CN106086231A - The Pyrosequencing primer of qualitative detection STAT4 gene type to and test kit - Google Patents
The Pyrosequencing primer of qualitative detection STAT4 gene type to and test kit Download PDFInfo
- Publication number
- CN106086231A CN106086231A CN201610773331.8A CN201610773331A CN106086231A CN 106086231 A CN106086231 A CN 106086231A CN 201610773331 A CN201610773331 A CN 201610773331A CN 106086231 A CN106086231 A CN 106086231A
- Authority
- CN
- China
- Prior art keywords
- stat4
- test kit
- primer
- amplimer
- plasmid
- 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.)
- Pending
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
- 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
- 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
- C12Q1/6869—Methods for sequencing
-
- 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)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
Abstract
The present invention relates to the Pyrosequencing primer pair of a kind of qualitative detection STAT4 gene type.Described primer is to including forward amplimer, reverse amplimer, sequencing primer, and 5 ' ends of described reverse amplimer carry out biotin labeling respectively.The invention still further relates to the Manganic pyrophosphate complex initiation test kit of a kind of qualitative detection STAT4 gene type.Described test kit includes amplimer, PCR reactant liquor, sequencing primer, uracil dna glycosylase and Taq polymerase.The present invention has the advantage that testing result is accurate, specificity is high, the detection cycle is short, simple to operate and can effectively meet Clinical Laboratory requirement；Can monitor that reaction process, response time is short, PCR primer simple process can go up pyrophosphoric acid sequencer and high flux sample detection, and ratio goldstandard method, the i.e. higher advantage of capillary electrophoresis sequencing sensitivity in real time additionally, also have.
Description
Technical field
The present invention relates to external nucleic acid detection technique field, particularly relate to Jiao of a kind of qualitative detection STAT4 gene type
Phosphoric acid sequencing primer to and test kit.
Background technology
Hepatocarcinoma is the malignant tumor that whole world fatality rate is in the 3rd, is also Chinese common a kind of malignant disease.The whole world
There are about 700,000 people every year and die from hepatocarcinoma.In hepatocarcinoma, hepatocellular carcinoma (HepatoCellular Carcinoma, HCC) is most common,
The investigation of 2000 of international cancer research institution shows, in HCC patient, 80% has with opposite sex hepatitis virus and hepatitis C virus
Close.History questionnaire is bright, and in the hepatocarcinoma patient of China, more than 80% has hepatitis B medical history.The data issued according to Ministry of Public Health, in
State's current total has 93,000,000 Hepatitis B patients, accounts for more than 1/3rd of whole world hepatitis B patient sum.
After China's universal hepatitis B vaccine injection in 1992, onset of liver cancer rate is existing to be declined.But the people being born before, about
8%～9% in hepatitis B surface antigen, (HBsAg) is positive.Therefore, China's Mainland will be one in following 40～50 years hepatocarcinoma
Individual important public health problem.Owing to primary hepatocarcinoma (HCC) patient occurs that clinical manifestation often belongs to terminal stage of a disease, mortality rate
Height, therefore, examination high risk factor is for the risk of the concurrent HCC of patient of dlinial prediction hepatitis B virus infection, beneficially disease
Early discovery and treatment.
Signal transduction and activity factor (STAT) family are class transcription factor of discovered in recent years, including STAT1,
STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6.STAT family is mediating cytokine, growth factor signal path
The important transcription factor of upper startup protein transcription, they all play important work in the cellular elements event of many signal paths
With, such as cell differentiation, cell proliferation and survival.
STAT4 is positioned at chromosome 2q32.2-2q32.3；Being T cell, NK cell, dendritic cell, core macrophage etc. is exempted from
The crucial element of IL-12/STAT4/IFN-γ signal transduction path in epidemic disease regulating cell, is that IL-12 induces immunoregulation
Cell produces the key regulator of IFN-γ, the immunoreation of mediation IL-12 and the differentiation of regulatory T-cell.Foreign study is demonstrate,proved
Real, STAT4 gene pleiomorphism participates in the generation of various autoimmune disease.
STAT4 is conducted by signal, makes interleukin 12 induction produce IFN-γ.IFN-γ is a kind of pleiotropic cellular
The factor, has important effect in host defense.Due to STAT4 gene pleiomorphism, the IFN-γ of activation may reduce it
Antiviral and anti-tumor activity, confirmed by research: the allelic single nucleotide polymorphism of G in STAT4 and hepatitis B cancer
The excessive risk become is relevant.
In December, 2012, genetic institute of Fudan University, genetic engineering National Key Laboratory professor Yu Long, " from
So hereditism " " the Genetic variants in STAT4and HLA-DQ that delivers on (Nature Genetics) magazine
Genes confer risk of hepatitis B virus related hepatocellular carcinoma ", determine
STAT4 and the HLA-DQ gene of people is the crucial tumor susceptibility gene of hepatitis B patient suffering from hepatic cancer.
Yu Long seminar contacted by 30 seminars at home and abroad, and 66 scholars carry out Co-operation research, have collected domestic 7 ground
District, amount to the hemocyte DNA sample of 11799 example hepatitis B patients.Including 5480 examples have hepatitis B pathological changes hepatocarcinoma case and
6319 examples have hepatitis B medical history but without the collator of hepatocarcinoma.Whole-genome association technology comparison is used to analyze this two groups of crowds
Whole genome sequence in the gene frequency of nearly 730,000 mononucleotide polymorphic sites, finally at STAT4 gene and HLA-
The susceptibility loci significantly associated with hepatitis B canceration risk it is found that in DQ gene cluster.
STAT4 gene is the crucial tumor susceptibility gene of hepatitis B patient suffering from hepatic cancer, studies further for the mankind and how to reduce liver
Cancer onset risk, treatment hepatitis B and hepatocarcinoma indicate new direction.By detecting the typing of STAT4 gene, examination hepatocarcinoma easy
Touching group, thus in advance Susceptible population is carried out corresponding pai n nursing and preventive measure, reduce onset of liver cancer risk.Therefore enter
The gene type detection in row STAT4 site has very important meaning for prevention hepatitis B canceration.
Manganic pyrophosphate complex initiation (Pyrosequencing) is that a kind of DNA sequencing based on polymerization principle (that is, determines DNA center
The order of thuja acid) method, belong to a new generation's DNA sequence analysis technology, possess the energy that a large amount of samples are carried out sequencing analysis simultaneously
Power, and there is high flux, specificity height, quick, the directly perceived and advantage of low cost.Its ultimate principle is, enzymatic same by 4 kinds
Enzyme cascade chemiluminescence reaction in one reaction system, takes turns in sequencing reaction each, only adds a kind of dNTP, if this dNTP with
Template is matched, and polymerase just can be incorporated in primer strand and discharge the pyrophosphoric acid group (PPi) of equimolar number, PPi
Can be eventually converted into visible light signal, and be converted into a peak value by PyrogramTM, the height of each peak value is mixed in reaction
The nucleotide number entered is directly proportional；It is subsequently adding lower a kind of dNTP, continues the synthesis of DNA；Finally by analyzing peak value
Situation, reaches to measure the purpose of DNA sequence.But, prior art does not the most utilize pyrosequencing techniques to detect STAT4 base
Product because of typing.
At present, in the prior art, use the PCR-direct sequencing of the title having " goldstandard " that STAT4 gene is examined
Surveying, but the sensitivity of the method is the highest, can only detect that mutant cell ratio is in the tumor tissues of more than 10-20% and periphery
Blood, is less than tumor tissues and the peripheral blood of 10% for mutant cell ratio, and Standard PCR-direct sequencing is the most helpless；
Additionally, the method there is also, cost is high, detect cycle length and the shortcoming of complex operation.
Summary of the invention
In order to solve said method detection STAT4 genotyping process in exist sensitivity the highest, detection cycle length, operation
The technical problem that loaded down with trivial details and cost is high, the present invention provides a kind of sensitivity height, high specificity, detection cycle short, simple to operate also
Effectively meet Clinical Laboratory requirement qualitative detection STAT4 gene type Pyrosequencing primer to and test kit.
The invention provides the Pyrosequencing primer pair of a kind of qualitative detection STAT4 gene type, described primer is to bag
Include:
STAT4 forward amplimer:
5’-GGAAAGGTTCACACTTGACTGTT-3’(SEQ ID NO.1)；
The reverse amplimer of STAT4:
5’-CCCCTGAAATTCCACTGAAATAAG-3’(SEQ ID NO.2)；
STAT4 sequencing primer: 5 '-AAAAGTTGGTGACCAAAATG-3 ' (SEQ ID NO.3)；
Wherein, 5 ' ends of the reverse amplimer of described STAT4 carry out biotin labeling.
Present invention also offers the Manganic pyrophosphate complex initiation test kit of a kind of qualitative detection STAT4 gene type, described test kit
Including:
PCR reactant liquor, described PCR reactant liquor contains STAT4 forward amplimer:
5’-GGAAAGGTTCACACTTGACTGTT-3’；
The reverse amplimer of STAT4:
5’-CCCCTGAAATTCCACTGAAATAAG-3’；
STAT4 sequencing primer: 5 '-AAAAGTTGGTGACCAAAATG-3 '；
Wherein, 5 ' ends of the reverse amplimer of described STAT4 carry out biotin labeling.
In a kind of preferred embodiment of the described test kit of present invention offer, described test kit also includes:
STAT4 positive reference substance 1, it is the STAT4 wild homozygote matter being inserted with nucleotide sequence shown in SEQ ID NO.5
Grain；
SEQ ID NO.5:
5’-ACACATATGGAAAGGTTCACACTTGACTGTTAATACGGATGTCTTTGAAGGTAGTGGTGTGGATGG
AGGTAAGGAAAAAAGAAGTGGGATAAAAAGAAGTTTGTAATTAAAAAGCTACATGTATATTATGATCTACTTTATGG
AAAATTACATGAGTGTGTATGCAGTAAAAGTATGAAAAGTTGGTGACCAAAATGTGAATAGTGGTTATCTTATTTCA
GTGGAATTTCAGGGGATTTTTTTTCTTTCTTCTTAGACTTTTCATTATCATTTGACTTTTTACAAAGATTTGCATTA
TTTAAGCAATCAGAAAGAAATTATAAAGCTATTTTCATCATAACAAAAATTCCATTGGTAAAAAATTTTTAATTAAT
TTACATAATGTGCAAAAATTAGAAAATTAGAACTCCTAAAGCAAGAAGTGGAAAAATTATTCCAATCTGAAGAAATA
AAACCATTCTCTGATGACTTGGAAACTGAAGGGATTGCTAATAGCTC-3’；
STAT4 positive reference substance 2, its for described STAT4 wild homozygote plasmid be inserted with nucleoside shown in SEQ ID NO.6
The plasmid mixture of the STAT4 no mutant homozygote plasmid composition of acid sequence；
STAT4 positive reference substance 3, it is the STAT4 no mutant homozygote matter being inserted with nucleotide sequence shown in SEQ ID NO.6
Grain；
SEQ ID NO.6:
5’-ACACATATGGAAAGGTTCACACTTGACTGTTAATACGGATGTCTTTGAAGGTAGTGGTGTGGATGG
AGGTAAGGAAAAAAGAAGTGGGATAAAAAGAAGTTTGTAATTAAAAAGCTACATGTATATTATGATCTACTTTATGG
AAAATTACATGAGTGTGTATGCAGTAAAAGTATGAAAAGTTGGTGACCAAAATGTTAATAGTGGTTATCTTATTTCA
GTGGAATTTCAGGGGATTTTTTTTCTTTCTTCTTAGACTTTTCATTATCATTTGACTTTTTACAAAGATTTGCATTA
TTTAAGCAATCAGAAAGAAATTATAAAGCTATTTTCATCATAACAAAAATTCCATTGGTAAAAAATTTTTAATTAAT
TTACATAATGTGCAAAAATTAGAAAATTAGAACTCCTAAAGCAAGAAGTGGAAAAATTATTCCAATCTGAAGAAATA
AAACCATTCTCTGATGACTTGGAAACTGAAGGGATTGCTAATAGCTC-3’；
Wherein, plasmid vector is pMD18-T plasmid；STAT4 no mutant homozygote described in described STAT4 positive reference substance 2
The quantity of plasmid and described STAT4 wild homozygote plasmid is than for 1:1.
In a kind of preferred embodiment of the described test kit of present invention offer, described test kit also includes: quality-control product
(control oligo) and blank product, the sequence of described quality-control product is: TAYGGTTTGCA (SEQ ID NO.4)；Described
Blank product are ultra-pure water；The sequence of quality-control product is by one section of oligonucleotide chain of QIAGEN design synthesis, is used for detecting
The property indices of PyroMark Q24 sequenator.
In described PCR reactant liquor, other components are conventional 10x PCR Buffer, dNTPS and H2O, each component is routinely
Volume ratio configuration (10x PCR Buffer, dNTPs, H2In O and reactant liquor, the volume ratio of primer is 5:3:37.5:1).
Other reagent and conventional reagent that solution is PCR and DNA Manganic pyrophosphate complex initiation in described test kit, as being polymerized by DNA
Enzyme, adenosine triphosphate sulfurylase, luciferase and the enzymatic mixture of bisphosphatase composition, by 5'-phosphosulfate and fluorescein
The substrate mixture of composition, uracil dna glycosylase, Taq polymerase etc..
Present invention also offers primer as above to preparation answering in the reagent detecting STAT4 gene type
With.
Compared to prior art, the Pyrosequencing primer of the qualitative detection STAT4 gene type that the present invention provides to and
Test kit has the advantages that
One, by utilize Manganic pyrophosphate complex initiation law technology devise highly sensitive and that specificity is good primer to and reagent
Box so that described test kit detect STAT4 gene type time, have qualitative accurately, the highly sensitive and advantage of high specificity；
Additionally, also have, sample treatment is simple, sequencing steps simple, order-checking speed is fast, half an hour completes once to go up machine and reacts, directly
The advantage providing detection site frequency analysis and visual result；
Two, by utilize Manganic pyrophosphate complex initiation law technology devise highly sensitive and that specificity is good primer to and reagent
Box so that described test kit, when detecting STAT4 gene type, can monitor that reaction process, response time is short, PCR primer in real time
Simple process can go up pyrophosphoric acid sequencer, easy and simple to handle and high flux sample detection, and ratio goldstandard method, i.e. capillary
Electrophoresis tube sequencing sensitivity is higher, is particularly suited for mutation analysis and Clinical Laboratory；
Three, by being provided with blank product, positive reference substance and quality-control product in described test kit so that described reagent
Box, when detecting STAT4 gene type, can preferably guarantee the accuracy of testing result.
Accompanying drawing explanation
Fig. 1 is the Manganic pyrophosphate complex initiation figure of clinical sample STAT4 wild type；
Fig. 2 is the Manganic pyrophosphate complex initiation figure of clinical sample STAT4 sudden change heterozygous；
Fig. 3 is the Manganic pyrophosphate complex initiation figure of clinical sample STAT4 mutant homozygous type；
Fig. 4 is the Manganic pyrophosphate complex initiation figure of clinical quality-control product control oligo；
Fig. 5 is the Manganic pyrophosphate complex initiation figure of clinical STAT4 blank product；
Fig. 6 to Fig. 8 is the Manganic pyrophosphate complex initiation figure of many groups of STAT4 design primer；Wherein the sequencing result of Fig. 6 and Fig. 7 is forbidden
Really, the sequencing result of Fig. 8 is true and reliable.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with the accompanying drawings and embodiment, right
The present invention is described in further detail.Should be appreciated that specific embodiment described herein only in order to explain the present invention, and
It is not used in the restriction present invention.
Embodiment 1: the preparation of test kit
One, primer and the design of probe and synthesis
For the mutational site that people's STAT4 gene Selection is special, use PyroMark Assay Design2.0 software, if
Meter primer；Wherein amplimer and sequencing primer first pass through PAGE purification, then through HPLC purification, wherein the 5 ' of SEQ ID NO.2
End carries out biotin labeling.
Table 1. mutational site and type
| Mutation | Base change |
| STAT4(rs2242480) | G>T |
Extension increasing sequence such as table 2:
Table 2. specificity amplification primer and primer sequence
Two, reference substance selects
The one section of oligonucleotide chain TAYGGTTTGCA control oligo using synthetic is quality-control product；
DNase/RNase-Free water is blank product.
Three, PCR reactant liquor composition
Table 3.PCR reactant liquor forms
| Material name | Volume (μ L) |
| 10x PCR Buffer | 5 |
| dNTP | 3 |
| STAT4 forward amplimer | 1 |
| H<sub>2</sub>O | 37.5 |
| Cumulative volume | 46.5μL |
Embodiment 2: the use of test kit
One, sample detection
Dissolve primer dry powder (after primer dissolves, effect duration is 1 month).System is prepared: take PCR reactant liquor according to template number,
Add solvent primer, uracil dna glycosylase, Taq DNA polymerase, subpackage system, add sample DNA, blank product
Or positive reference substance is template, form PCR reaction system.PCR amplification is carried out according to PCR response procedures.
The each main component of STAT4 system is as follows:
The each main component of table 4.STAT4 system
This system response procedures is as follows:
Table 5.PCR response procedures
After having expanded, agarose gel inspection PCR result, to carry out next step program.
Two, Manganic pyrophosphate complex initiation
Carry out sequencing procedures according to Manganic pyrophosphate complex initiation standard operating procedure, mainly comprise the following steps: the preparation and purification of sample, so
After sample after purification added upper machine order-checking in the MIX containing annealing liquid and sequencing primer.In Manganic pyrophosphate complex initiation instrument agent bin
Add the corresponding dATP of operation program, dTTP, dCTP, dGTP, enzymatic mixture, substrate mixture.Quality-control product control oligo
Carrying sequencing primer, ultimate density is 0.2 μM.
Three, result judges
Quality-control product base recall rate is 100%；Blank product recall rate is 0.
Four, quality control standard
All kinds of comparison quality-control product judged result such as following tables:
Table 6. quality-control product standard testing result
| Reference substance | Standard test result | |
| 1 | Quality-control product | Peak height peak type is normal, sequence is accurate |
| 2 | Blank product | Base recall rate is 0 |
Five, result report:
As shown in Figure 1, Figure 2 and Figure 3, the criterion of sample result is as follows for result:
Sample detection result reported by table 9.
| Pattern detection result | Report result | |
| 1 | STAT4 (G >=90%, T≤10%) | Wild type |
| 2 | STAT4 (G≤10%, T >=90%) | Mutant homozygous type |
| 3 | STAT4 (40%≤G≤60%, 40%≤T≤60%) | Sudden change heterozygous |
Fig. 1 is shown that the wild type of STAT4 in clinical sample testing result, and Fig. 2 is shown that clinical sample detection knot
The sudden change heterozygous of STAT4 in Guo, Fig. 3 is shown that the mutant homozygous type of STAT4 in clinical sample testing result, Fig. 4 and Fig. 5
It is shown that quality-control product control oligo and the Manganic pyrophosphate complex initiation figure of STAT4 blank product in Clinical detection result respectively；
Fig. 6 to Fig. 8 is shown that the Manganic pyrophosphate complex initiation design sketch of STAT4 many groups primer of design, and wherein the primer of Fig. 8 is optimal, is
STAT4 primer selected in our product.
The Pyrosequencing primer of the qualitative detection STAT4 gene type that the present invention provides to and test kit have and following have
Benefit effect:
One, by utilize Manganic pyrophosphate complex initiation law technology devise highly sensitive and that specificity is good primer to and reagent
Box so that described test kit detect STAT4 site time, have qualitative accurately, the highly sensitive and advantage of high specificity；This
Outward, also have that sample treatment is simple, sequencing steps simple, order-checking speed is fast, half an hour complete once go up machine react, directly to
The advantage going out detection site frequency analysis and visual result；
Two, by utilize Manganic pyrophosphate complex initiation law technology devise highly sensitive and that specificity is good primer to and reagent
Box so that described test kit, when detecting STAT4 site, can monitor reaction process, the response time is short, PCR primer is simple in real time
Process can go up pyrophosphoric acid sequencer, easy and simple to handle and high flux sample detection, and ratio goldstandard method, i.e. capillary tube electricity
Swimming sequencing sensitivity is higher, is particularly suited for mutation analysis and Clinical Laboratory；
Three, by being provided with blank product, positive reference substance and quality-control product in described test kit so that described reagent
Box, when detecting STAT4 site, can preferably guarantee the accuracy of testing result.
The foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention, every utilize this
The equivalent flow process conversion that bright description is made, or directly or indirectly it is used in other relevant technical field, the most in like manner wrap
Include in the scope of patent protection of the present invention.
SEQUENCE LISTING
<110>help bio tech ltd in Changsha three
<120>Pyrosequencing primer of qualitative detection STAT4 gene type to and test kit
<130> 2016
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213>artificial sequence
<400> 1
ggaaaggttc acacttgact gtt 23
<210> 2
<211> 24
<212> DNA
<213>artificial sequence
<400> 2
cccctgaaat tccactgaaa taag 24
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
aaaagttggt gaccaaaatg 20
<210> 4
<211> 11
<212> DNA
<213>artificial sequence
<400> 4
tayggtttgc a 11
<210> 5
<211> 498
<212> DNA
<213>artificial sequence
<400> 5
acacatatgg aaaggttcac acttgactgt taatacggat gtctttgaag gtagtggtgt 60
ggatggaggt aaggaaaaaa gaagtgggat aaaaagaagt ttgtaattaa aaagctacat 120
gtatattatg atctacttta tggaaaatta catgagtgtg tatgcagtaa aagtatgaaa 180
agttggtgac caaaatgtga atagtggtta tcttatttca gtggaatttc aggggatttt 240
ttttctttct tcttagactt ttcattatca tttgactttt tacaaagatt tgcattattt 300
aagcaatcag aaagaaatta taaagctatt ttcatcataa caaaaattcc attggtaaaa 360
aatttttaat taatttacat aatgtgcaaa aattagaaaa ttagaactcc taaagcaaga 420
agtggaaaaa ttattccaat ctgaagaaat aaaaccattc tctgatgact tggaaactga 480
agggattgct aatagctc 498
<210> 6
<211> 498
<212> DNA
<213>artificial sequence
<400> 6
acacatatgg aaaggttcac acttgactgt taatacggat gtctttgaag gtagtggtgt 60
ggatggaggt aaggaaaaaa gaagtgggat aaaaagaagt ttgtaattaa aaagctacat 120
gtatattatg atctacttta tggaaaatta catgagtgtg tatgcagtaa aagtatgaaa 180
agttggtgac caaaatgtta atagtggtta tcttatttca gtggaatttc aggggatttt 240
ttttctttct tcttagactt ttcattatca tttgactttt tacaaagatt tgcattattt 300
aagcaatcag aaagaaatta taaagctatt ttcatcataa caaaaattcc attggtaaaa 360
aatttttaat taatttacat aatgtgcaaa aattagaaaa ttagaactcc taaagcaaga 420
agtggaaaaa ttattccaat ctgaagaaat aaaaccattc tctgatgact tggaaactga 480
agggattgct aatagctc 498
Claims (7)
1. the Pyrosequencing primer pair of a qualitative detection STAT4 gene type, it is characterised in that described primer is to including:
STAT4 forward amplimer:
5’-GGAAAGGTTCACACTTGACTGTT-3’；
The reverse amplimer of STAT4:
5’-CCCCTGAAATTCCACTGAAATAAG-3’；
STAT4 sequencing primer: 5 '-AAAAGTTGGTGACCAAAATG-3 '；
Wherein, 5 ' ends of the reverse amplimer of described STAT4 carry out biotin labeling.
2. the Manganic pyrophosphate complex initiation test kit of a qualitative detection STAT4 gene type, it is characterised in that described test kit includes:
PCR reactant liquor, described PCR reactant liquor contains STAT4 forward amplimer:
5’-GGAAAGGTTCACACTTGACTGTT-3’；
The reverse amplimer of STAT4:
5’-CCCCTGAAATTCCACTGAAATAAG-3’；
STAT4 sequencing primer: 5 '-AAAAGTTGGTGACCAAAATG-3 '；
Wherein, 5 ' ends of the reverse amplimer of described STAT4 carry out biotin labeling.
Test kit the most according to claim 2, it is characterised in that described test kit also includes:
STAT4 positive reference substance 1, it is the STAT4 wild homozygote plasmid being inserted with nucleotide sequence shown in SEQ ID NO.5；
STAT4 positive reference substance 2, its for described STAT4 wild homozygote plasmid be inserted with nucleotides sequence shown in SEQ ID NO.6
The plasmid mixture of the STAT4 no mutant homozygote plasmid composition of row；
STAT4 positive reference substance 3, it is the STAT4 no mutant homozygote plasmid being inserted with nucleotide sequence shown in SEQ ID NO.6；
Wherein, plasmid vector is pMD18-T plasmid.
Test kit the most according to claim 3, it is characterised in that STAT4 described in described STAT4 positive reference substance 2 dashes forward
Become the quantity of homozygote plasmid and described STAT4 wild homozygote plasmid ratio into 1:1.
Test kit the most according to claim 2, it is characterised in that described test kit also includes: quality-control product and blank
Product, the sequence of described quality-control product is: TAYGGTTTGCA.
Test kit the most according to claim 5, it is characterised in that described blank product are ultra-pure water.
7. the application to being used for detecting in the reagent of STAT4 gene type in preparation of the primer described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610773331.8A CN106086231A (en) | 2016-08-30 | 2016-08-30 | The Pyrosequencing primer of qualitative detection STAT4 gene type to and test kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610773331.8A CN106086231A (en) | 2016-08-30 | 2016-08-30 | The Pyrosequencing primer of qualitative detection STAT4 gene type to and test kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106086231A true CN106086231A (en) | 2016-11-09 |
Family
ID=57224066
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610773331.8A Pending CN106086231A (en) | 2016-08-30 | 2016-08-30 | The Pyrosequencing primer of qualitative detection STAT4 gene type to and test kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106086231A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107586840A (en) * | 2017-10-25 | 2018-01-16 | 长沙三济生物科技有限公司 | For detecting the primer pair and kit of hepatitis B canceration tumor susceptibility gene polymorphism |
| CN108315420A (en) * | 2018-04-04 | 2018-07-24 | 广西中医药大学附属瑞康医院 | A kind of kit for detecting hepatitis B canceration polymorphism |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191334A (en) * | 2011-06-09 | 2011-09-21 | 广州益善生物技术有限公司 | Signal transducer and activator of transcription (STAT)4 gene polymorphism detection specific primer and liquid-phase chip |
| CN102277443A (en) * | 2011-09-16 | 2011-12-14 | 协和干细胞基因工程有限公司 | Kit for detecting tacrolimus and cyclosporine A individual medicine administration associated single nucleotide polymorphism (SNP) loci and amplification method and detection method |
| CN103667434A (en) * | 2012-09-25 | 2014-03-26 | 浙江爱易生物医学科技有限公司 | Kit for detecting susceptibility genes of lupus erythematosus |
| CN103834638A (en) * | 2012-11-27 | 2014-06-04 | 复旦大学 | Single nucleotide polymorphic site rs7574865 related to liver cancer susceptibility and application thereof |
| CN105154569A (en) * | 2015-05-14 | 2015-12-16 | 长沙三济生物科技有限公司 | Primer pair and kit for detecting VKORC1 (vitamin K epoxide reductase complex subunit 1) genotyping by pyrosequencing |
| CN105154568A (en) * | 2015-05-14 | 2015-12-16 | 长沙三济生物科技有限公司 | Primer pair and kit for detecting CYP3A4 genotyping by pyrosequencing |
| CN105176991A (en) * | 2015-05-14 | 2015-12-23 | 长沙三济生物科技有限公司 | Primer pair and kit for detecting CYP3A5 genotype with pyrosequencing method |
| CN105177159A (en) * | 2015-05-14 | 2015-12-23 | 长沙三济生物科技有限公司 | Primer pair and kit for detecting ALDH2 (Aldehyde Dehydrogenase 2) genotype with pyrosequencing method |
-
2016
- 2016-08-30 CN CN201610773331.8A patent/CN106086231A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191334A (en) * | 2011-06-09 | 2011-09-21 | 广州益善生物技术有限公司 | Signal transducer and activator of transcription (STAT)4 gene polymorphism detection specific primer and liquid-phase chip |
| CN102277443A (en) * | 2011-09-16 | 2011-12-14 | 协和干细胞基因工程有限公司 | Kit for detecting tacrolimus and cyclosporine A individual medicine administration associated single nucleotide polymorphism (SNP) loci and amplification method and detection method |
| CN103667434A (en) * | 2012-09-25 | 2014-03-26 | 浙江爱易生物医学科技有限公司 | Kit for detecting susceptibility genes of lupus erythematosus |
| CN103834638A (en) * | 2012-11-27 | 2014-06-04 | 复旦大学 | Single nucleotide polymorphic site rs7574865 related to liver cancer susceptibility and application thereof |
| CN105154569A (en) * | 2015-05-14 | 2015-12-16 | 长沙三济生物科技有限公司 | Primer pair and kit for detecting VKORC1 (vitamin K epoxide reductase complex subunit 1) genotyping by pyrosequencing |
| CN105154568A (en) * | 2015-05-14 | 2015-12-16 | 长沙三济生物科技有限公司 | Primer pair and kit for detecting CYP3A4 genotyping by pyrosequencing |
| CN105176991A (en) * | 2015-05-14 | 2015-12-23 | 长沙三济生物科技有限公司 | Primer pair and kit for detecting CYP3A5 genotype with pyrosequencing method |
| CN105177159A (en) * | 2015-05-14 | 2015-12-23 | 长沙三济生物科技有限公司 | Primer pair and kit for detecting ALDH2 (Aldehyde Dehydrogenase 2) genotype with pyrosequencing method |
Non-Patent Citations (5)
| Title |
|---|
| JIANG, DE-KE等: ""Genetic variants in STAT4 and HLA-DQ genes confer risk of hepatitis B virus–related hepatocellular carcinoma"", 《NATURE GENETICS》 * |
| QIAGEN: ""PyroMark® Control Oligo Handbook"", 《WWW.QIAGEN.COM》 * |
| 缪倩: ""IRF7、STAT4基因与系统性红斑狼疮的关联研究"", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 邵秀玲等: "《植物病原生物现代检测技术及应用》", 30 November 2015, 北京：中国标准出版社 * |
| 郑维等: "《汉英医学分子生物学实验方法》", 31 March 2005, 北京：中国协和医科大学出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107586840A (en) * | 2017-10-25 | 2018-01-16 | 长沙三济生物科技有限公司 | For detecting the primer pair and kit of hepatitis B canceration tumor susceptibility gene polymorphism |
| CN108315420A (en) * | 2018-04-04 | 2018-07-24 | 广西中医药大学附属瑞康医院 | A kind of kit for detecting hepatitis B canceration polymorphism |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Comprehensive one-step molecular analyses of mitochondrial genome by massively parallel sequencing | |
| Mesner et al. | Bubble-chip analysis of human origin distributions demonstrates on a genomic scale significant clustering into zones and significant association with transcription | |
| CN103602752B (en) | Primer set and kit for detecting rare deletion type thalassemia | |
| CN108220413B (en) | Fluorescent multiplex amplification kit for combined detection of human Y chromosome STR and Indel loci and application thereof | |
| CN102876793B (en) | Application of single nucleotide polymorphism rs2058660 to detecting susceptibility gene of leprosy | |
| Hou et al. | Gene expression profiling to predict and assess the consequences of therapy-induced virus eradication in chronic hepatitis C virus infection | |
| CN106119386A (en) | The PCR kit of detection people's JAK2 gene mutation | |
| CN105154569A (en) | Primer pair and kit for detecting VKORC1 (vitamin K epoxide reductase complex subunit 1) genotyping by pyrosequencing | |
| CN105154568A (en) | Primer pair and kit for detecting CYP3A4 genotyping by pyrosequencing | |
| Liang et al. | Common genetic variants in candidate genes and risk of familial lymphoid malignancies | |
| CN106086231A (en) | The Pyrosequencing primer of qualitative detection STAT4 gene type to and test kit | |
| CN105177159A (en) | Primer pair and kit for detecting ALDH2 (Aldehyde Dehydrogenase 2) genotype with pyrosequencing method | |
| CN106701903A (en) | Reagent kit for detecting mitochondrial heteroplasmy and detection method | |
| El-Bendary et al. | Association of genetic polymorphisms of chemokines and their receptors with clearance or persistence of hepatitis C virus infection | |
| CN106244710A (en) | The Pyrosequencing primer of qualitative detection HLA DQ gene type to and test kit | |
| CN103571923A (en) | Detection probes and detection liquid phase chip for BIM gene deletion mutation | |
| Pichon et al. | Analysis and annotation of DNA methylation in two nonhuman primate species using the Infinium Human Methylation 450K and EPIC BeadChips | |
| CN116042874A (en) | Primer probe combination and kit for detecting multi-drug resistance genes of mycobacterium tuberculosis | |
| CN107586840A (en) | For detecting the primer pair and kit of hepatitis B canceration tumor susceptibility gene polymorphism | |
| Moser et al. | Quantifying mixed populations of drug-resistant human immunodeficiency virus type 1 | |
| CN105274197A (en) | Quality control method and kit used for biological sample nucleic acid detection | |
| CN106244709A (en) | The Pyrosequencing primer of qualitative detection ApoE gene type to and test kit | |
| JP7199045B2 (en) | METHOD FOR ACQUIRING INFORMATION ON BREAST CANCER PROGNOSTICS, BREAST CANCER PROGNOSTIC DETERMINATION DEVICE, AND COMPUTER PROGRAM | |
| CN105176991A (en) | Primer pair and kit for detecting CYP3A5 genotype with pyrosequencing method | |
| CN106047990A (en) | Method for detecting SNP genotype/mutation of PCR product based on sequencing by synthesis of double nucleotides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161109 |