One group of primer for detecting SNP (single nucleotide polymorphism) site rs9263726, crRNA (CRISPR (clustered regularly interspaced short palindromic repeat sequences) RNA) sequence and application of primer

A technology of rs9263726 and loci, applied in the field of genetic testing, can solve problems such as difficult SNP types for operators, insignificant allelic differences, difficult allele types, etc., to achieve high specificity of detection results, easy judgment, fast effect

Active Publication Date: 2020-03-27
广州白云山拜迪生物医药有限公司
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Such a method shows a certain specificity in double-tube detection, but in some cases, especially in the presence of heterozygotes, the difference in detecting different alleles (alleles) is still not significant, and it is difficult to intuitively judge the detection of samples, etc. allele type
The second method is mainly used for multiple detection of different genes, but it is difficult to implement at the SNP site (wild type/mutant type)
In addition to the interference of the specific response due to the high homology be...

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  • One group of primer for detecting SNP (single nucleotide polymorphism) site rs9263726, crRNA (CRISPR (clustered regularly interspaced short palindromic repeat sequences) RNA) sequence and application of primer
  • One group of primer for detecting SNP (single nucleotide polymorphism) site rs9263726, crRNA (CRISPR (clustered regularly interspaced short palindromic repeat sequences) RNA) sequence and application of primer
  • One group of primer for detecting SNP (single nucleotide polymorphism) site rs9263726, crRNA (CRISPR (clustered regularly interspaced short palindromic repeat sequences) RNA) sequence and application of primer

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Embodiment 1

[0023] Embodiment 1 synthetic RPA primer

[0024] Specific RPA amplification primers were designed according to the sequence adjacent to the SNP site rs9263726 of the HLA-B*5801 gene, as described below. RPA primers were dissolved in RNase-free purified water to form a stock solution (24 μM), and stored at -20°C after aliquoting.

[0025] (1) rs9263726 wild-type fragment RPA amplification template

[0026]

[0027] (2) rs9263726 mutant RPA amplification template

[0028] Note: The box is the SNP site; the underline is the complementary sequence of the RPA primer

[0029] (3) Entrust Guangzhou Bolais Company to synthesize RPA primers according to the above sequences, and the specific primer sequences are shown in Table 1. The synthesized RPA primers were dissolved in RNase-free purified water to form a stock solution (24 μM), and stored at -20°C after aliquoting.

[0030] Table 1: RPA primer sequences for amplifying SNP site rs9263726

[0031]

Embodiment 2

[0032] Embodiment 2, synthetic crRNA

[0033] (1) Entrust Guangzhou Bolaisi Company to synthesize crRNA according to the above sequence, and the sequence information of crRNAs is shown in Table 2. The synthesized crRNA was dissolved in RNase-free purified water to form a 10X stock solution (225nM), and stored at -20°C after aliquoting.

[0034] Table 2: Sequences of synthetic CrRNAs

[0035]

[0036] (2) The base pairing information between CrRNAs and target RNA is as follows:

[0037]

Embodiment 3

[0038] Example 3. Synthesis of ssRNA reporter probes used by the Cas13 enzyme of the present invention: commissioned Guangzhou Bolaisi to synthesize two ssRNA reporter probes. See Table 3 for sequence specific information. The synthesized ssRNA reporter probes were dissolved in RNase-free purified water to form a stock solution (1.25 μM), and stored at -20°C after aliquoting.

[0039] Table 3: Specific sequence information of reporter probes used by Cas13 protease

[0040]

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Abstract

The invention provides a system for detecting the SNP (single nucleotide polymorphism) site rs9263726 of a HLA-B*5801 gene. The system comprises a primer for detecting the SNP site rs9263726, two (CRISPR (clustered regularly interspaced short palindromic repeat sequences) RNA) sequences, two different CRISPR/cas13 protein systems and reporter probes with different fluorescent marks. When the system disclosed by the invention is used for detecting a mutation type of the SNP site rs9263726 of the HLA-B*5801 gene, the system is convenient in operation, and each detection sample only needs to be carried out in a single tube; a whole reaction process is carried out at a temperature of about 37 DEG C instead of a delicate temperature control element and complex temperature change just like PCR (Polymerase Chain Reaction) amplification, and therefore, the system is suitable for grassroots units to carry out detection by relatively cheap instant detection equipment; and compared with a PCR technology, the system has a higher speed, can obtain an obvious detection signal in 30 min, is high in detection result specificity and can easily judge the type of an allele.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a group of amplification primers for detecting PSORS1C1 gene polymorphic site rs9263726 and application thereof. 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 correlation between the HLA-B*5801 allele and 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). Studies have shown that there is a strong correlation between the HLA-B*5801 allele and the severe dermatitis side effects caused by allopu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156Y02A50/30
Inventor 丘力功李润明符美娟赵海莲丁妹元
Owner 广州白云山拜迪生物医药有限公司
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