CN105039513B - For nonalcoholic fatty liver correlation target gene pleiomorphism detecting method and its primer and kit - Google Patents
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Abstract
The invention belongs to technical field of gene detection, nonalcoholic fatty liver correlation target gene pleiomorphism detecting method and its primer are used for more particularly to one kind, and kit, the present invention provides detection method and primer for nonalcoholic fatty liver correlation target gene, gene primer of the invention has corresponding specificity, it is of convenient length, transcription is efficient, it not will form secondary structure, primer itself is less than the complementation of continuous 4 bases, the complementation of continuous 4 bases is less than between primer, primer end can be modified, detection method has the characteristics that first, primer is short in PRC process synthesis cycle, it can synthesize and finish within one week；Second, multiple SNP detection can be done, testing cost is reduced；Third, sample size wider range, 100 to 1000 sample size are all well suited for；4th, detection site success rate and accuracy rate > 95%.
Description
Technical field
The invention belongs to technical field of gene detection, and in particular to one kind is used for nonalcoholic fatty liver correlation target gene
Pleiomorphism detecting method and its primer and kit.
Background technique
Except non-alcohol fatty liver (NAFLD) refers to caused by alcohol and other specific damage liver factors, with more
Unrestrained property liver cell Macrovesicular steatosis is the clinical pathology syndrome of main feature, including simple fatty liver and by its differentiation
Steatohepatitis (NASH) and cirrhosis, insulin resistance and genetic predisposition and its onset relation it is close.With fat and
Diabetes it is high-incidence, NAFLD has become common one of the chronic liver disease in China, seriously endangers people's health.
Research shows that NAFLD and SNP have substantial connection, the SNP is for single nucleotide polymorphism (single
Nucleotide polymorphism, SNP) refer to DNA sequence caused by a single nucleotide variation at the genomic level
Column polymorphism.NAFLD falls ill in access, and there are many genes to influence, however detects the methods of these gene pleiomorphisms there are many kind,
Such as sequencing analysis method, but its is at high cost, flux is low, and detection cycle is longer；For another example nucleic acid electrophoresis method, to sample requirement
It is high；There are also TaqMan probe methods, more complex to probe design, and the design cycle is long.
Summary of the invention
The first purpose of this invention is: providing a kind of for the inspection of nonalcoholic fatty liver correlation target gene polymorphism
The primer of survey method.
To achieve the goals above, the scheme that the present invention uses is as follows:
For the primer of nonalcoholic fatty liver correlation target gene pleiomorphism detecting method,
The related target gene is three classes, respectively insulin sensitivity gene, participates in oxidation activity metabolism and cell
Factor pathway gene, and participate in adipose metabolism gene；
The insulin sensitivity gene includes for PPARG gene, ADIPOQ gene, ADIPOR2 gene；
The participation oxidation activity metabolism and cell factor pathway gene are FDFT1 gene, tnf gene, AGTR1 base
Cause, PNPLA3 gene；
The participation adipose metabolism gene is PEMT gene, MTTP gene；
The primer of the PPARG gene its nucleotide sequence SEQ ID NO:1
The primer of the ADIPOQ gene its nucleotide sequence SEQ ID NO:2
The primer of the ADIPOR2 gene its nucleotide sequence SEQ ID NO:3
The primer of the FDFT1 gene its nucleotide sequence SEQ ID NO:4
The primer of the tnf gene its nucleotide sequence SEQ ID NO:5
The primer of the AGTR1 gene its nucleotide sequence SEQ ID NO:6
The primer of the PNPLA3 gene its nucleotide sequence SEQ ID NO:7
The primer of the PEMT gene its nucleotide sequence SEQ ID NO:8
The primer of the MTTP gene its nucleotide sequence SEQ ID NO:9.
Amplimer nucleotides sequence list is as follows
Extension primer nucleotides sequence list is as follows
Compared with the existing technology, gene primer of the invention has corresponding specificity, is of convenient length, and transcription is efficient, no
It will form secondary structure, primer itself is less than the complementation of continuous 4 bases, the complementation of continuous 4 bases is less than between primer, is drawn
Object end can be modified.
Second object of the present invention is, provides a kind of for the inspection of nonalcoholic fatty liver correlation target gene polymorphism
Survey method, includes the following steps,
S1, related target gene sample is chosen, the concentration of configuration sample gene is 5~10ng/ μ l；
S2, PCR amplification；It is expanded, is obtained containing the related target gene using relevant cdna sample DNA as template
PCR product；
The nucleotides sequence that the PCR amplification uses is classified as the primer of PPARG F/R gene；ADIPOQ F/R gene draws
Object, the primer of ADIPOR2F/R gene, the primer of FDFT1F/R gene；The primer of TNF F/R gene；AGTR1F/R gene draws
Object；The primer of PNPLA3F/R gene；The primer of PEMTF/R gene；The primer of MTTP F/R gene；
The purifying of S3, PCR product；1U phosphatase SAP enzyme and 1U exon Ⅰ type are added in every 10 μ lPCR product, at 37 DEG C
50~70min of warm bath, then deactivation 15min at 75 DEG C；
S4, single base extension；5 μ l SNaPshot Multiplex Kit are added in extension system
(ABI), then the PCR product of 2 μ l purifying, 1 μ l extension primer mixture, 2 μ l ultrapure waters carry out extension thermocycling program, obtain
To extension products；
S5, extension products purifying, 1U phosphatase SAP enzyme is added in every 10 μ l extension products, 37 DEG C of warm bath 50~
70min, then deactivation 15min at 75 DEG C；
S6, by extension products ABI3500 sequencer, take the extension products of 0.5 μ l after purification, 0.5 μ be added
LLiz120size standard internal standard and Hi-Di formamide mix, and are denaturalized after five minutes at 95 DEG C, place ice face 5min, then use
ABI3500 sequenator uses 2 software records initial data of 3500Series Software on ABI3500 sequenator, uses
The analysis of 5.0 software of GeneMapper.
Change as one kind of the present invention for nonalcoholic fatty liver correlation target gene pleiomorphism detecting method
Into, in S2 step PCR amplification step be specially amplification PCR reaction system be added 1x GC-I buffer, 3.0mM Mg2+,
0.3mM dNTP, 1U HotStarTaqpolymerase (Qiagen Inc.), 1 μ l sample DNA and 1 μ l PCR primer are passed through
95 DEG C of processing 2min of the first step；Second step is according to 94 DEG C, 20s, 65 DEG C, 40s, 72 DEG C, 1.5min carry out 11 circular treatments, the
Three steps are according to 94 DEG C, 20s, 59 DEG C, 30s, 72 DEG C, 1.5min carry out 24 circular treatments, the 4th 72 DEG C of step processing 2min, finally
4 DEG C of processing.
Change as one kind of the present invention for nonalcoholic fatty liver correlation target gene pleiomorphism detecting method
Concentration into, the 1 μ l PCR primer wherein every primer is 1uM.
Change as one kind of the present invention for nonalcoholic fatty liver correlation target gene pleiomorphism detecting method
Into in 96 DEG C of processing 1min, then the condition for extending thermocycling program in S4 step is: according to 96 DEG C, 10s, 52 DEG C, 5s, 60
DEG C, 30s, carry out 28 circulations, it is last: 4 DEG C of processing.
The primer concentration of PPARG gene is 0.4uM in the S4 step, and the concentration of remaining each primer is 0.8uM.
Had the following advantages that referring now to prior art, first, primer is short in PRC process synthesis cycle, one week it
Interior can synthesize finishes；Second, multiple SNP detection can be done, testing cost is reduced；Third, sample size wider range,
100 to 1000 sample size is all well suited for；4th, detection site success rate and accuracy rate > 95%.
Third object of the present invention is, provides a kind of for the inspection of nonalcoholic fatty liver correlation target gene polymorphism
The kit of survey includes following reagent:
(1) PCR amplification reagent, including PCR primer, archaeal dna polymerase, dNTPs, PCR reaction buffer；The PCR amplification
Primer is that sequence is
Amplimer nucleotides sequence list is as follows
(2) PCR product purified reagent, including SAP and exonuclease I；
(3) single base extension reagent, comprising: extension primer, single base extension enzyme, ddNTPs, extension buffering
Liquid；The single base extension enzyme is Taq DNA polymerase；Extension primer nucleotides sequence list is as follows
One or more in shown primer, the ddNTPs carries out fluorescent marker.
Compared with the existing technology, the multiple primers of kit of the invention are simultaneously present in mixed liquor, are tied without influencing
Fruit accuracy；The multiple gene pleiomorphisms of nonalcoholic fatty liver, save the cost and time can be detected simultaneously.
Specific embodiment
The present invention and its advantages are made below in conjunction with specific embodiment and nucleotides sequence list further detailed
Illustrate, still, a specific embodiment of the invention is not limited thereto.
For the primer of nonalcoholic fatty liver correlation target gene pleiomorphism detecting method,
The related target gene is three classes, respectively insulin sensitivity gene, participates in oxidation activity metabolism and cell
Factor pathway gene, and participate in adipose metabolism gene；The insulin sensitivity gene include for PPARG gene,
ADIPOQ gene, ADIPOR2 gene；It is FDFT1 gene that the participation oxidation activity, which is metabolized with cell factor pathway gene,
Tnf gene, AGTR1 gene, PNPLA3 gene；
The participation adipose metabolism gene is PEMT gene, MTTP gene；
The primer of the PPARG gene its nucleotide sequence SEQ ID NO:1
The primer of the ADIPOQ gene its nucleotide sequence SEQ ID NO:2
The primer of the ADIPOR2 gene its nucleotide sequence SEQ ID NO:3
The primer of the FDFT1 gene its nucleotide sequence SEQ ID NO:4
The primer of the tnf gene its nucleotide sequence SEQ ID NO:5
The primer of the AGTR1 gene its nucleotide sequence SEQ ID NO:6
The primer of the PNPLA3 gene its nucleotide sequence SEQ ID NO:7
The primer of the PEMT gene its nucleotide sequence SEQ ID NO:8
The primer of the MTTP gene its nucleotide sequence SEQ ID NO:9.
Amplimer nucleotides sequence list is as follows
Extension primer nucleotides sequence list is as follows
The present invention has collected 194 research cases, and wherein B ultrasound confirms NAFLD92, normal 102.To all samples into
Row SNP parting, site are the site tnf gene rs361525, the site PPARG gene rs10865710, ADIPOQ gene
The site rs1501299, the site FDFT1 gene rs2645424, the site AGTR1 gene rs3772622, MTTP gene rs3816873
Site, the site PNPLA3 gene rs738409, the site ADIPOR2 gene rs767870, the site PEMT gene rs7946.
Its nucleotides sequence list
Gene primer of the invention has corresponding specificity, is of convenient length, and transcription efficiently, not will form secondary structure,
Primer itself is less than the complementation of continuous 4 bases, the complementation of continuous 4 bases is less than between primer, primer end can be modified.
One kind of the invention is used for nonalcoholic fatty liver correlation target gene pleiomorphism detecting method, includes the following steps,
S1, related target gene sample is chosen, the concentration of configuration sample gene is 5~10ng/ μ l；
S2, PCR amplification；It is expanded, is obtained containing the related target gene using relevant cdna sample DNA as template
PCR product, the 1 μ l PCR primer wherein every primer concentration be 1uM；
The nucleotides sequence that the PCR amplification uses is classified as the primer of PPARG F/R gene；ADIPOQ F/R gene draws
Object, the primer of ADIPOR2F/R gene, the primer of FDFT1F/R gene；The primer of TNF F/R gene；AGTR1F/R gene draws
Object；The primer of PNPLA3F/R gene；The primer of PEMTF/R gene；The primer of MTTP F/R gene；
PCR amplification step is specially that 1x GC-I buffer, 3.0mM Mg2 is added in amplification PCR reaction system in S2 step
+, 0.3mM dNTP, 1U HotStarTaq polymerase (Qiagen Inc.), 1 μ l sample DNA and 1 μ l PCR primer, warp
Cross 95 DEG C of processing 2min of the first step；Second step is according to 94 DEG C, 20s, 65 DEG C, 40s, 72 DEG C, 1.5min carry out 11 circular treatments,
Third step is according to 94 DEG C, 20s, 59 DEG C, 30s, 72 DEG C, 1.5min carry out 24 circular treatments, the 4th 72 DEG C of step processing 2min, most
4 DEG C of processing afterwards.
Amplification PCR reaction system concrete configuration is as follows:
The setting of multiplex PCR cyclic program
The processing of multi-primers:
Invitrogen synthesizes dry powder primer (1OD)
Add diluted at 100 μM of mother liquor according to the nmol number of dry powder primer
With mother liquor plus diluted at 1 μM of reaction working solution
The concentration of each pair of primer in multiple PCR primer: (μM)
| rs361525F/R | rs7946F/R | rs2645424F/R | rs3816873F/R | rs767870F/R |
| 1 | 1 | 1 | 1 | 1 |
| rs3772622F/R | rs10865710F/R | rs1501299F/R | rs738409F/R | |
| 1 | 1 | 1 | 1 |
Table above indicates final concentration (μM) of each pair of primer in multiple PCR primer working solution, to dilute the more of 100ul
For weight PCR reaction working solution, the dilution of 91ul is added in the centrifuge tube of 1.5ml, then draws diluted 100 μM
The multiple PCR primer that every, object mother liquor respectively adds 1ul that 100ul can be obtained reacts working solution.
The purifying of S3, PCR product；1U shrimp alkali phosphatase SAP enzyme and 1U exon Ⅰ type are added in every 10 μ lPCR product,
37 DEG C of 50~70min of warm bath, then deactivation 15min at 75 DEG C；
PCR product purifying configuration such as following table
| Configuration | Volume (μ l) | The amount (U) of addition |
| PCR product | 10μl | |
| SAP enzyme (1U/ μ l) | 5μl | 5U |
| ExonucleaseI enzyme (20U/ μ l) | 0.1μl | 2U |
| Total volume | 15.1μl |
S4, single base extension；5 μ l SNaPshot Multiplex Kit are added in extension system
(ABI), then the PCR product of 2 μ l purifying, 1 μ l extension primer mixture, 2 μ l ultrapure waters carry out extension thermocycling program, obtain
To extension products；
Single base extension configuration such as following table
Note: SNaPshot Multiplex Kit is the sequencing combined complete that ABI company aims at SNP exploitation
The primer concentration of PPARG gene is 0.4uM in step, and the concentration of remaining each primer is 0.8uM.
The concentration of each extension primer is as follows: (μM) in extension primer mixture
| rs361525SR | rs7946SR | rs2645424SR | rs3816873SR | rs767870SR |
| 0.8 | 0.8 | 0.8 | 0.8 | 0.8 |
| rs3772622SF | rs10865710SR | rs1501299SR | rs738409SF | |
| 0.8 | 0.4 | 0.8 | 0.8 |
Table above indicates final concentration (μM) of each site extension primer in multi-job liquid, to dilute 100ul's
For multiple extension primer working solution, the dilution of 93.2ul, 100 μ that then will have been diluted are added in the centrifuge tube of 1.5ml
Every, the extension primer mother liquor of M is respectively plus the multiple extension of 100ul can be obtained in 0.8ul (rs10865710SR primer adds 0.4ul)
Primer reacts working solution.
In 96 DEG C of processing 1min, then the condition for extending thermocycling program in step is: according to 96 DEG C, 10s, 52 DEG C, 5s,
60 DEG C, 30s carry out 28 circulations, last: 4 DEG C of processing.
Extension program see the table below
S5, extension products purifying, 1U phosphatase SAP enzyme is added in every 10 μ l extension products, 37 DEG C of warm bath 50~
70min, then deactivation 15min at 75 DEG C；
S6, by extension products ABI3500 sequencer, take the extension products of 0.5 μ l after purification, 0.5 μ be added
LLiz120size standard internal standard and Hi-Di formamide mix, and are denaturalized after five minutes at 95 DEG C, place ice face 5min, then use
ABI3500 sequenator uses 2 software records initial data of 3500Series Software on ABI3500 sequenator, uses
The analysis of 5.0 software of GeneMapper.
The present invention has the following advantages that, first, primer is short in PRC process synthesis cycle, can synthesize and finish within one week；
Second, multiple SNP detection can be done, testing cost is reduced；Third, sample size wider range, 100 to 1000 sample
Amount is all well suited for；4th, detection site success rate and accuracy rate > 95%.
A kind of kit for nonalcoholic fatty liver correlation target gene polymorphic detection of the present invention includes following examination
Agent:
(1) PCR amplification reagent, including PCR primer, archaeal dna polymerase, dNTPs, PCR reaction buffer；The PCR amplification
Primer is that sequence is
Amplimer nucleotides sequence list is as follows
(2) PCR product purified reagent, including SAP and exonuclease I；
(3) single base extension reagent, comprising: extension primer, single base extension enzyme, ddNTPs, extension buffering
Liquid；The single base extension enzyme is Taq DNA polymerase；Extension primer nucleotides sequence list is as follows
One or more in shown primer, the ddNTPs carries out fluorescent marker.
The multiple primers of kit of the invention are simultaneously present in mixed liquor, without influencing result accuracy；It can be same
When detect the multiple gene pleiomorphisms of nonalcoholic fatty liver, save the cost and time.
Claims (2)
1. being used for the primer of nonalcoholic fatty liver correlation target gene pleiomorphism detecting method, it is characterised in that:
The related target gene is three classes, respectively insulin sensitivity gene, participates in oxidation activity metabolism and cell factor
Pathway gene, and participate in adipose metabolism gene；
The insulin sensitivity gene includes for PPARG gene, ADIPOQ gene, ADIPOR2 gene；
The described participation oxidation activity metabolism and cell factor pathway gene be FDFT1 gene, TNF gene, AGTR1 gene,
PNPLA3 gene；
The participation adipose metabolism gene is PEMT gene, MTTP gene；
Its nucleotide sequence of the primer of the PPARG gene is as shown in SEQ ID NO:1
Its nucleotide sequence of the primer of the ADIPOQ gene is as shown in SEQ ID NO:2
Its nucleotide sequence of the primer of the ADIPOR2 gene is as shown in SEQ ID NO:3
Its nucleotide sequence of the primer of the FDFT1 gene is as shown in SEQ ID NO:4
Its nucleotide sequence of the primer of the TNF gene is as shown in SEQ ID NO:5
Its nucleotide sequence of the primer of the AGTR1 gene is as shown in SEQ ID NO:6
Its nucleotide sequence of the primer of the PNPLA3 gene is as shown in SEQ ID NO:7
Its nucleotide sequence of the primer of the PEMT gene is as shown in SEQ ID NO:8
Its nucleotide sequence of the primer of the MTTP gene is as shown in SEQ ID NO:9.
2. being used for the kit of nonalcoholic fatty liver correlation target gene polymorphic detection, which is characterized in that including following examination
Agent:
(1) PCR amplifing reagent, including PCR primer, DNA polymerase, dNTPs, PCR reaction buffer；The PCR primer
It is that sequence is
Its nucleotide sequence of the primer of the PPARG gene is as shown in SEQ ID NO:1
Its nucleotide sequence of the primer of the ADIPOQ gene is as shown in SEQ ID NO:2
Its nucleotide sequence of the primer of the ADIPOR2 gene is as shown in SEQ ID NO:3
Its nucleotide sequence of the primer of the FDFT1 gene is as shown in SEQ ID NO:4
Its nucleotide sequence of the primer of the TNF gene is as shown in SEQ ID NO:5
Its nucleotide sequence of the primer of the AGTR1 gene is as shown in SEQ ID NO:6
Its nucleotide sequence of the primer of the PNPLA3 gene is as shown in SEQ ID NO:7
Its nucleotide sequence of the primer of the PEMT gene is as shown in SEQ ID NO:8
Its nucleotide sequence of the primer of the MTTP gene is as shown in SEQ ID NO:9；
(2) PCR product purification reagent, including SAP and exonuclease I；
(3) single base extension reagent, comprising: extension primer, single base extension enzyme, ddNTPs, extension buffer solution；Institute
Stating single base extension enzyme is TaqDNA polymerase；The extension primer is as follows one or more in primer:
Its nucleotide sequence of the primer of the PPARG gene is as shown in SEQ ID NO:19
Its nucleotide sequence of the primer of the ADIPOQ gene is as shown in SEQ ID NO:20
Its nucleotide sequence of the primer of the ADIPOR2 gene is as shown in SEQ ID NO:21
Its nucleotide sequence of the primer of the FDFT1 gene is as shown in SEQ ID NO:22
Its nucleotide sequence of the primer of the TNF gene is as shown in SEQ ID NO:23
Its nucleotide sequence of the primer of the AGTR1 gene is as shown in SEQ ID NO:24
Its nucleotide sequence of the primer of the PNPLA3 gene is as shown in SEQ ID NO:25
Its nucleotide sequence of the primer of the PEMT gene is as shown in SEQ ID NO:26
Its nucleotide sequence of the primer of the MTTP gene is as shown in SEQ ID NO:27；
The ddNTPs carries out fluorescent marker.
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| CN105420391A (en) * | 2015-12-31 | 2016-03-23 | 博奥生物集团有限公司 | Complete set primer for detecting SNP site related with nutrient metabolism, absorption, and preference |
| CN105586385A (en) * | 2016-03-10 | 2016-05-18 | 北京中科唯新生物医学研究所有限公司 | Phosphatidyl ethanolamine N-methyltransferase activity detecting method |
| CN107630084A (en) * | 2016-07-18 | 2018-01-26 | 昆山德诺瑞尔生物科技有限公司 | The polygenic combination detection kit of nonalcoholic fatty liver disease |
| JP7106489B2 (en) * | 2019-04-23 | 2022-07-26 | ジェネシスヘルスケア株式会社 | How to determine the risk of fatty liver |
| CN110079597A (en) * | 2019-05-29 | 2019-08-02 | 阿吉安(福州)基因医学检验实验室有限公司 | The susceptible related gene inspecting reagent kit of nonalcoholic fatty liver |
| CN113025701B (en) * | 2021-03-10 | 2022-11-08 | 北京科力丹迪生物医疗科技有限公司 | Early screening method and kit for non-alcoholic fatty liver disease susceptibility gene |
| CN114231620B (en) * | 2021-12-31 | 2023-03-10 | 吉林大学第一医院 | Application of UQCC1 gene polymorphism in diagnosis of moderate and severe MAFLD (myeloproliferative disorder) |
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