Visualized CYP2C19 genotyping detection kit

A technology of CYP2C19 and detection kit, which is applied in the field of molecular biotechnology and genetic detection, can solve the problems of high price and achieve the effects of reducing toxic and side effects, avoiding cross-contamination, and distinguishing characteristics

Inactive Publication Date: 2016-11-30
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

[0004] At present, the existing CYP2C19 genotyping detection kits on the market are basically PCR amplification technology based on fluorescent signal detection, which require real-time fluorescent PCR equipment or other equipment, which are relatively expensive and are not conducive to medical treatment in poor facilities. unit use
At the same time, in clinical practice, there is no closed-tube method that can directly use ordinary PCR cycle instruments to detect CYP2C19 genotyping.

Method used

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  • Visualized CYP2C19 genotyping detection kit
  • Visualized CYP2C19 genotyping detection kit
  • Visualized CYP2C19 genotyping detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Primer Probe

[0025] The corresponding primers and probes are designed according to the specificity of the CYP2C19 gene *2 and *3 sites respectively, and the detection results are determined by two pairs of specific primers and probes for the *2 sites and *3 sites respectively, and the primers The rationale for the location and method of design as figure 1 shown.

[0026] The CYP2C19 genotyping detection kit includes 4 primers, 7 conventional probes, 2 nano-gold modified probes, a reaction solution, and an enzyme solution.

[0027] Primer, probe and nano gold probe sequence such as primer 1-primer 4, probe 1-probe 7, nano gold probe 1-nano gold probe 2, or the complementary sequence of the above sequence:

[0028] Primer 1: CCA GAG CTT GGC ATA TTG TAT CTA TAC CTT TAT TAA ATG C; SEQ ID NO.1

[0029] Primer 2: CCA TCG ATT CTT GGT GTT CTT TTA CTT TCT CCA A; SEQ ID NO.2

[0030] Primer 3: TCT GCT CCA TTA TTT TCC AGA AAC GTT TCG A; SEQ ID NO.3

[0031] Primer...

Embodiment 2

[0045] Example 2 CYP2C19 gene *2 and *3 site typing detection sensitivity

[0046] In this example, the CYP2C19 genotyping detection kit described in Example 1 was used to detect different concentrations of CYP2C19 gene *2 and *3 site typing templates to verify the visual detection method stated in the present invention feasibility.

[0047] Reaction conditions:

[0048] The detection of each sample in the kit consists of 4 tubes of reaction, respectively containing primers and probes for the four genotypes, and the total volume of each tube reaction is 20 μL. CYP2C19 gene *2 and *3 site detection reaction system consists of: 10mM Tris buffer (pH 8.5), 1μM primer (primer 1 sequence: 5'-CCA GAG CTT GGC ATA TTG TAT CTA TAC CTT TAT TAAATG C-3' , SEQ ID NO.1; Primer 2 sequence: 5'-CCA TCG ATT CTT GGT GTT CTT TTA CTT TCTCCAA-3', SEQ ID NO.2; Primer 3 sequence: 5'-TCT GCT CCA TTA TTT TCC AGA AAC GTT TCGA-3', SEQ ID NO.3; Primer 4 sequence: 5'-C ACT GTA AGT GGT TTC TCA GGA AGC AAA...

Embodiment 3

[0050] Example 3 CYP2C19 gene *2 and *3 site typing detection matching genotype sample detection

[0051] In this example, the CYP2C19 genotyping detection kit described in Example 1 was used to detect the genomic DNA templates of different types of peripheral blood samples, and there were 3 samples, and the typing results were: *1*1 , *1*1, *1*2 are used to verify the feasibility of the visual detection method stated in the present invention to detect actual samples.

[0052] Reaction conditions:

[0053]The detection of each sample in the kit consists of 4 tubes of reaction, respectively containing primers and probes for the four genotypes, and the total volume of each tube reaction is 20 μL. CYP2C19 gene *2 and *3 site detection reaction system consists of: 10mM Tris buffer (pH 8.5), 1μM primer (primer 1 sequence: 5'-CCAGAG CTT GGC ATA TTG TAT CTA TAC CTT TAT TAAATG C-3', SEQ ID NO.1; Primer 2 sequence: 5'-CCA TCG ATT CTT GGT GTT CTT TTA CTT TCT CCAA-3', SEQ ID NO.2; Prim...

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Abstract

The invention relates to a CYP2C19 genotyping detection kit, which comprises 4 primers, 7 conventional probes, and 2 probes connected with nano gold particles. The primers are SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4; the conventional probes are SEQ ID NO.5 to SEQ ID NO. 11, or reverse complement sequences of SEQ ID NO. 5 to SEQ ID NO. 11; the base composition of the probes linked to the gold nanoparticle is as shown in SEQ ID NO. 12 to SEQ ID NO. 13, or reverse complement sequences of SEQ ID NO. 12 to SEQ ID NO. 13. The CYP2C19 genotyping detection kit can realize visualized, low-cost, and highly sensitive CYP2C19 genotyping assays.

Description

technical field [0001] The invention belongs to the fields of molecular biotechnology and gene detection, in particular to a CYP2C19 genotyping detection kit. Background technique [0002] The drug-metabolizing enzyme encoded by the CYP2C19 gene is an important member of the cytochrome P450 isoenzyme and one of the important enzymes involved in drug metabolism in vivo. Due to genetic polymorphisms, the enzyme metabolic activity encoded by the CYP2C19 gene has significant differences among different individuals. There are at least 18 gene polymorphisms in the CYP2C19 gene, among which c.681G>A and c.636G>A are the two most common allelic types in the Chinese population. These mutations can cause the enzymatic activity part of the CYP2C19 gene Loss, resulting in the weakening of its ability to metabolize substrates, thus causing individual differences in the metabolic rate of related drugs, and ultimately leading to individual differences in the efficacy of related drug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/6858C12Q2600/106C12Q2600/156C12Q2521/101C12Q2521/301C12Q2563/137
Inventor 王建平
Owner 广州市宝创生物技术有限公司
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