CN103882113B - Kit for predicting susceptibility of ankylosing spondylitis - Google Patents

Abstract

The invention discloses a kit for predicting susceptibility of ankylosing spondylitis, and belongs to the technical field of medical biology. A genome deoxyribonucleic acid (DNA) of a host cell is extracted, the genotype of a DDX39B gene SNP site of an experimenter is determined, and the susceptibility of the experimenter on the ankylosing spondylitis is predicted. The kit has the advantages that the correlation of the DDX39B gene polymorphic site and the ankylosing spondylitis is elaborated for the first time, and a method for predicting the susceptibility of the ankylosing spondylitis is provided. The method can be applied to prevention, auxiliary diagnosis and treatment of the ankylosing spondylitis, and also can be applied to research and development of a new drug.

Description

A kit for predicting susceptibility to ankylosing spondylitis

technical field

The invention relates to a kit for predicting the susceptibility of ankylosing spondylitis, more specifically, predicting the susceptibility of subjects to ankylosing spondylitis by determining the polymorphism of AS-related gene DDX39B, and the method can be used for diseases Auxiliary diagnosis, treatment and new drug development belong to the field of biotechnology.

Background technique

Ankylosing spondylitis (AS) is an autoimmune disease that mostly occurs in young adults aged 16-40, with a male to female ratio of about 4 to 10:1. Lesions often first occur in the sacroiliac joints, and a small number of severe cases show rigidity of the entire spine. In addition, some patients have different degrees of extra-spinal lesions such as hip joints, eyes, lungs, cardiovascular, and kidneys. The incidence of AS in the Caucasian population is about 1% to 3%, and the prevalence of AS in my country is about 0.2-0.6%, of which more than 60% of the patients are accompanied by hip joint involvement, resulting in more than 20% of AS patients with disability, inflammation It mainly involves the bony attachment points of joint capsules, tendons and ligaments, resulting in localized joint adhesions and ankylosis, and limited mobility. Clinically, there is still a lack of drugs that can significantly alleviate and control the development of the disease. AS is a polygenic disease with obvious genetic predisposition. Although it is generally believed that genetic and immune factors play a leading role in the pathogenesis of AS, the exact etiology and pathogenesis are still unclear.

At present, the genetic etiology research of AS mostly uses SNP as the association analysis method of genomic markers, which is effective and has been proved. SNP refers to the DNA sequence polymorphism caused by a single nucleotide variation at the chromosomal genome level. The frequency in the population needs to be >1%. SNPs are biallelic markers. 70.1% of this single base variation is the same type Conversion between bases: such as G/A or T/C, 29.1% are transversions between purine and pyrimidine. C (cytosine) is the most variable site in the human genome, because most of it is methylated cytosine, which can be converted to T (thymine) by spontaneous deamination, and SNP contains 80-90 of known polymorphisms %, is the most common genetic variation.

The distribution of SNPs in single genes and across the genome is heterogeneous due to selection pressure for survival. The number of SNPs in the non-coding region of genes is 4 times that of the coding region, and the total number can reach 3 million. SNP is characterized by its high density (1 per 1kb on average), strong representation (SNPs located inside the gene may directly affect protein structure or expression level), good genetic stability (compared with microsatellite polymorphism), Ease of automatic analysis (since SNPs are mostly biallelic markers in the population, they can be directly typed by "+/- or 1/0") and other characteristics have become good genetic markers.

In 1973, Brewerton et al. first discovered the human leukocyte antigen-B27 (HLA-B27) strongly related to AS. With the progress of research, other susceptibility genes related to AS such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) have been identified one after another.

The DDX39B gene contains 11 exons, is a member of the DEAD box family and can encode 3 mRNAs, but NR_037852.1 cannot be translated into protein. DEAD box family proteins have a highly conserved Asp-Glu-Ala-Asp (DEAD) sequence, which plays a very important role in the metabolic process of RNA, and can participate in the splicing of mRNA, the process of RNA passing through nuclear pores and the degradation of RNA, etc. . DDX39B is an essential factor in the process of U2 small nuclear riboprotein-mediated cleavage of pre-mRAN, and also mediates the process of RNA passing through the nuclear pore into the cytoplasm. So far, there are no research results on the association of DDX39B gene with AS.

Contents of the invention

The main purpose of the present invention is to provide a method for predicting susceptibility to ankylosing spondylitis.

The second object of the present invention is to provide a kit for predicting susceptibility to ankylosing spondylitis, including PCR primers and a kit containing the primers.

To achieve the above object, the present invention adopts the following technical solutions:

The nucleic acid sequence for detecting the susceptibility to ankylosing spondylitis is the base sequence shown in SEQ ID No.1 in the sequence table. The +401 position is the mutation site, which is marked with the letter "R". The nucleic acid sequence is the full-length sequence of the DDX39B gene. Figure 1 is a schematic diagram of the structure of the DDX39B gene and its polymorphic variation sites. The figure contains 2 exons, and the rs144802800 site is marked in the corresponding position of the seventh intron region in the DDX39B gene map.

A method for detecting the susceptibility of ankylosing spondylitis, detecting the genotype of the rs144802800 polymorphic site of the DDX39B gene of the subject. When the genotype is CC, the susceptibility of the subject is the lowest; when carrying the T allele, the subject is affected Subject's susceptibility increased.

A set of primers for detecting the susceptibility of ankylosing spondylitis can be specifically amplified to obtain the nucleotide sequence SEQ ID No.1 for detecting the susceptibility of ankylosing spondylitis, and the nucleotide sequences of the primers are sequence table SEQ ID No.2 And shown in sequence table SEQ ID No.3.

The present invention provides a diagnostic kit for detecting the susceptibility of ankylosing spondylitis, which contains the primer pair for specifically amplifying DDX39B gene rs144802800 of the present invention and the conventional components, reagents and buffers of the kit for PCR amplification detection etc. Those skilled in the art are familiar with these conventional components and detection methods. All components and contents in the kit of the present invention are as follows:

A test kit for detecting ankylosing spondylitis susceptibility genes, consisting of the following reagents:

(1) 10 μL 10×PCR buffer;

(2) 2 μL 10mM dNTP mixture;

(3) 2μL TaqDNA polymerase, 2unit/μL;

(4) 1 μL F1 primer, which is the nucleotide sequence shown in SEQ ID NO.2, with a concentration of 10 pmol/μL;

(5) 1 μL R1 primer, which is the nucleotide sequence shown in SEQ ID NO.3, with a concentration of 10 pmol/μL;

(6) 8 μL 10×LC-Green Plus saturated fluorescent dye;

(7) 2 μL oligonucleotide internal reference, consisting of 0.5 μL each of 4 internal reference primers, with a concentration of 10 pmol/μL, wherein the upstream primer F of the low-temperature oligonucleotide internal reference is the nucleotide sequence shown in SEQ ID NO.4, The low-temperature oligonucleotide internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.5, the high-temperature oligonucleotide internal reference upstream primer F is the nucleotide sequence shown in SEQ ID NO.6, and the high-temperature oligonucleotide Acid internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.7;

(8) 64 μL pure water.

The method of use is as follows:

(1) PCR amplification: Amplify the 6th intron region fragment of DDX39B gene by PCR, prepare the mixture: 10×PCR reaction buffer 1μL, 10mM/LdNTP0.2μL, TaqDNA polymerase 0.2μL, 10pM/L 0.1 μL of upstream primer, 0.1 μL of 10pM/L downstream primer, 0.8 μL of 1×LC-Green Plus saturated fluorescent dye, 0.2 μL of oligonucleotide internal reference (0.05 μL each for high and low temperature oligonucleotide internal reference upstream and downstream primers) ( See Table 1 for the sequence), 1 μL of genomic DNA, and add pure water to 10 μL. The PCR reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 5 s, a total of 45 cycles, and a total extension at 72°C for 2 min. Before high-resolution melting curve analysis, denaturation and renaturation treatment: 95°C for 30s, 25°C for 2min, 94°C for 30s, 24°C for 4min. Before PCR, 20 μL of paraffin oil was added to each system to prevent the liquid from volatilizing.

(2) Genotype determination: transfer the PCR product into a special 96-well plate for HRM, perform HRM analysis on the Light Scanner TMHR-I96, analyze the collected curve with the Light Scanner Call IT software, and determine the gene according to the difference of the melting curve type.

Use of the rs144802800 polymorphic site of DDX39B gene in the preparation of reagents or medicines for diagnosing or treating ankylosing spondylitis.

The assay method of the present invention measures the genomic DNA derived from human beings, and the sample source is not limited, such as: body fluid (blood, ascites, urine, etc.), tissue cells (such as liver tissue), and the like. Genomic DNA can be prepared by extracting and purifying these samples. Adjust the concentration of genomic DNA to make it as consistent as possible. Using genomic DNA as a template, nucleic acid fragments containing DDX39B gene mutation sites can be amplified to obtain a large number of samples for determination. The sample obtained by amplifying the DNA fragment containing the DDX39B gene variation point is particularly suitable as a measurement material.

When gene-assisted diagnosis is performed, the present invention is preferably applicable to an auxiliary diagnostic reagent for determining the presence of a mutation type of the DDX39B gene, and the auxiliary diagnostic reagent includes a specific reagent as an essential component corresponding to the method for determining the mutation type of the DDX39B gene. Appropriate specific reagents, such as DNA fragments and/or primers for PCR amplification steps, are selected according to the assay method employed.

The advantages of the present invention are: the present invention clarifies for the first time the correlation between DDX39B gene polymorphism site and AS, and provides a method for predicting AS susceptibility, which can be used for the prevention, auxiliary diagnosis and treatment of AS, and can also for new drug development.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that the public has a deeper understanding of the content of the invention, and is not a limitation of the present invention. All equivalent replacements in the field made according to the disclosure of the present invention belong to this invention. protection scope of the invention.

Description of drawings

Figure 1 DDX39B gene structure and schematic diagram of rs144802800 site

Figure 2 is the genotyping map of the DDX39B gene variation site by the HRM method

Figure 3 is the sequencing map of the DDX39B gene variation site

Detailed ways

The abbreviations used to represent reagents in the following examples are as follows:

10×PCR buffer: 10mM Tris-HCl (pH=8.3), 500mM potassium chloride (KCl), 10mM magnesium chloride (MgCl ₂ ), 0.01% (W/V) gelatin

dNTP: deoxynucleoside triphosphate

EDTA: disodium ethylenediaminetetraacetic acid

TE: 10mM Tris-HCl (pH=7.5), 1mM EDTA (pH=8.0)

Example 1: Collection of blood samples and extraction of genomic DNA:

1. Case selection:

The selected cases were selected according to the revised diagnostic criteria in New York in 1984. A total of 136 unrelated AS patients (age: 14-44 years old, average 24 years old) from Jilin area were selected, and 148 healthy control volunteers (age: 39 years old) from the same area were selected. -72 years, with an average of 46 years). All subjects were of Han nationality and signed written informed consent. This study was approved by the Beijing Hospital of the Ministry of Health and the Ethical Review Committee of the Institute of Gerontology of the Ministry of Health. .

2. According to the following method, human genomic DNA was prepared.

1. Add 1000 μL of erythrocyte lysate to a labeled 1.5mLEP tube, then add 400 μL of LEDTA anticoagulant blood (invert and mix 3-5 times before adding anticoagulant blood), invert and mix well, and let stand at room temperature for 10 minutes;

2. After centrifuging at 13000rpm for 30 seconds, remove the supernatant;

3. Add 480 μL of nucleic acid lysate to the obtained precipitate, flick the tube wall, mix well, add 20 μL of proteinase K (dilute proteinase K 20 times with cleavage solution), invert and mix well, incubate at 65°C for 10 minutes, ( Mix up and down from time to time to make sure there are no clots);

4. Take it out and cool it down to room temperature, add 300 μL of protein precipitation solution, mix thoroughly by inverting, let it stand for 10 minutes, and centrifuge at 13000 rpm for 2 minutes;

5. Transfer the supernatant to a new EP tube, add 670 μL of pre-cooled isopropanol, and mix thoroughly by inverting (more than 10 times), it can be seen that the linear DNA gradually forms small clumps, and centrifuge at 13,000 rpm for 2 minutes;

6. Discard the supernatant and ensure that the precipitate remains in the EP tube, add 670 μL of 70% ethanol, mix up and down, and centrifuge at 13,000 rpm for 2 minutes;

7. Discard the supernatant to evaporate the ethanol in the tube;

8. Add TE solution (400 μL), fully dissolve, analyze the concentration and purity of the extracted genomic DNA, draw part of the DNA solution as the working solution, correct the concentration to 20ng/μL, store at 4°C for later use, and place the remaining genomic DNA at - Store in refrigerator at 20°C.

Embodiment 2 Identification of SNP

The present invention simultaneously detects the genotype of the DDX39B gene rs144802800 (its allelic site is C/T) by using PCR-high resolution melting curve (HRM) analysis method and PCR sequencing technology.

1. Determination of PCR-HRM primers

The DNA base sequence (Seq ID No.1) near rs144802800 of the DDX39B gene was retrieved from Genebank, and the primers were designed under Oligo7.0 software. The target fragment is located in the 7th intron region of the DDX39B gene, with a total length of 52bp. The specific primer sequences of the sense strand F1 and the antisense strand R1 are determined as follows:

F1: 5'-AGCTGGCGTCTGAGGTAGCAC-3' (Seq ID No.2)

R1: 5'-GCATGTCTGGCAAGTTTGATTTCTG-3' (Seq ID No.3)

2. PCR reaction system and reaction conditions

Amplify a partial fragment of the second intron region of the DDX39B gene by PCR. The PCR reaction system is: 1μL of 10×PCR reaction buffer, 0.2μL of 10mM/LdNTP, 0.2μL of TaqDNA polymerase, 0.1μL of 10pM/L upstream primer, 10pM/L 0.1 μL downstream primer, 0.8 μL 10×LC-Green Plus saturated fluorescent dye, 0.2 μL oligonucleotide internal control (0.05 μL each for high and low temperature oligonucleotide internal control primers and downstream primers) (see Table 1 for sequence), genome DNA 1μL, add deionized water to 10μL. During PCR, 20 μL of paraffin oil was added to each system to prevent the liquid from volatilizing. The PCR reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 5 s, a total of 45 cycles, and a total extension at 72°C for 2 min. Before high-resolution melting curve analysis, denaturation and renaturation treatment: 95°C for 30s, 25°C for 2min, 94°C for 30s, 24°C for 4min.

Table 1: High and low temperature oligonucleotide internal reference primer sequences, annealing temperature and product fragment length

3. HRM genotype determination

Transfer the PCR product into a 96-well plate dedicated to HRM, and perform HRM analysis on Light Scanner TMHR-I 96: start at 40°C, collect the melting curve at a slope of 0.3°C/s, and end at 98°C, use Light Scanner Call IT software The collected curves were analyzed to determine the genotype.

4. Sequencing to determine genotype

Three samples were randomly selected from the obtained individuals of different genotypes for sequencing verification. The sequencing sample was re-amplified by PCR, and the sequencing primer sequence was: F2: 5'-TAGGGGTCGATGGTGTGTGAGAG-3' (Seq ID No.8), R2: 5'-GGATGCTGAGGTGGAAGG-3' (Seq ID No.9), the amplified fragment 497 bp long. The total PCR reaction system is 30 μL, including: Genomic DNA 2 μL, 10×PCRBuffer 3 μL, 10mMdNTP 0.5 μL, TaqDNA polymerase (5U/μL) 0.5 μL, upstream and downstream primers (10pM/μL) each 0.5 μL, pure water supplemented to a total volume of 30 μL . The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min and then entering the main cycle, denaturation at 95°C for 20 s, annealing at 60°C for 20 s, extension at 72°C for 20 s, 35 cycles, and extension at 72°C for 2 min. The PCR products were detected by 8% polyacrylamide gel electrophoresis, and the gel imaging system was observed to pass the inspection and then sent to the BGI Genomics Sequencing Department for sequencing verification.

The correlation of embodiment 3 gene SNP and ankylosing spondylitis (AS)

1. Statistical method:

Statistical methods: The Hardy-Weinberg balance test was used to study the group representativeness of the samples. Using the Pearson chi-square test in SPSS11.0 software to calculate the distribution frequency of alleles and genotypes of DDX39B gene rs144802800 between the AS case group and the normal control group, the AS risk OR value and its 95% CI are credible Interval, with P<0.05 as the standard of significant difference.

2. Results: The distribution of the genotype and allele frequency of rs144802800 on the DDX39B gene located in the 6p21.3 region between the cases and the control group is shown in Table 2.

Table 2 Distribution of genotype and allele frequency of DDX39B gene rs144802800 in case-control group

Note: OR: odds ratio; CI: confidence interval. *A allele is the risk allele for AS. Subjects were divided into AS risk allele (GA) carriers and non-risk allele (GG) carriers. the

It can be seen from Table 2 that the A allele of DDX39B gene rs144802800, that is, the T allele on its DNA complementary strand, has a significantly higher distribution frequency in the patient population than in a healthy normal population ( 21.3%vs.8.8%), there was a significant difference (P=0.003), and the OR value of the A allele was 2.598, 95%CI: 1.321-5.109; risk allele (GA) carriers in AS and Among the non-risk allele (GG) carriers, the distribution frequency of the risk genotype in the case group was significantly higher than that in the control group (P<0.001), which indicated that the DDX39B gene rs144802800 was positively correlated with the prevalence of AS, which may increase risk of developing AS.

Implement 4 detection kits

Prepare a kit for detecting AS-related risks, including a primer pair that can amplify the DDX39B gene SNPrs144802800, and other PCR-HRM corresponding reagents. The kit of the present invention is used for detection of 10 people, and is stored at -20°C in the dark, and its components, contents and sources include:

10 μL 10×PCR buffer (Pharmacia),

2 μL 10mM dNTP mixture (Pharmacia),

2 μL TaqDNA polymerase (2unit/μL) (Takara)

1 μL F1 (SEQ ID No.2) (10pM/μL)

1 μL R1 (SEQ ID No.3) (10pM/μL) primer,

8 μL 10×LC-Green Plus saturated fluorescent dye (Idaho, USA),

2 μL oligonucleotide internal reference, consisting of 0.5 μL each of 4 kinds of internal reference primers, the concentration is 10 pmol/μL, wherein the upstream primer F of the low temperature oligonucleotide internal reference is the nucleotide sequence shown in SEQ ID NO.4, low temperature oligonucleotide The downstream primer R of the nucleotide internal reference is the nucleotide sequence shown in SEQ ID NO.5, the upstream primer F of the high-temperature oligonucleotide internal reference is the nucleotide sequence shown in SEQ ID NO.6, and the downstream primer of the high-temperature oligonucleotide internal reference is Primer R is the nucleotide sequence shown in SEQ ID NO.7;

64 μL pure water.

This kit can detect the rs144802800 polymorphism of DDX39B gene after PCR-HRM detection.

The polymorphism of the rs144802800 site of the DDX39B gene of AS patients and normal people is determined by using the kit of the invention. Genomic DNA from AS patients and 10 normal individuals were randomly selected.

1. Method:

1. PCR amplification: Amplify the fragment of the sixth intron region of the DDX39B gene by PCR, and prepare a mixture: 1 μL of 10×PCR reaction buffer, 0.2 μL of 10mM/LdNTP, 0.2 μL of TaqDNA polymerase, 10pM/L upstream Primer 0.1 μL, 10pM/L downstream primer 0.1 μL, 1×LC-Green Plus saturated fluorescent dye 0.8 μL, oligonucleotide internal control 0.2 μL (high and low temperature oligonucleotide internal reference upstream and downstream primers each 0.05 μL) (sequence See Table 1), Genomic DNA 1μL, add pure water to 10μL. The PCR reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 5 s, a total of 45 cycles, and a total extension at 72°C for 2 min. Before high-resolution melting curve analysis, denaturation and renaturation treatment: 95°C for 30s, 25°C for 2min, 94°C for 30s, 24°C for 4min. Before PCR, 20 μL of paraffin oil was added to each system to prevent the liquid from volatilizing.

2. Genotype determination: transfer the PCR product into a special 96-well plate for HRM, perform HRM analysis on Light Scanner TMHR-I96, use Light Scanner Call IT software to analyze the collected curve, and determine the genotype according to the difference of melting curve .

2. Results:

The results showed that 3 cases of AS patient samples with DDX39B gene rs144802800 polymorphism genotype were GA and 7 cases of GG; the genotype of control group was 1 case of GA and 9 cases of GG. The kit of the present invention can effectively detect the genotype of the rs144802800 polymorphic site of the subject's DDX39B gene. When the genotype is CC, the subject's susceptibility is the lowest; when carrying the T allele, the subject's susceptibility increases high.

Examples of the utility of the present invention:

The detection method of the DDX39B gene polymorphism of the present invention can be used for analyzing the T allele of the rare variation site on the DDX39B gene of the human autosome 6p21. It is used for the auxiliary diagnosis of AS and the assessment of individual AS risk, so as to facilitate the early intervention and treatment of AS.

Utilizing the present invention, the base variation at the rs144802800 site of the DDX39B gene, as one of the biomarkers, can be used for the screening of molecular targets for drug design, to help find active molecules that can regulate the expression of DDX39B, and promote the development of new AS drugs.

The nucleic acid sequence and AS-related sites for detecting the polymorphism of the DDX39B gene established by the invention can be highly sensitive and specific applied to a kit for auxiliary diagnosis of the AS gene.

As mentioned above, it was concluded that the polymorphism at the rs144802800 site of the DDX39B gene was significantly associated with AS. Therefore, the determination of this polymorphism according to the present invention can be used for gene-assisted diagnosis of AS.

The present invention describes the new AS-related mutation site of DDX39B gene, and provides a method for determining the polymorphism of HCP gene. Moreover, according to the present invention, only a small amount of DNA samples is enough to determine the polymorphism of the gene. As a result, the present invention provides a gene-aided diagnosis method for determining AS-related gene polymorphisms.

Claims (3)

1. A group of primers for detecting the susceptibility of ankylosing spondylitis, characterized in that: the nucleotide sequence for detecting the susceptibility of ankylosing spondylitis shown in the sequence table SEQ ID No.1 can be amplified, and the nucleotide sequence of the primers They are respectively shown in the sequence table SEQ ID No.2 and the sequence table SEQ ID No.3. 2. A test kit for detecting ankylosing spondylitis susceptibility genes, characterized in that it consists of the following reagents: (1) 10 μL 10×PCR buffer; (2) 2 μL 10mM dNTP mixture; (3) 2μL TaqDNA polymerase, 2unit/μL; (4) 1 μL F1 primer, which is the nucleotide sequence shown in SEQ ID NO.2, the concentration is 10 pmol/μL; (5) 1 μL R1 primer, which is the nucleotide sequence shown in SEQ ID NO.3, the concentration is 10 pmol/μL; (6) 8 μL 10×LC-Green Plus saturated fluorescent dye; (7) 2 μL oligonucleotide internal reference, consisting of 0.5 μL each of 4 kinds of internal reference primers, with a concentration of 10 pmol/μL, wherein the upstream primer F of the low temperature oligonucleotide internal reference is the nucleotide sequence shown in SEQ ID NO.4, The low-temperature oligonucleotide internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.5, the high-temperature oligonucleotide internal reference upstream primer F is the nucleotide sequence shown in SEQ ID NO.6, and the high-temperature oligonucleotide Acid internal reference downstream primer R is the nucleotide sequence shown in SEQ ID NO.7; (8) 64 μL pure water. 3. the purposes of primer described in claim 1 in the preparation diagnosis ankylosing spondylitis reagent, the nucleotide sequence of described primer is respectively shown in sequence listing SEQ ID No.2 and sequence listing SEQ ID No.3.