CN108048555B - Kit for detecting osteoporosis - Google Patents

Abstract

The kit for detecting the osteoporosis comprises a probe and a primer which can specifically detect rs 10048745. Whether the subject suffers from osteoporosis can be detected by the kit, so that a clinician is guided to provide a preventive scheme or a therapeutic scheme for the subject. And the operation is simple, the experimental method is adopted, and the cost is low.

Description

kit for detecting osteoporosis

Technical Field

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

Background

Osteoporosis, is a group of bone diseases caused by various reasons, bone tissues have normal calcification, calcium salts and matrixes are in normal proportion, and metabolic bone lesions are characterized by the reduction of the amount of bone tissues in unit volume. In most osteoporosis, the reduction of bone tissue is mainly due to increased bone resorption. Is characterized by pain in the skeleton and susceptibility to fracture.

The clinical manifestation is pain, the most common symptom of primary osteoporosis, most common in low back pain, accounting for 70% -80% of pain patients. Pain spreads laterally along the spine, pain is relieved when lying on the back or sitting, pain is aggravated when standing upright or sitting for a long time, and pain is aggravated when bending down, coughing and defecating are forcefully performed. Generally, bone pain occurs when more than 12% of the bone mass is lost. In senile osteoporosis, the vertebral body is compressed and deformed, the spine is anteverted, and muscle fatigue and even spasm produce pain. More recently, compression fracture of thoracic and lumbar vertebrae can also cause acute pain, and the spinous processes of the corresponding parts can have strong tenderness and percussion pain. When the corresponding spinal nerves are pressed, the pain of limbs caused by radiation, sensory and motor disturbance of both lower limbs, intercostal neuralgia and sternal pain can be similar to angina pectoris. The pressure on the spinal cord and cauda equina also affects the function of the bladder and rectum. Degenerative osteoporosis is the most common and most serious complication.

Examination of osteoporosis is mainly, at a laboratory level, (1) blood calcium, phosphorus and alkaline phosphatase in primary osteoporosis, serum calcium, phosphorus and alkaline phosphatase levels are usually normal, and alkaline phosphatase levels may increase months after fracture. (2) Blood parathyroid hormone should be examined for secondary osteoporosis except for parathyroid function. Blood parathyroid hormone levels may be normal or elevated in patients with primary osteoporosis. (3) Markers for bone turnover osteoporosis patients are partially serologically biochemical indicators reflecting the state of bone turnover (including bone formation and bone resorption) and these biochemical measures include: bone-specific alkaline phosphatase (reaction bone formation), tartrate-resistant acid phosphatase (reaction bone resorption), osteocalcin (reaction bone formation), type i procollagen peptide (reaction bone formation), uropyridinoline and deoxypyridinoline (reaction bone resorption), and N-C-terminal cross-linked peptide of type i collagen (reaction bone resorption). (4) The normal ratio of morning urine calcium/creatinine is 0.13 + -0.01, while excessive urine calcium displacement increases the ratio, indicating increased bone resorption.

The second is auxiliary examination, which is divided into the following aspects, bone imaging examination and bone density examination, wherein the bone fracture and other pathological changes, such as osteoarthritis, intervertebral disc diseases and spondylolisthesis, can be found by taking X-ray films of pathological change parts. When the bone mass is decreased (low bone density), the increase of bone transparency, the decrease of trabecular bone and the increase of the gap are observed, the trabecular bone disappears, the bone structure is fuzzy, but the bone mass is usually decreased by more than 30 percent. In general, biconcave deformity of the vertebral body is observed, and the anterior edge of the vertebral body is collapsed to form a wedge-shaped change, which is also called compression fracture and is commonly seen in 11 th and 12 th thoracic vertebrae and 1 st and 2 nd lumbar vertebrae. Bone density detection is a prediction index of fracture. Measuring the bone density at any position can be used for evaluating the overall fracture occurrence risk; measuring bone density at a particular site can predict the risk of a localized fracture.

In the prior art, CN 102534019 a discloses a kit, which screens a high risk group of women susceptible to osteoporosis by detecting the genotype of 5 snp sites closely related to the occurrence of osteoporosis, and finally evaluates the risk level of osteoporosis susceptibility from the gene level according to the gene detection result of each subject, so as to guide climacteric women to effectively prevent osteoporosis.

CN 106755522A discloses a kit for detecting juvenile osteoporosis, and the gene comparison shows that DKK1 gene has obvious mutation site, namely 721 site, in juvenile osteoporosis patients and normal people. By detecting the mutation site, particularly 1 pair of specific primer pairs is provided, the condition that the human body suffers from juvenile osteoporosis can be specifically identified. Compared with the prior art, the detection method has the advantages of convenience in detection, accuracy and rapidness, and is suitable for popularization and use.

CN 107043811A discloses that CFAP20 gene can be used as molecular marker for early diagnosis of osteoporosis. Differentially expressed genes in osteoporosis patients were studied using high throughput sequencing and QPCR experiments. The research result shows that whether the subject suffers from osteoporosis can be judged by detecting the expression of the CFAP20 gene in blood.

Based on the limitation of means for detecting osteoporosis in the prior art, the search for specific molecular markers related to the early diagnosis and prognosis of osteoporosis has profound significance for realizing the early diagnosis and individualized treatment of osteoporosis.

Disclosure of Invention

The invention provides a kit for detecting rs10048745 genotype, and the detection result can be used for the auxiliary analysis of osteoporosis.

The first purpose of the invention is to provide a kit for detecting the genotype of the SNP locus rs10048745 of a gene, wherein the kit comprises a primer pair and a PCR detection reaction reagent.

According to the kit of the present invention, there is provided a method of diagnosing a susceptibility to osteoporosis in an individual, the method comprising: and detecting the polymorphism state of the rs10048745 locus of the individual, wherein the polymorphism state does not show the characteristic of low bone density when the locus is G, and the mutation genotype A causes the reduction of the bone density value, thereby finally causing the occurrence of osteoporosis.

The invention provides a method for detecting whether rs10048745 polymorphism exists in a sample, which comprises the following steps: (1) and amplifying the sample by using a kit designed by us to obtain a PCR amplification product.

The invention detects whether the single nucleotide polymorphism exists in the amplification product by utilizing the TAQMAN technology. The basic technical route of the invention is as follows: a method for identifying SNP molecular markers in rs10048745 polymorphism comprises the steps of extracting genomic DNA of a sample, carrying out PCR amplification by using the kit provided by the invention, amplifying a target fragment, detecting by using a TAQMAN technology, finding the existence of SNP, and confirming the existence and the position of the SNP by using a DNA sequencing method.

The sequence of a detection fragment of the rs10048745 site is shown as SEQ ID NO: 1 is shown. The length is 201bp, and the specific sequence is as follows: atttctggta aaactgaaag caagaaaagc agggtgctag cccctgtggg actgagggtg gaggctgggg gagtttgggt gccatcctcc agtgacagat ggatggacctt tcatctaaga gaaaggagga gacacgttgg caaatcagcc tcaagcctaa gattgcttgt gaagcaatca taaggaggaa caaaaacaga

it will be clear to those skilled in the art that there are many analytical methods available for detecting the frequency of single nucleic acid polymorphism distributions present in intronic sequences of a gene. 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 convenient to use and has high sensitivity, and which contains PCR-specific amplification primers and PCR reaction solutions (e.g., conventional components such as reagents, buffers, etc.) for PCR amplification detection, etc., and these conventional components and detection methods are well known to those skilled in the art. In the detection of mutation sites by using the TAQMAN technology to detect amplification products, some conventional reagents required by the TAQMAN are also required.

Specifically, the invention provides a PCR kit for detecting SNP of rs10048745, which is characterized by comprising a primer 1, a primer 2, a probe 3, a probe 4 and PCR reaction liquid, wherein 5 'ends of the probe 3 and the probe 4 are labeled with reporter groups, 3' ends of the probe 3 and the probe 4 are labeled with fluorescence quenching groups, and the 5 'end reporter group of the probe 4 is different from the 5' end reporter group of the probe 3.

Wherein the 5' end labeled reporter group may be selected from: FAM, HEX, TET, JOE, VIC, R0X, Cy3, Cy5, MAR, JUP, SAT, PLU, or NEP, and is not limited to the above groups; the fluorescence quenching group marked at the 3' end can be selected from: TAMRA, Eclipse, BHQ1, BHQ2, BHQ3, DABCYL, MGB, and is not limited to the above groups.

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

PCR amplification, wherein the used amplification primer sequences are as follows:

forward primer 1: atttctggta aaactgaaag;

Reverse primer 2: tctgtttttg ttcctcctta;

Specific probe sequence 3: agtgacagat a gatggacctt, wherein the 5 'segment of the sequence is marked by FAM, the 3' end is marked by MGB,

Specific probe sequence 4: agtgacagat g gatggacctt, in which the 5 'segment of the sequence is labeled with VIC and the 3' end is labeled with MGB.

The invention has the advantages and beneficial effects that:

The invention discovers that the rs10048745 polymorphism is related to osteoporosis for the first time, and whether the subject suffers from osteoporosis can be judged by detecting the rs10048745 polymorphism in the subject, so that a clinician is guided to provide a prevention scheme or a treatment scheme for the subject. And the operation is simple, the experimental method is adopted, and the cost is low.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are experimental procedures without specific conditions noted, or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

EXAMPLE 1 obtaining molecular markers for SNPs

study samples were obtained from a number of study and physical examination centers. All studies were approved by the ethics committee and informed consent was obtained from the study subjects. We performed a global genome association analysis (GWAS) on samples from six different lineages with lumbar and femoral cervical density measurements and combined the data for meta analysis. In addition, the meta analysis results of the lumbar and femoral neck bone density of the European blood group in the GEFOS-seq public database are collected, and the two parts of data are merged for joint analysis to mutually verify the correlation results.

first part internal sample meta analysis

1. Selection of samples

We performed GWAS meta analysis earlier in seven samples with lumbar and femoral cervical density measurements. Because the FHS sample coincides with the GEFOS-seq sample, the FHS sample is removed from the internal sample in order to ensure the independence of the two samples. The remaining six samples have the following basic information:

(1) omaha osteoporosis study specimen (OOS): OOS samples included a cross-sectional study of 1000 randomized participants.

(2) Kansassin city osteoporosis study sample (KCOS): the KCOS samples included a cross-sectional study of 2286 randomized participants.

(3) Indiana brittle fracture study sample (IFS): IFS samples were obtained from the dbGAP database and included 1493 pre-menopausal sisters of European descent in a cross-sectional study of participants.

(4) Chinese osteoporosis research sample (COS): the COS sample included 1627 cross-sectional studies of randomized participants.

(5) African american female health preliminary observation study (WHI-AA): the WHI-AA sample was part of a female health initial observation study (WHI) and contained 845 african american participants, available from the dbGAP database.

(6) Preliminary observation study of health of hispanic women (WHI-HIS): the WHI-HIS sample, also part of the WHI, contained 446 hispanic participants, available from the dbGAP database.

2. Phenotypic measurement and modeling

Bone density values were measured by DXA bone densitometer. The significance of covariates such as gender, age squared, height, and the top 10 principal components (for measuring population stratification effect) calculated from the genomic data were screened using a step regression method. The residuals of the original bone density values after covariate correction were normalized using the quantile of the normal distribution. The normalized residuals are used for downstream correlation analysis.

3. Genotyping and quality control

All six samples were typed using a high throughput genotyping chip. Quality control is realized at a sample level and an SNP level. At the sample level, sex was inferred by analyzing the X chromosome using the plink software and compared to sex in a questionnaire. Individuals of non-conforming gender were deleted from the data. At the SNP level, SNPs that do not meet the Ha-Weathering equilibrium are deleted from the data. When the population extreme value exists, the genotype of the main component is checked, and the extreme value is removed.

4. Genotype complementation defect

All samples were genotypically complemented using sequencing data for the thousand human genome (2013 version 5 months). First, haplotype data of 4 types of individuals sequenced from the thousand genome site (european ancestry 503, east asian ancestry 504, african ancestry 661, and mixed american ancestry 347) were downloaded and used as the genotyping reference template for each sample. Secondly, the alleles of the reference genome (i.e., thousand human genome data) and the target genome (i.e., GWAS sample) are tested for identity, and those that do not match will be deleted from the target genome. Finally, the genotype complementation is carried out by using software FISH, and the algorithm has the advantages of high operation speed, small occupied memory, no need of phase splitting (phasing) of the target genome and the like. And setting software parameters by default.

5. association analysis and meta analysis in a single sample

In each sample, the normalized phenotypic residual and the genetic association between the typed and complemented genotypes were examined by a genetic accumulation model. In the unrelated samples, the genetic association was examined using a linear regression model, the software being MACH2 QTL. For IFS family samples, the correlation of genotypes was calculated using a mixed linear model. The correlation results of the six samples were combined together to perform meta analysis. The model used was a sample-size weighted fixed effect model and the software used was METAL.

Collection of second part GEFOS-seq meta analysis results

The GEFOS-seq research organization performed sequencing and genotype-complementation based bone density GWIS analysis in over 50,000 participants of European descent. Sample information, quality control and statistical analysis are detailed in GEFOS-seq official network. Briefly, this study includes three sections: 1. whole genome sequencing of 2,882 subjects in the UK10K study; 2. whole genome sequencing was performed on 3,549 subjects out of 5 cohort studies; 3. the whole genome genotype was complemented for 26,534 subjects. All samples were meta analyzed and some results were published. Here, we downloaded the meta-analysis results of this fraction with sample volume up to 32,965 through GEFOS-seq official network.

SNP inclusion criteria

For the meta analysis results of the first six internal samples, we adopted quality control criteria for SNP inclusion as: gene heterogeneity I2< 50%. For the second part of the GEFOS-seq meta analysis results, we adopted the inclusion criteria of quality control as follows: gene heterogeneity I2< 50%; ② Minimum Allele Frequency (MAF) > 1%. For SNPs that meet both quality controls, we included them in the next study.

Combined analysis

From the meta analysis of the two samples we obtained two parts of z values: z1 and z 2. Then, we use the weighted stationary effect meta analysis model to perform the joint analysis, and the formula is as follows:

In the formula, n1 and n2 respectively refer to the sample amounts of the inner sample and the GEFOS-seq sample. In case the null hypothesis holds, i.e. there is no correlation, the statistical value Z follows a normal distribution or an approximately normal distribution. The GWAS significance level was set at 5.0x 10-8.

Conclusion

In the internal sample study, a total of 7,484 subjects were covered. No heterogeneous individuals were detected in each sample using principal component analysis. After PCA is used for correcting population stratification, the expansion coefficients of lumbar vertebra and femoral neck bone density genome are respectively 1.03 and 1.02 which are lower than a warning value, and no obvious population stratification effect exists. In the results of the GEFOS-seq meta analysis, a total of 32,965 subjects covered 10,586,901 SNPs, and after quality control screening, a total of 7,434,754 SNPs were included in the next combined analysis.

The combined analysis of the two parts found many sites associated with lumbar and femoral cervical bone density. Some sites not only reached genome-wide significance levels in the GEFOS-seq samples (GWS, p <5.0x10-8), but were also validated repeatedly in the internal samples (p < 0.05). Furthermore, we have also discovered new sites that reach significant levels throughout the genome: 2p13.3(rs10048745, p ═ 3.94x 10-8).

the SNP rs10048745 at this site correlates most significantly with lumbar vertebrae density (sample p1 ═ 0.02, GEFOS-seq sample p2 ═ 1.03x10-6, pooled sample p12 ═ 3.94x 10-8). The detailed results are shown in table 1 below:

table 1rs10048745 results data

As can be seen from the data in table 1, there is a mutation of a/G for the rs10048745 site, which exhibits a low bone density property when it is a and does not exhibit a low bone density property when it is G. The mutant genotype a leads to a decrease in bone density values and ultimately to the development of osteoporosis, which is statistically extremely significant (p ═ 3.94x10-8), and thus osteoporosis can be detected by detecting the site genotype.

Example 2 specific sample detection

1. Selecting a research sample;

The osteoporosis patient blood sample used in the present invention was obtained from the first hospital affiliated with suzhou university, and a total of 100 patients were confirmed. The inclusion criteria for study samples were women aged 18-70 years; confirming diagnosis through bone density detection and X-ray detection; the functional disorder of main organs is avoided, and the blood is normal, and the functions of liver and kidney and heart are basically normal; complete follow-up information can be obtained. All study subjects were hamsters without kindred relationship, mainly from eastern China and its surrounding areas geographically. Blood of 100 normal groups was used as control. And collecting and organizing basic information, clinical information and treatment information of the patients brought into the patients, and signing an informed consent form according to an informed consent principle.

2. Extracting genome DNA;

The sample was collected in an EDTA anticoagulated blood collection tube in an amount of 2ml from the peripheral venous blood of the subject, centrifuged to separate serum and blood cells, and stored in a freezer at-80 ℃. The peripheral blood genome DNA is extracted by adopting a centrifugal adsorption column capable of specifically binding DNA and a unique buffer system, and the operation is carried out according to a conventional method. 50ng of DNA is usually obtained with a purity (UV 2600D:2800D) of around 1.8.

3. Preparing a PCR amplification system:

And adding to each DNA the nucleotide sequence of SEQ ID NO: 2-5, performing PCR reaction on the mixture of the primers and the probes to respectively type each SNP, wherein the required reagents comprise 1 mul of the DNA sample of the person to be examined with the total volume of 10 mul, and the rest is 50 ng/mul, and the required reagents comprise Taqman Genotyping Master Mix5 mul, ddH203.5 mul 1, a fluorescent probe and a primer mixture of 0.5 mul.

4. PCR amplification procedure:

a first step of PCR: 60 ℃ for 35 s; and a second step of PCR: 96 deg.C for 10 min; and a third step of PCR: 93 ℃ for 20 s; and a fourth step of PCR: 60 ℃ for 95 s; and a fifth step of PCR reaction: the third step and the fourth step are cycled for 5 times, and then the temperature is 60 ℃ for 25 s. An ABI Stepone light-exposure quantitative PCR instrument (Applied Biosystems, Applied Biotechnology, Inc., USA) was used.

5. The experimental results are as follows:

After the PCR reaction, the PCR reaction was analyzed using Stepone software V2.1, and the 200 cases of results were classified, and 3 genotypes were identified in total according to the probe of the present invention: SNP rs10048745 three genotypes AA, GG and AG.

6. Accuracy analysis of the kit

In order to verify the detection accuracy of the kit, 200 samples were subjected to sequencing verification. 189 cases of SNP rs10048745 genotype GG, 10 cases of AG genotype and 1 case of AA genotype; wherein, both the AG genotype and the AA genotype are patients with osteoporosis, so the detection accuracy of the kit reaches 100 percent.

The kit has the value that only peripheral blood is needed without other tissue samples, SNP sites are detected through the simplest and most specific amplification primers, and then the condition of a patient is evaluated through an SNP spectrum, so that the kit is stable, convenient and accurate to detect, and greatly improves the sensitivity and specificity of disease diagnosis, and therefore, the kit is put into practice and can help guide the screening of high-risk people and more effective individualized treatment.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Sequence listing

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Claims (1)

1, SEQ ID NO: 2-5 in the preparation of a kit for detecting osteoporosis.