CN108949968B - A kind of mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit and its preparation method and application - Google Patents
A kind of mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit and its preparation method and application Download PDFInfo
- Publication number
- CN108949968B CN108949968B CN201811034967.6A CN201811034967A CN108949968B CN 108949968 B CN108949968 B CN 108949968B CN 201811034967 A CN201811034967 A CN 201811034967A CN 108949968 B CN108949968 B CN 108949968B
- Authority
- CN
- China
- Prior art keywords
- seq
- sequence
- primer
- tnf
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to field of biotechnology, and in particular to a kind of mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit and its preparation method and application.The kit includes for expanding the detection reagent of risk genes TNF-α CR polymorphism relevant to liver fibrosis, onset of liver cancer risk, positive reference substance and negative controls composition, and the detection reagent includes primer sequence, PNA sequence and the PCR reaction solution for expanding the site rs1800629 of TNF-α gene.Kit of the present invention is used to detect mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphism has many advantages, such as that high sensitivity, specificity is high, easy to operate, result is reliable, and detection can be completed in 1 hour, and result interpretation is simply objective.
Description
Technical field
The invention belongs to biomedical clinical detection technique fields, and in particular to a kind of mankind's liver fibrosis, liver cancer risk
Gene TNF-α polymorphic detection kit and its preparation method and application.
Background technique
Liver fibrosis refers to liver cell by inflammatory stimulus or when necrosing, and hyperplasia and the degradation of extracellular matrix lose
Balance, and then lead to the pathologic process of connective tissue abnormal deposition in liver.Primary carcinoma of liver occupies the 5th of common cancer,
Liver cancer about is died of more than 600,000 people every year, is number three in global cancer-related death, China suffers from liver cancer case and accounts for the whole world
50% or more, for liver cancer timely diagnosing and treating for improve survival be of great significance.
Liver fibrosis process is related to various kinds of cell and cell factor, forms a complicated network system and regulates and controls liver fibrosis
Occurrence and development.Promoting fibrosis factor TNF-α is considered as most important cell factor in inflammation associated tumour, multinomial to grind
Studying carefully proves that it participates in the occurrence and development process of hepatic injury, liver fibrosis and liver cancer pathologic.Research shows that TNF-α gene promoter
SNP (including -1031, -863, -857, -376, -308, -238) can influence the expression of TNF-α.Mostly research shows that TNF-α-
308 (G > A) are related with liver cancer genetic neurological susceptibility, have clinical detection meaning, and after -308 site G are replaced by A, TNF-α turns
Record efficiency will increase 6~7 times, significantly rise the expression quantity of TNF-α.The SNP of TNF-α -308 (G > A) is numbered
Rs1800629, positioned at the promoter region of TNF-α.About rs1800629 allele population diversity in each document of NCBI
Middle statistical data shows Asian A allele genotype accounting 2.2~14.6%.Document shows normal in Zhuang nationality in Guangxi crowd
A allele accounting 6.16% in control group, and HCC patient's accounting 17.16% carry the individual of TNF-α -308A allele
The risk for suffering from HCC is 3.153 times for carrying G allele individual.The detection of high frequency SNP has the detection of primary carcinoma of liver
Important value.
There are many kinds of the common methods detected at present for SNP, including PCR- direct sequencing, PCR- pyrosequencing
Method, PCR- gene chips, PCR- electrophoretic analysis, PCR- high-resolution melting curve method, ApoE gene method,
PCR- restriction fragment length polymorphism method, fluorescence quantitative PCR method, nest-type PRC-sonde method, in situ hybridization (ISH) etc..Most
It is PCR sequencing PCR for common method, this method expense is low, but time-consuming, sensitivity is not high；High-resolution melting curve method is to setting
It is relatively high for requiring, it is unfavorable for clinical expansion；Traditional fluorescent PCR method is clinically applied relatively broad, but detects micro spirit
Sensitivity is not high.Therefore, we establish it is a kind of it is easy to operate, cost is not high, the fluorescent PCR method of higher sensitivity detection liver fiber
Change, the polymorphism of liver cancer risk genes.
Summary of the invention
The present invention is in view of the deficiencies of the prior art, and it is an object of the present invention to provide a kind of mankind's liver fibrosis, liver cancer risk genes
TNF-α polymorphic detection kit and its preparation method and application.
For achieving the above object, the technical solution adopted by the present invention are as follows:
It is a kind of for mankind's liver fibrosis, the primer and PNA group of liver cancer risk genes TNF-α polymorphic detection, it is described to draw
Object and PNA group include sequence forward primer as shown in SEQ ID NO.1, sequence reverse primer as shown in SEQ ID NO.2,
Sequence as shown in SEQ ID NO.3 and 5 ' ends carry the identification primers of VIC fluorophors, sequence as shown in SEQ ID NO.4 and
5 ' ends carry the identification primer of FAM fluorophor, sequence as shown in SEQ ID NO.5 and 3 ' ends carry BHQ quenching group
PNA, sequence as shown in SEQ ID NO.6 and 3 ' ends carry the PNA of BHQ quenching groups, sequence as shown in SEQ ID NO.7 in
Ginseng forward primer, sequence internal reference reverse primer as shown in SEQ ID NO.8, sequence are as shown in SEQ ID NO.9 and 5 ' ends are taken
Internal reference identification primer with ROX fluorophor, sequence is as shown in SEQ ID NO.10 and 3 ' hold the internal reference for carrying BHQ quenching groups
PNA。
A kind of mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit, including detection reagent, the positive
Reference substance and negative controls；The detection reagent includes: sequence forward primer, sequence such as SEQ as shown in SEQ ID NO.1
Reverse primer, sequence shown in IDNO.2 are as shown in SEQ ID NO.3 and 5 ' ends carry the identification primer of VIC fluorophor, sequence
It arranges as shown in SEQ ID NO.4 and 5 ' the end carrying identification primers of FAM fluorophors, sequence is as shown in SEQ ID NO.5 and 3 '
End carries the PNA of BHQ quenching group, sequence as shown in SEQ ID NO.6 and 3 ' ends carry the PNA of BHQ quenching group, sequence such as
Internal reference forward primer shown in SEQ ID NO.7, sequence internal reference reverse primer, sequence such as SEQ as shown in SEQ ID NO.8
Shown in ID NO.9 and 5 ' ends carry the internal reference identification primer of ROX fluorophor, sequence as shown in SEQ ID NO.10 and 3 ' ends are taken
Internal reference PNA and PCR reaction solution with BHQ quenching group；
The positive reference substance includes: the recombination of the rs1800629-G of gene containing TNF-α, TNF-α gene rs1800629-A
The mixture of plasmid and the recombinant plasmid containing reference gene；
The negative control is the recombinant plasmid without reference gene and target gene site.
In above scheme, in the detection reagent, sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.7,
The concentration of primer described in SEQ ID NO.8 is 100~400nM, sequence such as SEQ ID NO.3, SEQ ID NO.4, SEQ ID
The concentration of primer described in NO.9 is 50~100nM, and sequence is as described in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.10
The concentration of PNA is 50~100nM.
In above scheme, the PCR reaction solution includes Premix qPCR MIX and TE buffer.
In above scheme, the negative controls are PUC-19 plasmid empty carrier.
Above-mentioned mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit are fine for mankind liver in preparation
Application in dimensionization, liver cancer risk genes TNF-α polymorphic detection product.The specifically used method of the kit includes as follows
Step:
(1) genomic DNA of human peripheral to be measured is extracted as sample to be detected using genome DNA extracting reagent kit；
(2) fluorogenic quantitative detection: sample to be detected and detection reagent are added into reaction tube, while positive controls are set
And negative control group, reaction tube is then placed in fluorescent PCR instrument and carries out pcr amplification reaction, and acquires FAM, VIC and ROX fluorescence
Signal；
(3) after pcr amplification reaction, result judgement is carried out according to fluorescence signal initial line situation collected.
In above scheme, it is 0.1~100ng/ μ L that the sample DNA to be detected, which is diluted to concentration, in step (1), then into
Row pcr amplification reaction.
In above scheme, the condition of step (2) described pcr amplification reaction are as follows: 95 DEG C of 1 minute initial denaturations；15 circulations: 95
DEG C 5 seconds, 61 DEG C 32 seconds, do not collect fluorescence；30 circulation: 95 DEG C 5 seconds, 61 DEG C 32 seconds, acquire fluorescence.
In above scheme, the criterion of step (3) described result judgement are as follows: 1. when positive control has FAM, VIC, ROX glimmering
Optical signal initial line, negative control is without FAM, VIC, ROX fluorescence initial line, when detecting sample has ROX fluorescence signal initial line, determines real
Success is tested, subsequent parting judgement can be carried out；2. judging TNF-α gene according to FAM, VIC fluorescence signal of detection sample
The parting of rs1800629: VIC fluorescence signal initial line, TNF-α gene rs1800629 parting are homozygous wildtype；FAM fluorescence letter
Number initial line, TNF-α gene rs1800629 parting are homozygous mutant；The equal initial line of VIC, FAM fluorescence signal, TNF-α gene
Rs1800629 parting is heterozygous mutant.
Kit of the present invention is the gene pleiomorphism kit based on ARMS-PNA- fluorescence quantifying PCR method, and technology is former
Reason is as shown in Figure 1.It include 2 pairs of common amplimers in detection reagent described in this kit, 3 identifications with fluorophor are drawn
Object, 3 PNA sequences matched with identification primer.Two identification primers 3 ' end for cls gene to be checked is SNP site alkali to be measured
Base, 5 ' hold mark fluorescent groups, wherein mutant primer flag F AM fluorophor, wild primer mark VIC fluorophor.In addition,
The end of identification primer 5 ' the label ROX fluorophor of reference gene.PNA (peptide nucleic acids, peptide nucleic acid) is a kind of
Replace the DNA analog of sugared phosphate backbone with polypeptide backbone, can be identified by way of base pairing and combines DNA or RNA
Sequence forms stable double-spiral structure, as PNA and complementary DNA exact matching, because its PNA is peptide chain amide 2- amino second
Base glycine bond energy enough prevents DNA cloning.PNA sequence 3 ' end label quenching group BHQ, can with identification primer match make not with
The identification primer that template strand combines can not discharge fluorescence signal.When amplified reaction occurs, common amplimer will contain SNP site
Sequence amplified from genomic DNA come, if identification primer be detached from PNA sequence in conjunction with template strand, identify that primer can be released
Fluorescence signal is put, with the increase of amplification cycles number, more and more to identify primer in conjunction with template strand, fluorescence signal is gradually put
Greatly；It can keep pairing that can not discharge fluorescence with PNA sequence if identification primer and template strand mismatch.Similarly, internal reference is detected
Identification primer can discharge ROX fluorescence signal in the reaction.VIC and ROX is presented in homozygous wildtype genotype in final detection result
FAM and ROX fluorescence initial line is presented in fluorescence initial line, homozygous mutant genotype, and it is glimmering that FAM, VIC and ROX is presented in heterozygous genotype
Light initial line.
Beneficial effects of the present invention are as follows:
(1) kit of the present invention can be rapid qualitative detection TNF-α gene pleiomorphism, directly sentence by the way that fluorescence is linear
Break as a result, it is possible to locus gene type to be measured be detected rapidly and sensitively, to accurately carry out base to human TNF-alpha gene's polymorphism
Because of parting；Using the identification primer of specificity fluorescent label, in a hole PCR, carry out PCR reaction gene can be completed
Parting detection (do not need a point hole be mutated respectively, wild or internal reference detection), realize each gene polynorphisms point
Only detection need to can be completed by a multi-PRC reaction in type, greatly reduce testing cost, improve detection efficiency；
(2) detection reagent of kit of the present invention is to prepare the finished product completed, it is only necessary to sample to be tested be added
PCR fluorescence reaction is carried out, detection time had not only been saved but also had improved detection efficiency；Also contain in detection reagent of the present invention simultaneously
There is reference gene primer, by detecting reference gene, false negative caused by sample extraction and false positive issue can be enable to have
Effect avoids, and guarantees the accuracy of testing result；
It (3) can be by by carrying the specificity identification primer of fluorophor and sequence label in kit of the present invention
The PNA for carrying quenching group combines that fluorescence is made to be in cancellation state, in PCR reaction process, carries the mirror of fluorophor
Release fluorescence generates fluorescence curve progress result interpretation after determining primer and gene template to be detected specific binding；It is glimmering using carrying
The specificity identification primer of light combines the PNA, internal control primer, internal control that carry quenching group to identify primer, internal control PNA, mentions significantly
The high specificity and accuracy of Genotyping, it is easy to operate, result is reliable, detection and genotyping can be completed in 1 hour；
(4) contain 2 pairs of common amplimers in kit of the present invention, it can be by the sequence containing SNP site from gene
It amplifies to come and be further enriched in group DNA, parting efficiency can be greatly improved, and then can detecte the gene of lower concentration
Group sample, kit of the present invention is minimum to carry out polymorphic detection to 0.1ng genomic DNA；
(5) kit of the present invention is suitable for common fluorescent PCR instrument, has sensitivity and specificity height, false positive low
The advantages that, while cheap, economical and convenient, conducive to the application on hospital clinical.
Detailed description of the invention
Fig. 1 is the detection schematic diagram of kit of the present invention.
Fig. 2 is the testing result amplification curve of kit minimum detection limit of the present invention (0.1ng/ μ L).
Fig. 3 is using kit of the present invention to TNF-α gene rs1800629 homozygous wildtype amplification curve.
Fig. 4 is using kit of the present invention to TNF-α gene rs1800629 heterozygous mutant amplification curve.
Fig. 5 is using kit of the present invention to TNF-α gene rs1800629 homozygous mutant amplification curve.
Fig. 6 is the testing result figure of the homozygous wild sample in the site TNF-α gene rs1800629 in embodiment 4.
Fig. 7 is the testing result figure of the site TNF-α gene rs1800629 heterozygous mutant sample in embodiment 4.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but it is of the invention
Content is not limited solely to the following examples.
Embodiment 1
It is a kind of for quickly detecting the kit of mankind's liver fibrosis, liver cancer risk genes TNF-α gene pleiomorphism, detect
Reagent is directed to the rs1800629 polymorphic site of TNF-α gene, and detection reagent includes: sequence is that the forward direction of SEQ ID NO.1 is drawn
(5 ' ends carry VIC fluorescent base to the identification primer that object, sequence are the reverse primer of SEQ ID NO.2, sequence is SEQ ID NO.3
Group), identification primer that sequence be SEQ ID NO.4 (5 ' end carrying FAM fluorophor), the PNA that sequence is SEQ ID NO.5
(3 ' ends carry BHQ quenching group), the PNA that sequence is SEQ ID NO.6 (3 ' ends carry BHQ quenching group), sequence SEQ
The internal reference that the internal reference forward primer of ID NO.7, sequence are the internal reference reverse primer of SEQ ID NO.8, sequence is SEQ ID NO.9
Identification primer (5 ' ends carry ROX fluorophor), (3 ' ends carry BHQ and base are quenched the internal reference PNA that sequence is SEQ ID NO.10
Group), PCR reaction solution (Premix qPCR MIX and TE buffer).Except above-mentioned detection reagent, kit further includes 1 positive
Reference substance and 1 negative controls, positive reference product are to include TNF-α gene rs1800629 (G), TNF-α gene
The recombinant plasmid of rs1800629 (A) and the mixture of the recombinant plasmid comprising reference gene, negative controls are without internal reference base
The recombinant plasmid of cause and target gene site.
For the primer and PNA sequence of TNF-α gene rs1800629 site primer, SEQ ID NO.1 and SEQ ID
NO.2 amplified production is that 139bp, SEQ ID NO.3 and SEQ ID NO.2 amplified production are 72bp, SEQ ID NO.4 and SEQ
It is 138bp that ID NO.1 amplified production, which is 105bp, SEQ ID NO.7 and SEQ ID NO.8 amplified production, SEQ ID NO.9 with
SEQ ID NO.8 amplified production is 119bp.
Upstream amplification primer SEQ ID NO.1:5 '-TCCTGCATCCTGTCTGGAAG-3 '
Downstream amplification primer SEQ ID NO.2:5 '-CAAGCATCAAGGATACCCCT-3 '
Wild identification primer SEQ ID NO.3:5 '-VIC-ATAGGTTTTGAGGGGCATGG-3 '
Mutation identification primer SEQ ID NO.4:5 '-FAM-GAGGCTGAACCCCGTCCT-3 '
Wild PNA sequence SEQ ID NO.5:5 '-CCATGCCCCTCAAAACCTAT-BHQ-3 '
It is mutated PNA sequence SEQ ID NO.6:5 '-GGGACGGGGTTCAGCCTC-BHQ-3 '
Internal reference upstream primer SEQ ID NO.7:5 '-CGGGACCTGACTGACTACCT-3 '
Internal reference downstream primer SEQ ID NO.8:5 '-GGCCATCTCTTGCTCGAAGT-3 '
Internal reference identifies primer SEQ ID NO.9:5 '-ROX-TCCAGGGCGACGTAGCACAG-3 '
Internal reference PNA sequence SEQ ID NO.10:5 '-CTGTGCTACGTCGCCCTGGA-BHQ-3 '
Mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit detection reagent in further include
PCR reaction solution (Premix qPCR MIX and TE buffer).Except above-mentioned detection reagent, kit further includes 1 positive control
Product and 1 negative controls, positive reference substance are to include TNF-α gene rs1800629 (G), TNF-α gene rs1800629 (A)
Recombinant plasmid and the recombinant plasmid comprising reference gene mixture, negative controls be without reference gene and target gene
The recombinant plasmid (PUC-19 plasmid empty carrier) in site.The above carrier sequence is not enumerate herein in detail known to those skilled in the art
Thin sequence.
Embodiment 2
A kind of mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit, including detection reagent, the positive
Control and negative control；Each constituent such as the following table 1 of the detection reagent, the composition of the kit as shown in Table 2:
1 detection reagent of table composition
| Component | Proportion |
| Premix qPCR MIX | 25μL |
| SEQ ID NO.1 | 100~400nM |
| SEQ ID NO.2 | 100~400nM |
| SEQ ID NO.3 | 50~100nM |
| SEQ ID NO.4 | 50~100nM |
| SEQ ID NO.5 | 50~100nM |
| SEQ ID NO.6 | 50~100nM |
| SEQ ID NO.7 | 100~400nM |
| SEQ ID NO.8 | 100~400nM |
| SEQ ID NO.9 | 50~100nM |
| SEQ ID NO.10 | 50~100nM |
| TE buffer | Complement to 30 μ L |
2 kit forms of table
Embodiment 3
Using the minimum detection limit of kit test sample prepared by embodiment 2, following operating procedure is specifically included:
(1) 5 human peripheral blood genomes are extracted using Tiangen biochemical biotechnology company's whole blood DNA extracts kit
DNA, concrete operation step are carried out in strict accordance with kit specification, obtain human genome DNA's sample to be detected, and sample is dilute
It releases to 2ng/ μ L, 0.5ng/ μ L, 0.1ng/ μ L, 0.05ng/ μ L.
(2) eight connecting leg of PCR is taken, the sample of 20 μ L gradient dilutions is added in hole respectively；It is added and is uniformly mixed in each hole
30 μ L of detection reagent；Eight connecting legs are shaken into centrifugation and mix reaction solution；
(3) eight connecting legs are put into 7500 fluorescent PCR instrument of ABI, carry out augmentation detection, PCR reaction condition such as table 3:
3 PCR response procedures of table
(4) result is analyzed:
Analyze 4 concentration gradient testing results of 5 samples: 2~0.1ng/ μ L can correct interpretation and the (examination of value≤17 Ct
One of agent box examination criteria), the 0.05ng/ μ L of 3 samples can correct interpretation but value > 17 Ct.It repeats to detect 20 samples
0.1ng/ μ L normal parting and can meet examination criteria, and lowest detection is limited to 0.1ng/ μ L (detection figure result such as Fig. 2 institute
Show).
Embodiment 4
Sample to be tested is detected using kit prepared by embodiment 2, specifically includes following operating procedure:
(1) 120 human peripheral blood genomes are extracted using Tiangen biochemical biotechnology company's whole blood DNA extracts kit
DNA, concrete operation step are carried out in strict accordance with kit specification, obtain human genome DNA's sample to be detected (0.1~
100ng/μL)；
(2) eight connecting leg of PCR is taken, 2 μ L samples to be detected are added in hole respectively, is added 2 μ L positive reference substances in a hole, one
2 μ L negative controls are added in hole, supplement the ddH of 18 μ L2O to 20 μ L；Uniformly mixed 30 μ of detection reagent is added in each hole
L；Eight connecting legs are shaken into centrifugation and mix reaction solution；
(3) eight connecting legs are put into 7500 fluorescent PCR instrument of ABI, carry out augmentation detection, PCR reaction condition such as table 3；
(4) result is analyzed:
Reference result interpretation table (as shown in table 4) and result interpretation are with reference to figure (as seen in figures 3-5), 120 samples of interpretation
Genotype；
Homozygous wild 111, sample of the site TNF-α rs1800629, testing result such as Fig. 6；
9, heterozygous mutant sample, the site TNF-α rs1800629, testing result such as Fig. 7；
The site TNF-α rs1800629 does not detect homozygous mutation sample；
4 result interpretation of table
| TNF-αrs1800629 | FAM(-A) | VIC(-G) | ROX (internal reference) |
| GG (homozygous wildtype) | – | + | + |
| AG (heterozygous mutant) | + | + | + |
| AA (homozygous mutant) | + | – | + |
Above-mentioned 120 sample fluorescence quantitative detection results and sequencing result 100% are consistent.The above result shows that this kit
It is reliable for mankind's liver fibrosis, liver cancer risk genes polymorphic detection result.
Obviously, above-described embodiment is only intended to clearly illustrate made example, and is not the limitation to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation or change therefore amplified
It moves within still in the protection scope of the invention.
Sequence table
<110>Wuhan Kang Lu Biotechnology Ltd.
<120>a kind of mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit and preparation method thereof and
Using
<160> 10
<210> 1
<211> 20bp
<212> DNA
<213>artificial sequence
400 > 1 of <
tcctgcatcc tgtctggaag 20
<210> 2
<211> 20bp
<212> DNA
<213>artificial sequence
400 > 2 of <
caagcatcaa ggatacccct 20
<210> 3
<211> 20bp
<212> DNA
<213>artificial sequence
400 > 3 of <
ataggttttg aggggcatgg 20
<210> 4
<211>18bp
<212> DNA
<213>artificial sequence
400 > 4 of <
gaggctgaac cccgtcct 18
<210> 5
<211>20bp
<212> DNA
<213>artificial sequence
400 > 5 of <
ccatgcccct caaaacctat 20
<210> 6
<211>18bp
<212> DNA
<213>artificial sequence
400 > 6 of <
gggacggggt tcagcctc 18
<210> 7
<211>20bp
<212> DNA
<213>artificial sequence
400 > 7 of <
cgggacctga ctgactacct 20
<210> 8
<211>20bp
<212> DNA
<213>artificial sequence
400 > 8 of <
ggccatctct tgctcgaagt 20
<210> 9
<211>20bp
<212> DNA
<213>artificial sequence
400 > 9 of <
tccagggcga cgtagcacag 20
<210> 10
<211>20bp
<212> DNA
<213>artificial sequence
400 > 10 of <
ctgtgctacg tcgccctgga 20
Claims (10)
1. primer and PNA group, which is characterized in that the primer and PNA group are drawn including sequence forward direction as shown in SEQ ID NO.1
Object, sequence reverse primer as shown in SEQ ID NO.2, sequence are as shown in SEQ ID NO.3 and 5 ' ends carry VIC fluorophor
Identification primer, sequence as shown in SEQ ID NO.4 and 5 ' end carry FAM fluorophors identification primer, sequence such as SEQ ID
Shown in NO.5 and 3 ' ends carry the PNA of BHQ quenching group, sequence as shown in SEQ ID NO.6 and 3 ' ends carry BHQ quenching group
PNA, sequence internal reference forward primer as shown in SEQ ID NO.7, sequence internal reference as shown in SEQ ID NO.8 reversely draw
Object, sequence are as shown in SEQ ID NO.9 and 5 ' ends carry internal reference the identification primer, sequence such as SEQ ID of ROX fluorophor
Shown in NO.10 and 3 ' ends carry the internal reference PNA of BHQ quenching group.
2. a kind of detection kit, which is characterized in that including detection reagent, positive reference substance and negative controls；
The detection reagent includes: sequence forward primer as shown in SEQ ID NO.1, sequence are as shown in SEQ ID NO.2
Reverse primer, sequence are as shown in SEQ ID NO.3 and 5 ' ends carry identification primer, the sequence such as SEQ ID of VIC fluorophor
Shown in NO.4 and 5 ' ends carry the identification primer of FAM fluorophor, sequence as shown in SEQ ID NO.5 and 3 ' ends carry BHQ and quench
The PNA of group, sequence go out as shown in SEQ ID NO.6 and 3 ' ends carry PNA, the sequence such as SEQ ID NO.7 of BHQ quenching groups
Shown in internal reference forward primer, sequence internal reference reverse primer as shown in SEQ ID NO.8, sequence as shown in SEQ ID NO.9
And 5 ' end carry ROX fluorophor internal reference identification primer, sequence as shown in SEQ ID NO.10 and 3 ' end carry BHQ base is quenched
The internal reference PNA and PCR reaction solution of group；
The positive reference substance includes: the recombinant plasmid of the rs1800629-G of gene containing TNF-α, TNF-α gene rs1800629-A
With the mixture of the recombinant plasmid containing reference gene；
The negative control is the recombinant plasmid without reference gene and target gene site.
3. the detection kit according to shown in claim 2, which is characterized in that in the detection reagent, sequence such as SEQ ID
NO.1, SEQ ID NO.2, SEQ ID NO.7, primer described in SEQ ID NO.8 concentration be 100 ~ 400nM, sequence such as SEQ
ID NO.3, SEQ ID NO.4, primer described in SEQ ID NO.9 concentration be 50 ~ 100 nM, sequence such as SEQ ID NO.5,
The concentration of PNA described in SEQ ID NO.6, SEQ ID NO.10 is 50 ~ 100 nM.
4. the detection kit according to shown in claim 2, which is characterized in that the PCR reaction solution includes Premix qPCR
MIX and TE buffer.
5. the detection kit according to shown in claim 2, which is characterized in that the negative controls are that PUC-19 plasmid is unloaded
Body.
6. any one of claim 2 ~ 5 detection kit is in preparation for detecting mankind's liver fibrosis, liver cancer risk genes
Application in TNF-α polymorphism product.
7. applying according to claim 6, which is characterized in that specific detection method includes the following steps:
(1) genomic DNA of human peripheral to be measured is extracted as sample to be detected using genome DNA extracting reagent kit；
(2) fluorogenic quantitative detection: sample to be detected and detection reagent are added into reaction tube, while positive controls and yin are set
Property control group, then reaction tube, which is placed in fluorescent PCR instrument, carries out pcr amplification reaction, and acquires FAM, VIC and ROX fluorescence signal；
(3) after pcr amplification reaction, result judgement is carried out according to fluorescence signal initial line situation collected.
8. application according to claim 7, which is characterized in that be diluted to the sample DNA to be detected in step (1) dense
Degree is 0.1 ~ 100ng/ μ L, then carries out pcr amplification reaction.
9. application according to claim 7, which is characterized in that the condition of step (2) described pcr amplification reaction are as follows: 95 DEG C
1 minute initial denaturation；15 circulation: 95 DEG C 5 seconds, 61 DEG C 32 seconds, do not collect fluorescence；30 circulation: 95 DEG C 5 seconds, 61 DEG C 32
Second, acquire fluorescence.
10. application according to claim 7, which is characterized in that the criterion of step (3) described result judgement are as follows: 1. work as sun
Property control group have FAM, VIC, ROX fluorescence signal initial line, negative control group detects sample without FAM, VIC, ROX fluorescence initial line
When originally having ROX fluorescence signal initial line, determines Success in Experiment, subsequent parting judgement can be carried out；2. according to detection sample FAM,
VIC fluorescence signal judges the parting of TNF-α gene rs1800629: VIC fluorescence signal initial line, and TNF-α gene rs1800629 divides
Type is homozygous wildtype；FAM fluorescence signal initial line, TNF-α gene rs1800629 parting are homozygous mutant；VIC, FAM fluorescence
The equal initial line of signal, TNF-α gene rs1800629 parting are heterozygous mutant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811034967.6A CN108949968B (en) | 2018-09-06 | 2018-09-06 | A kind of mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811034967.6A CN108949968B (en) | 2018-09-06 | 2018-09-06 | A kind of mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108949968A CN108949968A (en) | 2018-12-07 |
| CN108949968B true CN108949968B (en) | 2019-08-09 |
Family
ID=64475989
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811034967.6A Active CN108949968B (en) | 2018-09-06 | 2018-09-06 | A kind of mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108949968B (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101240322A (en) * | 2007-02-06 | 2008-08-13 | 上海主健生物工程有限公司 | Kit for detecting femaleosteoporosis susceptible inheritance risk |
| CN104293920B (en) * | 2014-09-17 | 2016-05-04 | 武汉康录生物技术有限公司 | A kind of kit and using method thereof for fast detecting mankind VKORC1 and CYP2C9 gene pleiomorphism |
| CN104212904B (en) * | 2014-09-17 | 2016-05-04 | 武汉康录生物技术有限公司 | A kind of kit and using method thereof for fast detecting mankind CYP2C19 gene pleiomorphism |
| CN105441568B (en) * | 2016-01-06 | 2019-01-08 | 武汉海吉力生物科技有限公司 | For detecting primer, probe and the kit of mankind BCR-ABL fusion T315I mutation |
-
2018
- 2018-09-06 CN CN201811034967.6A patent/CN108949968B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN108949968A (en) | 2018-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107058538B (en) | Primer composition, kit composed of primer composition and application of kit | |
| CN108977531B (en) | A kind of human hypertension's risk genes polymorphic detection kit and its preparation method and application | |
| CN109055367B (en) | A kind of mankind ALDH2 genetic polymorphism detection kit and its preparation method and application | |
| CN114085903B (en) | Primer pair probe combination product for detecting mitochondria 3243A & gtG mutation, kit and detection method thereof | |
| CN109055368B (en) | A kind of human vein's thrombotic risk gene F5 and PAI-1 polymorphic detection kit and its preparation method and application | |
| CN109055530A (en) | A kind of mankind's aspirin resistance genetic polymorphism detection kit and its preparation method and application | |
| WO2021136559A1 (en) | Method for distinguishing human dna based on fluorescent quantitative pcr technology | |
| CN106399479B (en) | A kind of SNP parting kit for the detection of type-2 diabetes mellitus tumor susceptibility gene | |
| CN106967719B (en) | Application of long-chain non-coding RNA as prostate cancer molecular marker | |
| CN108265113A (en) | ALDH2 genetic polymorphism detection kits | |
| CN109207611A (en) | One kind SNP marker relevant to sheep heat character and its detection kit and application | |
| JP2021171052A (en) | Microrna serum marker, primer set, kit, and their use to identify sex of sturgeon | |
| CN103374574B (en) | CYP4V2 gene mutant and application thereof | |
| CN108949968B (en) | A kind of mankind's liver fibrosis, liver cancer risk genes TNF-α polymorphic detection kit and its preparation method and application | |
| TWI402502B (en) | Genome detection system | |
| CN107058486B (en) | Primer group and kit for detecting glycolytic related genotyping of nasopharyngeal carcinoma | |
| CN113322317A (en) | Primer pair, probe set and kit for mitochondrial obesity gene mutation detection | |
| CN111057763B (en) | Kit for detecting susceptibility genotyping of human breast cancer, and use method and application thereof | |
| CN109182492B (en) | A kind of Citrin Defect Disease-causing gene detection primer and kit | |
| CN109055366A (en) | A kind of human asthma's risk genes polymorphic detection kit and its preparation method and application | |
| CN109182474A (en) | A kind of CYP2D6 detection in Gene Mutation primer and kit | |
| AU2021102364A4 (en) | Fluorescent pcr primer, probe and kit for detecting bovine rhinitis b virus | |
| CN106755489A (en) | High sensitivity detection method of gene mutation and used kit | |
| LU102676B1 (en) | A Biomarker for Severe Asthma and Application Thereof | |
| CN114457151B (en) | Detection kit for detecting phenylalanine hydroxylase gene mutation and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |