CN108034715A - A kind of kit for being used to detect osteoporosis - Google Patents

Abstract

The present invention is used for the kit for detecting osteoporosis, and the kit includes the probe and primer of energy specific detection rs12591780.It can detect whether subject suffers from osteoporosis by this kit, so as to instruct clinician to provide prevention scheme or therapeutic scheme to subject.And easy to operate, experimental method, it is of low cost.

Description

A kind of kit for being used to detect osteoporosis

Technical field

The invention belongs to field of biological detection, in particular belongs to a kind of kit for being used to detect osteoporosis.

Background technology

Osteoporosis, that is, osteoporosis, is one group of osteopathy caused by many reasons, and bone tissue has normal calcification, calcium salt It is in normal rates with matrix, the metabolic bone disease with the characteristics of the reduction of unit volume inner bone tissues amount becomes.In most osteoporosises In, caused by the reduction of bone tissue increases mainly due to bone absorption.By skeleton pain, be easy to fracture characterized by.

Clinical manifestation is pain, and the most common symptom of primary osteoporosis is common with lumbago, accounts in pain patients 70%~80%.Pain is lain on the back or pain relief during seat along backbone to two side diffusions, when upright after stretch or stand long, sitting When the pain increased, bend over, cough, defecate firmly when aggravate.Ostalgia is may occur in which during general bone loss more than 12%.Old bone During matter osteoporosis, Vertebral Compression deformation, flexion of vertebral column, muscular fatigue even spasm, produces pain.Recently Thoracolumbar disk compressibility bone Folding, can also produce Acute Pain, the spinous process of corresponding site can have strong tenderness and percussion pain.If oppress corresponding spinal nerve Four limbs Rediating Pain, double lower limb sensorimotor obstacle, intercostal neuralgia, retrosternal pain can be produced similar to angina pectoris.If oppress ridge Marrow, cauda equina nerve have an effect on bladder, rectum function.Degeneration osteoporosis is most common and the complication of most serious.

The inspection of osteoporosis in the aspect of laboratory mainly it is following some:(1) blood calcium, phosphorus and alkaline phosphatase are in original In hair property osteoporosis, serum calcium, phosphorus and alkaline phosphatase levels are typically normal, several months alkaline phosphatase after fracture Level can increase.(2) blood parathyroid hormone should check secondary osteoporosis except parathyroid function.Primary sclerotin Osteoporosis person's blood parathyroid hormone level can normally or rise.(3) the label patients with osteoporosis part-blood of bone renewal Clear biochemical indicator of learning can change (including bon e formation and bone information) state with reactive bone, these Biochemistry measurement indexs include：Bone is special Different alkaline phosphatase (reaction bon e formation), Tartrate resistant acid phosphatase (reaction bone information), osteocalcin (reaction bon e formation), I type virgin rubber former peptide (reaction bon e formation), Pyridinoline and Deoxypyridinoline (reaction bone information), the N-C- ends of Type I collagen are handed over Join peptide (reaction bone information).(4) urina sanguinis calcium/creatinine ratio normal ratio is 0.13 ± 0.01, and urinary calcium discharge capacity crosses at most ratio increasing Height, prompts have bone absorption rate increase may.

Secondly auxiliary examination, and it is divided into the following aspects, 1. bone imageological examination and bone density absorb diseased region X-ray film x-ray can be found that fracture and other lesions, such as osteoarthritis, disc disease and spondylolisthesis.Sclerotin is reduced (low bone density) takes the photograph visible bone bright degree increase during piece, and bone trabecula is reduced and its gap is broadening, and row bone trabecula disappears, bone structure It is fuzzy, but usually need to decline more than 30% just in bone amount it is observed that.Generally visible centrum concave-concave deformation, anterior margin of vertebral body collapse Wedge shaped change, also known as compression fracture, are common in the 11st, 12 thoracic vertebraes and the 1st, 2 lumbar vertebraes.2. Bone mineral density Bone mineral density is The prediction index of fracture.The bone density at what position is measured, can be used for assessing overall fracture degree of causing danger；Measure particular portion The bone density of position can predict the danger that local fracture occurs.

In the prior art, it is close by detection and osteoporosis generation to disclose a kind of kit by CN 102534019A Cut relevant 5 mononucleotide polymorphism sites genotype come examination go out easily suffer from osteoporosis women people at highest risk, And finally according to the genetic test result of each person under inspection from the susceptible risk class of gene aspect assessment osteoporosis, refer to Lead the climacteric women targetedly effectively generation of pre- anti-osteoporosis.

CN 106755522A disclose a kind of kit for the detection of juvenile form osteoporosis, are compared by gene It was found that DKK1 genes are 721 sites there is obvious mutational site in juvenile form sufferers of osteoporosis face and normal population.It is logical Cross and detect this mutational site, 1 be especially to provide pair specific primer pair, can suffer from children when specific identification human body Model year osteoporosis symptoms.The detection method has easy to detect, quickly and accurately advantage relative to the method for the prior art, fits It is suitable for and promotes the use of.

CN 107043811A disclose the molecular marker that CFAP20 genes can be early diagnosed as osteoporosis. Utilize the difference expression gene in high-flux sequence and QPCR experimental studies patients with osteoporosis.The achievement in research shows can To judge whether subject suffers from osteoporosis by detecting in blood CFAP20 expression conditions.

Based on the limitation for the means for detecting osteoporosis in the prior art, osteoporosis early diagnosis and prognosis are found Relevant specificity molecular marker has far reaching significance to the early diagnosis and individualized treatment of realizing osteoporosis.

The content of the invention

The present invention provides the kit for detecting rs12591780 genotype, testing result can be used in osteoporosis The assistant analysis of disease.

First purpose of the present invention is to provide the kit for detecting gene SNP site rs12591780 genotype, The kit includes primer pair and PCR detection reaction reagents.

According to the kit of the present invention, the side that a kind of osteoporosis neurological susceptibility to individual is diagnosed can be provided Method, this method are：The polymorphism status in the rs12591780 sites of individual is detected, low bone density is not shown when site is G Characteristic, when mutated-genotype A result in the reduction of bone density value, so as to ultimately result in the generation of osteoporosis.

The present invention provides a kind of detect in sample to include such as with the presence or absence of the method for rs12591780 polymorphisms, this method Lower step：(1) using our self-designed kit amplified samples, pcr amplification product is obtained.

The present invention, which is utilized in TAQMAN technology for detection amplified productions, whether there is single nucleotide polymorphism.The present invention's is basic Technology path is：A kind of method of SNP marker in identification rs12591780 polymorphisms, by the genome for extracting sample DNA, is carried out PCR amplification using kit provided by the invention, expands purpose fragment, be detected using TAQMAN technologies, sent out Existing SNP exists, and the presence of the SNP and existing position are confirmed using DNA sequencing method.

The detection fragment sequence in rs12591780 sites such as SEQ ID NO：Shown in 1.Length is 261bp, and particular sequence is such as Under：agagtgccat aaccacctac ggattcactc ttcgctgtgc catgccagtc aactatgccc ttccctacta tccttcttcc r ccaccattcc tctgactgtc aggctcagca aacagcttgc agttggtcaa tgagtgctta ccaagccccg gctctgtgcc cagcatcatg caaagcataa atggacatgg acaacagtct taaactactt cctacccaca agcagctcac aaacttgttg ggaagaaagt agaacaagga； Wherein r is mutated for a/g.

Those skilled in the art is aware that have many analysis methods to can be used in detection gene intron sequence existing Single nucleotide polymorphism distribution frequency.These technologies include：DNA sequencing, PCR-SSCP, PCR-HPLC, PCR-TAQMAN etc..

Present invention especially provides a kind of detection kit of the above-mentioned SNP of detection easy to use, high sensitivity, it contains There are PCR specific amplification primers and the PCR reaction solution (such as reagent, buffer solution general components) for PCR amplification detection etc., ability These known general components of field technique personnel and detection method.Using TAQMAN technology for detection amplified production detection mutation position Point, it is also necessary to some required conventional reagents of TAQMAN etc..

Specifically, the present invention provides a kind of PCR kit of the SNP of detection rs12591780, it is characterized in that by drawing Thing 1, primer 2, probe 3, probe 4 and PCR reaction solution are formed, wherein, the 5' ends of the probe 3 and probe 4 are marked with report base Group, 3' ends are marked with fluorescent quenching group, and the 5' ends reporter group of probe 4 is different from the 5' ends reporter group of probe 3.

Wherein, the reporter group of 5' ends mark may be selected from：FAM、HEX、TET、JOE、VIC、R0X、Cy3、Cy5、MAR、 JUP, SAT, PLU or NEP, and it is not limited to above-mentioned group；The fluorescent quenching group of 3' ends mark may be selected from：TAMRA、Eclipse、 BHQ1, BHQ2, BHQ3, DABCYL, MGB, and it is not limited to above-mentioned group.

Preferably, the reporter group of 5' ends mark is FAM or the fluorescent quenching group of VIC, 3' end mark is MGB.

PCR amplification:Used amplimer sequence is:

Forward primer 1:agagtgccat aaccacctac；

Reverse primer 2:tccttgttct actttcttcc；

Specific probe sequence 3:Tccttcttcc a ccaccattcc, the wherein section of sequence 5 ' are marked using FAM, and 3 ' ends are used MGB is marked,

Specific probe sequence 4:Tccttcttcc g ccaccattcc, the wherein section of sequence 5 ' are marked using VIC, and 3 ' ends are used MGB is marked.

The advantages of the present invention：

Present invention firstly discovers that rs12591780 polymorphisms are related to osteoporosis, by detecting in subject The polymorphic implementations of rs12591780, it can be determined that whether subject suffers from osteoporosis so that instruct clinician give by Examination person provides prevention scheme or therapeutic scheme.And easy to operate, experimental method, it is of low cost.

Embodiment

With reference to specific embodiment, the present invention is further explained.It is to be understood that these embodiments are merely to illustrate the present invention Rather than limit the scope of the invention.The experimental method of actual conditions is not specified in the following example, or according to manufacturer Proposed condition.Unless otherwise stated, otherwise percentage and number are calculated by weight.

Unless otherwise defined, all professional and scientific terms used in text and meaning known to one skilled in the art Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in the present invention.Described in text Preferred implement methods and materials be for illustrative purposes only.

The acquisition of embodiment 1SNP molecular labelings

Research sample is obtained from multiple researchs and medical center.All researchs passed the examination ＆ approval of Ethics Committee, and Obtain the informed consent of research object.We are to from six different blood lineages having a lumbar vertebrae and femoral neck bone density value measures Sample has carried out full-length genome correlation analysis (GWAS), and merging data carries out meta analyses.In addition, we also have collected The meta analysis results of European descent crowd lumbar vertebrae and femoral neck bone density in GEFOS-seq public databases, and by two Divided data, which merges, carries out Conjoint Analysis so that association results are mutually authenticated.

Part I internal specimen meta is analyzed

1st, the selection of sample

Early period, we have carried out meta points of GWAS in seven samples that there is lumbar vertebrae and femoral neck bone density value to measure Analysis.Wherein, Yin Fulei Framinghams heart disease research (FHS) sample is overlapped with GEFOS-seq samples, in order to ensure two parts sample Independence, we reject FHS samples from internal specimen.Remaining six sample essential informations are as follows：

(1) Omaha osteoporosises research sample (OOS)：Grind in the cross section that OOS samples include 1000 random participants Study carefully.

(2) Kansas City osteoporosis research sample (KCOS)：KCOS samples include the horizontal stroke of 2286 random participants Section is studied.

(3) state of Indiana fragility fractures research sample (IFS)：IFS samples are obtained by dbGAP databases, bag Cross-sectional study of the premenopausal sister to participant of 1493 European descents is included.

(4) Chinese osteoporosis research sample (COS)：COS samples include the cross section of 1627 random participants Research.

(5) the non-preliminary observational study of descendants American's women's health (WHI-AA)：WHI-AA samples are that women's health is tentatively seen A part for research (WHI) is surveyed, 845 non-descendants American participants is contained, can be obtained by dbGAP databases.

(6) the preliminary observational study of Hispanic women's health (WHI-HIS)：WHI-HIS samples are also a part of WHI, 446 Hispanic participants are contained, can be obtained by dbGAP databases.

2nd, phenotype measurement and modeling

Bone density value is measured by DXA borne densitometers.Use stepping Return Law examination gender, age, age square, height And the conspicuousness of the covariant such as preceding 10 principal components (being used to weigh population layering effect) calculated from genomic data.It is former Residual error of the beginning bone density value after covariant corrects carrys out normal state using the quantile of normal distribution.Residual error after normal state is used Analyzed in downstream associative.

3rd, Genotyping and Quality Control

All six samples use high throughput gene typing chips parting.Quality Control is realized in sample aspect and SNP aspects. In sample aspect, gender is inferred using plink software analysis X chromosome, and compare with the gender in questionnaire.Gender The individual not being inconsistent is deleted from data.In SNP aspects, the SNP for not meeting Ha-temperature balance is deleted from data.When there are crowd During extremum, the genotype of principal component is checked, and reject extremum.

4th, genotype is filled a vacancy

Genotype is carried out using the sequencing data (in May, 2013 version) of thousand human genomes to all samples to fill a vacancy.First, Haplotype data (503 people of European descent, the East Asia blood lineage 504 of the sequencing individual of 4 class ethnic groups are downloaded from thousand human genome websites 347 people of people, 661 people of African descent and mixing American blood lineage), respectively mould is referred to as the Genotyping of respective sample Plate.Secondly, one is done to the allele of reference gene group (i.e. thousand human genome data) and target gene group (i.e. GWAS samples) Cause property is examined, and examines the person of not being inconsistent to be deleted from target gene group.Finally, carry out genotype using software FISH to fill a vacancy, this Algorithm has the advantages that arithmetic speed is fast, and committed memory is small, without shifting to an earlier date split-phase (phasing) to target gene group.Software is joined Number default setting.

5th, the association analysis and meta analyses in single sample

In each sample, the genetic association between the phenotype residual sum parting of normal state and the genotype filled a vacancy out passes through something lost Cumulative model is passed to examine.In unrelated sample, genetic association is examined using linear regression model (LRM), software MACH2QTL.It is right In IFS family samples, then using the degree of association of mixed linear model calculating genotype.The association results of six samples merge one Rise and carry out meta analyses.Model used is the fixed-effect model of sample size weighting, and software used is METAL.

The collection of Part II GEFOS-seq meta analysis results

GEFOS-seq researchs are organized in has carried out sequencing and genotype is filled a vacancy in more than 50,000 European descent participants Based on bone density GWAS meta analysis.Sample information, Quality Control and statistical analysis refer to GEFOS-seq official websites.In short It, this research includes three parts content：1st, 2,882 research objects in studying UK10K carry out genome sequencing；2nd, it is right 3,549 research objects in 5 cohort studies carry out genome sequencing；3rd, full genome is carried out to 26,534 research objects Group genotype is filled a vacancy.All samples have all carried out meta analyses, and partial results external disclosure.Here, we pass through GEFOS-seq has downloaded official website the meta analysis results that this part sample size is up to 32,965.

SNP inclusive criterias

For the meta analysis results of six internal specimens of Part I, the quality control standard that the SNP that we take is included is： Allelic heterogeneity I²<50%.For Part II GEFOS-seq meta analysis results, what we took includes quality control standard then For：1. allelic heterogeneity I²<50%；2. minimum gene frequency (MAF)>1%.For meeting two parts Quality Control SNPs, we are included in research in next step.

Conjoint Analysis

By the meta analysis results of two parts sample, we have respectively obtained two parts z values：z₁And z₂.Then, Wo Menyun Conjoint Analysis is carried out with weighting fixed effect meta analysis models, formula is as follows：

In formula, n₁And n₂Refer to the sample size of internal specimen and GEFOS-seq samples respectively.In the case where null hypothesis is set up, When i.e. there is no associating, statistical value Z Normal Distributions or approximate normal distribution.The GWAS levels of signifiance are set to 5.0x10^-8。

Conclusion

In internal specimen research, 7,484 research objects are covered altogether.Do not examined using principal component analysis in each sample Go out heterogeneous individual.After PCA correction population layerings, lumbar vertebrae and the femoral neck bone density genome coefficient of expansion are 1.03 respectively With 1.02, less than warning value, illustrate no obvious population layering effect.

In GEFOS-seq meta analysis results, one shares 32,965 research objects, covers 10,586,901 SNPs, after Quality Control screens out, one, which shares 7,434,754 SNPs, is included into the Conjoint Analysis of next step.

Two-part Conjoint Analysis is found that many and lumbar vertebrae and the relevant site of femoral neck bone density.Some sites are not only Reach the full-length genome level of signifiance (GWS, p in GEFOS-seq samples<5.0x10^-8), also repeated in internal specimen Verify (p<0.05).Furthermore it has been found that reach the new site of the full-length genome level of signifiance：15q14 (rs12591780, p=3.70x10^-8)。

SNP rs12591780 and associating for lumbar spine bmd on the site is the most notable (it was found that sample p₁= 6.82x10^-4, GEFOS-seq samples p₂=1.19x10^-5, merge sample p₁₂=3.70x10^-8).Detailed results such as table 1 below：

Table 1rs12591780 result datas

It can be seen that from the data of table 1 for rs12591780 sites, there are the mutation of G/A, when it is A, table It is now low bone density characteristic, does not show low bone density characteristic when it is G.Mutated-genotype A result in the drop of bone density value Low, so as to ultimately result in the generation of osteoporosis, this rise is statistically extreme significant (p=3.70x10^-8), Therefore osteoporosis can be detected by detecting the loci gene type.

2 specific pattern detection of embodiment

1. study the selection of sample；

Patients with osteoporosis blood sample sample used in the present invention comes from First Affiliated Hospital of Soochow University,Suzhou, totally 100 Patient diagnosed's sample.The inclusion criteria for studying sample is as follows:Age is the women of 18-70 Sui；Examined through Bone mineral density and X-ray Survey is made a definite diagnosis；Dysfunction without main organs, blood routine, hepatic and renal function and cardiac function are normal；Can obtain completely with Visit information.All research objects are the Chinese Han Population of consanguinity-less relation, geographically essentially from North China and its surrounding area.With 100 normal population blood are control.Sufferer is included to above-mentioned, compiles its essential information, clinical information and treatment letter Breath, and sign informed consent form according to Principles in Informed Consent.

2. the extracting of genomic DNA；

Sample centrifuges serum and blood using the peripheric venous blood 2ml of the heparin tube collection research object of EDTA anti-freezings - 80 DEG C of refrigerators are stored in after cell.Using the centrifugal adsorbing column that can specifically bind DNA and unique buffer system, extraction Peripheral blood genomic DNA, operates according to a conventional method.Usually lead to 50ng DNA, purity (ultraviolet 2600D:2800D) 1.8 Left and right.

3rd, PCR amplification system is prepared：

And SEQ ID NO are separately added into each DNA：The mixed liquor of primer and probe shown in 2-5 is cooked PCR reactions, is come Parting is carried out to each SNP respectively, required reagent includes Taqman Genotyping Master Mix5 μ l, ddH₂0 3.5μ 1st, 0.5 μ l of fluorescence probe and primer mixed liquor, remaining is the 1 μ l of examinate's DNA sample, 10 μ l of cumulative volume of 50ng/ μ l.

4th, PCR amplification program：

PCR reacts the first step：60℃、35s；PCR reacts second step：96℃、10min；PCR reacts the 3rd step：93℃、 20s；PCR reacts the 4th step：60℃、95s；PCR reacts the 5th step：Circulate the 4th step of the 3rd step 5 times, then 60 DEG C, 25s.Adopt Dashed forward Fluorescent Quantitative PCR instrument (Applied Biosystems, Applied Biotechnology Co., Ltd of the U.S.) with ABI Stepone.

5th, experimental result：

PCR after reaction, is analyzed using Stepone software V2.1, and obtain 200 results are carried out Classification, the probe according to the present invention, which is divided into, discerns 3 kinds of genotype：Tri- kinds of frequency of genotypes AA of SNP rs12591780, GG and AG.

6th, the analysis of the accuracy of kit

For the accuracy of verification kit detection, sequence verification is carried out to 200 samples.SNP rs12591780 genotype GG has 162, and AG genotype has 30, and AA genotype has 8；Wherein AG genotype and AA genotype are that osteoporosis is suffered from Person, therefore the accuracy of kit detection has reached 100%.

The value of this kit is only to need peripheral blood without other tissue samples, by most simplifying and special Amplimer detects SNP site, then composes evaluation patient profiles by SNP, not only stablizes, easy to detect, and accurately, greatly improves The Sensitivity and Specificity of medical diagnosis on disease, therefore this kit is put into and is put into practice, can help to instruct people at highest risk screening and More effective individualized treatment.

The specific embodiment of the present invention is described in detail above, but it is only used as example, and the present invention is not intended to limit In particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and to replace In generation, is also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and repair Change, all should be contained within the scope of the invention.

Sequence table

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Claims (6)

1. A kind of 1. kit for being used to detect osteoporosis, it is characterised in that：The kit includes can specific detection The probe and primer of rs12591780.
2. 2. kit as claimed in claim 2, it is characterised in that：The kit includes SEQ ID NO：Described in 2-5 Primer sets and probe sequence.
3. 3.SEQ ID NO：Primer sets and probe sequence described in 2-5 are in the kit for osteoporosis detection is prepared Using.
4. 4.rs12591780 the purposes of the target spot as detection osteoporosis.
5. 5.rs12591780 preparing for the application in the kit of osteoporosis detection.
6. 6. a kind of method for detecting osteoporosis, includes the use of claim 1-2 any one of them kits.