CN108220425B - It is a kind of for detecting the kit of osteoporosis - Google Patents

Abstract

The kit for detecting osteoporosis of the invention comprises probes and primers capable of specifically detecting rs2380128. Whether the subject suffers from osteoporosis can be detected through the kit, so as to guide the clinician to provide the subject with a prevention plan or a treatment plan. Moreover, the operation is simple, the experimental method is low, and the cost is low.

Description

A kit for detecting osteoporosis

technical field

The invention belongs to the field of biological detection, in particular to a kit for detecting osteoporosis.

Background technique

Osteoporosis, or osteoporosis, is a group of bone diseases caused by a variety of reasons. Bone tissue has normal calcification, and the ratio of calcium salt to matrix is normal. It is a metabolic bone lesion characterized by a decrease in the amount of bone tissue per unit volume. . In most cases of osteoporosis, the loss of bone tissue is mainly due to increased bone resorption. Characterized by bone pain and susceptibility to fractures.

The clinical manifestation is pain. The most common symptom of primary osteoporosis is low back pain, which accounts for 70% to 80% of pain patients. The pain spreads along the spine to both sides. The pain is relieved when lying on the back or sitting, and the pain is aggravated when standing upright or standing for a long time or sitting for a long time, and it is aggravated when bending over, coughing, and defecation. Generally, bone pain occurs when the bone mass is lost by more than 12%. In senile osteoporosis, the vertebral body is compressed and deformed, the spine bends forward, muscle fatigue and even spasm, resulting in pain. Recent thoracolumbar compression fractures can also cause acute pain, and the corresponding parts of the spinous processes of the spine may have strong tenderness and percussion pain. If the corresponding spinal nerve is compressed, it can produce radiating pain in the extremities, sensorimotor disturbance in the lower extremities, intercostal neuralgia, and retrosternal pain similar to angina pectoris. If the spinal cord and cauda equina are compressed, the function of the bladder and rectum will also be affected. The most common and serious complication of degenerative osteoporosis.

The examination of osteoporosis mainly includes the following points on the laboratory level: (1) blood calcium, phosphorus and alkaline phosphatase In primary osteoporosis, serum calcium, phosphorus and alkaline phosphatase levels are usually Normally, the alkaline phosphatase level may increase several months after the fracture. (2) Serum parathyroid hormone should be checked for parathyroid function except for secondary osteoporosis. The level of parathyroid hormone in the blood of patients with primary osteoporosis can be normal or elevated. (3) Bone renewal markers Some serum biochemical indicators in patients with osteoporosis can reflect the state of bone turnover (including bone formation and bone resorption). These biochemical measurement indicators include: bone-specific alkaline phosphatase (response to bone formation) , anti-tartrate acid phosphatase (response to bone resorption), osteocalcin (response to bone formation), type Ⅰ procollagen peptide (response to bone formation), urinary pyridinoline and deoxypyridinoline (response to bone resorption), N-C of type Ⅰ collagen - Terminally cross-linked peptides (response to bone resorption). (4) The normal morning urine calcium/creatinine ratio is 0.13±0.01, and the ratio increases when the urinary calcium excretion is excessive, suggesting that the bone resorption rate may increase.

The second is auxiliary examination, which is divided into the following aspects: Bone imaging examination and bone density ① Take X-ray films of the lesion. X-rays can find fractures and other lesions, such as osteoarthritis, intervertebral disc disease, and spondylolisthesis. Osteopenia (low bone density) shows increased brightness of bone penetration, reduction of bone trabeculae and widening of gaps, disappearance of transverse bone trabeculae, and blurred bone structure in radiographs of osteopenia (low bone density). . In general, biconcave deformation of the vertebral body can be seen, and the anterior edge of the vertebral body collapses into a wedge shape, also known as compression fracture, which is commonly seen in the 11th and 12th thoracic vertebrae and the 1st and 2nd lumbar vertebrae. ②Bone density detection Bone density detection is a predictor of fracture. The bone density at which part is measured can be used to assess the overall risk of fracture; the bone density at a specific part can be used to predict the risk of local fracture.

In the prior art, CN 102534019 A discloses a kit to screen for osteoporosis by detecting the genotypes of 5 single nucleotide polymorphism sites closely related to the occurrence of osteoporosis According to the genetic test results of each subject, the risk level of osteoporosis susceptibility is evaluated from the genetic level, and guides menopausal women to effectively prevent the occurrence of osteoporosis.

CN 106755522 A discloses a kit for juvenile osteoporosis detection. Through gene comparison, it is found that there is an obvious mutation site in the DKK1 gene between juvenile osteoporosis patients and normal population, which is the 721 site . By detecting this mutation site, especially a pair of specific primers provided, it is possible to specifically identify the human body suffering from juvenile osteoporosis symptoms. Compared with the methods in the prior art, the detection method has the advantages of convenient detection, accuracy and speed, and is suitable for popularization and use.

CN 107043811 A discloses that CFAP20 gene can be used as a molecular marker for early diagnosis of osteoporosis. Differentially expressed genes in osteoporosis patients were investigated using high-throughput sequencing and QPCR experiments. The results of this study indicate that it is possible to determine whether a subject suffers from osteoporosis by detecting the expression of the CFAP20 gene in the blood.

Based on the limitations of the methods for detecting osteoporosis in the prior art, finding specific molecular markers related to the early diagnosis and prognosis of osteoporosis has far-reaching significance for the early diagnosis and individualized treatment of osteoporosis.

SUMMARY OF THE INVENTION

The invention provides a kit for detecting rs2380128 genotype, and the detection result can be used for auxiliary analysis of osteoporosis.

The first object of the present invention is to provide a kit for detecting the genotype of the gene SNP site rs2380128, the kit including primer pairs and PCR detection reaction reagents.

According to the kit of the present invention, a method for diagnosing an individual's susceptibility to osteoporosis can be provided, the method is: detecting the polymorphism status of the individual's rs2380128 site, and when the site is G, it does not show Low bone density characteristics, when the mutant genotype A leads to a decrease in bone density, which eventually leads to the occurrence of osteoporosis.

The present invention provides a method for detecting whether there is rs2380128 polymorphism in a sample. The method comprises the following steps: (1) using a kit designed by ourselves to amplify the sample to obtain a PCR amplification product.

The invention utilizes TAQMAN technology to detect whether there is a single nucleotide polymorphism in the amplification product. The basic technical route of the present invention is: a method for identifying SNP molecular markers in the rs2380128 polymorphism, by extracting the genomic DNA of the sample, using the kit provided by the present invention to perform PCR amplification, amplifying the target fragment, and using TAQMAN technology to carry out Detect, find the existence of SNP, and confirm the existence and location of the SNP by DNA sequencing method.

The detection fragment sequence of rs2380128 site is shown in SEQ ID NO:1. The length is 201bp, and the specific sequence is as follows:

where r is the a/g mutation.

Those skilled in the art are well aware that there are many analysis methods that can be used to detect the distribution frequency of single nucleic acid polymorphisms in gene intron sequences. These techniques include: DNA sequencing, PCR-SSCP, PCR-HPLC, PCR-TAQMAN, etc.

The present invention particularly provides a detection kit for detecting the above-mentioned SNP which is easy to use and has high sensitivity, which contains PCR specific amplification primers and PCR reaction liquid (such as reagents, buffers and other conventional components) for PCR amplification detection etc. Those skilled in the art are familiar with these conventional components and detection methods. When using TAQMAN technology to detect amplification products and detect mutation sites, some conventional reagents required by TAQMAN are also required.

Specifically, the present invention provides a PCR kit for detecting the SNP of rs2380128, which is characterized in that it is composed of primer 1, primer 2, probe 3, probe 4 and PCR reaction solution, wherein the probe 3 and The 5' end of probe 4 is labeled with a reporter group, and the 3' end is marked with a fluorescence quencher group, and the 5' end reporter group of probe 4 is different from the 5' end reporter group of probe 3.

Wherein, the reporter group labeled at the 5' end can be selected from: FAM, HEX, TET, JOE, VIC, ROX, Cy3, Cy5, MAR, JUP, SAT, PLU or NEP, and is not limited to the above groups; the 3' end The labeled fluorescence quenching group can be selected from: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3, DABCYL, MGB, and is not limited to the above groups.

Preferably, the reporter group labeled at the 5' end is FAM or VIC, and the fluorescent quencher group labeled at the 3' end is MGB.

PCR amplification: the amplification primer sequence used is:

Forward primer 1: tataacccca ataccttggg

Reverse primer 2: tccaacacct ggggtcaagc

Specific probe sequence 3: caacacagtg a gatactgtct, wherein the 5' segment of the sequence is marked with FAM, and the 3' end is marked with MGB,

Specific probe sequence 4: caacacagtg g gatactgtct, wherein the 5' segment of the sequence is marked with VIC, and the 3' end is marked with MGB.

Advantages and beneficial effects of the present invention:

The present invention discovers for the first time that the rs2380128 polymorphism is related to osteoporosis, and by detecting the polymorphism of rs2380128 in the subject, it can be judged whether the subject suffers from osteoporosis, thereby guiding clinicians to give the subject Offer prevention or treatment options. Moreover, the operation is simple, the experimental method is low, and the cost is low.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not indicated in the following examples, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

The acquisition of embodiment 1 SNP molecular marker

Study samples were obtained from multiple research and medical examination centers. All studies were approved by the ethics committee and informed consent was obtained from the research subjects. We performed a genome-wide association analysis (GWAS) of samples from six different ancestry with BMD measurements at the lumbar spine and femoral neck and pooled the data for meta-analysis. In addition, we also collected the meta-analysis results of lumbar spine and femoral neck BMD in the European ancestry population in the GEFOS-seq public database, and combined the two parts of data for joint analysis to mutually verify the association results.

The first part internal sample meta-analysis

1. Sample selection

Previously, we performed a GWAS meta-analysis in seven samples with BMD values measured at the lumbar spine and femoral neck. Among them, because the Framingham Heart Study (FHS) samples overlapped with the GEFOS-seq samples, in order to ensure the independence of the two samples, we excluded the FHS samples from the internal samples. The basic information of the remaining six samples is as follows:

(1) Omaha Osteoporosis Study Sample (OOS): The OOS sample included a cross-sectional study of 1000 random participants.

(2) Kansas City Osteoporosis Study Sample (KCOS): The KCOS sample included a cross-sectional study of 2286 random participants.

(3) Indiana Fragility Fracture Study Sample (IFS): The IFS sample was obtained through the dbGAP database and included a cross-sectional study of 1493 premenopausal sister-pair participants of European ancestry.

(4) Chinese Osteoporosis Study Sample (COS): The COS sample included a cross-sectional study of 1627 random participants.

(5) African-American Women's Health Initial Observational Study (WHI-AA): The WHI-AA sample is part of the Women's Health Initial Observational Study (WHI), which includes 845 African-American participants and can be obtained through the dbGAP database .

(6) Hispanic Women's Health Preliminary Observational Study (WHI-HIS): The WHI-HIS sample is also part of the WHI, including 446 Hispanic participants, which can be obtained through the dbGAP database.

2. Phenotype measurement and modeling

Bone density was measured by DXA bone densitometer. Covariates of sex, age, age squared, height, and the top 10 principal components calculated from genomic data (to measure population stratification effects) were screened for significance using stepwise regression. Covariate-adjusted residuals of raw BMD values were normalized using quantiles of a normal distribution. The normalized residuals were used for downstream association analysis.

3. Genotyping and quality control

All six samples were typed using a high-throughput genotyping array. Quality control is implemented at the sample level and at the SNP level. At the sample level, use plink software to analyze the X chromosome to infer gender, and compare it with the gender in the questionnaire. Gender-inconsistent individuals were removed from the data. At the SNP level, SNPs not in Harbin-Wen equilibrium were removed from the data. When there are extreme values in the population, check the genotypes of the principal components and remove the extreme values.

4. Genotype imputation

Genotype imputation was performed on all samples using the sequencing data of 1000 Genomes (May 2013 version). First, download the haplotype data of sequenced individuals of 4 types of individuals from the Thousand Genomes website (503 individuals of European descent, 504 individuals of East Asian descent, 661 individuals of African descent, and 347 individuals of mixed American descent), and use them as their respective samples genotyping reference template. Secondly, a consistency test is performed on the alleles of the reference genome (ie, the thousand genome data) and the target genome (ie, the GWAS sample), and those that do not match will be deleted from the target genome. Finally, use the software FISH to carry out genotype imputation. This algorithm has the advantages of fast calculation speed, small memory usage, and no need for phasing the target genome in advance. The software parameters are set by default.

5. Association analysis and meta-analysis in single sample

Within each sample, normalized phenotypic residuals and genetic associations between typed and imputed genotypes were tested by genetic additive models. In unrelated samples, genetic associations were tested using a linear regression model with the software MACH2QTL. For the IFS family samples, the mixed linear model was used to calculate the correlation degree of genotype. The association results of the six samples were combined for meta-analysis. The model used is a fixed effect model weighted by sample size, and the software used is METAL.

The second part is the collection of GEFOS-seq meta-analysis results

The GEFOS-seq research group performed sequencing and genotype imputation-based bone density GWAS meta-analysis in more than 50,000 participants of European ancestry. See GEFOS-seq official website for details of sample information, quality control and statistical analysis. In short, this study consists of three parts: 1. Whole genome sequencing of 2,882 subjects in the UK10K study; 2. Whole genome sequencing of 3,549 subjects in 5 cohort studies; 3. Whole genome sequencing of 26,534 subjects The whole genome genotype imputation was performed on 100 subjects. All samples were meta-analyzed, and some of the results have been made public. Here, we downloaded the meta-analysis results of this part of the sample size up to 32,965 through the GEFOS-seq official website.

SNP inclusion criteria

For the meta-analysis results of the six internal samples in the first part, the quality control standard for SNP inclusion we adopted was: gene heterogeneity I ² <50%. For the results of the second part of the GEFOS-seq meta-analysis, the inclusion quality control criteria we adopt are: ①gene heterogeneity I ² <50%; ②minimum allele frequency (MAF)>1%. For SNPs that meet the two-part quality control, we will include them in the next step of research.

conjoint analysis

Through the meta-analysis results of two samples, we obtained two z-values: z ₁ and z ₂ . Next, we use the weighted fixed-effects meta-analysis model for joint analysis, the formula is as follows:

In the formula, n ₁ and n ₂ refer to the sample sizes of internal samples and GEFOS-seq samples, respectively. When the null hypothesis is established, that is, when there is no association, the statistical value Z follows a normal distribution or an approximately normal distribution. The GWAS significance level was set at 5.0x10 ^-8 .

in conclusion

A total of 7,484 subjects were included in the internal sample study. Application of principal component analysis within each sample did not detect heterogeneous individuals. After applying PCA to correct population stratification, the genome expansion coefficients of BMD at the lumbar spine and femoral neck were 1.03 and 1.02, respectively, which were lower than the warning value, indicating that there was no obvious population stratification effect. In the GEFOS-seq meta-analysis results, a total of 32,965 research subjects covered 10,586,901 SNPs. After quality control screening, a total of 7,434,754 SNPs were included in the next joint analysis.

The combined two-part analysis identified numerous loci associated with BMD at the lumbar spine and femoral neck. Some loci not only reached genome-wide significance level in GEFOS-seq samples (GWS, p<5.0x10 ^-8 ), but also got repeated validation in internal samples (p<0.05). In addition, we also found a new locus reaching genome-wide significance: 20q13.33 (rs2380128, p=3.44x10 ^-8 ).

The SNP rs2380128 located at this site had the most significant association with lumbar bone density (found sample p ₁ =0.0644, GEFOS-seq sample p ₂ =1.46x10 ^-5 , pooled sample p ₁₂ =3.44x10 ^-8 ). The detailed results are shown in Table 1 below:

Table 1 rs2380128 result data

It can be seen from the data in Table 1 that for the rs2380128 site, there is an A/G mutation. When it is G, it shows low bone density characteristics, and when it is A, it does not show low bone density characteristics. Mutant genotype G leads to a decrease in bone mineral density, which ultimately leads to the occurrence of osteoporosis. This increase is statistically extremely significant (p=3.44x10 ^-8 ), so it can be detected by detecting this locus Genotype to detect osteoporosis.

Embodiment 2 Concrete sample detection

1. Selection of research samples;

The blood samples of osteoporosis patients used in the present invention come from the First Affiliated Hospital of Soochow University, a total of 100 confirmed patient samples. The inclusion criteria of the research samples are as follows: women aged 18-70; confirmed by bone density test and X-ray test; no major organ dysfunction, blood routine, liver and kidney function and heart function are basically normal; can obtain a complete Follow-up information. All the subjects in this study were unrelated Han people, geographically mainly from East China and its surrounding areas. The blood of 100 normal people was used as the control. For the above-mentioned included patients, their basic information, clinical information and treatment information were collected and sorted out, and informed consent was signed according to the principle of informed consent.

2. Extraction of genomic DNA;

Samples 2ml of peripheral venous blood from the research subjects was collected using EDTA anticoagulated blood collection tubes, and the serum and blood cells were separated by centrifugation and stored in a -80°C refrigerator. Genomic DNA from peripheral blood was extracted by using a centrifugal adsorption column that can specifically bind DNA and a unique buffer system, and operated according to conventional methods. Usually 50ng of DNA can be obtained, and the purity (UV 2600D:2800D) is around 1.8.

3. Prepare the PCR amplification system:

And each DNA was added to the mixture of primers and probes shown in SEQ ID NO: 2-5 for PCR reaction to type each SNP respectively. Reagents required include Taqman Genotyping Master Mix 5 μl, ddH ₂ 03.5 μl, 0.5 μl of fluorescent probe and primer mixture, and 1 μl of 50 ng/μl human DNA sample under examination for the rest, with a total volume of 10 μl.

4. PCR amplification program:

The first step of PCR reaction: 60°C, 35s; the second step of PCR reaction: 96°C, 10min; the third step of PCR reaction: 93°C, 20s; the fourth step of PCR reaction: 60°C, 95s; the fifth step of PCR reaction: Cycle the third step and the fourth step 5 times, then 60°C, 25s. ABI Stepone fluorescent quantitative PCR instrument (Applied Biosystems, USA Applied Biotechnology Co., Ltd.) was used.

5. Experimental results:

After the PCR reaction was finished, the Stepone software V2.1 was used for analysis, and the obtained 200 cases were classified. According to the probe of the present invention, three genotypes were identified: three genotypes AA, AG and GG of SNP rs2380128.

6. Analysis of the accuracy of the kit

In order to verify the accuracy of the kit detection, 200 samples were sequenced for verification. There were 188 cases of SNP rs2380128 genotype AA, 10 cases of AG genotype, and 2 cases of GG genotype; the AG and GG genotypes were all osteoporosis patients, so the detection accuracy of the kit reached 100%.

The value of this kit is that it only needs peripheral blood and no other tissue samples, detects SNP sites through the most streamlined and specific amplification primers, and then evaluates the patient’s condition through the SNP spectrum. It is not only stable, convenient, and accurate, greatly improving The sensitivity and specificity of disease diagnosis, so putting this kit into practice can help guide the screening of high-risk groups and more effective individualized treatment.

The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention shall fall within the scope of the present invention.

sequence listing

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Claims (1)

1. The use of the primer set and probe sequence described in SEQ ID NO: 2-5 in the preparation of a kit for osteoporosis detection.