CN108949965B - Application of the SNP marker in type-2 diabetes mellitus diagnosis - Google Patents
Application of the SNP marker in type-2 diabetes mellitus diagnosis Download PDFInfo
- Publication number
- CN108949965B CN108949965B CN201810958625.7A CN201810958625A CN108949965B CN 108949965 B CN108949965 B CN 108949965B CN 201810958625 A CN201810958625 A CN 201810958625A CN 108949965 B CN108949965 B CN 108949965B
- Authority
- CN
- China
- Prior art keywords
- primer
- diabetes mellitus
- gene
- type
- snp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses application of the SNP marker in type-2 diabetes mellitus diagnosis.Present invention firstly discovers that enteric microorganism B.coprocola bacterial strain EDV01869.1 gene SNP site genotype is related with type-2 diabetes mellitus, pass through the genotype of EDV01869.1 gene 424 SNP in detection excrement genome, it may determine that whether patient suffers from type-2 diabetes mellitus, prompt the early clinical diagnosis that above-mentioned SNP site can be applied to type-2 diabetes mellitus.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to application of the SNP marker in type-2 diabetes mellitus diagnosis, specifically
The SNP marker for being related to EDV01869.1 gene
Background technique
Diabetes are a kind of common metabolic disease, disease incidence and inherent cause, life style, dietary structure and age
Etc. factors it is related.The Pathologic Characteristics shown according to patient, diabetes can be divided into two classes: type-1 diabetes mellitus, and also known as insulin relies on
Property diabetes and type-2 diabetes mellitus (Diabetes mellitus type 2, T2DM), also known as adult-onset diabetes,
The latter one account for 90% or more.The main feature of type-2 diabetes mellitus are as follows: the amount of insulin secretion of pancreatic beta cell declines and insulin
Effect decline, i.e. insulin resistance (insulin resistance, IR).
Single nucleotide polymorphism (single nucleotide polymorphism, SNP), i.e., it is single in DNA sequence
The polymorphism of DNA sequence dna caused by the mutation of a nucleotide, it is with shapes such as the transversion of single base, conversion, insertion and missings
Formula exists.As third generation molecular labeling, SNP marker has very big development potentiality, is widely used in biology, agronomy, doctor
The various fields such as, biological evolution domain, in the diagnosing and treating etc. of molecular genetics, pharmacogenetics, medical jurisprudence and disease
Aspect plays an important role.
Currently, the diagnosis of type-2 diabetes mellitus relies primarily on glucose in urine, urine ketone, urinary albumin, blood electrolyte, blood urea nitrogen and flesh
The laboratories such as acid anhydride check and various auxiliary examinations, but it is costly and uncomfortable that type-2 diabetes mellitus is diagnosed currently based on blood sugar test
It is researched and developed a kind of for passing through susceptibility loci in universal screening based on the limitation that the prior art early diagnoses type-2 diabetes mellitus
The product of detection type-2 diabetes mellitus has great importance for the early diagnosis of type-2 diabetes mellitus.
Summary of the invention
In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide one kind can be used for diagnosing type-2 diabetes mellitus it is easy
The SNP site of perception.Type-2 diabetes mellitus is diagnosed by detection SNP genotype with early warning.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides a kind of SNP marker, the SNP site is enteron aisle B.coprocola bacterial strain EDV01869.1
The 424th of gene.
Further, the SNP marker sports c.A424G.
The present invention provides application of the SNP marker in the product of preparation diagnosis type-2 diabetes mellitus neurological susceptibility, the SNP
Site is the 424th of B.coprocola bacterial strain EDV01869.1 gene.
Further, in type 2 diabetes patient, the 424th of EDV01869.1 gene sports G by A.
Further, the product includes the reagent for detecting the 424th genotype of SNP marker.
Further, the reagent includes the primer and/or non-specific amplification of specific amplification EDV01869.1
The primer of EDV01869.1.
Further, the primer sequence of the specific amplification EDV01869.1 gene is non-as shown in NO.1~6 SEQ ID
The primer sequence of specific amplification EDV01869.1 gene is as shown in NO.7~8 SEQ ID.
The present invention provides a kind of product for diagnosing type-2 diabetes mellitus, the product includes the reagent for detecting SNP marker,
The reagent is able to detect the 424th genotype of B.coprocola bacterial strain EDV01869.1 gene.
Further, the product includes chip, kit or nucleic acid film item.
Further, the reagent is selected from:
Identify the oligonucleotide probe of the SNP marker；Or
Expand the primer of the SNP marker.
Further, the primer includes specificity amplification primer and/or non-specific amplification primer.
Further, the specificity amplification primer sequence is as shown in NO.1~6 SEQ ID, non-specific amplification primer sequence
Column are as shown in NO.7~8 SEQ ID.
The present invention provides application of the said goods in the tool of preparation diagnosis type-2 diabetes mellitus.
In the present invention, the EDV01869.1 gene reference gene order that NCBI is provided is as shown in SEQ ID NO.9.
In the present invention, those skilled in the art can be detected by detecting any method or technique of SNP site
The SNP site of EDV01869.1 gene.The method includes but be not limited to solidifying with single-strand conformation polymorphism (SSCP), denatured gradient
Gel electrophoresis (DDGE), digestion amplification polymorphism sequence (CAPS), ApoE gene (allele-specific PCR,
) etc. AS-PCR the detection method for being representative based on gel electrophoresis and with direct Sequencing, DNA chip, denaturing high-performance liquid
Phase chromatography (DHPLC), mass spectrum detection, high-resolution solubility curve (HRM) etc. be the high throughput of representative, the degree of automation compared with
High detection method.
SSCP refers to single stranded DNA since base sequence difference can cause space conformation difference, this species diversity will lead to identical
Or the difference of close length single stranded DNA electrophoretic mobility, so as to be had by native polyacrylamide gel electrophoresis
The detection of effect ground.PCR-SSCP is the method that SSCP is used for the detection in Gene Mutation of pcr amplification product, the DNA fragmentation of PCR amplification
Under denaturant conditions, so that double-stranded DNA amplified fragments is untwisted by high-temperature process and maintain single-chain state, further progress is non-
Denaturing polyacrylamide gel electrophoresis.Currently, PCR-SSCP technology is widely used in the every field of molecular biology.
When DEEG carries out electrophoresis using double chain DNA molecule in the gel containing a certain concentration denaturing gradient agent, due to depositing
Double chain DNA molecule in mutation and the double chain DNA molecule there is no mutation untwist at different positions and are distinguished.DGGE can be examined
The DNA fragmentation for being up to 1kb is surveyed, if SNP appears in generating unit just and decomposes in the region of DNA domain of rotation, recall rate can be up to
100%.
CAPS is also known as PCR-RFLP.CAPS is to combine round pcr with RFLP technology, using DNA fragmentation in digestion
The variation of base on site carries out digestion to the pcr amplification product of the DNA fragmentation using corresponding restriction enzyme, thus
Generate polymorphism.Derivative digestion amplification polymorphism (derived cleaved amplified polymorphic sequence,
It dCAPS by introducing base mismatch, Created Restriction Site on primer, and then is realized polymorphic on the basis of CAPS technology
Property detection.CAPS and dCAPS connected applications are actually used in any SNP of detection.
The principle of AS-PCR is can not to be repaired due to Taq archaeal dna polymerase to the mispairing for the single base for being located at the end primer 3'
It is multiple, when the base of the end primer 3' and the allelic complementation of SNP site match clock synchronization, amplified reaction could occur；As primer 3'
The allele of the base of end and SNP site not complementary pairing when, then amplified reaction cannot occur.Currently, having there is base
In the certain methods of AS-PCR improvement, such as four draw
Object expands Refracting Mutation system PCR (tetra-primer amplification refractory mutation
System PCR, Tetra-primer ARMS-PCR), fragment length difference allele specific pcr (fragment length
Discrepant allele specific PCR, FLDAS-PCR), multiple alleles specific amplified (PCR
Amplification of multiple specific alleles, PMASA) etc..
Direct Sequencing detection SNP is that most directly reliable method, recall rate are up to 100%, and representing sequencing technologies has burnt phosphorus
Acid sequencing (Pyrosequencing), taqman technology, micro sequence (SNaPshot) etc..This method is by comparing in different samples
Same gene or genetic fragment pcr amplification product sequencing result, or weight the oriented sequence label of sequencing analysis
Site (STS) and EST (EST) detect SNP.Can by after PCR product purification and recovery by being connected on carrier
It is sequenced, PCR product can also be directly sequenced.Pass through the comparison of sequence, it will be able to accurately detect the mutation of SNP
Type and position.
DNA chip is otherwise known as genetic chip, biochip.This method according to known SNP site design two kinds or
Designed probe is fixed on special carrier by a variety of probes；DNA to be measured and has been fixed after fluorochrome label
Probe hybridized；According to base pair complementarity, there are the sequences of single base mismatch to hybridize with probe, passes through hybridization
Elution otherness, that is, fluorescence power between molecule detects SNP site.
DHPLC is a kind of method that new SNP is detected that grown up on the basis of DGGE and SSCP, is close
The ION PAIR Reverse Phase chromatography carried out under the conditions of DNA unwinding temperature, the solid phase in this method chromatographic column is its deciding factor,
Solid phase has single stranded DNA and double-stranded DNA different affinity.There are the pcr amplification products of SNP site to pass through heat denatured
After annealing, nucleic acid molecules form homologous and heterologous double chain DNA molecule due to different sources.In partial denaturation temperature,
The unwinding temperature of allogeneic dna sequence DNA double-strand it is lower and be easier to untwist be it is single-stranded, first flowed out from chromatographic column, with homoduplex DNA point
From in this way SNP relatively can be detected by absorption peak peak type and the number at peak.
Matrix-assisted laser desorption ionization (matrix as-sisted laser desorption
Ionization time of flight, MAL-DI-TOF) it is currently used for detecting main a kind of side in the mass spectrography of SNP
Method.There are mainly three types of different detection modes by MALDI-TOF: (one) single base primer extension (singlenu-cleotide
Primer extend, SNuPE), if the end primer 3' in SNP site upstream, and 4 kinds of ddNTPs all in the presence of, primer will be single
Base extends, and the ddNTP of different quality is introduced according to SNP site base type, can also be introduced on primer or ddNTP attached
Add quality, Lai Tigao accuracy and resolution ratio.(2) polybase base primer extension (primer oligonucleotide base
Extension, PROBE), if the end primer 3', in SNP site upstream, and there are a kind of ddNTP and 3 kinds of dNTPs, at first
With ddNTP pairing base at, primer extend simultaneously terminates at this, due to SNP site difference and generate with different molecular weight
Product.Increasingly automated and high-throughput analysis may be implemented in PROBE method in conjunction with DNA chip.(3) peptide nucleic acid probe
(peptide nucleic acids, PNA) hybrid method, the single stranded DNA after denaturation is hybridized with PNA, utilizes Mass Spectrometer Method
The quality difference of technology analysis hybrid product.
HRM, which combines face by the instrument of conventional fusion Curve Technique, saturated fluorescence dyestuff and acquisition fluorescence signal, to be developed
Come, without the probe of specificity, PCR product is analyzed by a kind of saturated fluorescence dyestuff.This method is molten by DNA double chain
The variation of solution curve can reflect the difference of nucleotide identity.Saturated fluorescence dyestuff is added in PCR reaction, it is glimmering during PCR
Photoinitiator dye can be embedded in DNA double chain, and when the DNA double chain embedded with dyestuff is untwisted, instrument will adopt fluorescence signal
Collection, and then show melting curve.As soon as will appear different melting curve shapes if there is a base to be changed in sequence
Shape and Tm value can rapidly analyze known and unknown sample using software.
Any one or several methods may be selected to detect SNP site in those skilled in the art, as long as SNP site may be implemented
Detection.
In the present invention, a kind of product diagnosing type-2 diabetes mellitus neurological susceptibility, the product include detection EDV01869.1
The reagent of gene SNP site.Wherein, when the genotype of the SNP site of EDV01869.1 is G, the neurological susceptibility of type-2 diabetes mellitus
Increase.The product of the invention may include using the reagent of any technology detection SNP site genotype known in the art, only
It is wanted to be able to detect SNP site genotype in sample.
In the present invention, the product for diagnosing type-2 diabetes mellitus can be chip, array, kit or nucleic acid film item.It is described
Chip includes solid phase carrier；And it is fixed on the oligonucleotide probe on the solid phase carrier, the oligonucleotide probe includes
Specifically correspond to the SNP site of EDV01869.1 gene recited above.The chip can be used for detecting
Multiple SNP sites including the SNP site of EDV01869.1 gene (such as one relevant to type-2 diabetes mellitus neurological susceptibility or more
One or more SNP sites of a gene).
The nucleic acid film item includes substrate and the oligonucleotide probe that is fixed in the substrate；The substrate, which can be, appoints
What is suitable for the substrate of immobilized oligonucleotide probe, such as nylon membrane, nitrocellulose filter, polypropylene screen, sheet glass, silica gel crystalline substance
Piece, miniature magnetic bead etc..
In the present invention, the oligonucleotide probe for the SNP site of EDV01869.1 gene can be DNA, RNA,
DNA-RNA chimera, PNA or other derivatives.There is no limit as long as complete specific hybrid and mesh for the length of the probe
Nucleotide sequence specific binding, any length is ok.The length of the probe can be as short as 25,20,15,13 or 10 alkali
Base length.Equally, the length of the probe can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.
Since different probe lengths has different influences to hybridization efficiency, signal specificity, the length of the probe is typically at least
14 base-pairs, longest are usually no more than 30 base-pairs, and the length complementary with purpose nucleotide sequence is with 15-25 base-pair
Most preferably.The probe self-complementary sequences are most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
In the present invention, the solid phase carrier includes plastic products, microparticle, membrane carrier etc..The plastic products can lead to
It crosses non-covalent or physical absorption mechanism to combine with antibody or proteantigen, most common plastic products are made of polystyrene
Small test tube, globule and micro-reaction plate；The microparticle is the microballoon or particle aggregated by high polymer monomer, and diameter is mostly
Micron easily can form chemical coupling with antibody (antigen), binding capacity is big with the functional group in conjunction with protein due to having；Institute
Stating membrane carrier includes the miillpore filters such as nitrocellulose filter, glass fibre element film and nylon membrane.
In some embodiments in the present invention, the testing product carries out the easy of type-2 diabetes mellitus in human experimenter
It is used in the in-vitro method of perception evaluation and/or diagnosis and/or prediction, wherein the product includes for carrying out the following steps
Proper implements:
(1) at least one type with the EDV01869.1 gene SNP site in the genome DNA sample is evaluated
And/or it is horizontal；
(2) by the biomarkcr data of the human experimenter evaluated in (1) and healthy and/or illness people
Biomarkcr data be compared, come make the human experimenter type-2 diabetes mellitus risk assessment and/or diagnosis
And/or prediction.
In the present invention, term " genotype " refers to the identity for being present in individual or the allele in sample.Typically,
It refers to the genotype of individual relevant to interested specific gene；In polyploid individual, refer to that individual carries gene etc.
Which kind of combination of position gene.
In some embodiments, the kit is relevant to type-2 diabetes mellitus neurological susceptibility in identification human experimenter
It is used in the in-vitro method of SNP, the kit includes the proper implements for carrying out the following steps:
(1) at least one SNP of the EDV01869.1 gene in the nucleic acid samples of the detection human experimenter, wherein
At least one SNP implies the neurological susceptibility of type-2 diabetes mellitus；
(2) the SNP haplotype of the identification human experimenter, wherein the SNP haplotype includes the inspection in (1)
The SNP of survey.
In the present invention, when referring to detection kit, term " proper implements " refers to any for reaching specified
Purpose any technological means.As the non-limitative example of this proper implements, can enumerate reagent and/or material and/
Or process and/or specification and/or software etc..All kits of the present invention may include that packaging appropriate and specification are used
In being used in method disclosed herein.
In the present invention, " nucleic acid " means both DNA and RNA, both exists with any possible configuration, i.e., with double-strand
(ds) form of nucleic acid, or in the form of (ss) nucleic acid, or in the form of a combination thereof (part ds or ss).Such nucleic acid is corresponding
In at least two continuous deoxyribonucleotides or ribonucleotide of the nucleotide for optionally including at least one modification.
Nucleic acid can also be modified (such as thiophosphoric acid, H- phosphate, alkyl phosphoric acid in the level of the key of nucleotide part
Ester), (such as α-oligonucleotides) or PNA or 2 '-O- alkylribose are modified in the level of main chain).It is every in these modifications
One kind can combine generation, as long as there is at least one phosphate in nucleic acid.The nucleic acid can be it is natural or synthesis,
Oligonucleotides, polynucleotides, nucleic acid fragment, rRNA, mRNA, transfer RNA, the core obtained by enzymatic amplification technology
Acid.The enzymatic amplification technology is, for example, that PCR (polymerase chain reaction) and its RT-PCR derivative form, PCR (it is anti-to repair chain
Answer), 3SR (self-sustained sequence replication), the NASBA amplification of nucleic acid sequence (rely on), TMA (amplification of transcriptive intermediate).
In the present invention, term " primer " refers to naturally occurring oligonucleotides (such as restriction fragment) or is synthetically produced
Oligonucleotides can be used as the starting point of primer extension product synthesis, when in condition appropriate (such as buffer, salt, temperature
Degree and pH) and the reagent (such as the polymerase for relying on DNA or dependenc RNA) there are nucleotide and for nucleic acid polymerization condition
Under, the primer extension product is complementary with nucleic acid chains (template or target sequence).
Further, the detection detection reagent includes the primer and/or probe for SNP site.The reagent further includes
For in the prior art it has been reported that diagnosis type-2 diabetes mellitus susceptible gene SNP site primer and/or probe.It will be a kind of
Or the detection primer and/or probe of multiple SNP sites of several genes are placed in identical product by detecting a variety of SNP sites
The case where index Combining diagnosis type-2 diabetes mellitus neurological susceptibility, is also contained within protection scope of the present invention.
In the present invention, " array " or " microarray " is that hybridised arrays original part is ordered in matrix, the hybridization battle array
Column original part such as polynucleotide probe (such as oligonucleotides) or bonding agent (such as antibody).The matrix can be solid-based
Matter, for example, glass or silica slide, pearl, fibre optics binder or semi-solid matrix, such as nitrocellulose filter.Core
Nucleotide sequence can be DNA, RNA or in which any arrangement.Microarray can be from being produced from known type-2 diabetes mellitus neurological susceptibility
It is prepared by gene-special oligonucleotide probe.Array contains the different oligonucleotide probes of each gene SNP site,
Array can also may also comprise containing control, the control appropriate of the one or more of Non-specific hybridization on microchip.
The advantages of the present invention:
Present invention firstly discovers that enteric microorganism B.coprocola bacterial strain EDV01869.1 gene SNP site genotype
It is related with type-2 diabetes mellitus.One kind is proposed on this basis by detection SNP site to prejudge type-2 diabetes mellitus neurological susceptibility risk
The product of property.Testing product of the invention has good sensitivity, stability and specificity.
Product of the invention to normal population carry out type-2 diabetes mellitus neurological susceptibility screening, can effectively play Risk-warning,
The effect of early diagnosis.
Detailed description of the invention
Fig. 1 shows that EDV01869.1 gene clusters analyze result figure；
Fig. 2 shows the SNP site of the method detection EDV01869.1 gene specific using Sanger sequencing.
Specific embodiment
Below with reference to specific embodiment further illustrate the present invention, the embodiment of the present invention for explaining only the invention,
It is not intended to limit protection scope of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment screens EDV01869.1 gene in enteric microorganism B.coprocola bacterium relevant to type-2 diabetes mellitus
SNP site
1, research object
Type-2 diabetes mellitus group: 72 type-2 diabetes mellitus first visit patients that division of endocrinology, hospital accepts for medical treatment are randomly selected, mark is included in
It is quasi-: to meet " WHO1999 diabetes diagnostic criterion ".
Normal group: choosing healthy volunteer 56.
All research objects know to this research and endorsed informed consent form.
2, the extraction of excrement genomic DNA
Take the fecal sample 200g for being stored in -80 DEG C of refrigerators, using Tiangeng excrement genome DNA extracting reagent kit according to
The operating process of " Tiangeng excrement extracting genome DNA specification " extracts fecal sample genomic DNA.
3, DNA purity detecting
DNA purity: OD260/OD280 ratio is the index for measuring protein contamination degree in DNA sample.High quality
DNA sample, OD260/OD280 value is 1.8 or so.
4, PCR amplification target gene
(1) design of primers
Pcr amplification product is designed according to the coded sequence of EDV01869.1 gene in Genbank, it is far raw by Beijing day brightness
The synthesis of object Science and Technology Ltd..The synthesized specific primer sequence for capableing of specific amplified target gene (secondary PCR primer) is such as
Under:
1) forward primer 5 '-TTCCCTCACCGTCAGAAAGC-3 ' (SEQ ID NO.1),
Reverse primer 5 '-AGATGGTGGCTGCTTCTTCC-3 ' (SEQ ID NO.2)
2) forward primer 5 '-CCCGGGCATTTTGGTTCAAG-3 ' (SEQ ID NO.3)
Reverse primer 5 '-GCTTTCTGACGGTGAGGGAA-3 ' (SEQ ID NO.4)
3) forward primer 5 '-TGAACGTCGTGACCGTTTCT-3 ' (SEQ ID NO.5)
Reverse primer 5 '-TCCGTCTACTGGACCGAAGT-3 ' (SEQ ID NO.6)
The synthesized specific primer sequence for capableing of non-specific amplification target gene (PCR primer) is as follows:
Forward primer 5 '-TAGGACATCAGGCGTACGGA-3 ' (SEQ ID NO.7)
Reverse primer 5 '-CTAAGCCAGCACGGTCTAGG-3 ' (SEQ ID NO.8)
(2) amplification mode selects pre-designed non-specific amplification by way of secondary amplification using regular-PCR
Primer carries out a PCR (20 μ l system), and reaction system is shown in Table 1, reaction condition: 94 DEG C of 3min, (94 DEG C of 30s, 58 DEG C of 30s, 72
DEG C 2min) * 40 circulations, 72 DEG C of 5min.Three pairs of pre-designed specificity amplification primers are selected to carry out secondary PCR (30 μ l
System), reaction system is shown in Table 2, reaction condition: 94 DEG C of 3min, (94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min) * 40 circulations, and 72
℃5min。
1 PCR reaction systems of table
| Reagent | Volume |
| Forward primer | 0.4μl |
| Reverse primer | 0.4μl |
| Tiangeng 2 × Pfu PCR MasterMix | 10μl |
| Template | 1μl |
| ddH2O | Supply 20 μ l |
2 secondary PCR reaction system of table
| Reagent | Volume |
| Forward primer | 0.6μl |
| Reverse primer | 0.6μl |
| Tiangeng 2 × Pfu PCR MasterMix | 15μl |
| Template | 1μl |
| ddH2O | Supply 30 μ l |
5, testing goal band
Using the method (deposition condition: 1% glue of plain agar sugar gel electrophoresis；1 × TAE electrophoretic buffer；90v, 30 points
Clock) testing goal band.
6, PCR product is sequenced
PCR product is sequenced by Sanger, and positive anti-primer is sequenced obtains aim sequence simultaneously.
7, sequencing result is analyzed
Phylogenetic analysis is done to obtained sequencing result with MEGA7 and RAxML, with the SNP in SeqMan statistical series.
8, result
As a result as shown in Figure 1, blue triangles represent Healthy People in figure, red spots represent diabetic, cluster knot
Fruit is obviously divided into two classes, and the first kind is mainly diabetic, and Healthy People concentrates on the second class, and right figure is corresponding mutational site
Analysis, the first kind are nearly all mutated at 424, and all there is no mutation, OR (odds ratio) at 424 for the second class
Value is that 6.22, Fei Sheer accurately examines p=8.87 × 10-4, show B.coprocola bacterium EDV01869.1 gene in patient of diabetes
There is marked difference between person and Healthy People.
As shown in Fig. 2, the analysis of EDV01869.1 gene mutation site is as a result, refer to base with the EDV01869.1 that NCBI is provided
Because of sequence alignment, there are 67 the 424th in gene to be all mutated in 68 diabetics in the first kind, by A alkali
Base mutation has become glutamic acid from lysine for G base, corresponding amino acid, belongs to missense mutation, and strong in the second class
Health people does not mutate in the 424th of gene.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Sequence table
<110>PLA Academy of Military Sciences's military medical research institute
<120>application of the SNP marker in type-2 diabetes mellitus diagnosis
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ttccctcacc gtcagaaagc 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
agatggtggc tgcttcttcc 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cccgggcatt ttggttcaag 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gctttctgac ggtgagggaa 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tgaacgtcgt gaccgtttct 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tccgtctact ggaccgaagt 20
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
taggacatca ggcgtacgga 20
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ctaagccagc acggtctagg 20
<210> 9
<211> 1908
<212> DNA
<213> Bacteroides coprocola
<400> 9
atggaacaac ccatttctta cgaattgttg aataaaatag atagcccgga agatttgcgt 60
catcttccta tcgagtcttt acctgaggtc tgtcgggagc tcagggataa aatcattgat 120
gaactttctc gcaatcccgg gcattttggt tcaagtttag gagtaataga attaactgtt 180
gcacttcatt atatattcaa taccccctat gaccgcattg tatgggacgt aggacatcag 240
gcgtacggac ataagatact gactgaacgt cgtgaccgtt tctgtacaaa cagaaaacta 300
aacggtttat gcccttttcc ctcaccgtca gaaagcgaat atgatacgtt tacctgtgga 360
catgcatcca actctatctc cgtagctcta ggcatggctg ttgctgcggc ccgcaaaggt 420
gaaaaaaacc gccatgtagt agccgtaatc ggggatggtt cgatgagtgg cgggctggct 480
tttgaaggat tgaataatgc gtcttctact cccaacaatt tgctgattat cttgaacgat 540
aacaacatgg cgatcgatcg gagcgtagga ggaatgaaac aatatctgct aaatctccaa 600
acatcagaag gatacaaccg actgagatat aacatctcaa agaagctaca tcagtgggga 660
gttctgaatg aagaacgccg gaaaagctta atccgtttca acaatagcct gaaatctgtt 720
cttacccaac aacagaacat ttttgaggga atgaatatcc gctacttcgg tccagtagac 780
ggacacgatg tttttgctct ggcaaaagta ctgaaagaaa ttaaggacat gaagggaccg 840
aaactacttc atatccatac aatcaaaggc aaaggattca aacctgcgga agaagcagcc 900
accatctggc atgctccagg catattcgat aaagaaactg gagaaagaat cgtaaatgat 960
acaagtggta tgccaccttt attccaggat gtgtttggca aaaccttact ggaactggca 1020
aaaaagaacg acaaaattat tggagtgact ccagccatgc ctactggctg ttctatgaat 1080
atcttaatga aagctatgcc cgaccgtgca ttcgatgtag gcattgccga aggacatgcc 1140
gtaacttttt cgggaggtat ggccaaagag ggattactgc ctttctgtaa catttactcc 1200
tcgttcatgc aacgggcata cgacaatatt atccacgaca tagccataca gaaactgaat 1260
gtagtgttat gcctagaccg tgctggctta gtaggagaag acggtccaac gcatcatggc 1320
gtattcgatc tggcgtattt acgtcctatc cctaacctaa caatagcttc accttacgac 1380
gaacaggaac tgcgccggct gatgtataca gcacagcttc ccgaccaagg tccttttgtt 1440
atccgctatc ctcgcggacg aggtgtgctg accaactggg aatgtccact taaagctgtt 1500
gaagtgggaa agggcagaaa actgaaagac ggttccgacc tggctgtaat cacaatcggt 1560
cctatcggaa atacagcagc caaagctatt acagaagccg agaatactca taattgcagc 1620
attgcccact acgatctccg ttttttaaaa ccactggatg aagcattatt acacgagata 1680
ggaaaaaaat tctcacgtat cattacaata gaagatggag tgctcaaagg tggaatggga 1740
agtgctgttc tggaatttat gtcagacaat aactacaccc ctatggtaaa gcgtatgggt 1800
attcctgacc attttataca acatggtcct gtaaatgctc tttactccat ttgtggtatc 1860
gatcaaacag gtatatataa tacattaatt catattctga actcatga 1908
Claims (9)
1. a kind of application of reagent for detecting SNP marker in the product of preparation diagnosis type-2 diabetes mellitus neurological susceptibility, feature
It is, the SNP marker is the 424th of enteron aisle B.coprocola bacterial strain EDV01869.1 gene, sports c.A424G.
2. application according to claim 1, which is characterized in that the reagent includes specific amplification EDV01869.1 gene
Primer and/or non-specific amplification EDV01869.1 gene primer.
3. application according to claim 2, which is characterized in that the primer sequence of the specific amplification EDV01869.1 gene
Column are as shown in NO.1 ~ 6 SEQ ID, the primer sequence of the non-specific amplification EDV01869.1 gene such as institute of SEQ ID NO.7 ~ 8
Show.
4. a kind of product for diagnosing type-2 diabetes mellitus, which is characterized in that the product includes described in detection claim 1
The reagent of SNP marker.
5. product according to claim 4, which is characterized in that the product includes chip, kit or nucleic acid film item.
6. product according to claim 4, which is characterized in that the reagent is selected from:
Identify the oligonucleotide probe of the SNP marker；Or
Expand the primer of the SNP marker.
7. product according to claim 6, which is characterized in that the primer includes specificity amplification primer and/or non-spy
Specific amplification primers.
8. product according to claim 7, which is characterized in that the specificity amplification primer sequence such as SEQ ID NO.1 ~
Shown in 6, non-specific amplification primer sequence is as shown in NO.7 ~ 8 SEQ ID.
9. application of the described in any item products of claim 4 ~ 8 in the tool of preparation diagnosis type-2 diabetes mellitus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810958625.7A CN108949965B (en) | 2018-08-22 | 2018-08-22 | Application of the SNP marker in type-2 diabetes mellitus diagnosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810958625.7A CN108949965B (en) | 2018-08-22 | 2018-08-22 | Application of the SNP marker in type-2 diabetes mellitus diagnosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108949965A CN108949965A (en) | 2018-12-07 |
| CN108949965B true CN108949965B (en) | 2019-09-03 |
Family
ID=64473573
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810958625.7A Active CN108949965B (en) | 2018-08-22 | 2018-08-22 | Application of the SNP marker in type-2 diabetes mellitus diagnosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108949965B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101960019A (en) * | 2007-04-03 | 2011-01-26 | 国家科学研究中心 | FTO gene polymorphisms associated to obesity and/or type II diabetes |
| CN105567851A (en) * | 2016-02-29 | 2016-05-11 | 北京泱深生物信息技术有限公司 | Diabetes-related molecular marker |
| CN105969889A (en) * | 2016-07-06 | 2016-09-28 | 四川省人民医院 | Use of SNP (single nucleotide polymorphism) locus of MUC3B gene |
-
2018
- 2018-08-22 CN CN201810958625.7A patent/CN108949965B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101960019A (en) * | 2007-04-03 | 2011-01-26 | 国家科学研究中心 | FTO gene polymorphisms associated to obesity and/or type II diabetes |
| CN105567851A (en) * | 2016-02-29 | 2016-05-11 | 北京泱深生物信息技术有限公司 | Diabetes-related molecular marker |
| CN105969889A (en) * | 2016-07-06 | 2016-09-28 | 四川省人民医院 | Use of SNP (single nucleotide polymorphism) locus of MUC3B gene |
Non-Patent Citations (1)
| Title |
|---|
| 人类肠道宏基因组SNP模式与疾病的关联研究;陈垚文;《中国博士学位论文全文数据库 医药卫生科技辑》;20171115;摘要，第13-23、25-48、65页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108949965A (en) | 2018-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6238866B1 (en) | Detector for nucleic acid typing and methods of using the same | |
| CN108441565B (en) | Fluorescence labeling multiplex amplification kit for 37 STR loci of human Y chromosome and application thereof | |
| CN100588953C (en) | Method for detecting mononucleotide polymorphism with biochip | |
| KR102044683B1 (en) | Pepetide nucleic acid probe for identifying Coxiella burnetii and Method for identifying Coxiella burnetii using the same | |
| JP5662293B2 (en) | SNP for diagnosing attention deficit / hyperactivity disorder and microarray and kit including the same | |
| CA2964265A1 (en) | Polymerase chain reaction primers and probes for mycobacterium tuberculosis | |
| KR101777161B1 (en) | A Multiplex SNP marker composition and a method for diagnosis or prediction of canine hip dysplasia using same marker | |
| CN110564861B (en) | Fluorescent marker composite amplification kit for human Y chromosome STR locus and InDel locus and application thereof | |
| WO2012171990A1 (en) | Discrimination of blood type variants | |
| KR101206028B1 (en) | Method for diagnosing a breast cancer using a breast cancer specific polymorphic sequence, polynucleotide specific to a breast cancer and microarray immobilized with the polynucleotide | |
| CN110628920A (en) | Fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome and application thereof | |
| US11535897B2 (en) | Composite epigenetic biomarkers for accurate screening, diagnosis and prognosis of colorectal cancer | |
| US7833710B2 (en) | Polynucleotide associated with breast cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing breast cancer using the same | |
| CN108949965B (en) | Application of the SNP marker in type-2 diabetes mellitus diagnosis | |
| KR101100437B1 (en) | A polynucleotide associated with a colon cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a colon cancer using the polynucleotide | |
| CN108676871B (en) | Diagnostic marker for type II diabetes | |
| KR102124770B1 (en) | Composition for early predicting or diagnosing hip dysplasia in dog | |
| CN109750098B (en) | ATP7B gene large fragment deletion detection kit and detection method | |
| CN113215241A (en) | Detection primer group for risk early warning of cardiovascular and cerebrovascular diseases and application thereof | |
| KR20200129600A (en) | Pepetide nucleic acid probe for genotyping Helicobacter pylori and Method using the same | |
| KR101858962B1 (en) | Method and kit for identifying variety of Persimmon using single nucleotide polymorphism markers | |
| KR102185440B1 (en) | Composition for early predicting or diagnosing hypercholesterolemia in dog | |
| KR100647304B1 (en) | 2 A polynucleotide associated with a type II diabetes mellitus comprising single nucleotide polymorphism microarray and diagnostic kit comprising the same and method for analyzing polynucleotide using the same | |
| KR102167792B1 (en) | Composition for early predicting or diagnosing hypercalcemia in dog | |
| KR102194880B1 (en) | SNP marker for prediction of dog's diabetes risk and prediction method using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |