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Rapid detection kit for CYP2C9 and VKORC1 genotype based on POCT mode

A kit and gene technology, applied in the field of molecular biology, can solve problems such as time-consuming, difficult to achieve, and unsatisfactory 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

Therefore, the cost of these two detection methods is high, the steps are cumbersome and time-consuming, and it is difficult to promote them clinically.
Although the PCR-RFLP method reduces the cost of instruments and reagents in the sequencing method and gene chip method, its operation is complicated and there are many steps, and it is not suitable for clinical use.
The Chinese patent application whose publication number is CN107447035A adopts the PCR-fluorescent probe method, although the cost of instruments and reagents in the sequencing method and gene chip method is reduced, but because the reaction system can only amplify the purified genomic DNA sample, so Multi-step multi-tube operation must be adopted, which is not ideal for clinical promotion
[0008] Therefore, these technologies are difficult to solve the urgent problem of clinical detection of warfarin drug users, and it is difficult to realize the fast and accurate requirements in the POCT mode

Method used

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  • Rapid detection kit for CYP2C9 and VKORC1 genotype based on POCT mode
  • Rapid detection kit for CYP2C9 and VKORC1 genotype based on POCT mode
  • Rapid detection kit for CYP2C9 and VKORC1 genotype based on POCT mode

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Configuration of PCR reaction solution for detecting CYP2C9 and VKORC1 genes

[0087] The PCR reaction solution prepared in this example is a 23.5 μL system, and the raw materials and their final concentrations are as follows:

[0088] DNA polymerase 0.05U / μL;

[0089] dNTPs 0.2mM;

[0090] 5×Reaction buffer 1.1×;

[0091] MgCl 2 2.5mM;

[0092] Cell lysate 1×;

[0093] Upstream primer 0.5μM;

[0094] Downstream primer 0.5μM;

[0095] Mutant probe 0.5μM;

[0096] Wild type probe 0.5μM;

[0097] Make up the ultrapure water to 23.5μL.

[0098] Among them, the reaction buffer is Colorless Reaction Buffer; dNTPs, MgCl 2 , 5x Colorless Reaction Buffer, DNA polymerase using GoTaq DNA Polymerase were all purchased from Shanghai Promega Biological Products Co., Ltd.

[0099] The cell lysate is composed of sodium lauryl sulfate and polyethylene glycol octyl phenyl ether;

[0100] Wherein, the concentration of sodium lauryl sulfate is 0.0005 to 0.015% (w / v); the concentration of polyethy...

Embodiment 2

[0101] Example 2 LNA modified probe improves typing accuracy

[0102] LNA is an oligonucleotide derivative, which 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 the annealing temperature by 3 to 8°C. The probes developed in this kit are modified with LNA. According to the software prediction, the Tm value of the modified wild-type probe and the mutant probe combined with the template is increased by about 3°C. In order to prove whether the difference between the LNA modified probe and the unmodified probe can improve the typing success rate, we conducted the following comparative experiment. The PCR reaction system is shown in Table 2-1. Three genotypes are detected respectively, and each genotype is repeated in three groups. The reaction procedure is shown in Table 1.

[0103] Table 1

[0104]

[0105] The probe primer s...

Embodiment 3

[0140] Example 3 Accuracy Test

[0141] This kit uses oral mucosal exfoliated cells as the amplified sample, but the living habits and gene sequences of the subjects are diverse, which may interfere with the reagents. Therefore, in this experiment, the reagents were tested in large quantities by expanding the number of sampling personnel to verify the accuracy of reagent detection. We use sterile swabs to collect oral mucosal exfoliated cells from three different genotypes for testing (the genotypes of the testers are confirmed by sequencing), and the sampling method refers to the attachment figure 1 Operation process, the total number of samples is 600, and the reagent formula is shown in Table 3-1 below.

[0142] Table 3-1. Formulation of PCR reaction system

[0143]

[0144] Experimental results:

[0145] Table 3-2. Statistics of accuracy of the three genotypes

[0146]

[0147]

[0148] According to Table 3-2, the detection accuracy of the reagents at the two sites of CYP2C9*3 and...

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Abstract

The present invention provides a rapid detection kit for CYP2C9 and VKORC1 genotypes based on a POCT mode. The kit contains a fluorescent quantitative PCR primer and probe for detecting CYP2C9 and VKORC1 genotypes, and a cell lysate, wherein the sequence of the PCR primer is shown as SEQ ID NO.1-2 and SEQ ID NO.5-6, and the sequence of the probe is shown as SEQ ID NO.3-4 and SEQ ID NO.7-8. The kitcan realize real-time detection without DNA purification. A sample can be directly added into a reagent for a PCR reaction, the kit is especially suitable for rapid accurate detection of samples withlow 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 riskof medicine administration.

Description

Technical field [0001] The invention relates to the field of molecular biology, in particular to a rapid detection kit for CYP2C9 and VKORC1 genotypes based on a POCT model. Background technique [0002] Warfarin is a coumarin-based oral anticoagulant that is currently widely used clinically. It has the dual effects of anticoagulation and thrombolysis. Warfarin can be used for anticoagulant treatment of patients after mechanical heart valve replacement, non-valvular atrial fibrillation and deep vein thrombosis. However, warfarin has a narrow range of anticoagulant therapeutic index. If the dose is insufficient to achieve the therapeutic effect, a slight overdose will cause bleeding, which can endanger the life of the patient. At the same time, there are obvious individual differences in the plasma concentration and efficacy of warfarin, even if the same individual requires different doses in different periods. The dose of warfarin is affected by genetic polymorphisms. Studies ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2531/113C12Q2563/107C12Q2561/101
Inventor 罗德朋贺庭祯向霄熊伟黎帮勇钟越刘黎崔奇新董锐
Owner 重庆京因生物科技有限责任公司
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