CN108220424A - A kind of method and kit for detecting IL28 gene locis - Google Patents
A kind of method and kit for detecting IL28 gene locis Download PDFInfo
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- CN108220424A CN108220424A CN201810113560.6A CN201810113560A CN108220424A CN 108220424 A CN108220424 A CN 108220424A CN 201810113560 A CN201810113560 A CN 201810113560A CN 108220424 A CN108220424 A CN 108220424A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
A kind of method and kit for detecting IL28 gene locis, the present invention relates to molecular biology and medical domain, to provide a kind of more accurately kit and method of IL28 genotype detections, the present invention provides a species-specific primers, specific probe sequence and report sequences.The kit prepared using the special primer and probe sensitive, quickly low can detect curative effect related SNP type in people's whole blood sample, and reliable experimental evidence, guiding clinical treatment selection can be provided for interferon medication outcome prediction.
Description
Technical field
The present invention relates to molecular biology and medical domain, and more specifically, the present invention relates to a kind of interferon medicine curative effects
The kit of SNP site, by the mononucleotide polymorphism site for detecting the relevant IL28 genes of interferon medicine curative effect simultaneously
(SNP) genotype assesses the effect of interferon is in hepatitis C patients and other diseases use.
Background technology
Hepatitis C (hepatitis C) is mainly passed as pandemic communicable disease in a kind of world wide
Broadcast mode is blood born, and needle thorn, drug abuse etc. are also its common route of infection in addition.It is most of after infection Hepatitis C Virus
Patient is gradually developed to chronic hepatitis C, if regular effective therapy intervention cannot be obtained, about 1/3 chronic hepatitis C is suffered from
Person will be finally to advanced lesions such as hepatic sclerosis, liver failures.For many years, the treatment of interferon joint Ribavirin (PR)
Mode is always to treat the standard scheme (standard of care, SOC) of hepatitis C.
Current many researchs prompting, viral hepatitis type C prognosis and outcome receive the effect of antiviral therapy and host
IL-28B gene pleiomorphisms, HCV genotype, Baseline viral rna level (baseline viral load) and degree of hepatic fibrosis
Etc. closely related.
Ge etc. first reported IL-28B genes and the correlation of hepatitis C infection person's prognosis, to different the third types of ethnic group
Hepatitis is found after carrying out genetic test：IL-28B gene mononucleotide polymorphisms site rs12979860 and continued viral
Response is significantly correlated, its gene phenotype of IL-28B rsl2979860 sites has C/C, C/T and T/T, and wherein C/C is protectiveness base
Because of type.Rsl2979860 has stronger relevance with virological response, and whether wherein C/C genotype obtains continued viral with patient
It is highly relevant to learn response rate.In standard regimens therapeutic process is received, carry C/C genotype patient can obtain it is higher
Continued viral response rate is probably 2~3 times of other genotype.Meanwhile the distribution of C/C genotype has apparent ethnic difference
It is different, in non-HCV infection person, C/C genotype accounting highest in the yellow race, more than 90%.Rs12979860C/C type ratios
Higher, the spontaneous clearance rate of virus of patients with chronic hepatitis C is higher, and rs12979860 genotype is not with obtaining quick virus
The continued viral response rate of the hepatitis C patients of response is related, and C/C types are respectively with T/T type continued viral response rates
87% and 29%.And the detection and primer and corresponding specific probe and reporter probe to IL-28B genotype have it is very big
Relationship.
Invention content
To provide a kind of kit that can precisely detect IL-28 genotype, the present invention provides a species-specific primers, specifically
Probe sequence and report sequence.
Technical solution provided by the invention is as follows：
A kind of kit for detecting IL28 gene locis, which is characterized in that the kit includes following sequence
Primer：
Preferably, the kit further includes specific probe, and the specific probe sequence is as follows：
Preferably, the kit further includes reporter probe, and the reporter probe sequence is as follows：
| Reporter probe 1 | GAACCAGGGT TGAATTGCAC | It is the reporter probe of the magnetic bead of IL28B |
A kind of method for detecting IL28B gene locis, the method include the steps：PCR reactions are carried out first,
Realize amplification；OLA reactions, label and connection are carried out again；Then hybridization reaction is carried out, finally carries out the analysis of genotype.
Preferably, the PCR reaction systems are as follows：2x qiagen Hotstar MM 5ul, primer mix1ul,
DNA sample 2ul, aqua sterilisa 2ul；PCR reaction conditions be 95 DEG C, 15min, carry out 30 cycle 94 DEG C, 30 seconds, 60 DEG C, 30
Second, 72 DEG C, 30 seconds；72 DEG C, 7 minutes, 4 DEG C of maintenances.
Preferably, OLA reactions are as follows：2xOLA master mix are prepared, OLA master mix and PCR is anti-
Product is answered to mix, coupled reaction is carried out after mixing.
Preferably, 2xOLA master mix are prepared to include：10x Taq Ligase buffer 2ul,40000U/ml
Taq DNA Ligase 0.25ul, wild-type probe mix 1ul, saltant type probe mix 2ul, deionized water 4.75ul.
Preferably, coupled reaction condition is as follows：96 DEG C of 2min, the 94 DEG C of 15s, 37 DEG C of 1min of 30 cycles；4 DEG C of maintenances.
Preferably, the washing procedure process of hybridization reaction is as follows：The magnetic bead of reporter probe is selected, is resuspended, then dilutes most
It up to 100u/ul, with 2X Tm hybridization buffer, is added in magnetic bead to every hole after mixing, adds 1-5ul
OLA reaction and 25ul dH2O carry out PCR reactions to each hole：96 DEG C of 90s, 37 DEG C of 30min, siphon away supernatant, use 1x
Magnetic bead is resuspended in Tm hybridization buffer, and magnetically attractive 30-60s siphons away supernatant again, repeats with 1x Tm
Magnetic bead is resuspended in hybridization buffer, and magnetically attractive 30-60s siphons away supernatant for the third time, with 1x Tm hybridization
Magnetic bead is resuspended in buffer, incubates 15min at 37 DEG C, adds in 50ul reaction products to LUMINEX and analyze.
Preferably, the not washing process of hybridization reaction is as follows：1st, magnetic bead is selected, and is resuspended；2nd, magnetic bead is diluted to 100u
/ ul, with 2X Tm hybridization buffer, oscillation mixing；3rd, it adds in magnetic bead mix to every hole；4th, sample is added
In product to every hole；5th, PCR reactions are carried out：96 DEG C of 90s, 37 DEG C of 30min；6th, prepare 6ug/ml SAPE in 1x
hybridization buffer；7th, 100ul SAPE mix, mixing are added；8th, 37 DEG C of incubation 15min；9th, 100ul is added in extremely
It is analyzed in 37 DEG C of luminex.
The present invention is relevant using interferon medicine curative effect for infection with hepatitis C virus patient and other Diseases
SNP site designs special primer and probe, separable detect the type of the SNP site.Using above-mentioned special primer and probe system
Standby kit sensitive, quickly low can detect curative effect related SNP type in people's whole blood sample, can be that interferon medication curative effect is pre-
It surveys and reliable experimental evidence is provided, guiding clinical treatment selection.
Specific embodiment
A kind of kit, the kit is to coming the gene loci of interferon medicine curative effect, i.e. IL28 in human genome DNA
That is rs12979860.Design specific primer and wild type/saltant type probe, the MagPlex- to the corresponding reporter probe of coupling
TAG magnetic beads carry out hybridization reaction, are detected on 200 instruments of luminex by chromogenic reaction.Pass through the reading to signal value
The base type of each SNP site of interpretation is taken to be judged that wherein specific primer sequences are as follows：
Specific probe sequence is as follows：
Reporter probe sequence is as follows：
Reaction process is as follows：
PCR reactions are carried out in PCR reaction tubes, the system of reaction is 10 μ l of total volume, includes 2x qiagen Hotstar
MM 5ul, primer mix 1ul, DNA sample 2ul, aqua sterilisa 2ul.
It is reacted in ABI9700 type PCR amplification instruments, reaction condition is 95 DEG C, 15min, carries out the 94 of 30 cycles
DEG C, 30 seconds, 60 DEG C, 30 seconds, 72 DEG C, 30 seconds；72 DEG C, 7 minutes, 4 DEG C of maintenances.
OLA reacts：Prepare 2xOLA master mix：10x Taq Ligase buffer 2ul,Taq DNA Ligase
(40,000U/ml) 0.25ul, wild-type probe mix (100nM each) 1ul, saltant type probe mix (2.5uM each) 2ul,
Deionized water 4.75ul.OLA master mix are mixed with reaction product：2xOLA master mix 10ul, the PCR of amplification
Product 5ul, sterile deionized water 5ul.Piping and druming mixing up and down, covers reaction tube, connect in ABI9700 type PCR amplification instruments
It is reversed to answer.96 DEG C of 2min, the 94 DEG C of 15s, 37 DEG C of 1min of 30 cycles；4 DEG C of maintenances.
Hybridization reaction：The MagPlex-TAG magnetic beads of corresponding reporter probe are selected, and are resuspended.Each magnetic bead is mixed, and dilute
It releases to 100u/ul, with 2X Tm hybridization buffer, oscillation mixing 20s.25ul magnetic bead mix is added in every
Kong Zhong.(2500 pearls/each reaction should be provided).1-5ul OLA reaction and 25ul dH2O are added to each hole.
The volume of H2O is adjusted, makes total volume close to 50ul.It closes the lid, carries out PCR reactions：96℃90s,37℃30min.It is put in magnetic
30s-60s in power plate, is sucked magnetic bead.Supernatant carefully is siphoned away, does not siphon away magnetic bead.With 1x Tm hybridization buffer
MagPlex-TAG magnetic beads, magnetically attractive 30-60s is resuspended in 75ul.Supernatant carefully is siphoned away, does not siphon away magnetic bead.It repeats with 1x Tm
MagPlex-TAG magnetic beads, magnetically attractive 30-60s is resuspended in hybridization buffer 75ul.Carefully siphon away supernatant.Use 75ul
1x Tm hybridization buffer (include 2-8ug/ml SAPE), and magnetic bead is resuspended, and 15min is incubated at 37 DEG C.At 37 DEG C
In, it adds in 50ul reaction products to LUMINEX and analyzes.
Interpretation of result
It is compareed by plasmid and water, obtains background signal, when detecting sample results, after subtracting background, numerical value is more than 200
For positive reaction.
Risk related SNP type can be provided in examining report as a result, reference gene is wild type gene.Detect IL28B bases
Because the corresponding result of rs12979860 saltant types is shown as TT, the corresponding result of heterozygous is shown as CT, the corresponding knot of wild type
Fruit is shown as CC.CYP1A2(734C>A) the corresponding result of gene rs762551 saltant types is shown as AA, the corresponding knot of heterozygous
Fruit is shown as AC, and the corresponding result of wild type is shown as CC.
Sequence table
<110>Guangzhou and health medical technology Co., Ltd
<120>A kind of method and kit for detecting IL28 gene locis
<130> PJ1810003.06
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence (" artificial sequence ")
<400> 1
gcttatcgca tacggctag 19
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (" artificial sequence ")
<400> 2
caggctcagg gtcaatca 18
<210> 3
<211> 38
<212> DNA
<213>Artificial sequence (" artificial sequence ")
<400> 3
tcaaactctc aattcttact taatctcccc gaaggcgc 38
<210> 4
<211> 38
<212> DNA
<213>Artificial sequence (" artificial sequence ")
<400> 4
tttacaaatc taatcacact atacctcccc gaaggcgt 38
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (" artificial sequence ")
<400> 5
gaaccagggt tgaattgcac 20
Claims (10)
1. a kind of kit for detecting IL28 gene locis, which is characterized in that the kit includes drawing for following sequence
Object：
2. the kit of detection IL28 gene locis as described in claim 1, which is characterized in that the kit further includes
Specific probe, the specific probe sequence are as follows：
3. the kit of detection IL28 gene locis as described in claim 1, which is characterized in that the kit further includes
Reporter probe, the reporter probe sequence are as follows：
A kind of 4. method for detecting IL28 gene locis, which is characterized in that the method includes the steps：It carries out first
PCR reacts, and realizes amplification；OLA reactions, label and connection are carried out again；Then hybridization reaction is carried out, finally carries out point of genotype
Analysis.
5. the method for detection IL28 gene locis as claimed in claim 4, which is characterized in that the PCR reaction systems are such as
Under：2x qiagen Hotstar MM 5ul, primer mix 1ul, DNA sample 2ul, aqua sterilisa 2ul；PCR reaction conditions are
95 DEG C, 15min carry out 30 94 DEG C, 30 seconds, 60 DEG C, 30 seconds, 72 DEG C, 30 seconds recycled；72 DEG C, 7 minutes, 4 DEG C of maintenances.
6. the method for detection IL28B gene locis as claimed in claim 4, which is characterized in that the OLA reactions are as follows：
2xOLA master mix are prepared, OLA master mix with PCR reaction products are mixed, coupled reaction is carried out after mixing.
7. the method for detection IL28 gene locis as claimed in claim 6, which is characterized in that prepare 2xOLA master mix
Include：The Taq DNA Ligase 0.25ul of 10x Taq Ligase buffer 2ul, 40000U/ml, wild-type probe
Mix 1ul, saltant type probe mix 2ul, deionized water 4.75ul.
8. the method for detection IL28 gene locis as claimed in claim 6, which is characterized in that coupled reaction condition is as follows：96
DEG C 2min, the 94 DEG C of 15s, 37 DEG C of 1min of 30 cycles；4 DEG C of maintenances.
9. a kind of method of SNP site genotype detection for detecting IL28B curative effects as claimed in claim 4, which is characterized in that
The washing procedure process of hybridization reaction is as follows：The magnetic bead of reporter probe is selected, is resuspended, then dilution is up to 100u/ul, uses
2X Tm hybridization buffer, after mixing add in magnetic bead to every hole in, addition 1-5ul OLA reaction and
25ul dH2O carries out PCR reactions to each hole：96 DEG C of 90s, 37 DEG C of 30min, siphon away supernatant, with 1x Tm
Magnetic bead is resuspended in hybridization buffer, and magnetically attractive 30-60s siphons away supernatant again, repeats with 1x Tm
Magnetic bead is resuspended in hybridization buffer, and magnetically attractive 30-60s siphons away supernatant for the third time, with 1x Tm hybridization
Magnetic bead is resuspended in buffer, incubates 15min at 37 DEG C, adds in 50ul reaction products to LUMINEX and analyze.
10. a kind of method of SNP site genotype detection for detecting IL28B curative effects as claimed in claim 4, feature exist
In the not washing process of hybridization reaction is as follows：1st, magnetic bead is selected, and is resuspended；2nd, magnetic bead is diluted to 100u/ul, with 2X Tm
Hybridization buffer, oscillation mixing；3rd, it adds in magnetic bead mix to every hole；4th, it adds in sample to every hole；5、
Carry out PCR reactions：96 DEG C of 90s, 37 DEG C of 30min；6th, prepare 6ug/ml SAPE in 1x hybridization buffer；7th, add
Add 100ul SAPE mix, mixing；8th, 37 DEG C of incubation 15min；9th, it adds in 100ul to 37 DEG C of luminex and analyzes.
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| CN201810113560.6A CN108220424A (en) | 2018-02-05 | 2018-02-05 | A kind of method and kit for detecting IL28 gene locis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964791B (en) * | 2019-12-26 | 2023-08-15 | 贵州中医药大学第二附属医院 | Method for detecting single nucleotide polymorphism and corresponding kit |
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