Screening kit for detecting high myopia
A high myopia, kit technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem of not finding high myopia causative genes and other problems
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Embodiment 1
[0042] Example 1 Sample collection and extraction of genomic DNA
[0043] The samples are cases collected by the National Natural Science Foundation of China (project number: 30900809, 30871350) undertaken by the Sichuan Provincial Key Laboratory of Human Disease Gene Research, Sichuan Academy of Medical Sciences Sichuan Provincial People's Hospital. A total of 697 cases of high myopia patients were collected, with an average age of 39.7±11.3 years, of which 38.6% were male, and 1086 cases of controls (normal controls, the inclusion criteria were: over 40 years old, long-term close work, diopter>-1.0D.), The mean age was 62.7±10.2 years old, and 37.7% of them were male. All subjects were of Han nationality and signed informed consent, and this study was also approved by the ethics committee.
[0044] Genomic DNA was prepared from human peripheral blood according to the following method. In the presence of anticoagulant EDTA, 5 ml of human peripheral blood collected was centr...
Embodiment 2
[0045] Example 2 PCR amplification and genotype analysis of rs9510902, rs9318086, rs3794338, rs7325450 and rs7331047 single nucleotide polymorphism DNA fragments
[0046] The present invention adopts the Snapshot genotyping technology to detect five sites of rs9510902, rs9318086, rs3794338, rs7325450 and rs7331047.
[0047] The experimental steps are as follows:
[0048] 1. PCR: Denaturation at 95°C for 5 minutes; 35 cycles at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 45 seconds; extension at 72°C for 7 minutes, and incubation at 4°C.
[0049] 2. Purification 1: Take 1-3 μl of the PCR products in step 1 (depending on the number of PCRs to be mixed, the total amount is 9 μl, add 3 μl of the mixed solution (SAP 2 μl, 1:10 Exol enzyme 1 μl). 1:10 Exol enzyme Preparation: 1 volume Exol enzyme, 1 volume SAP 10Xbuffer, 8 volumes ddH 2 O. Program: 37°C for 1h, 75°C for 15min.
[0050] 3. Snapshot reaction: Snapshot MULTIPLEX 1μl, purified PCR mi...
Embodiment 3
[0101] Correlation between embodiment 3 SNP loci and high myopia
[0102] Statistical methods: SPSS13.0 software was used for analysis, and the Hardy-Weinberg equilibrium test was tested. Quantitative variables were tested by t test, and quality variables were tested by chi-square test. Two-sided P<0.05 was considered statistically significant. The result is as follows:
[0103] 1) Basic clinical data of cases and controls
[0104]
[0105] Note: **, P<0.01.
[0106] 2) SNP polymorphisms are closely related to high myopia
[0107] rs9510902:
[0108]
[0109] Without considering the traditional risk factors of high myopia, the A allele can significantly increase the risk of high myopia (OR=1.36, 95% CI=1.19-1.55), that is, the risk of high myopia increases by 1.36 times, and The lower limit of its 95% CI is 1.19>1, and it can be determined that the A allele of rs9510902 is a risk factor; the homozygous carrier (AA) will increase the risk of the disease by 1.87 times...
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