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Rapid detection kit for CYP2C19*2 or *3 genotype based on POCT mode

A CYP2C19 and genotyping technology, applied in the field of molecular biology, can solve problems such as environmental pollution, difficulty in meeting the requirements of rapid and accurate CYP2C19 gene detection, and unsuitability for clinical promotion.

Inactive Publication Date: 2019-01-22
重庆京因生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This open operation is likely to cause environmental pollution and affect the accuracy of the results, so it is not suitable for clinical promotion
The current conventional detection technology is difficult to meet the requirements of rapid and accurate CYP2C19 gene detection for the population carrying the CYP2C19 mutation gene in China

Method used

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  • Rapid detection kit for CYP2C19*2 or *3 genotype based on POCT mode
  • Rapid detection kit for CYP2C19*2 or *3 genotype based on POCT mode
  • Rapid detection kit for CYP2C19*2 or *3 genotype based on POCT mode

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1 Cell lysate performance test

[0111] PCR is an extremely sensitive technique for the detection of trace amounts of DNA. At present, the main samples of painless and non-invasive detection methods in clinical practice include oral swabs, hair, nails, oral saliva and body cavity fluid, etc. After the contained DNA is extracted and purified, it can be added to the PCR reaction system for reaction, which requires a lot of manpower, material resources, and financial resources. In order to solve this problem, and considering that the amplification effect after directly adding cells is not ideal, and it is not suitable for clinical testing, the inventors tried to add a certain concentration of cell lysate. And the comparative experiment of adding cell lysate and not adding cell lysate was carried out. (Note: Exfoliated cells are used as templates)

[0112] The reagent formula is shown in Table 1-1 and Table 1-2. 100 replicates were prepared for each of the three ...

Embodiment 2

[0146] Example 2 LNA modified probe improves typing accuracy

[0147] LNA is an oligonucleotide derivative that has a similar structure to DNA / RNA, so it can effectively recognize and bind DNA and RNA. After LNA is used to modify oligonucleotides, it can increase the thermal stability of primers or probes and increase their annealing temperature by 3-8°C. The probes developed in this kit are modified with LNA. According to the software prediction, the Tm values ​​of the modified wild-type probes and mutant probes combined with the template are all increased by about 4°C. In order to fully demonstrate the difference between LNA-modified probes and non-LNA-modified probes, the following comparative experiments were carried out. The PCR systems are shown in Table 2-1 and Table 2-2, respectively, to detect wild homozygotes, heterozygotes and mutant homozygotes, each Three replicates were performed for each genotype, and the reaction procedures are shown in Table 1.

[0148] The ...

Embodiment 3

[0188] Example 3 Primer Probe Optimum Ratio Optimization Experiment

[0189] After the specific primers and probes are screened and confirmed, the concentration of primers and probes in the PCR reaction system needs to be optimized (using oral cells as templates). The PCR reaction system corresponding to the primer and probe concentration is shown in Table 3-1.

[0190] Table 3-1 CYP2C19*2 Site Primer Probe Concentration Experiment PCR Reaction Overall System Formula Table

[0191]

[0192]

[0193] Table 3-2 CYP2C19*3 Site Primer Probe Concentration Experiment PCR Reaction Overall System Formula Table

[0194]

[0195] The final concentrations of sodium lauryl sulfate and polyethylene glycol octylphenyl ether in the above PCR reaction system are 0.005% w / v and 0.01% w / v.

[0196] Experimental results:

[0197] 1. CYP2C19*2

[0198] (1) Primer concentration gradient experiment (concentration one, concentration two, concentration three)

[0199] a. The statistical...

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Abstract

The invention provides a rapid detection kit for CYP2C19*2 or *3 genotype based on a POCT mode. The kit contains at least a fluorescent quantitative PCR primer and probe for detecting a CYP2C19*2 (rs4244285, SEQ ID NO: 1-4) or CYP2C19*3 (rs4986893, SEQ ID NO: 5-8) genotype of and a cell lysate. In addition, the kit may comprise dNTPs, DNA polymerase, Mg<2+>, a reaction buffer, a standard positivetemplate, a sampling rod, a sample collection tube and the like. The kit can realize real-time detection without DNA purification. A sample can be directly added into a reagent for a PCR reaction, thekit is especially suitable for rapid accurate detection of samples with low DNA content (such as oral exfoliated cells), and the detection accuracy is 99% or more. The detection sensitivity is high,and the detection limit of genomic DNA as low as 0.125 ng can be accurately detected. The whole detection takes a short time, and a detection result can be obtained within one hour and provides a medicine use basis for a doctor at the first time, thereby reducing the risk of medicine administration.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a POCT mode-based rapid detection kit for CYP2C19*2 or *3 genotype. Background technique [0002] Cytochrome P450 (Cytochrome P450) isozymes are a superfamily composed of a series of structurally and functionally related enzymes, including CYP1, 2, and 3 families. CYP2C19 is an important enzyme of the CYP2C family, which plays an important role in drug metabolism in vivo. [0003] The CYP2C19 gene has genetic diversity, and there are differences in enzyme activity among different individuals, resulting in large differences in the metabolism of drugs among different individuals, resulting in three phenotypes in the population: fast metabolizer, intermediate metabolizer and slow metabolizer. CYP2C19 gene polymorphism is mainly caused by two mutations: one mutation reduces enzyme activity; one mutation enhances enzyme activity. Among them, CYP2C19*2 (681G>A, rs4244285) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 贺庭祯罗德朋黎帮勇向霄熊伟钟越刘黎董锐崔奇新杨园
Owner 重庆京因生物科技有限责任公司
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