Detecting reagent predicting susceptivity of ankylosing spondylitis
A technique for ankylosing spondylitis and susceptibility, which can be used in the determination/examination of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., and can solve the problem that TNXB gene has not been reported yet.
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Embodiment 1
[0041] Example 1: Collection of blood samples and extraction of genomic DNA:
[0042] 1. Case selection:
[0043] The selected cases were selected according to the revised diagnostic criteria in New York in 1984. A total of 275 unrelated AS patients (age: 8-71 years old, average 27 years old) from Ningxia area were selected, and 260 healthy control volunteers (age: 18 years old) from the same area were selected. -21 years old, average 19 years old). 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. .
[0044] 2. According to the following method, human genomic DNA was prepared.
[0045] 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 stan...
Embodiment 2
[0053] Embodiment 2: identification and identification of SNP
[0054] The present invention adopts PCR-high resolution melting curve (HRM) analysis method and PCR sequencing technology to simultaneously detect the genotype at position 32038541 (the allelic site is G / A) of the intron region of TNXB gene. figure 2 It is the genotyping map of the TNXB gene variation site by the HRM method, image 3 It is the sequencing map of TNXB gene mutation site.
[0055] 1. Determination of PCR-HRM primers
[0056] The DNA base sequence (SEQ ID No.1) near the position 32038541 was retrieved from Genebank, and the primer design was completed under Oligo7.0 software. The target fragment is located in the intron region of the TNXBB gene, the full length is 51bp, and the specific primer sequence is as follows:
[0057] F1: 5'-TTTCCGGCCAAATCACAGCCAAATC-3' (SEQ ID No. 2)
[0058] R1: 5'-TTTTTTCCTCTTACCCAGGAGCACA-3' (SEQ ID No. 3)
[0059] 2. PCR reaction system and reaction conditions
[0...
Embodiment 3
[0067] Embodiment 3: the correlation of gene SNP and ankylosing spondylitis (AS)
[0068] 1. Statistical method:
[0069] 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 in the intron region 32038541 of the TNXB gene between the AS case group and the normal control group, the AS risk OR value and its 95% CI confidence interval, with P<0.05 as the standard of significant difference.
[0070] 2. Results:
[0071] The distribution of the genotype and allele frequency of the intron region 32038541 of the TNXB gene located in the 6p21.3 region between the cases and the control group is shown in Table 2.
[0072] Table 2 The distribution of genotype and allele frequency at position 32038541 in the intron region of TNXB gene between cases and control groups
[0073]
[0074] Note: OR: odds ra...
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