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SLCO1B1 genetype rapid detection kit based on POCT mode

A technology of SLCO1B1T521C and a kit, which is applied in the field of molecular biology, can solve the problems of complex operation, difficulty in solving the urgent problem of statin drug users, and numerous steps

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

Although this method is low in cost, short in time and simple in result analysis, it needs to purify the patient's genomic DNA and use the DNA as a template for typing detection.
[0008] These technologies are difficult to solve the urgent problem of clinical detection of statin drug users, and it is difficult to realize the POCT mode

Method used

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  • SLCO1B1 genetype rapid detection kit based on POCT mode
  • SLCO1B1 genetype rapid detection kit based on POCT mode
  • SLCO1B1 genetype rapid detection kit based on POCT mode

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1 is used to detect the configuration of the PCR reaction solution of SLCO1B1 gene

[0085] 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:

[0086] DNA polymerase 0.05U / μL;

[0087] dNTPs 0.2mM;

[0088] 5× Reaction Buffer 1.1×;

[0089] MgCl 2 2.5mM;

[0090] Cell lysate 1×

[0091] Upstream primer 0.5μM;

[0092] Downstream primer 0.5μM;

[0093] Mutant probe 0.5μM;

[0094] Wild-type probe 0.5μM;

[0095] Make up to 23.5 μL with ultrapure water.

[0096] Among them, the reaction buffer is Colorless Reaction Buffer; dNTPs, MgCl 2 , 5x ColorlessReaction Buffer, and DNA polymerase using GoTaq DNA Polymerase were all purchased from Shanghai Promega Biological Products Co., Ltd.

[0097] The cell lysate is composed of sodium lauryl sulfate and polyethylene glycol octylphenyl ether;

[0098] Wherein, the concentration of sodium lauryl sulfate is 0.0005-0.01...

Embodiment 2

[0099] Example 2 LNA modified probe improves typing accuracy

[0100] LNA is an oligonucleotide derivative that has a similar structure to DNA / RNA, so it can recognize and bind DNA and RNA powerfully. 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 system is shown in Table 2-1, and wild homozygotes, heterozygotes and mutant homozygotes were detected respectively, and three sets were made for each genotype. Repeat, see Table 1 for the reaction procedure.

[0101] Table 1

[0102]

[0103...

Embodiment 3

[0132] Example 3 Primer probe concentration optimization experiment

[0133] Table 3-1 Primer probe concentration determination experiment PCR reaction total system formula table

[0134]

[0135] Experimental results:

[0136] 1) Ct value statistics:

[0137] Table 3-2 Statistics on the average Ct value of each formulation at the T521C / A388G site

[0138]

[0139] Note: "-" indicates no Ct value.

[0140] 2) End point fluorescence value (EPF) statistics:

[0141] Table 3-3 Statistics of the mean value of endpoint fluorescence value (EPF) of each formulation at T521C / A388G site

[0142]

[0143]

[0144] In this example, the reagents at the two sites of SLCO1B1T521C / A388G were measured. Under the conditions of probe concentrations of 0.5 μM for wild probes and 0.5 μM for mutant probes, the typing and amplification results of different concentration systems of forward / reverse primers, And tested the genotyping amplification results of optimizing the wild-type p...

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Abstract

The invention provides an SLCO1B1 genetype rapid detection kit based on the POCT mode. The kit contains fluorogenic quantitative PCR primers, probes and a cell lysis buffer used for detecting the SLCO1B1 genetype, wherein the sequences of the PCR primers are as shown in SEQ ID NO.1-2 and SEQ ID NO.5-6, and the sequences of the probes are as shown in SEQ ID NO.3-4 and SEQ ID NO.7-8. The kit can realize instant detection, DNA purification is not needed, a sample can be directly added into a reagent for carrying out the PCR reaction, the kit is particularly suitable for rapidly and accurately detecting samples (such as shedding buccal cells) with low content of DNA, the detection accuracy rate achieves 99% or above, the detection sensitivity is high, and 0.125ng genomic DNA can be accuratelydetected to the minimum; and the whole detection consumes short time, a detection result can be obtained within 1h, an administration basis can be provided for a doctor at first time, and thus the administration risk of patients is reduced.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a POCT mode-based rapid detection kit for SLCO1B1 genotype. Background technique [0002] The SLCO1B1 gene is an important member of the coding genes of the organic anion transporting polypeptides (OATPs) family, including 15 exons and 14 introns, encoding a total of 691 amino acids, and plays an important role in human drug metabolism. Studies have found that OATP1B1 can interact with cyclosporine, fibrates, rifampin, etc., and can also participate in the transport of statins. Among them, statin drugs are clinically recognized as the most effective and high-efficiency lipid-lowering drugs, which can competitively inhibit endogenous cholesterol synthesis rate-limiting HMG-CoA reductase (β-hydroxymethylglutaryl-CoA reductase), Blocking the intracellular valonate metabolic pathway, reducing intracellular cholesterol synthesis, lowering blood lipids, and thus being widely used in t...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6883C12Q2600/106C12Q2600/156C12Q2563/107C12Q2527/125C12Q2531/113
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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