Visualized HLA-B*5801 genotyping detection kit

A detection kit and genotyping technology, applied in the field of molecular biotechnology and genetic detection, can solve the problems of high price and achieve the effects of low cost, strong applicability, and stable storage conditions

Active Publication Date: 2017-05-24
广州市宝创生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the existing HLA-B*5801 allele typing detection kits on the market are basically PCR amplification technology based on fluorescent signal detection, which requires real-time fluorescent PCR equipment, which is relatively expensive and is not conducive to facility conditions. poor medical unit use
At the same time, in clinical practice, so far there is no closed-tube method that can directly use ordinary PCR cycle instruments to achieve visual typing of HLA-B*5801 alleles

Method used

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  • Visualized HLA-B*5801 genotyping detection kit
  • Visualized HLA-B*5801 genotyping detection kit
  • Visualized HLA-B*5801 genotyping detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Primer Probe

[0027] The corresponding primers and probes are specifically designed according to the SNP sites of the HLA-B*5801 gene, and the detection results are determined by a pair of specific primers and probes for the sites, and the position and method of the primer design principle such as figure 1 shown.

[0028] The HLA-B*5801 genotyping detection kit includes 2 primers, 4 conventional probes, 2 nano-gold modified probes, a reaction solution, and an enzyme solution.

[0029] Primer, probe and nano gold probe sequence such as primer 1-primer 2, probe 1-probe 4, nano gold probe 1-nano gold probe 2, or the complementary sequence of the above sequence:

[0030] Primer 1: GGA CCC CAG CTC CTT AAC A; SEQ ID NO.1

[0031] Primer 2: CCA TGT GGC AAA GTC GG; SEQ ID NO.2

[0032] Probe 1: CGC GCC GAG GGT CCC CCC C; SEQ ID NO.3

[0033] Probe 2: CGC GCC GAG GAT CCC CCC C; SEQ ID NO.4

[0034] Probe 3: AGA GTT TCC TCG GAG; SEQ ID NO.5

[0035] Probe 4: GTC...

Embodiment 2

[0042] Example 2 HLA-B*5801 gene SNP site typing detection sensitivity

[0043] In this embodiment, the HLA-B*5801 genotyping detection kit described in Example 1 is used to detect the templates of the SNP site typing of the HLA-B*5801 gene at different concentrations, which is used to verify the statement of the present invention The feasibility of the visual detection method.

[0044] Reaction conditions:

[0045] The detection of each sample in the kit consists of 2 tubes of reaction, which respectively contain primers and probes for two genotypes, and the total volume of each tube reaction is 20 μL. The HLA-B*5801 gene SNP site detection reaction system consists of: 10mM Tris buffer (pH 8.5), 1 μM primer (primer 1 sequence: SEQ ID NO.1; primer 2 sequence: SEQ ID NO.2), 1 μM probe Needle (probe 1 sequence: SEQ ID NO.3; probe 2 sequence: SEQ ID NO.4; probe 3 sequence: SEQ ID NO.5; probe 4 sequence: SEQ ID NO.6), 0.1μM nano-gold probe Needle (probe 1: SEQ ID NO.7; probe 2:...

Embodiment 3

[0047] Example 3 HLA-B*5801 gene SNP site typing detection matching genotype sample detection

[0048] In this example, the HLA-B*5801 genotyping detection kit described in Example 1 was used to detect the genomic DNA templates of peripheral blood samples of different types, and the typing results were: mutant heterozygous type (sample 1 ), wild homozygous type (sample 2), mutant homozygous type (sample 3), used to verify the feasibility of the visual detection method stated in the present invention to detect actual samples.

[0049] Reaction conditions:

[0050] The detection of each sample in the kit consists of 2 tubes of reaction, which respectively contain primers and probes for two genotypes, and the total volume of each tube reaction is 20 μL. The HLA-B*5801 gene SNP site detection reaction system consists of: 10mM Tris buffer (pH 8.5), 1 μM primer (primer 1 sequence: SEQ ID NO.1; primer 2 sequence: SEQ ID NO.2), 1 μM probe Needle (probe 1 sequence: SEQ ID NO.3; probe...

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Abstract

The invention relates to an HLA-B*5801 genotyping detection kit. The kit comprises two primers, four conventional probes, and two probes connected with gold nanoparticles. The primers are shown in SEQ ID NO.1 and SEQ ID NO.2. The conventional probes are shown in SEQ ID NO.3 to SEQ ID NO.6 or reverse complementary sequences of SEQ ID NO.3 to SEQ ID NO.6. The base compositions of the probes connected with the gold nanoparticles are shown in SEQ ID NO.7 and SEQ ID NO.8 or reverse complementary sequences of SEQ ID NO.7 and SEQ ID NO.8. The HLA-B*5801 genotyping detection kit can achieve visualized, low-cost and high-sensitivity HLA-B*5801 genotyping detection.

Description

technical field [0001] The invention belongs to the field of molecular biotechnology and gene detection, in particular to an HLA-B*5801 genotyping detection kit. Background technique [0002] Allopurinol is the first-line drug for the treatment of gout and is currently the only xanthine oxidase inhibitor. The drug can cause serious side effects, including Stevens-John syndrome (SJS) and toxic epidermal necrosis (TEN). The strong association of the HLA-B*5801 allele with allopurinol-induced SJS or TEN was first discovered in the Han population by scholars in Taiwan, China, and confirmed by multiple research groups in Thailand, Singapore, Hong Kong, and China in the Han population , the HLA-B*5801 allele frequency in the Han people is 9-15% (geographical differences). [0003] Studies have shown that there is a strong correlation between the HLA-B*5801 allele and the severe dermatitis side effects caused by allopurinol, especially in the Han people, as high as 100%, and almo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 王建平潘腾飞
Owner 广州市宝创生物技术有限公司
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