |
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| Title |
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| Linsong Tang等.Synthetic lethality: A promising therapeutic strategy for hepatocellular carcinoma.Cancer Letters.2020,第476卷第120-128页. * |
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