CN108753931A - A kind of CDX2 gene expressions fluorescent quantificationally PCR detecting kit and method - Google Patents
A kind of CDX2 gene expressions fluorescent quantificationally PCR detecting kit and method Download PDFInfo
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- CN108753931A CN108753931A CN201810571634.0A CN201810571634A CN108753931A CN 108753931 A CN108753931 A CN 108753931A CN 201810571634 A CN201810571634 A CN 201810571634A CN 108753931 A CN108753931 A CN 108753931A
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Abstract
The invention discloses specificity trip primer, β-actin probes, people's CDX2 genetic tests standard items and the β-actin standard items of a kind of CDX2 gene expressions fluorescent quantificationally PCR detecting kit, including the specific primer of CDX2, CDX2 probes, β-actin.The copy number of CDX2 and β-actin is surveyed by the other sorting of double calibration curve methods, and the ratio F=N of the two is calculatedt‑CDX2/Nt‑β‑actin, it is denoted as the relative expression quantity of CDX2, clinical guidance is provided for tumour patient adjuvant chemotherapy.Kit can be stablized with the relative expression quantity of specific detection CDX2 genes, sensitivity height, high specificity, result.
Description
Technical field
The present invention relates to a kind of CDX2 gene expressions fluorescent quantificationally PCR detecting kit and methods.
Background technology
Colon cancer is common malignant tumor of digestive tract, incidence including China majority state and area be in
The trend that existing incidence rises, and its death rate is higher.In past ten years, due to the appearance of novel auxiliary chemotherapy regimen, III
Phase colorectal cancer patients are greatly improved without disease (DFS) survival rate, but this effective adjuvant chemotherapy method
Success is not converted into early stage (I or II phases) colorectal cancer patients and obtains more long DFS.CDX2 is the same source capsule of gene line tail type
A member in gene (caudal homeobox gene) family belongs to intestines idiosyncratic transcription factor, to normal and tumorous intestines
Epithelium all has relative specificity and sensibility.In recent years research shows that, the variation of CDX2 gene expressions and it is out of control to alimentary canal,
The occurrence and development of the tumours such as urogenital system play an important role.
Currently, the method for detection CDX2 expression is mainly immunohistochemistry, although the method principle is simple, process mistake is tested
In cumbersome, the reagent type needed is various, and test result needs veteran expert to carry out interpretation, sentence read result exist compared with
Big subjectivity limits the application of the method to a certain extent.
Invention content
The technical problem to be solved by the present invention is to overcome in the prior art detection CDX2 expression method it is relatively complicated lack
It falls into, a kind of CDX2 gene expressions fluorescent quantificationally PCR detecting kit and method is provided.
In order to solve the above technical problem, the present invention provides the following technical solutions：
In order to overcome defect present in background technology, the technical solution adopted by the present invention to solve the technical problems to be：
A kind of CDX2 gene expressions fluorescent quantificationally PCR detecting kit, including the specific primer of CDX2, CDX2 probes,
Specificity trip primer, β-actin probes, people's CDX2 genetic tests standard items and the reference gene examination criteria product of β-actin.
CDX2 sense primers：5'-CTGAGAAGTGTCCCAGAGCC-3'
CDX2 downstream primers：5'-TGGGTGACGGTGGGGTTTA-3'
CDX2 probes：5'-FAM-TTGAGTCCGGTGTCTTCCCTGCAA-MGB-3'
β-actin sense primers：5'-CACCATGGATGATGATATCGC-3'
β-actin downstream primers：5'-CATAGGAATCCTTCTGACCCA-3'
β-actin probes：5'-HEX-GGCTTCGCGGGCGACGAT-MGB-3'
The sequence of people's CDX2 genetic test standard items is：
CTGAGAAGTGTCCCAGAGCCCTTGAGTCCGGTGTCTTCCCTGCAAGCCTCAGTGCCTGGCTCTGT
CCCTGGGGTTCTGGGGCCAACTGGGGGGGTGCTAAACCCCACCGTCACCCA
The sequence of the reference gene examination criteria product is：
CACCATGGATGATGATATCGCCGCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCCGGCT
TCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGGCGTGA
TGGTGGGCATGGGTCAGAAGGATTCCTATG。
Further include PCR reaction reagents in kit：DNTP, reverse transcriptase, reverse transcription buffer, Taq enzyme, Μ NG enzymes,
QPCR buffer solutions, ddH2O etc..
The preparation method of standard items is：The vein peripheral blood of Healthy People is acquired, mRNA is extracted and carries out reverse transcription acquisition
CDNA carries out PCR amplification with above-mentioned primer, is then built into CDX2 standard plasmids and β-using the target gene fragment of acquisition
The standard plasmid of actin, and the concentration and purity of bioassay standard product.Establish CDX2 and β-actin's respectively using standard plasmid
Standard curve.
The method for detecting CDX2 gene expressions using the kit, includes the following steps：
1) flesh tissue sample, is acquired, extracts total serum IgE, and measure the concentration and purity of RNA；Sample can derive from stone
The flesh tissue of wax tissue, puncturing tissue or operation excision；
2), reverse transcription：Using the total serum IgE of extraction as template, reverse transcription obtains cDNA；
3), quantitative fluorescent PCR reacts, and carries out PCR by template of the cDNA of the standard plasmid of gradient dilution and sample respectively
Amplified reaction；The condition of fluorescent quantitation reaction is that 95 DEG C of pre-degenerations handle 5min；95 DEG C denaturation 15s, 58 DEG C annealing 30s, 40
Cycle；
4) C, is readtValue, the C obtained using gradient standard plasmidtThe standard that value draws CDX2 and β-actin respectively is bent
Line, and according to the C of sampletValue, the copy number for obtaining CDX2 and β-actin is calculated separately using standard curve, is calculated
The ratio F=N of CDX2 and β-actin copy numberst-CDX2/Nt-β-actin, the as relative expression quantity of CDX2, wherein Nt-CDX2For
The gene copy number of CDX2, Nt-β-actinFor the gene copy number of β-actin.
The beneficial effects of the invention are as follows：The kit of the present invention measures to obtain people CDX2 by the method for double standard curves
The relative expression quantity of gene, sensitivity height, high specificity, result are stablized, the CDX2 expression that can be used between the different cases of comparison
Height, offer guidance and suggestion for tumour patient adjuvant chemotherapy.
Description of the drawings
Attached drawing is used to provide further understanding of the present invention, and a part for constitution instruction, the reality with the present invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the standard curve of kit of the present invention and the testing result of 3 clinical samples；
Fig. 2 is the amplification curve of 3 clinical samples.
Specific implementation mode
Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings, it should be understood that preferred reality described herein
Apply example only for the purpose of illustrating and explaining the present invention and is not intended to limit the present invention.
Embodiment
A kind of CDX2 gene expressions fluorescent quantificationally PCR detecting kit, including the specific primer of CDX2, CDX2 probes,
Specificity trip primer, β-actin probes, people's CDX2 genetic tests standard items and the reference gene examination criteria product of β-actin.
CDX2 sense primers：5'-CTGAGAAGTGTCCCAGAGCC-3'
CDX2 downstream primers：5'-TGGGTGACGGTGGGGTTTA-3'
CDX2 probes：5'-FAM-TTGAGTCCGGTGTCTTCCCTGCAA-MGB-3'
β-actin sense primers：5'-CACCATGGATGATGATATCGC-3'
β-actin downstream primers：5'-CATAGGAATCCTTCTGACCCA-3'
β-actin probes：5'-HEX-GGCTTCGCGGGCGACGAT-MGB-3'
The sequence of people's CDX2 genetic test standard items is：
CTGAGAAGTGTCCCAGAGCCCTTGAGTCCGGTGTCTTCCCTGCAAGCCTCAGTGCCTGGCTCTGT
CCCTGGGGTTCTGGGGCCAACTGGGGGGGTGCTAAACCCCACCGTCACCCA
The sequence of the reference gene examination criteria product is：
CACCATGGATGATGATATCGCCGCGCTCGTCGTCGACAACGGCTCCGGCATGTGCAAGGCCGGCT
TCGCGGGCGACGATGCCCCCCGGGCCGTCTTCCCCTCCATCGTGGGGCGCCCCAGGCACCAGGGCGTGA
TGGTGGGCATGGGTCAGAAGGATTCCTATG。
Further include PCR reaction reagents in kit：DNTP, reverse transcriptase, reverse transcription buffer, Taq enzyme, Μ NG enzymes,
QPCR buffer solutions, ddH2O.
The preparation method of standard items is：The vein peripheral blood of Healthy People is acquired, mRNA is extracted and carries out reverse transcription acquisition
CDNA carries out PCR amplification with above-mentioned primer, is then built into CDX2 standard plasmids and β-using the target gene fragment of acquisition
The standard plasmid of actin, and the concentration and purity of bioassay standard product.Establish CDX2 and β-actin's respectively using standard plasmid
Standard curve.
The method for detecting CDX2 gene expressions using the kit, includes the following steps：
1), acquisition takes proper amount of fresh tumor tissues, Trizol methods to extract total serum IgE, and the survey of concentration and purity is carried out to RNA
Fixed, the concentration of RNA is more than 200ng, and A260/A280 is more than 1.9；
2), reverse transcription：Using the total serum IgE of extraction as template, reverse transcription obtains cDNA；
TaKaRa PrimeScriptTM RT-PCR Kit
A. RNA 500ng are taken, dNTP, oligo dT Primer and ddH is added2O is made into 5 μ l systems, enterprising in PCR instrument
Row denaturation annealing reaction, 65 DEG C of 5min, 4 DEG C of ∞；
B. 5 μ l, 5x PrimeScript of reaction solution after above-mentioned denaturation is annealed is addedTMB μ ffer 2.0 μ l, RNase
Inhibitor (40 Μ/μ l) 0.25 μ l, PrimeScriptTMRTase 0.25 μ l, ddH22.5 μ l of O, 10.0 μ l of total volume.
C. reaction condition：30 DEG C of 10min, 50 DEG C of 30min, 95 DEG C of 5min；
3), quantitative fluorescent PCR reacts, and carries out PCR by template of the cDNA of the standard plasmid of gradient dilution and sample respectively
Amplified reaction；The condition of fluorescent quantitation reaction is that 95 DEG C of pre-degenerations handle 5min；95 DEG C denaturation 15s, 58 DEG C annealing 30s, 40
Cycle；
4) C, is readtValue, the C obtained using gradient standard plasmidtThe standard that value draws CDX2 and β-actin respectively is bent
Line, as shown in Figure 1, and according to the C of sampletValue, the copy number for obtaining CDX2 and β-actin is calculated separately using standard curve,
The ratio F=N of CDX2 and β-actin copy numbers is calculatedt-CDX2/ Nt-β-actin, the as relative expression quantity of CDX2, wherein
Nt-CDX2For the gene copy number of CDX2, Nt-β-actinFor the gene copy number of β-actin.
Reaction system is：
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
With technical scheme described in the above embodiments is modified or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in the present invention's
Within protection domain.
<110>The accurate medical test Co., Ltd of Wuxi canonical
<120>A kind of CDX2 gene expressions fluorescent quantificationally PCR detecting kit and method
<160>8
<210>1
<211>20
<212>DNA
<213>Artificial Sequence
<400>1
CTGAGAAGTG TCCCAGAGCC 20
<210>2
<211>19
<212>DNA
<213>Artificial Sequence
<400>2
TGGGTGACGG TGGGGTTTA 19
<210>3
<211>24
<212>DNA
<213>Artificial Sequence
<400>3
TTGAGTCCGG TGTCTTCCCT GCAA 24
<210>4
<211>21
<212>DNA
<213>Artificial Sequence
<400>4
CACCATGGAT GATGATATCG C 21
<210>5
<211>21
<212>DNA
<213>Artificial Sequence
<400>5
CATAGGAATC CTTCTGACCC A 21
<210>6
<211>18
<212>DNA
<213>Artificial Sequence
<400>6
GGCTTCGCGG GCGACGAT 18
<210>7
<211>116
<212>DNA
<213>Artificial Sequence
<400>7
CTGAGAAGTG TCCCAGAGCC CTTGAGTCCG
GTGTCTTCCC TGCAAGCCTC AGTGCCTGGC
TCTGTCCCTG GGGTTCTGGG GCCAACTGGG
GGGGTGCTAA ACCCCACCGT CACCCA 116
<210>8
<211>164
<212>DNA
<213>Artificial Sequence
<400>8
CACCATGGAT GATGATATCG CCGCGCTCGT
CGTCGACAAC GGCTCCGGCA TGTGCAAGGC
CGGCTTCGCG GGCGACGATG CCCCCCGGGC
CGTCTTCCCC TCCATCGTGG GGCGCCCCAG
GCACCAGGGC GTGATGGTGG GCATGGGTCA
GAAGGATTCC TATG 164
Claims (5)
1. a kind of CDX2 gene expressions fluorescent quantificationally PCR detecting kit, which is characterized in that specific primer including CDX2,
Specificity trip primer, β-actin probes, people's CDX2 genetic tests standard items and the β-actin standards of CDX2 probes, β-actin
Product；
The sequence of CDX2 sense primers as shown in SEQ ID No.1,
The sequence of CDX2 downstream primers as shown in SEQ ID No.2,
The sequence of CDX2 probes as shown in SEQ ID No.3,
The sequence of β-actin sense primers as shown in SEQ ID No.4,
The sequence of β-actin downstream primers as shown in SEQ ID No.5,
The sequence of β-actin probes is as shown in SEQ ID No.6.
2. CDX2 gene expressions fluorescent quantificationally PCR detecting kit as described in claim 1, which is characterized in that the people
The sequence of CDX2 genetic test standard items is as shown in SEQ ID No.7, the sequence such as SEQ ID of the β-actin standard items
Shown in No.8.
3. using CDX2 gene expression fluorescent quantificationally PCR detecting kits detection CDX2 gene expressions described in claim 1
Method, which is characterized in that include the following steps：
1) flesh tissue sample, is acquired, extracts total serum IgE, and measure the concentration and purity of RNA；
2), reverse transcription：Using the total serum IgE of extraction as template, reverse transcription obtains cDNA；
3), quantitative fluorescent PCR reacts, and carries out PCR by template of the cDNA of the β-actin standard items of gradient dilution and sample respectively
Amplified reaction；
4) C, is readtValue, the C obtained using gradient standard plasmidtValue draws the standard curve of CDX2 and β-actin, and root respectively
According to the C of sampletValue, the copy number for obtaining CDX2 and β-actin is calculated separately using standard curve, CDX2 and β-is calculated
The ratio F=N of actin copy numberst-CDX2/Nt-β-actin, the as relative expression quantity of CDX2, wherein Nt-CDX2For the gene of CDX2
Copy number, Nt-β-actinFor the gene copy number of β-actin.
4. method as claimed in claim 3, feature exist, the condition of fluorescent quantitation reaction is that 95 DEG C of pre-degenerations handle 5min；
95 DEG C of denaturation 15s, 58 DEG C of annealing 30s, 40 recycle.
5. method as claimed in claim 3, feature exist, samples sources are in paraffin organization, puncturing tissue or operation excision
Flesh tissue.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113930436A (en) * | 2021-10-28 | 2022-01-14 | 吉林正业生物制品股份有限公司 | Double-standard-curve rabies virus positive standard plasmid and application thereof in quantitative detection of rabies virus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906288A (en) * | 2017-03-24 | 2017-06-30 | 福州艾迪康医学检验所有限公司 | Detect the primer and method of leukaemia CDX2 gene expression doses |
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2018
- 2018-06-06 CN CN201810571634.0A patent/CN108753931A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906288A (en) * | 2017-03-24 | 2017-06-30 | 福州艾迪康医学检验所有限公司 | Detect the primer and method of leukaemia CDX2 gene expression doses |
Non-Patent Citations (3)
| Title |
|---|
| 刘以俊 等: "急性白血病遗传学改变的预后分析及微小残留病检测", 《医学综述》 * |
| 李力等: "《实用生物医学概论教程》", 31 July 2016, 广西科学技术出版社 * |
| 许春伟 等: "5"-Aza-CdR对HT-29和LoVo结直肠癌细胞株中CDX2基因甲基化状态、mRNA表达及蛋白表达的影响", 《世界华人消化杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113930436A (en) * | 2021-10-28 | 2022-01-14 | 吉林正业生物制品股份有限公司 | Double-standard-curve rabies virus positive standard plasmid and application thereof in quantitative detection of rabies virus |
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Application publication date: 20181106 |