CN106929580B - Application of the IL12RB1 gene mononucleotide polymorphism rs2305740 in detection tuberculosis susceptibility - Google Patents
Application of the IL12RB1 gene mononucleotide polymorphism rs2305740 in detection tuberculosis susceptibility Download PDFInfo
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Abstract
The invention discloses application of the IL12RB1 gene mononucleotide polymorphism rs2305740 in detection tuberculosis susceptibility.Application provided by the present invention is whether the substance for detecting the single nucleotide polymorphism in the site rs2305740 suffers from the application in product lungy in preparation evaluation or the product of the tuberculous risk of auxiliary evaluation people to be measured, or in preparation diagnosis or auxiliary diagnosis people to be measured.The present inventor has found that there are significant correlations between IL12RB1 gene SNP-rs2305740 single nucleotide polymorphisms and resistive connection nuclearity by Chinese han population case-control study.The crowd of carrying IL12RB1-rs2305740GG genotype and " GGGAG " double body type onset risk when mycobacterium tuberculosis infects is lower.The present invention being associated between inflammatory and gene involved in immunity SNP and tuberculosis neurological susceptibility provides neodoxy, has substantial worth to the screening of tuberculosis tumor susceptibility gene and prevention.
Description
Technical field
The invention belongs to fields of biomedicine, are related to IL12RB1 gene mononucleotide polymorphism rs2305740 and tie in detection
Application in core neurological susceptibility, in particular to the site IL12RB1 gene mononucleotide polymorphism rs2305740 marker is in crowd couple
Application in the screening of tuberculosis Gene susceptibility and assessment.
Background technique
Tuberculosis (Tubercu1osis, TB) is a kind of global disease for seriously threatening human health, is had become as people
One of big infectious diseases killer of class three.The report of World Health Organization's whole world tuberculosis points out that about 1/3 people is by tuberculosis branch
Bacillus infection, wherein only 10% the infected falls ill.This mean that tuberculosis infected students whether fall ill with host-pathogen it
Between interaction, environment, heredity etc. have close relationship.Recently, many studies demonstrate that risk lungy and siberian crabapple
The polymorphism of system and inflammatory reaction related gene has close connection.
IL12RB1 gene is located at the area human chromosomal 19p13.1, and the protein of coding is to belong to cytagenin receptor
The I type transmembrane protein of superfamily.The albumen is considered as that IL12 receptor is multiple with low-affinity combination interleukin 12 (IL12)
Close a part of object.The development of the mutation damage IL-17 generation property T lymphocyte of the gene, and cause to mycobacteria and sand
The neurological susceptibility of door Salmonella infection increases.
With the development of genomics and the completion of mycobacterium tuberculosis genome sequencing, thousands of mononucleotide
The site polymorphism (SNP) is accredited from tubercular.Researcher draws out mycobacterium tuberculosis SNP system using these data
Spectrum is set and proposes the evolution hypothesis of mycobacterium tuberculosis pedigree.Although mycobacterium tuberculosis SNP is studied in methodology and gene
Group makes remarkable achievements on learning, but the relationship between many SNP and tuberculosis infection risk is but still known little about it,
Relationship especially between IL12RB1 gene SNP and tuberculosis risk is still unclear so far.
Accordingly, it is determined that the SNP of IL12RB1 gene and furtheing investigate its relationship between tuberculosis risk and seeming very
It is necessary to.
Summary of the invention
The object of the present invention is to provide IL12RB1 gene mononucleotide polymorphism rs2305740 in detection tuberculosis susceptibility
In application.
Application concretely two provided by the present invention, i.e., using A and using B.
Using A: the substance for detecting the single nucleotide polymorphism in the site rs2305740 is preparing evaluation or auxiliary evaluation
Application in the product of the tuberculous risk of people to be measured.
Wherein, the site rs2305740 be No. 19 chromosomes of human genome in the 18069426th from 5 ' ends
Nucleotide；The nucleotide in the site rs2305740 is A or G.
Further, it is described using A be the substance for detecting the single nucleotide polymorphism in the site rs2305740 and
The readable carrier of content shown in following (a1) is recorded in preparation evaluation or the tuberculous risk of auxiliary evaluation people to be measured
Product in application；(a1) site rs2305740 is the tuberculous risk of people to be measured of GG genotype lower than described
The site rs2305740 is the people to be measured (codominant inheritance model and dominant inheritance model) of AA genotype or AG genotype.
Using B: for detecting the substance of double body type M in preparation evaluation or the tuberculous risk of auxiliary evaluation people to be measured
Product in application.
Haplotype (Haplotype) is interrelated positioned at one group of item chromosome specific region, and is tended to whole
Body is hereditary to the combination of the mononucleotide polymorphic of offspring.Double body type is the haplotype pair on homologue.
Wherein, the mononucleotide polymorphic combination for constituting the haplotype of the double body type M is successively as follows: the site rs2305740,
The site rs401502, the site rs375947, the site rs17852635 and the site rs11575934.
The site rs2305740 be No. 19 chromosomes of human genome in from 5 ' ends the 18069426th nucleosides
Acid；The nucleotide in the site rs2305740 is A or G.
The site rs401502 be No. 19 chromosomes of human genome in from 5 ' ends the 18069603rd nucleotide；
The nucleotide in the site rs401502 is G or C.
The site rs375947 be No. 19 chromosomes of human genome in from 5 ' ends the 18069641st nucleotide；
The nucleotide in the site rs375947 is A or G.
The site rs17852635 be No. 19 chromosomes of human genome in from 5 ' ends the 18075765th nucleosides
Acid；The nucleotide in the site rs17852635 is A or G.
The site rs11575934 be No. 19 chromosomes of human genome in from 5 ' ends the 18075808th nucleosides
Acid；The nucleotide in the site rs11575934 is A or G.
Further, described for the substance for detecting double body type M and to record content shown in following (b1) using B
Readable carrier preparation evaluation or the tuberculous risk of auxiliary evaluation people to be measured product in application；(b1) it constitutes
2 haplotypes of the double body type M are the 2 of the tuberculous risk of the people to be measured double body type M described lower than composition of " GGGAG "
A haplotype is not the people to be measured of " GGGAG ".
The present invention also protects a kind of product, the substance containing the single nucleotide polymorphism for detecting the site rs2305740；
The product has the function of evaluation or the tuberculous risk of auxiliary evaluation people to be measured.
Wherein, the site rs2305740 be No. 19 chromosomes of human genome in the 18069426th from 5 ' ends
Nucleotide；The nucleotide in the site rs2305740 is A or G.
In the present invention, the substance of the single nucleotide polymorphism for detecting the site rs2305740 is specially primer
To first or complete single strand dna first；Sequence in primer pair first single stranded DNA as shown in sequence 1 in sequence table and sequence table
The composition of single stranded DNA shown in column 2；Complete single strand dna first single stranded DNA as shown in sequence 1 in sequence table, sequence table
Single stranded DNA shown in sequence 3 forms in single stranded DNA shown in middle sequence 2 and sequence table.
In the product, also containing the readable carrier for recording content shown in (a1) as above.
The present invention also protects a kind of product, contains the substance for detecting double body type M；Constitute the monomer of the double body type M
Type mononucleotide polymorphic combination it is successively as follows: the site rs2305740, the site rs401502, the site rs375947,
The site rs17852635 and the site rs11575934；The product has evaluation or the tuberculous risk of auxiliary evaluation people to be measured
The function of property.
The site rs2305740 be No. 19 chromosomes of human genome in from 5 ' ends the 18069426th nucleosides
Acid；The nucleotide in the site rs2305740 is A or G.
The site rs401502 be No. 19 chromosomes of human genome in from 5 ' ends the 18069603rd nucleotide；
The nucleotide in the site rs401502 is G or C.
The site rs375947 be No. 19 chromosomes of human genome in from 5 ' ends the 18069641st nucleotide；
The nucleotide in the site rs375947 is A or G.
The site rs17852635 be No. 19 chromosomes of human genome in from 5 ' ends the 18075765th nucleosides
Acid；The nucleotide in the site rs17852635 is A or G.
The site rs11575934 be No. 19 chromosomes of human genome in from 5 ' ends the 18075808th nucleosides
Acid；The nucleotide in the site rs11575934 is A or G.
In the present invention, the substance for detecting double body type M is specifically sensed by the site rs2305740
The substance of single nucleotide polymorphism, the substance of single nucleotide polymorphism for detecting the site rs401502, for detecting
The substance of the single nucleotide polymorphism in the site rs375947, the mononucleotide for detecting the site rs17852635 are more
The material composition of the substance of state property and the single nucleotide polymorphism for detecting the site rs11575934.
Wherein, the substance of the single nucleotide polymorphism for detecting the site rs2305740 is primer pair first or complete
Single strand dna first；It is single shown in sequence 2 in primer pair first single stranded DNA as shown in sequence 1 in sequence table and sequence table
Chain DNA composition；Complete single strand dna first single stranded DNA as shown in sequence 1 in sequence table, in sequence table shown in sequence 2
Single stranded DNA and sequence table in the composition of single stranded DNA shown in sequence 3.For detecting the mononucleotide in the site rs401502
The substance of polymorphism is primer pair B or complete single strand dna second；The primer pair B is single as shown in sequence 4 in sequence table
Single stranded DNA shown in sequence 5 forms in chain DNA and sequence table；The complete single strand dna second is by 4 institute of sequence in sequence table
Single stranded DNA composition shown in sequence 6 in single stranded DNA shown in sequence 5 and sequence table in the single stranded DNA that shows, sequence table.For examining
The substance for surveying the single nucleotide polymorphism in the site rs375947 is primer pair third or complete single strand dna third；It is described to draw
Object forms single stranded DNA shown in sequence 8 in the third single stranded DNA shown in sequence 7 in sequence table and sequence table；The complete list
The single stranded DNA as shown in sequence 7 in sequence table of ssdna molecule third, in sequence table in single stranded DNA and sequence table shown in sequence 8
The composition of single stranded DNA shown in sequence 9.The substance of single nucleotide polymorphism for detecting the site rs17852635 is primer
To fourth or complete single strand dna fourth；Sequence in primer pair fourth single stranded DNA as shown in sequence 10 in sequence table and sequence table
The composition of single stranded DNA shown in column 11；Complete single strand dna fourth single stranded DNA as shown in sequence 10 in sequence table, sequence
The composition of single stranded DNA shown in sequence 12 in single stranded DNA shown in sequence 11 and sequence table in table.It is described for detecting
The substance of the single nucleotide polymorphism in the site rs11575934 is primer pair penta or complete single strand dna penta；The primer pair
Single stranded DNA shown in sequence 14 forms in penta single stranded DNA shown in sequence 13 in sequence table and sequence table；It is described complete single-stranded
The single stranded DNA as shown in sequence 13 in sequence table of DNA molecular penta, in sequence table in single stranded DNA and sequence table shown in sequence 14
The composition of single stranded DNA shown in sequence 15.
In the product, also containing the readable carrier for recording content shown in (b1) as above.
In the present invention, the people to be measured is preferably from Chinese han population.
The present inventor has found IL12RB1 gene SNP-by Chinese han population case-control study
There are significant correlations between rs2305740 single nucleotide polymorphisms and resistive connection nuclearity.Carry IL12RB1-rs2305740GG
The crowd of genotype and " GGGAG " double body type onset risk when mycobacterium tuberculosis infects is lower.The present invention is inflammatory and exempts from
Being associated between epidemic disease related gene SNP and tuberculosis neurological susceptibility provides neodoxy, has to the screening of tuberculosis tumor susceptibility gene with prevention
Substantial worth.
Specific embodiment
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.
Agents useful for same unless otherwise specified, is purchased from Chinese medicines group chemical reagents corporation in following embodiments.
In following embodiments using Whole Blood Genomic DNA extracts kit (Beijing Tiangeng biochemical technology Co., Ltd,
DP304 kit) carry out Whole Blood Genomic DNA extraction.The screening of target SNP uses The International HapMap
(http://hapmap.ncbi.nlm.nih.gov) database.Chinese han population genotype data is counted from Haploview 4.2
(http://www.broad.mit.edu/haploview) is obtained according to library.Using the ground substance assistant laser solution of Sequenom company
The iPLEX kit analysed in time-of-flight mass spectrometry system (MALDI-TOF) carries out SNP genotyping.
The application of embodiment 1, IL12RB1 gene mononucleotide polymorphism rs2305740 in detection tuberculosis susceptibility
One, patient and grouping situation
The confirmed cases that case-control study according to the present invention selects the 3rd 09 hospital of liberation army to accept for medical treatment altogether
2039, be divided into two groups: (1) tuberculosis group: institute of tuberculosis, the entire PLA accept for medical treatment clinically through iconography, laboratory check and
Antituberculosis therapy and make a definite diagnosis active tuberculosis patient 1032, wherein male 605, female 427, average age (39.30 ±
19.29) year.Including pulmonary tuberculosis, tuberculous pleurisy, tuberculous pericarditis, tubercular meningitis, tubercular peritonitis, uropoiesis
Tying core, tuberculous osteoarthropathy, scrofula etc..(2) it non-tuberculosis control group: clinically checks and controls through iconography, laboratory
Therapeutic effect and make a definite diagnosis non-tuberculosis disease patient 1007, wherein male 534, female 473, average age (45.23 ± 24.23)
Year.
Two, human peripheral leucocytes extracting genome DNA
Detection in peripheral blood of patients underwent 2ml is acquired, is placed in anticoagulant tube.Using complete genome DNA extracts kit (Beijing Tiangeng
Biochemical Co., Ltd's product), leucocyte genomic DNA is extracted from 1ml peripheral blood to specifications, it is slow to be dissolved in 0.1 × TE
In fliud flushing (10mmol/L Tris-1mmol/LEDTA, pH8.0), stored for future use in -20 DEG C of refrigerators.
Three, the selection of Tag SNP
Using the genotype data of mankind's full-length genome SNP of HapMap database, the selection in label site is carried out.Screening
Label site follows the principle of linkage disequilibrium coefficient r2 > 0.8, i.e., label site and is had enough between site that it is represented
The linkage strength of strong linkage disequilibrium (r2 value > 0.8).4.2 program of Haploview is reused, is downloaded down to from HapMap
Chinese han population (CHB) genotype data come, carries out the selection in label site, and selected site only limits minimum allele
Frequency (minor allele frequency, MAF) > 0.05.
Four, SNP mass spectral analysis
Using in the Matrix Assisted Laser Desorption time-of-flight mass spectrometry system (MALDI-TOF) of Sequenom company
IPLEX kit carries out SNP genotyping.Its step is summarized as follows:
(1) multiple PCR primer design and amplification: including: 1 × reaction buffer, 10ng genome in 5 μ l reaction systems
DNA, 0.5U thermal starting Taq archaeal dna polymerase (HotStarTaq, Qiagen), 500 μm of ol deoxyribonucleoside triphosphates
(dNTPs), each site 100nmol upstream and downstream primer, is placed in 384 orifice plates, using ABI-9700PCR amplification instrument, in
After 94 DEG C of 15min activated dna polymerases, in 94 DEG C of denaturation 20s, 30s and 72 DEG C of extension 60s of 56 DEG C of annealing, recycle 45 times.PCR
Product is detected with 2.0% agarose gel electrophoresis.
(2) after removing unreacted primer and dNTPs:PCR amplification, 2 μ l 0.3U shrimp alkalinity phosphorus are added in the reaction system
Sour enzyme (shrimp alkaline phosphatase, SAP), constant temperature 20min carries out extra base abatement reaction at 37 DEG C,
85 DEG C of placement 5min are placed in again to be inactivated.
(3) Single base extension PCR reacts: applying iPLEX kit.The concentration of extension primer is adjusted, mass spectrum peak figure is reduced
Background noise after, in 9 μ l reaction systems, it is poly- that 100 μm of 0.2 μ l of ol ddNTP, the above-mentioned DNA of 0.04 μ l, 0.05 U is added
The extension primer of synthase, 625-1250nmol/L.First set 94 DEG C of 30s of PCR amplification instrument；Extension PCR reaction cycle is carried out again: being set
94 DEG C of 5s, 5 × (52 DEG C of 5s+80 DEG C of 5s), the circulation carry out 200 times；Then 94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s, the circulation are set
It carries out 40 times；Most 72 DEG C of extension 3min of postposition, set 4 DEG C of preservations.
(4) the extension product purified carries out MALDI-TOF-MS analysis: above-mentioned sample application 6mg resin desalting processing
Afterwards, it is transferred on the 384 hole mass spectrum chips of Sequenom using robotic arm point sample instrument, is separately set on 384 hole mass spectrum chips
Set the repetition quality-control sample in 4 holes.Genotyping is carried out to the mass spectrum chip after point sample in mass spectrum classification system.
(5) data acquisition and genotyping: different wave spectrum peak shapes represents the different quality of charge label.If produced
There are 2 mononucleotide polymorphism sites in object, there is 1 allele in each site, then the allele is homozygote；
If mass spectrograph captures 4 different wave spectrum peaks, 2 single nucleotide polymorphism are heterozygotes.Pass through operation interface
4.0 software package of Typer reads mass spectrum peak figure, obtains genotype data.
Five, statistical analysis
The statistics of all data uses Stata statistical package (version 10.0；StataCorp
LP, College Station, TX, USA) software, tuberculosis group adopts with non-tuberculosis control group gene frequency and genotypic difference
Use χ2It examines, shows there is significant difference as P < 0.05.Each SNP site of non-tuberculosis check sample uses Hardy-
Weinberg Equilibrium (HWE) balance uses database (http://ihg.gsf.de/cgi-bin/hw/hwal.pl)
In χ2Inspection is balanced inspection, and the standard for passing through Hardy-Weinberg balance check is indicated as P > 0.05.Akaike
Information standard be used to choose the most conservative genetic model of each SNP.Target SNP base is calculated by conditional QTL analysis
Because type odds ratio (OR) and 95% confidence interval (CI) and be aided with the age and gender is corrected.
Linkage disequilibrium (LD) intensity between each SNP site uses Lewontin normalisation coefft D according to conventional way
With linkage disequilibrium coefficient r2It indicates, monomer domain is divided using the default parameters of Haploview 4.2 software.Each
In monomer domain, using 2.1 software of PHASE based on Bayesian algorithm, it is inferred to the haplotype of each sample.Then use
HAPLO.STATS software carries out haplotyping.Specific method is: based on generalized linear model, and be aided with each specific admixture because
The correction of son carries out (global) of overall importance and analyzes for the Hap.Score of haplotype (haplotype-specific).Respectively
The haplotype of frequency < 0.03 in a monomer domain merges into other (others) items.It is replaced using 100000 times
(simulations) it examines and obtains observation P value, and calculate the Hap.Score value of each haplotype.Bayesian algorithm is obtained
Dual MCU system type (diplotype) data, it is same to carry out unconditional logistic regression analysis, and correct age and gender.
The double body type of a certain haplotype is divided into 3 classes: 0 part of copy sample, 1 part of copy sample and 2 parts of copy samples, with 0 part of copy sample
For reference, the tuberculosis susceptibility or resistive connection nuclearity of 1 part of copy sample and 2 parts of copies are analyzed.
Six, analysis of experimental results
By genotyping, 5 single nucleotide polymorphism (SNP) sites are selected from IL12RB1 gene, i.e.,
The site rs2305740, the site rs401502, the site rs375947, the site rs17852635 and the site rs11575934.It is described
The site rs2305740 be No. 19 chromosomes of human genome in from 5 ' ends the 18069426th nucleotide；It is described
The nucleotide in the site rs2305740 is A or G.The site rs401502 is in No. 19 chromosomes of human genome from 5 ' ends
Play the 18069603rd nucleotide；The nucleotide in the site rs401502 be G or C described in the site rs375947 be human gene
Group No. 19 chromosomes in from 5 ' ends the 18069641st nucleotide；The nucleotide in the site rs375947 is A or G.Institute
State the site rs17852635 be No. 19 chromosomes of human genome in from 5 ' ends the 18075765th nucleotide；It is described
The nucleotide in the site rs17852635 is A or G.The site rs11575934 is in No. 19 chromosomes of human genome from 5 ' ends
The 18075808th nucleotide is held；The nucleotide in the site rs11575934 is A or G.
Upstream and downstream primer in step 4 for detecting the site rs2305740 is primer 1 and primer 2, extension primer
For primer 3.Primer 1 and primer 2 are pair of primers, expand the product containing about 200-300bp before and after site to be measured, primer 3 is the 3rd
Primer, that is, Single base extension primer, about 15-20bp only extends 1 base on a chain of 200-300bp product.Finally
Genotype is corresponded to by the molecular size range that Mass Spectrometer Method 16-21bp extends son.
Primer 1:5'-ACGTTGGATGGGTTCAAACCTGCAGGGAAG-3 ' (sequence 1)；
Primer 2: 5'-ACGTTGGATGTGTCCCTACCTCTGTATGAC-3 ' (sequence 2)；
Primer 3:5'-CCTCTGTATGACATTGAGTAAGC-3 ' (sequence 3).
Upstream and downstream primer in step 4 for detecting the site rs401502 is primer 4 and primer 5, extension primer are
Primer 6.Primer 4 and primer 5 are pair of primers, expand the product containing about 200-300bp before and after site to be measured, primer 6 is the 3rd article
Primer, that is, Single base extension primer, about 15-20bp only extends 1 base on a chain of 200-300bp product.It is final logical
The molecular size range for crossing Mass Spectrometer Method 16-21bp extension corresponds to genotype.
Primer 4:5'-ACGTTGGATGGTCTTGCGGCGCAGTCAGG-3 ' (sequence 4)；
Primer 5:5'-ACGTTGGATGCGTATTGCATTGAATGGCAG-3 ' (sequence 5)；
Primer 6:5 '-CCTGTGGGCCAGGAC-3 ' (sequence 6).
Upstream and downstream primer in step 4 for detecting the site rs375947 is primer 7 and primer 8, extension primer are
Primer 9.Primer 7 and primer 8 are pair of primers, expand the product containing about 200-300bp before and after site to be measured, primer 9 is the 3rd article
Primer, that is, Single base extension primer, about 15-20bp only extends 1 base on a chain of 200-300bp product.It is final logical
The molecular size range for crossing Mass Spectrometer Method 16-21bp extension corresponds to genotype.
Primer 7:5'-ACGTTGGATGAACGGGACCACCATGTATTG-3 ' (sequence 7)；
Primer 8:5'-ACGTTGGATGCAGGCTGCCATTCAATGCAA-3 ' (sequence 8)；
Primer 9:5'-ctCCATTCAATGCAATACGTC-3 ' (sequence 9).
Upstream and downstream primer in step 4 for detecting the site rs17852635 is primer 10 and primer 11, and extension is drawn
Object is primer 12.Primer 10 and primer 11 are pair of primers, expand the product containing about 200-300bp before and after site to be measured, primer
12 be the 3rd article of primer i.e. Single base extension primer, about 15-20bp, only extends 1 alkali on a chain of 200-300bp product
Base.Genotype is corresponded to eventually by the molecular size range that Mass Spectrometer Method 16-21bp extends son.
Primer 10:5'-ACGTTGGATGCTCAGAGTGATCTTACCAGG-3 ' (sequence 10)；
Primer 11:5'-ACGTTGGATGAAGGAAGTTCCTGGAGCAAG-3 ' (sequence 11)；
Primer 12:5'-ggatCAAGTGGAGCAGCCC-3 ' (sequence 12).
Upstream and downstream primer in step 4 for detecting the site rs11575934 is primer 13 and primer 14, and extension is drawn
Object is primer 15.Primer 13 and primer 14 are pair of primers, expand the product containing about 200-300bp before and after site to be measured, primer
15 be the 3rd article of primer i.e. Single base extension primer, about 15-20bp, only extends 1 alkali on a chain of 200-300bp product
Base.Genotype is corresponded to eventually by the molecular size range that Mass Spectrometer Method 16-21bp extends son.
Primer 13:5'-ACGTTGGATGTCCAGGAACTTCCTTGGCTC-3 ' (sequence 13)；
Primer 14:5 '-ACGTTGGATGTCTGCCCCCTGGAGATGAAT-3 ' (sequence 14)；
Primer 15:5 '-cccgcATTCCAGCTCCGACGACGGC-3 ' (sequence 15).
Specifying information, chromosome location and the gene frequency of this five SNP is shown in Table 1.All sites are in non-tuberculosis pair
According to passed Hardy-Weinberg balance check (P > 0.01) in group sample.Although 5 SNP gene frequencies are being tied
There is no significant difference (table 1) between core group and control group, but under codominant inheritance model, conditional QTL analysis
Analysis find the site IL12RB1-rs2305740 GG genotype it is related to significant resistive connection nuclearity (P=0.029, OR 0.364,
95%CI 0.147-0.901, table 2).As shown in table 3, under recessive inheritance model, the site IL12RB1-rs2305740 (GG
Vs. (AA+AG)) genotype is related to significant resistive connection nuclearity (P=0.029, OR 0.364,95%CI 0.147-0.900),
And other 4 SNP either under dominant inheritance model or recessive inheritance model without significant tuberculosis risk or resistive connection
Nuclearity.
1 IL12RB1 gene SNP site information of table and gene frequency list
5 SNP genotype frequencies of IL12RB1 gene are between tuberculosis group and control group under 2 codominant inheritance model of table
Distribution
Under table 3 is dominant or recessive inheritance model 5 SNP genotype frequencies of IL12RB1 gene tuberculosis group and control group it
Between distribution
I.e. according to result above, it is known that: the site rs2305740 is that the tuberculous risk of people of GG genotype is lower than
The site rs2305740 is the people of AA genotype or AG genotype.
In order to further evaluate linkage disequilibrium intensity of 5 SNP two-by-two between site, we are had found using Haploview
One monomer being made of 5 SNP such as rs2305740, rs401502, rs375947, rs17852635 and rs11575934
Domain (table 4).Scores test of overall importance shows that the haplotype in the monomer domain has significantly between tuberculosis group and non-tuberculosis control group
Sex differernce (Global P=0.01856, df=3), this statistical difference are still set up after 100000 displacements
(Psim=0.01868, table 4).
Is contacted between 4 IL12RB1 gene common monomer type of table and tuberculosis risk
A.P value: difference of the haplotype frequency between tuberculosis group and control group
b.Psim: the difference after 100000 displacements
C. unconditional logistic regression analysis is subject to age and gender correction
D.NA: not applicable
We have also carried out Logistic recurrence to the relationship between IL12RB1 gene pleiomorphism double body type and tuberculosis risk
Analysis.As shown in table 5, there is quite high between 0 part of copy and 2 parts of copies for the double body type being made of " GGGAG " haplotype
Resistive connection nuclearity (P=0.030, OR 0.367,95%CI 0.148-0.908).
Is contacted between 5 IL12RB1 gene SNP double body type of table and tuberculosis risk
A. unconditional logistic regression analysis is aided with age and gender correction
B. inspection of overall importance
I.e. according to result above: 2 haplotypes for constituting double body type are the tuberculous risk of people of " GGGAG "
Lower than the people that 2 haplotypes for constituting double body type are not " GGGAG ".
The present invention has found that the site IL12RB1-rs2305740 is polymorphic by Chinese han population case-control study for the first time
Property is significant associated with resistive connection nuclearity, and IL12RB1-rs2305740GG genotype and " GGGAG " double body type carrier suffer from tuberculosis
The risk of disease is lower.This research achievement is screens and predicts that tuberculosis provides new way, to research and develop crowd's tuberculosis risk
Kit for screening provides theoretical foundation.
Claims (6)
1. the substance of the single nucleotide polymorphism for detecting the site rs2305740 is suffered from preparation evaluation or auxiliary evaluation people to be measured
Application in the product of risk lungy.
2. application according to claim 1, it is characterised in that: the application is described for detecting the site rs2305740
Single nucleotide polymorphism substance and record the readable carrier of content shown in following (a1) in preparation evaluation or auxiliary
Evaluate the application in the product of the tuberculous risk of people to be measured；(a1) site rs2305740 be GG genotype to
Survey to be measured people of the tuberculous risk of people lower than the site rs2305740 for AA genotype or AG genotype.
3. application according to claim 1 or 2, it is characterised in that: described for detecting the monokaryon glycosides in the site rs2305740
The substance of sour polymorphism is primer pair first or complete single strand dna first；The primer pair first is as shown in sequence 1 in sequence table
Single stranded DNA shown in sequence 2 forms in single stranded DNA and sequence table；The complete single strand dna first is by sequence 1 in sequence table
Shown in single stranded DNA, single stranded DNA composition shown in sequence 3 in single stranded DNA shown in sequence 2 and sequence table in sequence table.
4. the substance for detecting double body type M is in the product of preparation evaluation or the tuberculous risk of auxiliary evaluation people to be measured
Application；
The mononucleotide polymorphic combination for constituting the haplotype of the double body type M is successively as follows: the site rs2305740, rs401502
Site, the site rs375947, the site rs17852635 and the site rs11575934.
5. application according to claim 4, it is characterised in that: the application is described for detecting the substance of double body type M
And the readable carrier of content shown in following (b1) is recorded in preparation evaluation or the tuberculous wind of auxiliary evaluation people to be measured
Application in dangerous product；(b1) 2 haplotypes of the composition double body type M are that the people to be measured of " GGGAG " suffers from tuberculosis
Risk lower than 2 haplotypes for constituting the double body type M be not " GGGAG " people to be measured.
6. application according to claim 4 or 5, it is characterised in that: the substance for detecting double body type M is by for examining
The substance for the single nucleotide polymorphism for surveying the site rs2305740, the mononucleotide for detecting the site rs401502
The substance, described for detecting of the substance of polymorphism, single nucleotide polymorphism for detecting the site rs375947
The substance of the single nucleotide polymorphism in the site rs17852635 and mononucleotide for detecting the site rs11575934 are more
The material composition of state property；
The substance of single nucleotide polymorphism for detecting the site rs2305740 is primer pair first or complete single stranded DNA point
Sub- first；Single stranded DNA group shown in sequence 2 in primer pair first single stranded DNA as shown in sequence 1 in sequence table and sequence table
At；It is complete single strand dna first single stranded DNA as shown in sequence 1 in sequence table, single-stranded shown in sequence 2 in sequence table
The composition of single stranded DNA shown in sequence 3 in DNA and sequence table；
The substance of single nucleotide polymorphism for detecting the site rs401502 is primer pair B or complete single strand dna
Second；Single stranded DNA shown in sequence 5 forms in primer pair B single stranded DNA shown in sequence 4 in sequence table and sequence table；
Complete single strand dna second single stranded DNA as shown in sequence 4 in sequence table, single stranded DNA shown in sequence 5 in sequence table
It is formed with single stranded DNA shown in sequence 6 in sequence table；
The substance of single nucleotide polymorphism for detecting the site rs375947 is primer pair third or complete single strand dna
Third；Single stranded DNA shown in sequence 8 forms in the single stranded DNA shown in sequence 7 in sequence table of primer pair third and sequence table；
The single stranded DNA as shown in sequence 7 in sequence table of complete single strand dna third, single stranded DNA shown in sequence 8 in sequence table
It is formed with single stranded DNA shown in sequence 9 in sequence table；
The substance of single nucleotide polymorphism for detecting the site rs17852635 is primer pair fourth or complete single stranded DNA point
Sub- fourth；Single stranded DNA group shown in sequence 11 in primer pair fourth single stranded DNA as shown in sequence 10 in sequence table and sequence table
At；Complete single strand dna fourth single stranded DNA as shown in sequence 10 in sequence table, list shown in sequence 11 in sequence table
Single stranded DNA shown in sequence 12 forms in chain DNA and sequence table；
The substance of single nucleotide polymorphism for detecting the site rs11575934 is primer pair penta or complete single stranded DNA point
Son penta；Single stranded DNA group shown in sequence 14 in the single stranded DNA as shown in sequence 13 in sequence table of primer pair penta and sequence table
At；The single stranded DNA as shown in sequence 13 in sequence table of complete single strand dna penta, list shown in sequence 14 in sequence table
Single stranded DNA shown in sequence 15 forms in chain DNA and sequence table.
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| CN201710171540.XA CN106929580B (en) | 2017-03-21 | 2017-03-21 | Application of the IL12RB1 gene mononucleotide polymorphism rs2305740 in detection tuberculosis susceptibility |
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| CN201710171540.XA CN106929580B (en) | 2017-03-21 | 2017-03-21 | Application of the IL12RB1 gene mononucleotide polymorphism rs2305740 in detection tuberculosis susceptibility |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363799A (en) * | 2007-08-09 | 2009-02-11 | 上海主健生物工程有限公司 | Kit for evaluating medicine for tuberculosis |
| CN102808030A (en) * | 2012-08-21 | 2012-12-05 | 首都医科大学附属北京儿童医院 | Application of single nucleotide polymorphism rs3888188 to detection of tuberculosis susceptibility |
| CN103305594A (en) * | 2012-03-09 | 2013-09-18 | 上海市肺科医院 | Detection target, primer and reagent used for detecting susceptibility to tuberculosis |
-
2017
- 2017-03-21 CN CN201710171540.XA patent/CN106929580B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363799A (en) * | 2007-08-09 | 2009-02-11 | 上海主健生物工程有限公司 | Kit for evaluating medicine for tuberculosis |
| CN103305594A (en) * | 2012-03-09 | 2013-09-18 | 上海市肺科医院 | Detection target, primer and reagent used for detecting susceptibility to tuberculosis |
| CN102808030A (en) * | 2012-08-21 | 2012-12-05 | 首都医科大学附属北京儿童医院 | Application of single nucleotide polymorphism rs3888188 to detection of tuberculosis susceptibility |
Non-Patent Citations (1)
| Title |
|---|
| Haplotypes of IL-12Rb1 impact on the clinical phenotype ofhidradenitis suppurativa;Sophia Giatrakos等;《Cytokine》;20130402;第299页表1和3 |
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