CN110305952A - It is a kind of for detecting primer, probe compositions and its application method of fat and diabetes B - Google Patents
It is a kind of for detecting primer, probe compositions and its application method of fat and diabetes B Download PDFInfo
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Abstract
The present invention provides the primers for detecting fat and diabetes B, probe compositions, including for the primer and probe in the site rs4994 on 3 gene of ADR β, for the primer and probe in the site rs9465871 on CDKAL1 gene, the primer and probe for the site rs4994 on 3 gene of ADR β includes: the downstream primer of the upstream primer of ADR β 34994, ADR β 34994；The probe of ADR β 34994；Primer and probe for the site rs9465871 on CDKAL1 gene includes: the upstream primer of CDKAL1；The downstream primer of CDKAL1；The probe of CDKAL1.The present invention provides a kind of detection method for detecting convenient, accuracy rate is high, economical and practical obesity and diabetes B related mutation gene loci；Real-time quantitative PCR amplification is carried out to sample DNA by multiple groups gene primer, and obtains the genotype of subject by probe parting, achievees the effect that quickly to detect fat and diabetes B susceptibility gene mutation site.
Description
Technical field
The present invention relates to molecular Biological Detection fields, specifically, being related to Q-PCR method detection obesity and diabetes B
The amplimer and probe in the mutational site of tumor susceptibility gene and its application.
Background technique
Obesity refers to body fat total content excessively and (or) locally homeomorphic map as a kind of common Nutrition and Metabolism disease
Content is excessive, is mainly formed by chronic metabolic disease by h and E factor collective effect.With life style
Change, fat illness rate is in rapid increase trend, and has become global public health problem.WHO will within 2002
Obesity is classified as one of the global prime risk factor for causing human diseases to bear, and China, which has surmounted the U.S., at present becomes whole world fertilizer
The most country of fat population, thus prevent obesity prevalence be also following a period of time countries in the world face maximum it is public
One of health challenge.
Diabetes B is a kind of long-term metabolic disturbance diseases, and main performance is hyperglycemia, insulin resistance and lacks relatively
Weary insulin.Mostly caused by body inherent cause and outside environmental elements interaction, participated in pathogenetic process by polygenes,
Multi-step causes.In addition, expanding rapidly with fat troop, trend is consistent to be, diabetes B crowd also grows therewith.Therefore,
Obesity has been counted as harm health and the Major Risk Factors of diabetes occurs at present.Since obesity can cause insulin to support
It is anti-, the paracrisis of insulin can also be caused, to influence the effect that insulin adjusts blood glucose, makes blood glucose rise, and then occur
Diabetes, therefore fat or overweight people keeps normal people to be more susceptible to suffer from diabetes than weight.The obese patient of diabetes B subtracts
Light weight not only contributes to the control of blood glucose, can also reduce the generation of complication.Therefore, to control diabetes B and its phase
Complication is closed, it is imperative to solve problem of obesity.Many is the study found that the figure of obese patient is more related to diabetes B
Property, and have synergistic effect with obesity.Therefore people are improved to fat and diabetes B understanding, and to its pathogenesis
With the research of prevention and treatment, it has also become the important health problem of today's society.
Fat and diabetes the causes of disease are considerably complicated, research shows that inherent cause is in fat and diabetes mechanism
Particularly important and crucial effect is played, and is studied it has also been found that fat and diabetes are disease of multifactorial inheritance.With obesity
Candidate Gene Study deepens continuously, it has now been found that a gene loci is related with the generation of obesity more than 600, obtains
To (FTO) gene related to weight regulation closely related gene such as body fat and obesity, the leptin receptor confirmed extensively
(LEPR) gene, melanocortin-4 receptor (MC4R) gene, uncoupling protein (UCP2) gene, β3-adrenergicreceptor
(ADRB3) gene, peroxidase proliferator activated receptor γ (PPAR γ 2) gene etc..Meanwhile it is reported a 2 more than 100
Patients with type Ⅰ DM tumor susceptibility gene/site, wherein the tumor susceptibility gene verified repeatedly in not agnate and crowd specifically includes that carefully
5 regulator subunit associated protein 1 analog 1CDK5(CDKAL1 of born of the same parents' period element dependent kinase (CDK)), 7 analog of transcription factor
2(TCF7L2), insulin-like growth factor 2 mRNA binding protein (IGF2BP2), inward rectifyimg potassium channel subfamily member 11
(KCNJ11), Melatonin receptor 1B (MTNR1B) and ob gene (FTO) etc..Currently, only for the expansion inspection of fat or diabetes
It surveys, this method can only prompt a certain risk.In addition, other methods based on sequencing require sample size more, time-consuming, not side
Just flexibly carry out detection.Based on this, the present invention provides the primer of joint-detection obesity and diabetes B susceptibility gene mutation site
And probe combinations, using Q-PCR sonde method, detection efficiently, flexibly, plays screening to fat and 2 type glycosurias to a certain extent
Effect, also plays certain suggesting effect to the subject containing susceptibility gene mutation.
Summary of the invention
The present invention provides the primer and probe combination in joint-detection obesity and diabetes B susceptibility gene mutation site, adopts
With Q-PCR sonde method, detection efficiently, flexibly, plays the role of screening to fat and 2 type glycosurias to a certain extent, also to containing
The subject of susceptibility gene mutation plays certain suggesting effect.
The present invention provides the primers for detecting fat and diabetes B, probe compositions, including are directed to 3 base of ADR β
Because of the primer and probe in the upper site rs4994, for the primer and probe in the site rs9465871 on CDKAL1 gene, for ADR β
The primer and probe in the site rs4994 includes: on 3 genes
The upstream primer of ADR β 34994: 5 '-AGGCAACCTGCTGGTCATC-3 ' (SEQ ID NO.1)；
The downstream primer of ADR β 34994: 5 '-CAGCGAAGTCACGAACACG-3 ' (SEQ ID NO.2)；
The probe of ADR β 34994: 5 '-CCCGGACTCCGAGACT-3 ' (SEQ ID NO.3)；
Primer and probe for the site rs9465871 on CDKAL1 gene includes:
The upstream primer of CDKAL1: 5 '-AATCCAGTAAGTCAATCTTCAGTCT -3 ' (SEQ ID NO.4)
The downstream primer of CDKAL1: 5 '-CAGTAGAGGTGGAGGAAGTCG -3 ' (SEQ ID NO.5)
The probe of CDKAL1: 5 '-CTAACTCAAATTTCTCAGC -3 ' (SEQ ID NO.6)
Further, the composition further includes the primer and probe for the site rs6265 on BDNF gene, is specifically included:
The upstream primer of BDNF6265: 5 '-GCTTGACATCATTGGCTGACA -3 ' (SEQ ID NO.7)
The downstream primer of BDNF6265: 5 '-ACTTTCTGGTCCTCATCCAACAG -3 ' (SEQ ID NO.8)
The probe of BDNF6265: 5 '-TTTCGAACACGTGATAGA -3 ' (SEQ ID NO.9)
Further, the composition further includes the primer and probe for the site rs7566605 on INSIG2 gene, specific to wrap
It includes:
The upstream primer of INSIG26605: 5 '-CAATAGCCACTGCCAAGTACTTAACA -3 ' (SEQ ID NO.10)
The downstream primer of INSIG26605: 5 '-ACTGAAAACCACCCTGGTACAGA -3 ' (SEQ ID NO.11)
The probe of INSIG26605: 5 '-TGGATATTTGATGGTGGTC -3 ' (SEQ ID NO.12)
Further, on IGF2BP2 gene the site rs4402960 primer and probe, specifically include:
The upstream primer of IGF2BP2: 5 '-CCATTCCTTATCTGGGGCAT -3 ' (SEQ ID NO.13)
The downstream primer of IGF2BP2: 5 '-GTCTTGGAATCTAACAGCTCTATGG -3 ' (SEQ ID NO.14)
The probe of IGF2BP2: 5 '-TCTTCAATCTACTGTCCATC -3 ' (SEQ ID NO.15)
Further, the composition further includes the primer and probe for the site rs5219 on KCNJ11 gene, is specifically included:
The upstream primer of KCNJ11: 5 '-GCAGTTGCCTTTCTTGGACAC -3 ' (SEQ ID NO.16)
The downstream primer of KCNJ11: 5 '-GCATCATCCCCGAGGAATAC -3 ' (SEQ ID NO.17)
The probe of KCNJ11: 5 '-CCTGGGCTCGGCA -3 ' (SEQ ID NO.18)
Further, the composition further includes the primer and probe for the site rs10830963 on MTNR1B gene, specifically
Include:
The upstream primer of MTNR1B: 5 '-GCCCCCAGTGATGCTAAGAA-3 ' (SEQ ID NO.19)
The downstream primer of MTNR1B: 5 '-CACCTGCATAGGCAGAATATTCC-3 ' (SEQ ID NO.20)
The probe of MTNR1B: 5 '-CACACCATCTCCTATC-3 ' (SEQ ID NO.21)
The present invention also provides a kind of above-mentioned primers, the application method of probe compositions, comprise the following steps that
Step 1: extracting test dna；
Step 2:Q-PCR amplification, reads fluorescent value；
Step 3: result judgement.
Further, the step 2 specifically: mix test dna with primer, probe compositions, configuration Q-PCR amplification
System reads fluorescent value 1min before reacting at 60 DEG C；95 DEG C of initial denaturation 5min；95 DEG C of denaturation 15s, 60 DEG C of annealing extend 60s,
It recycles 40 times altogether；Fluorescent value 1min after 60 DEG C of reading reactions.
Further, the Q-PCR amplification system is as follows:
| Reagent | Additional amount |
| Premixed liquid | 10μL |
| 10 μM of upstream primers | 0.5μL |
| 10 μM of downstream primers | 0.5μL |
| 10 μM of probes | 1μL |
| Test dna | 10-100ng |
| ddH2O | Complementing to amplification system is 20 μ L. |
Further, the premixed liquid is TaqPath ProAmp Master Mix.
Technical problem to be solved by the present invention lies in provide a kind of to detect the obesity that convenient, accuracy rate is high, economical and practical
With the detection method of diabetes B related mutation gene loci；Real-time quantitative is carried out to sample DNA by multiple groups gene primer
PCR amplification, and by the genotype of probe parting acquisition subject, reach quickly detection obesity and diabetes B tumor susceptibility gene
The effect in mutational site.
Compared with prior art, the present invention combines fat and diabetes B people's susceptibility gene mutation site, rationally designs
Q-PCR primer and probe, in conjunction with the mutation of TaqMan method detection subject's obesity and diabetes B susceptibility gene mutation site
Situation.The present invention have it is easy to operate, sequencing is high, sample detection is flexible, identification is quick and result is accurate, avoid traditional sequencing
The identification methods such as technology or agarose electrophoresis technology take time and effort, are difficult to the disadvantages of distinguishing.
Detailed description of the invention
Fig. 1 is that Taqman sonde method detects gene mutation site technical principle schematic diagram；
Fig. 2 is genotypic results figure, and the result of detection: blue, red and green dot respectively indicate different genotype,
The sample of the expression parting failure of cross, the black square that fluorescence signal is 0 or relatively other samples are very low are that DNA profiling is not added
Blank control compare (NC).
Specific embodiment
It in order to enable those skilled in the art to better understand the solution of the present invention, below will be to the skill in the embodiment of the present invention
Art scheme is clearly and completely described, it is clear that and the described embodiment is only a part of the embodiment of the present invention, without
It is whole embodiments.
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description.
To achieve the above object, the embodiment of the present invention provides the following technical solutions, and proposes that a kind of Q-PCR method detection is fat
With the amplimer and probe in diabetes B susceptibility gene mutation site, the mutational site to be detected of the obesity-prone gene
Are as follows: the site rs4994 on ADRB3 gene, the site rs6265 on BDNF gene, the position rs7566605 on INSIG2 gene
Point；The site rs9465871 on the CDKAL1 gene in mutational site to be detected of the diabetes B tumor susceptibility gene, IGF2BP2 base
Because of the upper site rs4402960, the site rs5219 on KCNJ11 gene, the site rs10830963 on MTNR1B gene.
The quantitative PCR in obesity described in the embodiment of the present invention and diabetes B susceptibility gene mutation site amplification target draws
Object and probe sequence are as follows:
For the site rs4994 on 3 gene of ADR β:
The upstream primer of ADR β 34994: 5 '-AGGCAACCTGCTGGTCATC-3 '
The downstream primer of ADR β 34994: 5 '-CAGCGAAGTCACGAACACG-3 '
The probe of ADR β 34994-T1(FAM fluorescent marker): 5 '-CCCGGACTCCGAGACT-3 '
The probe of ADR β 34994-T2(VIC fluorescent marker): 5 '-CCTGGACTCCGAGACT -3 '
To the site rs6265 on BDNF gene:
The upstream primer of BDNF6265: 5 '-GCTTGACATCATTGGCTGACA -3 '
The downstream primer of BDNF6265: 5 '-ACTTTCTGGTCCTCATCCAACAG -3 '
The probe of BDNF6265-T1(FAM fluorescent marker): 5 '-TTTCGAACACGTGATAGA -3 '
The probe of BDNF6265-T2(VIC fluorescent marker): 5 '-TTTCGAACACATGATAGA -3 '
For the site rs7566605 on INSIG2 gene:
The upstream primer of INSIG26605: 5 '-CAATAGCCACTGCCAAGTACTTAACA -3 '
The downstream primer of INSIG26605: 5 '-ACTGAAAACCACCCTGGTACAGA -3 '
The probe of INSIG26605-T1(FAM fluorescent marker): 5 '-TGGATATTTGATGGTGGTC -3 '
The probe of INSIG26605-T2(VIC fluorescent marker): 5 '-TGGATATTTGATCGTGGTC -3 '
For the site rs9465871 on CDKAL1 gene:
The upstream primer of CDKAL1: 5 '-AATCCAGTAAGTCAATCTTCAGTCT -3 '
The downstream primer of CDKAL1: 5 '-CAGTAGAGGTGGAGGAAGTCG -3 '
The probe of CDKAL1-T1(FAM fluorescent marker): 5 '-CTAACTCAAATTTCTCAGC -3 '
The probe of CDKAL1-T2(VIC fluorescent marker): 5 '-CTAACTCAAGTTTCTCAGC -3 '
For the site rs4402960 on IGF2BP2 gene:
The upstream primer of IGF2BP2: 5 '-CCATTCCTTATCTGGGGCAT -3 '
The downstream primer of IGF2BP2: 5 '-GTCTTGGAATCTAACAGCTCTATGG -3 '
The probe of IGF2BP2-T1(FAM fluorescent marker): 5 '-TCTTCAATCTACTGTCCATC -3 '
IGF2BP2-T2 (probe of VIC fluorescent marker): 5 '-TCTTAAATCTACTGTCCATCC -3 '
For the site rs5219 on KCNJ11 gene:
The upstream primer of KCNJ11: 5 '-GCAGTTGCCTTTCTTGGACAC -3 '
The downstream primer of KCNJ11: 5 '-GCATCATCCCCGAGGAATAC -3 '
The probe of KCNJ11-T1(FAM fluorescent marker): 5 '-CCTGGGCTCGGCA -3 '
The probe of KCNJ11-T2(VIC fluorescent marker): 5 '-CCTGGGCTTGGCA -3 '
For the site rs10830963 on MTNR1B gene:
The upstream primer of MTNR1B: 5 '-GCCCCCAGTGATGCTAAGAA-3 '
The downstream primer of MTNR1B: 5 '-CACCTGCATAGGCAGAATATTCC-3 '
The probe of MTNR1B-T1(FAM fluorescent marker): 5 '-CACACCATCTCCTATC-3 '
The probe of MTNR1B-T2(VIC fluorescent marker): 5 '-TCACACCATCTGCT -3 '
The detection method of the embodiment of the present invention includes the following steps:
(1) saliva sample Genome DNA extraction extracts genomic DNA (note from fat and type 2 diabetic patient's buccal swab sample
Meaning: in order to guarantee that sample is not polluted by food or beverage, patient please don't feed and drink water in 30min before sampling)；
(2) it is added to PCR pipe in the following proportions by different reagents, for primer, the probe of the design of different genes site, seals
Lid, of short duration centrifugation make the solution on tube wall be collected into tube bottom, are configured to Q-PCR amplification system, and system is as follows:
| Reagent | Additional amount |
| Premixed liquid | 10μL |
| 10 μM of upstream primers | 0.5μL |
| 10 μM of downstream primers | 0.5μL |
| 10 μM of probes | 1μL |
| Test dna | 10-100ng |
| ddH2O | Complementing to amplification system is 20 μ L. |
It is configured to respectively the site rs6265 for the site rs4994 on ADRB3 gene, on BDNF gene according to above method,
The site rs7566605 on INSIG2 gene, the site rs9465871 on CDKAL1 gene, on IGF2BP2 gene
The site rs4402960, the site rs5219 on KCNJ11 gene, 7 kinds of the rs10830963 site primer on MTNR1B gene
(corresponding primer, probe is added according to the gene loci of Q-PCR amplification system detection) in Q-PCR amplification system.
(3) Q-PCR is expanded: with above-mentioned Q-PCR amplification system, carrying out Q-PCR amplification, the program of reaction are as follows: 60 DEG C
Read fluorescent value 1min before reacting；95 DEG C of initial denaturation 5min；95 DEG C of denaturation 15s, 60 DEG C of annealing extend 60s, and 40 are followed in total
Ring；Fluorescent value 1min after 60 DEG C of reading reactions；
(4) result judges: according to allelic gene typing as a result, the sample of different genotype is indicated with different colours, that is, can determine that
Fat and diabetes B susceptibility gene mutation site genotype detected in sample, the result of detection is as shown in Fig. 2, indigo plant
The dot of color (the dark point in 3, left side in Fig. 2), red (the dark point in 4, right side in Fig. 2) and green (4 light points in the middle part of Fig. 2)
Different genotype is respectively indicated, the sample of non-parting is indicated with cross, and black square is NC control.
If in amplification procedure, fluorophor can be made to fall off in advance point as shown in Figure 1, probe and DNA profiling are exactly matched
From generation fluorescence signal, quenching group then falls off from probe, fluorophor is quenched.If probe with DNA profiling not
Full matching, in amplification procedure, can make probe fall off, unstressed configuration signal.
Technical effect and advantage of the invention is as follows:
(1) easy to operate: to complete reaction and only need quantitative PCR instruments；
(2) efficiently, flexibly: reaction only needs 2~3 hours, and test sample quantity is unrestricted；
(3) accuracy rate is high: special for the primer and probe of the obesity of screening and the design of diabetes B susceptibility gene mutation site
The genotype by sample product is distinguished in strange land, is sequenced by Sanger, and discovery accuracy rate is 84.0 ± 6.5%；
The embodiment of the present invention combines fat and diabetes B people's susceptibility gene mutation site, rationally designs Q-PCR primer and spy
Needle, in conjunction with the catastrophe of TaqMan method detection subject's obesity and diabetes B susceptibility gene mutation site.In addition, this
Inventive embodiments have it is easy to operate, sequencing is high, sample detection is flexible, identification is quick and result is accurate, avoid traditional sequencing
The identification methods such as technology or agarose electrophoresis technology take time and effort, are difficult to the disadvantages of distinguishing.
Finally it should be noted that the above embodiments are merely illustrative of the technical scheme of the present invention and are not intended to be limiting thereof, to the greatest extent
Invention is explained in detail referring to above-described embodiment for pipe, it should be understood by a person of ordinary skill in the art that technology
Personnel read present specification after still can with modifications or equivalent substitutions are made to specific embodiments of the invention, but this
A little modifications are changed within all without departing from the present patent application accompanying claims protection scope.
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Claims (10)
1. a kind of primer for detecting fat and diabetes B, probe compositions, which is characterized in that the composition includes
For the primer and probe in the site rs4994, the primer for the site rs9465871 on CDKAL1 gene and spy on 3 gene of ADR β
Needle, the primer and probe for the site rs4994 on 3 gene of ADR β include:
The upstream primer of ADR β 34994: 5 '-AGGCAACCTGCTGGTCATC-3 ' (SEQ ID NO.1)；
The downstream primer of ADR β 34994: 5 '-CAGCGAAGTCACGAACACG-3 ' (SEQ ID NO.2)；
The probe of ADR β 34994: 5 '-CCCGGACTCCGAGACT-3 ' (SEQ ID NO.3)；
Primer and probe for the site rs9465871 on CDKAL1 gene includes:
The upstream primer of CDKAL1: 5 '-AATCCAGTAAGTCAATCTTCAGTCT -3 ' (SEQ ID NO.4)；
The downstream primer of CDKAL1: 5 '-CAGTAGAGGTGGAGGAAGTCG -3 ' (SEQ ID NO.5)；
The probe of CDKAL1: 5 '-CTAACTCAAATTTCTCAGC -3 ' (SEQ ID NO.6).
2. the primer according to claim 1 for detecting fat and diabetes B, probe compositions, which is characterized in that
The composition further includes the primer and probe for the site rs6265 on BDNF gene, is specifically included:
The upstream primer of BDNF6265: 5 '-GCTTGACATCATTGGCTGACA -3 ' (SEQ ID NO.7)；
The downstream primer of BDNF6265: 5 '-ACTTTCTGGTCCTCATCCAACAG -3 ' (SEQ ID NO.8)；
The probe of BDNF6265: 5 '-TTTCGAACACGTGATAGA -3 ' (SEQ ID NO.9).
3. the primer according to claim 1 for detecting fat and diabetes B, probe compositions, which is characterized in that
The composition further includes the primer and probe for the site rs7566605 on INSIG2 gene, is specifically included:
The upstream primer of INSIG26605: 5 '-CAATAGCCACTGCCAAGTACTTAACA -3 ' (SEQ ID NO.10)；
The downstream primer of INSIG26605: 5 '-ACTGAAAACCACCCTGGTACAGA -3 ' (SEQ ID NO.11)；
The probe of INSIG26605: 5 '-TGGATATTTGATGGTGGTC -3 ' (SEQ ID NO.12).
4. the primer according to claim 1 for detecting fat and diabetes B, probe compositions, which is characterized in that
The primer and probe in the site rs4402960 on IGF2BP2 gene, specifically includes:
The upstream primer of IGF2BP2: 5 '-CCATTCCTTATCTGGGGCAT -3 ' (SEQ ID NO.13)；
The downstream primer of IGF2BP2: 5 '-GTCTTGGAATCTAACAGCTCTATGG -3 ' (SEQ ID NO.14)；
The probe of IGF2BP2: 5 '-TCTTCAATCTACTGTCCATC -3 ' (SEQ ID NO.15).
5. the primer according to claim 1 for detecting fat and diabetes B, probe compositions, which is characterized in that
The composition further includes the primer and probe for the site rs5219 on KCNJ11 gene, is specifically included:
The upstream primer of KCNJ11: 5 '-GCAGTTGCCTTTCTTGGACAC -3 ' (SEQ ID NO.16)；
The downstream primer of KCNJ11: 5 '-GCATCATCCCCGAGGAATAC -3 ' (SEQ ID NO.17)；
The probe of KCNJ11: 5 '-CCTGGGCTCGGCA -3 ' (SEQ ID NO.18).
6. the primer according to claim 1 for detecting fat and diabetes B, probe compositions, which is characterized in that
The composition further includes the primer and probe for the site rs10830963 on MTNR1B gene, is specifically included:
The upstream primer of MTNR1B: 5 '-GCCCCCAGTGATGCTAAGAA-3 ' (SEQ ID NO.19)；
The downstream primer of MTNR1B: 5 '-CACCTGCATAGGCAGAATATTCC-3 ' (SEQ ID NO.20)；
The probe of MTNR1B: 5 '-CACACCATCTCCTATC-3 ' (SEQ ID NO.21).
7. the application method of any primer for detecting fat and diabetes B of claim 1-6, probe compositions,
It is characterised in that it includes steps are as follows:
Step 1: extracting test dna；
Step 2:Q-PCR amplification, reads fluorescent value；
Step 3: result judgement.
8. application method according to claim 7, which is characterized in that the step 2 specifically: by test dna and primer,
Probe compositions mixing, configures Q-PCR amplification system, and fluorescent value 1min before reacting is read at 60 DEG C；95 DEG C of initial denaturations
5min；95 DEG C of denaturation 15s, 60 DEG C of annealing extend 60s, recycle 40 times altogether；Fluorescent value 1min after 60 DEG C of reading reactions.
9. application method according to claim 8, which is characterized in that the Q-PCR amplification system is as follows:
10. the primer according to claim 9 for detecting fat and diabetes B, probe compositions, feature exist
In the premixed liquid is TaqPath ProAmp Master Mix.
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