CN103725781B - Kit and method for predicting susceptibility of ankylosing spondylitis - Google Patents

Abstract

The invention discloses a kit and a method for predicting the susceptibility of ankylosing spondylitis, belonging to the field of medical biotechnology. The susceptibility of a testee to the ankylosing spondylitis is predicted through extracting genome DNAs (deoxyribonucleic acids) from host cells and testing the genotype of an SNP (single nucleotide polymorphism) locus of an HCP5 gene of the testee. The kit and the method have the advantages that the correlation of the polymorphic locus of the HCP5 gene with the ankylosing spondylitis is described for the first time; the provided method can be used for prevention, auxiliary diagnosis and treatment of the ankylosing spondylitis as well as research and development of new drugs.

Description

A kit and method for predicting susceptibility to ankylosing spondylitis

technical field

The present invention relates to a reagent and method 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 HCP5, and the method can be used for Auxiliary diagnosis, treatment and new drug development of diseases 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, and 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 HCP5 (HLA Complex Protein 5, Human Leukocyte Antigen Complex Protein 5) gene is located at 6q21.3, with a total length of 2635bp, containing 2 exons, and its encoded product is a long fragment of RNA, which is located in the human leukocyte antigen III region , is closely related to the occurrence and development of immune response and autoimmune diseases.

At present, there are no research results on the association of HCP5 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, marked with the letter "Y". The nucleic acid sequence is the full-length sequence of the HCP5 gene. Figure 1 is a schematic diagram of the structure of the HCP5 gene and its polymorphic variation sites. The figure contains 2 exons, and the rs192776040 site is marked in the corresponding position of the second exon in the HCP5 gene map.

A method for detecting the susceptibility of ankylosing spondylitis, detecting the genotype of the rs192776040 site in the second exon region of the HCP5 gene of the subject. When the gene is CC, the susceptibility of the subject is the lowest; when carrying the T allele , the subject's susceptibility increased.

A set of primers for detecting the susceptibility of ankylosing spondylitis is characterized in that: the nucleotide sequence SEQ ID No.1 for detecting the susceptibility of ankylosing spondylitis can be amplified, and the nucleotide sequences of the primers are sequence Shown in list SEQ ID No.2 and sequence listing SEQ ID No.3.

The invention provides a diagnostic kit for detecting the susceptibility of ankylosing spondylitis, which contains the primer pair for specific amplification of the HCP5 gene +401 site of the invention and the conventional components and reagents of the kit for PCR amplification detection , buffer, 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) 2μL 10mMdNTP 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 fragment of the second exon region of the HCP5 gene by PCR, and prepare a mixture: 10×PCR reaction buffer 1 μL, 10mM/LdNTP 0.2 μL, TaqDNA polymerase 0.2 μL, 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 rs192776040 polymorphic site in the second exon region of HCP5 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 HCP5 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 HCP5 gene variation point is particularly suitable for use as a determination material.

When gene-assisted diagnosis is performed, the present invention is preferably applicable to the determination of auxiliary diagnostic reagents that exist according to the mutation type of HCP5 gene, and the auxiliary diagnostic reagent includes specific reagents as essential components corresponding to the method for determining the mutation type of HCP5 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 the correlation between HCP5 gene polymorphism site and AS for the first time, 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 is a schematic diagram of the HCP5 gene structure and its polymorphic variation sites

Figure 2 is the genotyping map of HCP5 gene variation sites by HRM method

Figure 3 is the sequencing map of the HCP5 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 118 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.

Identification and identification of embodiment 2 SNP

The present invention adopts PCR-high resolution melting curve (HRM) analysis method and PCR sequencing technology to simultaneously carry out the genotype analysis of the +401 site (its allelic site is C/T) in the third exon region of the HCP5 gene. detection.

1. Determination of PCR-HRM primers

The DNA base sequence (Seq ID №1) near rs192776040 was retrieved from Genebank, and the primer design was completed under Oligo7.0 software. The target fragment is located in the second exon region of the HCP5 gene, with a total length of 48bp. The sense strand F1 (+671bp-+691bp) and the antisense strand R1 (+685bp-+719bp) are determined. The specific primer sequences are as follows:

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

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

2. PCR reaction system and reaction conditions

Amplify the partial fragment of exon 2 of HCP5 gene by PCR. The PCR reaction system is: 1 μL of 10×PCR reaction buffer, 0.2 μL of 10mM/LdNTP, 0.2 μL of TaqDNA polymerase, 0.1 μL 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 special 96-well plate for HRM, and perform HRM analysis on Light Scanner TMHR-I96: 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 to analyze The collected curves were analyzed to determine the genotype. Fig. 2 is the genotyping diagram of HCP5 gene variation site by HRM method.

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'-GGGAAATTACCTGGAAATGCGACTGA-3' (SEQ ID NO.8), R2: 5'-GAGACCAGCGGGTGAGAAGAGGAG-3' (SEQ ID NO.9), the amplified fragment 270bp 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. Fig. 3 is the sequencing map of the HCP5 gene variation site.

Example 3 Correlation between gene SNP and ankylosing spondylitis (AS)

1. Statistical method:

The Hardy-Weinberg balance test was used to study the group representativeness of the sample. The Pearson chi-square test in SPSS11.0 software was used to calculate the distribution frequency of the alleles and genotypes of the HCP5 gene rs192776040 site between the AS case group and the normal control group, and the AS risk OR value and its 95% CI were credible Interval, with P<0.05 as the standard of significant difference.

2. Results: The distribution of the genotype and allele frequency of the rs192776040 site on the HCP5 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 frequencies of HCP5 gene rs192776040 between case and control groups

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

It can be seen from Table 2 that the T allele of HCP5 gene rs192776040, that is, the A allele on its DNA complementary strand, has a significantly higher distribution frequency in the patient population than in a healthy normal population ( 19.9% vs. 1.7%), there was a significant difference (P=1.55×10 ¹² ), and the OR value of the T site was 3.752, 95%CI:; risk allele (CT) carriers in AS and non- Among risk allele (CC) carriers, the distribution frequency of risk genotypes in the case group was significantly higher than that in the control group (P<0.05), which indicated that the HCP5 gene rs192776040 locus was positively correlated with the prevalence of AS, which may have Increased risk of developing AS.

Implement 4 detection kits

Prepare a kit for detecting AS-related risks, including a primer pair that can amplify the rs192776040 site of the HCP5 gene, 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 rs192776040 polymorphism in the second exon region of HCP5 gene through PCR-HRM detection.

The polymorphism of the rs192776040 site in the second exon region of the HCP5 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: preparation mixture: 10×PCR reaction buffer 1μL, 10mM/LdNTP 0.2μL, TaqDNA polymerase 0.2μL, 10pM/L upstream primer 0.1μL, 10pM/L downstream primer 0.1μL, 1×LC- Green Plus saturated fluorescent dye 0.8 μL, oligonucleotide internal reference 0.2 μL (high and low temperature oligonucleotide internal reference upstream and downstream primers 0.05 μL each) (see Table 1 for sequence), genomic DNA 1 μL, 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 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 the polymorphism of the rs192776040 site polymorphism in the second exon region of HCP5 gene in AS patient samples was 6 cases of CC type, 3 cases of CT type, and 1 case of TT type; 10 cases of genotype of control group were CC type . The kit of the present invention can effectively detect the genotype of the subject's rs192776040 site in the second exon region of the HCP5 gene. When the gene is CC, the subject's susceptibility is the lowest; when carrying the T allele, the subject's susceptibility Emotional heightened.

Examples of the utility of the present invention:

The detection method of the HCP5 gene polymorphism of the present invention can be used to analyze the T allele of the rare variation site on the HCP5 gene of the human autosome 6p21.3 region, that is, the A allele on its DNA complementary strand. 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 of the rs192776040 site of the HCP5 gene, as one of the biomarkers, can be used for the screening of molecular targets for drug design, to help find active molecules that regulate HCP5 expression, and promote the development of new AS drugs.

The nucleic acid sequence and AS-related sites for detecting HCP5 gene polymorphisms established by the invention can be applied to kits for AS gene-aided diagnosis with high sensitivity and specificity.

As mentioned above, it was concluded that the polymorphism at the rs192776040 site of the HCP5 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 invention describes the new AS-related mutation site of HCP5 gene, and provides a method for determining the polymorphism of HCP gene, and according to the 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.