CN104878084B - Detect kit of the oxaliplatin for colorectal cancer validity - Google Patents
Detect kit of the oxaliplatin for colorectal cancer validity Download PDFInfo
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- CN104878084B CN104878084B CN201510097019.7A CN201510097019A CN104878084B CN 104878084 B CN104878084 B CN 104878084B CN 201510097019 A CN201510097019 A CN 201510097019A CN 104878084 B CN104878084 B CN 104878084B
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
Abstract
The present invention relates to for detecting kit of the oxaliplatin for colorectal cancer validity, the kit includes the amplimer sequence of a pair of detection MAP4K1 gene copy numbers, the amplimer sequence and a pair of primer sequences for detecting reference gene GAPDH gene copy numbers of a pair of detection PIK3CA gene copy numbers.The kit designed by the present invention, efficiently and effectively can be detected to oxaliplatin for colorectal cancer validity, evaluate the patient to be checked if appropriate for application oxaliplatin as front-line chemotherapeutic agents, effective reference is provided for clinical application.
Description
[technical field]
The detection oxaliplatin of kit the present invention relates to to(for) colorectal cancer validity.
[background technology]
Colorectal cancer (CRC) is the big common malignant tumour in third place in the world, and causes the second largest original of death by cancer
Cause, serious threat human health (Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D.Global
cancer statistics.CA:a cancer journal for clinicians.2011；61(2):69-90.).It is near several
Over 10 years, the clinical effectiveness of colorectal cancer has been significantly improved, not only due to the progress of surgical technic, will also be attributed to chemotherapy
With interventional therapy (Moertel CG, Fleming TR, Macdonald JS, the et al.Levamisole and of targeted drug
fluorouracil for adjuvant therapy of resected colon carcinoma.The New England
journal of medicine.1990；322(6):352-358；Andre T,Boni C,Navarro M,et
al.Improved overall survival with oxaliplatin,fluorouracil,and leucovorin as
adjuvant treatment in stage II or III colon cancer in the MOSAIC
trial.Journal of clinical oncology:official journal of the American Society
of Clinical Oncology.2009；27(19):3109-3116；Hurwitz HI,Fehrenbacher L,
Hainsworth JD,et al.Bevacizumab in combination with fluorouracil and
leucovorin:an active regimen for first-line metastatic colorectal
cancer.Journal of clinical oncology:official journal of the American Society
of Clinical Oncology.2005；23(15):3502-3508；Van Cutsem E,Kohne CH,Lang I,et
al.Cetuximab plus irinotecan,fluorouracil,and leucovorin as first-line
treatment for metastatic colorectal cancer:updated analysis of overall
survival according to tumor KRAS and BRAF mutation status.Journal of clinical
oncology:official journal of the American Society of Clinical Oncology.2011；
29(15):2011-2019.).According to American National synthesis cancer network how-tos, it is proposed that II phase and the III high-risk trouble of phase colorectal cancer
Person is carrying out adjuvant chemotherapy after radical operation.MOSAIC and NSABP C-07 clinical test results are proved using difficult to understand husky
Sharp platinum gets the nod (Andre T, Boni C, Navarro M, et al.Improved as the scheme of a line NACT
overall survival with oxaliplatin,fluorouracil,and leucovorin as adjuvant
treatment in stage II or III colon cancer in the MOSAIC trial.Journal of
clinical oncology:official journal of the American Society of Clinical
Oncology.2009；27(19):3109-3116；Kuebler JP,Wieand HS,O'Connell MJ,et
al.Oxaliplatin combined with weekly bolus fluorouracil and leucovorin as
surgical adjuvant chemotherapy for stage II and III colon cancer:results from
NSABP C-07.Journal of clinical oncology:official journal of the American
Society of Clinical Oncology.2007；25(16):2198-2204.).However, in III phase colorectal cancer patients
Receive the adjuvant chemotherapy method containing oxaliplatin after 3 years disease-free survival rates still only 65% (Uncu D, Aksoy S,
Cetin B,et al.Results of adjuvant FOLFOX regimens in stage III colorectal
cancer patients:retrospective analysis of 667patients.Oncology.2013；84(4):
240-245).One effective therapeutic strategy be just to discriminate between may benefiting in oxaliplatin adjuvant chemotherapy method it is more or benefit compared with
Few PATIENT POPULATION, to CRC patient carry out personalized drug therapy, but at present still lack predictive marker oxaliplatin sensitivity and
The clinical verification of drug resistance.Therefore, clinical research be badly in need of can the easy method for carrying out prognosis and outcome prediction exactly, for knot
Rectal cancer patient postoperative provides important references.
Blocking effect of mitogen activated protein kinases kinase kinase kinase 1 (MAP4K1), it is in serine/threonine kinase sub-families STE20
A member, belong to a upstream stimulating factor of blocking effect of mitogen activated protein kinases (MAPK) signal transduction pathway, MAPK is one group
Can (including growth factor, hormone, ultraviolet good fortune be penetrated, DNA damage agent, inflammatory cytokine and ring by various kinds of cell external signal
Border stress wait) activation serine/threonine kinases.MAPK signal transduction pathways are highly conserved during biological evolution, from film by
Body arrives mapk kinase (MAPKK) and then to MAPK again to mapk kinase kinases (MAPKKK), in waterfall shape phosphorylation
Downstream kinase, in the end last position that intracellular signal transduction pathway Cao MAPK are in cytoplasmic compartment, it can be gone to after activation in core
Action target spot, the expression of regulatory gene.This activation model is present in yeast to mammal.It participates in a variety of lifes of cell
Thing scholarship and moral conduct is, including Apoptosis (Kyosseva SV.Mitogen-Activated Protein Kinase
Signaling.In:S.John, editor^, editors " .International Review of Neurobiology,
City；Academic Press；2004, p.201-20；Willaime-Morawek S, et al.C-jun N-terminal
kinases/c-Jun and p38pathways cooperate in ceramide-induced neuronal
apoptosis.Neuroscience2003；119:387-97.), break up and breed (Aouadi M, et al.p38Mitogen-
Activated Protein Kinase Activity Commits Embryonic Stem Cells to Either
Neurogenesis or Cardiomyogenesis.STEM CELLS 2006；24:1399-406；Aouadi M,et
al.Inhibition of p38MAPK Increases Adipogenesis From Embryonic to Adult
Stages.Diabetes 2006；55:281-9；Roux PP and Blenis J.ERX and p38MAPK-Activated
Protein Kinases:a Family of Protein Kinases with Diverse Biological
Functions.Microbiology and Molecular Biology Reviews2004；68:320-44.), the cell cycle
Regulation and control, the maintenance of cells survival and malignant transformation of cells etc..STE20 families are MAPKKK upstream kinases, mammal
STE20/ mitogen protein kinase kinase kinase kinases (MAP4K) families are by 28 kinds of related serine/threonines of its catalytic domain
Kinases forms.These kinases can be divided into two kinds of structured sorts according to the position of catalytic domain：Catalytic domain is located at the P21 activity of C-terminal
Protein kinase (PAKs) and the germinal center kinase (GCKs) positioned at N-terminal.GCKs lack PAKs in Cdc42/Rac phase interactions
N- terminal regulatory domains, and instead N- terminal catalytic domains and the extension of the C- ends of different length.C- ends are an inflexible rafters
Homologous region (CNH), referred to as citron rho interaction kinases (CRIK), the region plays an important role for kinase activity section.
Phosphatidylinositol 3-kinase (phosphatidylinositol 3-kinase, PI3K) family is that an albuminoid swashs
Enzyme, participate in the regulatory mechanism (Franke, Kaplan et al.1997) in various kinds of cell.PI3K is specifically catalyzed phosphatidyl
3 dis on inositol (phosphatidylinositol, PI) ring, caused corresponding inositol lipid material, such as 3,
4- diphosphonic acid phosphatidylinositols [PI (3,4) P2] and 3,4,5- triphosphoric acids phosphatidylinositols [PI (3,4,5) P3] are as the second letter
Make to combine and activate a variety of target proteins, formed a signal cascade compound, so as to adjust the propagation of cell, differentiation, survival and
(Katso, Okkenhaug et al.2001) such as migrations.PI3K families are divided into I type, II type according to substrate and catalytic subunit difference
With III type.Wherein, I types are with PI (phosphatidylinositol), PIP (Phosphatidylinositol 4-
Phosphate) and PIP2 (Phosphatidylinositol 4,5-bis-phosphate) is substrate, and II type is with PI and PIP
For substrate, III type PI3K is using PI as substrate (Djordjevic and Driscoll 2002).I type PI3K can be further divided into two
Individual subclass, the heterodimer being made up of one of regulation subunit p85 and catalytic subunit p110 α/βs/δ are IA types, regulation subunit p101
Heterodimer with catalytic subunit p110 γ compositions is IB types, and the two has different activation mechanisms and effect in cell.I type
Heterodimer is formed with regulation subunit with the catalytic subunit of III type, II types do not form heterodimer, have unique C2 domains.
Much researchs in human tumor show that the PI3K of I type is often over-expressed, and strengthen PI3K catalytic activity, increase simultaneously
The amount of its albumen synthesis, promote cell carcinogenesis, it is relevant with into cancer conversion.In colorectal cancer, the PIK3CA genes more than 25%
Expression quantity increases (Samuels, Wang et al.2004), shows that the PIK3CA of IA types plays important work in tumor development
With (Vivanco and Sawyers2002).
The present invention has found the copy number change of MAP4K1, PIK3CA gene in 142 parts of colorectal cancer patients tumor tissues
Use the effect of oxaliplatin is as front-line chemotherapeutic agents closely related with colorectal cancer patients Post operation.Therefore, the present invention adopts
With the label that MAP4K1, PIK3CA gene are colorectal cancer, summary draws a kind of method of curative effect evaluation, so as to be effectively
Colorectal cancer patients postoperative provides important references.Further, the present invention uses fluorescent quantitative PCR technique, using voluntarily
The internal reference and purpose primer for designing and optimizing, incorporate real-time fluorescence quantitative PCR reagent, and detection kit is made, convenient clinical
Detection uses.
[content of the invention]
It can be used in detecting reagent of the oxaliplatin for colorectal cancer validity it is an object of the invention to provide a kind of
Box.
The kit includes the amplimer sequence of a pair of detection MAP4K1 gene copy numbers, a pair of detection PIK3CA genes
The primer sequence of the amplimer sequence of copy number and a pair of detection reference gene GAPDH gene copy numbers.
Found in research process, MAP4K1 genes, PIK3CA gene copy numbers increase corresponding is death wind
Danger significantly improves.Experimentation can be found in specific embodiment.The kit designed by the present invention, can be efficiently and effectively to Austria
Husky sharp platinum detects for colorectal cancer validity, evaluates the patient to be checked if appropriate for application oxaliplatin as a line
Medicine is treated, effective reference is provided for clinical application.
Further, the kit also has following prioritization scheme：
The positive sequence such as SEQ ID NO of the amplimer sequence of described detection MAP4K1 gene copy numbers:Shown in 1,
Reverse sequence such as SEQ ID NO:Shown in 2.
The positive sequence such as SEQ ID NO of the amplimer sequence of described detection PIK3CA gene copy numbers:Shown in 3,
Reverse sequence such as SEQ ID NO:Shown in 4.
The positive sequence such as SEQ ID NO of the amplimer sequence of described detection reference gene GAPDH gene copy numbers:
Shown in 5, reverse sequence such as SEQ ID NO:Shown in 6.
Also include the real-time fluorescence quantitative PCR amplification system that 2 × SYBR Green qPCR Mix and ultra-pure water form.
Described real-time fluorescence quantitative PCR amplification system, every 20 μ L reaction systems are final concentration of：Detection MAP4K1 genes are copied
The amplimer sequence of shellfish number, the amplimer sequence and a pair of detection reference genes of a pair of detection PIK3CA gene copy numbers
The forward primer concentration of the primer sequence of GAPDH gene copy numbers is 2uM, and reverse primer concentration is 2uM, 2 × SYBR
Green qPCR Mix concentration is 10 μ L, and remaining is ultra-pure water.
[brief description of the drawings]
Fig. 1 is that the packet of MAP4K1, PIK3CA gene risk score carries out Kaplan-Meier curves
[embodiment]
Kit includes：2 × SYBR Green qPCR Mix, ultra-pure water and two pairs of qPCR detection primers (being shown in Table 1) compositions
Real-time fluorescence quantitative PCR amplification system.
1 three pairs of qPCR detection primers of table
Described real-time fluorescence quantitative PCR amplification system, every 20 μ L reaction systems are final concentration of：Forward and reverse each 4uM of primer,
The μ L of 2 × SYBR Green qPCR Mix 10, ultra-pure water complement to 20uL.
Described mM and μM be molar concentration unit, refer to the molal quantity of contained solute in every liter of solution.
The method of the variation prediction carcinoma of the rectum chemotherapeutic efficacy of present invention detection MAP4K1, PIK3CA gene copy number, mainly
Comprise the following steps：
(1) collect the surgery excision cancerous tissue sample of colorectal cancer patients and FFPE processing, tracking follow-up patient answer
Send out the time；
(2) histotomy and the DNA sample of tissue is extracted：With the DNA paraffin extraction agent boxes (DNA of Qiagen companies
FFPE kit, Qiagen, German) extracting DNA；
(3) DNA quality controls：With the DNA of Thermo Scientific Varioskan Flash ELIASAs detection extracting
Quality；
(4) quantitative PCR detection：The iQ5 quantitative PCR apparatus produced with Bio-rad companies detects the gene copy in DNA sample
Number；
(5) MAP4K1, PIK3CA gene are calculated in all samples relative to reference gene GAPDH copy number changing value；
(6) using Chi-square Test evaluation patients clinical feature difference, the difference of student's t test evaluation continuous variables, just
Can step judges as the index for distinguishing Patients on Recurrence；
(7) sample of collection is divided into training sample set, checking sample set, total sample set and carries out multivariable Cox ratios respectively
Example risk survival analysis, analysis and regulation age, sex, clinical stage and the tissue influence to result by stages.Draw corresponding base
The risk-benefit risks (HRs) and its 95% confidential interval (CI) of cause.
(8) assessed using Kaplan-Meier survivorship curves and be divided into patient using MAP4K1, PIK3CA gene copy number
Excessive risk group and low-risk group between prognosis difference, utilize the conspicuousness of log-rank inspection statistics.
Case is embodied
Following examples are merely to illustrate the present invention, rather than limitation the scope of the present invention.Unreceipted tool in embodiment
The experimental method of concrete conditions in the establishment of a specific crime, according to the condition proposed by manufacture kit production company or according to conventional laboratory conditions, such as
Sambrook et al., molecular cloning：Laboratory manual (New York：Cold Spring Harbor LaboratoryPress,
1989) condition described in.
1st, experimental subjects
The research object of the present embodiment is selected in June, 2006 to 142 collected during in December, 2011 through histology
It is accredited as the patient of colorectal cancer.Wherein 76 patients for being collected in the attached Ruijin Hospital of Shanghai Communications University are as statistical model
Training set, in addition 66 patients for being collected in attached tenth the People's Hospital of Shanghai Tongji University as checking the set pair analysis model tested
Card.
Include and exclusion standard：
(1) the equal initial diagnosis of all cases is colorectal cancer and did not receive radiation and chemotherapy before.
(2) in order to farthest reduce inter-sample difference, we only study the III phase large intestines performed the operation through radical excision
Cancer patient, and at least six cycle is used using oxaliplatin as a line NACT medicine, all patients are according to the U.S.
Cancer Joint Committee tumour node transfer Staging System (TNM) is carried out by stages.
(3) without other organ tumor medical histories；It is difficult lower family history：Without colorectal carcinoma history, no adenoma in 1~2 grade of relatives
Property polyp medical history and Familial Occurrence syndrome history, mainly including familial adenomatous polyposis (FAP), hereditary nonpolyposis
Colorectal cancer (HNPCC) etc.；
(4) we have looked back the medical records of patient and have collected clinical diagnosis information, including diagnosis date, clinical stage,
Knub position, histology by stages, pathological staging and treatment information.Patient's recurrence, dead information by medical records and phone with
Visit and obtain, follow-up in January, 2014.
2nd, experimental method
(1) using the tumor tissues sample of above-mentioned 142 colorectal cancer patients underwent operatives excision, the sample of each FFPE
Originally 50-100 sections are cut, are passed through after haematoxylin ＆ eosin (HE) dyeing by 3 pathologists diagosis.Three pathology doctors are scraped by hand
The tumour cell area that teacher unanimously thinks exceedes the section of section area 70%.Tumor tissue section, which is immersed in dimethylbenzene, to dewax,
With the QIAamp-DNA-FFPE-Tissue Kit of Qiagen companies, according to the explanation of kit, the DNA samples in tissue are extracted
This.DNA sample after extracting is stored in -20 DEG C of low temperature refrigerator.
(2) DNA extracted with the detection of Thermo Scientific Varioskan Flash ELIASAs concentration and matter
Amount.
(3) the iQ5 quantitative PCR apparatus produced with Bio-rad companies detects MAP4K1, PIK3CA base in 142 parts of DNA samples
The copy number of cause and internal reference GAPDH genes.Each sample draws 20ng genomic DNAs, utilizes SYBR Green kits
(Qiagen, German) carries out quantitative PCR, and the primer is shown in Table 1.PCR programs are：95 DEG C of denaturation 15s, 58 DEG C of primer annealings
20s, 72 DEG C of extension 20s.
3rd, interpretation of result
It can may cause the change of gene expression in view of many factors, while it is very difficult to assess these factors, therefore
It easy can not reflect exactly evaluates the effect of tumour.And the change of gene copy number and tumour are closely related, it is possible to make
For diagnosing tumor and the target of efficacy determination.The present invention by detect the important gene MAP4K1 related to colorectal cancer,
PIK3CA copy number changes, its copy number is become to the relative indicatrix for being turned to evaluate colorectal cancer curative effect, there is provided medication is joined
Examine.
(1) each sample sets three multiple holes, if the different circulation more than one of multiple holes Ct value differences, will be removed out experiment point
Analysis.Following (Livak K, the Schmittgen TD.Analysis of relative gene of copy number analysis process
expression data using real-time quantitative CR and the 2(-DeltaDelta C(T))
method.Methods 2001；25:402–8.)：1st, the average value that each sample target gene three repeats Ct values is calculated, simultaneously
Calculate the average value that corresponding reference gene three repeats；2nd, the average value of all sample target gene and reference gene is calculated；3rd, use
The target gene Average Ct values of each sample subtract the Average Ct values of all sample target gene, and as target gene △ Ct are interior
Join the same algorithm process of △ Ct；4th, the 2 of each gene is calculated- Δ Ct (target gene), also calculate the 2 of reference gene- Δ Ct (reference gene)；5th, mesh
Gene 2-ΔΔCt=2- Δ Ct (target gene)/- Δ Ct (reference gene).MAP4K1, PIK3CA gene copy number are shown in Table 2 in 142 samples.
(2) Clinical symptoms of 142 III phase colorectal cancer patients is listed in table 2 by sample analysis packet, training set
76 patient age intermediate values are 60 years old (scope is 24-79 year), and the intermediate value of follow up time is 57 months.19 patients during follow-up
Recur (25%), be not reaching to the intermediate value without recurrence existence.66 patient age intermediate values of checking collection are that (scope was in 30- in 63 years old
84 years old), the intermediate value of follow up time is 39 months.During follow-up, 19 deaths (28.8%).Training set is concentrated with checking suffers from
There is no significant difference between person's age, sex, clinical stage statistically, have significant difference between existence statistics, pass through
Subsequent analysis is carried out after adjustment.
The III phase colorectal cancer patients Clinical symptoms in 2. two sources of table
(3) relation between copy number of target genes and clinical effectiveness：We using through the age, sex, knub position and
Multivariate Cox model histology adjusts by stages after have evaluated between MAP4K1, PIK3CA gene copy number and Patients on Recurrence
Contact.In training set sample, patient MAP4K1 genes (HR 1.11,95%CI, 1.03-1.19；P=0.01) copy number increase
Patients on Recurrence risk significantly improve；In checking collection sample, the increased Patients on Recurrence risk (HR=of MAP4K1 gene copy numbers
1.16；95%CI, 1.01-1.32；P=0.04) also significantly improve；Total sample concentrate risk of relapse ratio be 1.14 (95%CI,
1.06to1.22；P=0.0002).PIK3CA gene copies numerical value is separately carried out statistical analysis by us by three period in arithmetrics, training
Collect (HR, 4.40,95%CI 1.00-19.30；P=0.049), checking collection (HR=3.66；95%CI, 1.03-13.06, P=
And total sample set (HR=3.93 0.045)；95%CI, 1.53-10.14, P=0.005) in PIK3CA gene copy numbers it is increased
Patients on Recurrence risk also significantly improves.
(4) in order to further evaluate the risk of recurrence predictive value of MAP4K1, PIK3CA gene pairs patient, We conducted
Risk score is analyzed.With reference to MAP4K1 genes, 142 patients are divided into recurrence excessive risk group using risk score threshold value (2.7)
With low-risk group, the PIK3CA genes of two groups of patients are analyzed respectively.By contrast, the risk-ratio of excessive risk group is low
2.7 times (95%CI, 1.10-4.28) of risk group, it is more than 94 months nothings without recurrence life cycle relatively low risk group within its 77 months
It is appreciably shorter to recur life cycle.
(5) gene copy number and the relation of life cycle are assessed using Kaplan-Meier survivorship curves, utilizes log-
The conspicuousness of rank inspection statistics, is shown in Fig. 1.Low-risk group has preferable prognosis than excessive risk group, and patient is through oxaliplatin chemotherapeutic
After when reaching identical life span, the survival probability of low-risk group is apparently higher than excessive risk group.Log-rank examine P values be
0.04, illustrate there is significant difference between two packets in model.
4th, the application in curative effect judgement
The surgery excision cancerous tissue sample of colorectal cancer patients to be checked is gathered, according to preceding method, extracts DNA sample, and
Copy number of MAP4K1, PIK3CA gene in the specimens DNA sample is detected, then, evaluating the patient to be checked is
No suitable application oxaliplatin provides reference as front-line chemotherapeutic agents for clinical application.
5th, detection kit
Kit of the present invention is formed by following reagent and (is shown in Table 3), and source is as follows, and kit of the present invention detects for 100 person-portions
Using -20 DEG C of preservations：
The kit forms of table 3
| Component | Volume (mL) | Source |
| 2×SYBR Green qPCR Mix | 1 | Dongsheng biology |
| Ultra-pure water | 1 | Dongsheng biology |
| Forward primer 3 (each 2uM) | 0.2 | Self-control |
| Reverse primer 3 (each 2uM) | 0.2 | Self-control |
Claims (3)
1. a kind of be used to detect kit of the oxaliplatin for colorectal cancer validity, it is characterised in that the kit includes one
To detecting the amplimer sequence of MAP4K1 gene copy numbers and the amplimer sequence of a pair of detection PIK3CA gene copy numbers
And the primer sequence of a pair of detection reference gene GAPDH gene copy numbers,
Detect the positive sequence such as SEQ ID NO of the amplimer sequence of MAP4K1 gene copy numbers:Shown in 1, reverse sequence is such as
SEQ ID NO:Shown in 2,
Detect the positive sequence such as SEQ ID NO of the amplimer sequence of PIK3CA gene copy numbers:Shown in 3, reverse sequence is such as
SEQ ID NO:Shown in 4,
Detect the positive sequence such as SEQ ID NO of the amplimer sequence of reference gene GAPDH gene copy numbers:Shown in 5, reversely
Sequence such as SEQ ID NO:Shown in 6.
2. kit as claimed in claim 1, it is characterised in that also including 2 × SYBR Green qPCR Mix and ultrapure
The real-time fluorescence quantitative PCR amplification system of water composition.
3. kit as claimed in claim 2, it is characterised in that described real-time fluorescence quantitative PCR amplification system, every 20 μ L
Reaction system is：The amplimer sequence of detection MAP4K1 gene copy numbers, a pair of amplifications for detecting PIK3CA gene copy numbers
The forward primer concentration of the primer sequence of primer sequence and a pair of detection reference gene GAPDH gene copy numbers is 2 μM, instead
It it is 2 μM to primer concentration, 2 × SYBR Green qPCR Mix are 10 μ L, and remaining is ultra-pure water.
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| US11897878B2 (en) | 2018-10-31 | 2024-02-13 | Gilead Sciences, Inc. | Substituted 6-azabenzimidazole compounds |
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| EP3873608A1 (en) | 2018-10-31 | 2021-09-08 | Gilead Sciences, Inc. | Substituted 6-azabenzimidazole compounds having hpk1 inhibitory activity |
| CN111321220A (en) * | 2018-12-14 | 2020-06-23 | 中国医学科学院肿瘤医院 | Composition, microarray and computer system for detecting sensitivity of radiotherapy and chemotherapy of rectal cancer |
| TWI826690B (en) | 2019-05-23 | 2023-12-21 | 美商基利科學股份有限公司 | Substituted eneoxindoles and uses thereof |
| CN112680517B (en) * | 2020-12-28 | 2023-06-02 | 广东南芯医疗科技有限公司 | Guiding method and kit for oxaliplatin personalized medicine genes |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11897878B2 (en) | 2018-10-31 | 2024-02-13 | Gilead Sciences, Inc. | Substituted 6-azabenzimidazole compounds |
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