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An HLA-B and allele technology, applied in the field of alleles, can solve the problems of inappropriate use, time-consuming and labor-intensive, low-throughput, etc., and achieve the effects of simple operation, high detection accuracy and high sensitivity
Inactive Publication Date: 2018-05-01
江苏美因康生物科技有限公司
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[0004] At present, the methods for detecting HLA-B*5801 alleles at home and abroad mainly include DNA direct sequencing and Taqman probe method. DNA direct sequencing is the current gold standard for allele detection, but it is time-consuming and laborious. , is a low-throughput detection method that is not suitable for a large number of samples; Taqman is a highly specific quantitative PCR technology, which is characterized by being suitable for the detection of a small number of alleles in large samples, and is a medium-throughput method. Allele detection, but for the simultaneous detection of multiple allele sites, the cost is high and it is not suitable for use
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Embodiment 1
[0046] Detection of human HLA-B*5801 allele
[0047] Follow the steps below:
[0048] (1) Genomic DNA extraction from human whole blood
[0049] Extract genomic DNA from whole blood with a DNA extraction kit, and adjust the concentration to 20-50ng / μl;
[0050] (2) Multiplex PCR reaction
[0051] (a) Find the target gene sequence, design and synthesize PCR primers for the mutation site,
[0052] (b) Prepare a 384-well reaction table based on the extracted sample, and indicate the primers used for the number of the corresponding DNA sample in each well,
[0053] (c) According to the table, add 1 μl of DNA template to each well of the 384-well plate, stick to the membrane, centrifuge at 2000 rpm for 10 seconds, and set aside.
[0054] (d) Prepare the PCR reaction solution according to the following table: (take 384 samples as an example)
[0055]
[0056] Note: The above 384-well reaction solution has 5% excess,
[0057] (e) Take a row of 12 tubes, add 133μl of prepared P...
Embodiment 2
[0086] Detection of 29 SNP sites of HLA-B*5801 allele in 10 patients with gout by time-of-flight mass spectrometry
[0087] (1) Genomic DNA extraction from human whole blood
[0088] Extract genomic DNA from whole blood with a DNA extraction kit, and adjust the concentration to 20-50ng / μl;
[0089] (2) Multiplex PCR reaction
[0090] (a) Find the target gene sequence, design and synthesize PCR primers for the allelic site information, and simultaneously detect 29 HLA-B*5801 alleles in the HLA-B gene. The designed primers are shown in the table below,
[0091] The following table shows the primers used for genotype detection of 29 sites in the HLA-B*5801 allele and the single-base extensions produced:
[0095] (b) Prepare a 384-well reaction table based on the extracted samples, indicating the number of the DNA sample corresponding to each well, the primers used...
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Abstract
The invention discloses a method for detecting human HLA-B*5801 alleles. The method comprises the following steps: 1) designing a sequence-specific primer to search a DNA sequence of a target gene; screening out proper primer pairs in accordance with detection sites of the target gene; and combining the various sites, so that various PCR reaction systems are defined; 2) extracting genome DNA of human peripheral blood cell samples, and conducting amplification via primers which are screened out; 3) conducting SAP treatment on amplification PCR products, implementing single base extension and conducting purifying, so that sample analytes are obtained; and 4) applying the sample analytes to a chip matrix, and implementing analysis via matrix-assisted laser desorption ionization time-of-flight, so that sample molecular weights are obtained; and detecting out corresponding site information of the alleles, thus completing genetic typing analysis. The method provided by the invention has theadvantages that 384 samples can undergo multiple detection via one chip, each system can accept 30 multiples of reactions to the greatest extent, and multiple HLA-B*5801 allele sites can be simultaneously detected.
Description
technical field [0001] The invention relates to a method for detecting human HLA-B*5801 alleles, in particular to a matrix-assisted laser desorption ionization time-of-flight mass spectrometry method for detecting human HLA-B*5801 genes, belonging to pharmacogenomics and gene diagnosis field. Background technique [0002] Human leukocyte antigen (HLA) is the gene complex with the highest allelic polymorphism among known genes so far. Nearly 3000 alleles have been found in the world, most of which are very rare. This gene is by far the most complex genetic polymorphism system in humans, and its polymorphism distribution has obvious ethnic characteristics. Studies on Han people from different countries and regions have shown that there is a strong correlation between the HLA-B*5801 gene and the severe skin adverse reactions caused by the drug allopurinol. In the non-Han population, the correlation between the drug and it is very low. [0003] Gout is a metabolic disease cau...
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