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Kit and method used for detecting polymorphisms of apolipoprotein E (ApoE) gene and solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene

A SLCO1B1521T, gene polymorphism technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of unsuitable detection of sites with low mutation frequency and a small number of samples, increasing experimental difficulty and complex steps and other problems, to achieve the effect of saving experimental consumables and labor costs, omitting nucleic acid extraction and purification steps, and ensuring accuracy and sensitivity

Pending Publication Date: 2020-05-22
上海未凡生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the detection methods for gene polymorphism mainly include: PCR-RFLP method, PCR-chip hybridization method, PCR-sequencing method and Taqman-qPCR method, among which: PCR-RFLP method mainly depends on the restriction enzyme cutting site of the target , the difficulty of the experiment increases, and the subsequent electrophoresis analysis is likely to cause laboratory contamination, which cannot be used in large-scale clinical applications; the PCR-chip hybridization method uses PCR technology and gene chip technology in combination, and there are also complex steps and laboratory cross-contamination. risk, low detection sensitivity and poor specificity, prone to false positive results; PCR-sequencing method is the gold standard for genetic detection, but its operation process is too long, the cost is too high, and the equipment is expensive and it is difficult to be suitable for large-scale application in hospitals; Taqman -qPCR method has high detection sensitivity, but often due to the site sequence, the detection specificity is not high, and the genotyping method is used for genotyping, which has certain requirements for the number of samples and polymorphism distribution, and is not suitable for detecting low mutation frequency sites and a small number of samples

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  • Kit and method used for detecting polymorphisms of apolipoprotein E (ApoE) gene and solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene
  • Kit and method used for detecting polymorphisms of apolipoprotein E (ApoE) gene and solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene
  • Kit and method used for detecting polymorphisms of apolipoprotein E (ApoE) gene and solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene

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Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1: Preparation of the kit for detecting ApoE and SLCO1B1 gene polymorphism according to the present invention

[0047] 1. Synthesize primers and probes for the four sites of ApoE 526C>T, ApoE A388T>C, SLCO1B1 388A>G and SLCO1B1 521T>C as shown in the following table:

[0048]

[0049] 2. Prepare the joint detection reaction solution of ApoE 526C>T and ApoE A388T>C as shown in the table below, including:

[0050] components Proportion The upstream primer of the sequence shown in SEQ ID NO.1 200nmol / L The downstream primer of the sequence shown in SEQ ID NO.2 200nmol / L The wild-type probe of the sequence shown in SEQ ID NO.3 200nmol / L The mutant probe of the sequence shown in SEQ ID NO.4 200nmol / L The upstream primer of the sequence shown in SEQ ID NO.5 200nmol / L The downstream primer of the sequence shown in SEQ ID NO.6 200nmol / L The wild-type probe of the sequence shown in SEQ ID NO.7 200nmol / L ...

Embodiment 2

[0058] The method for detecting ApoE and SLCO1B1 gene polymorphisms by using the above-mentioned kit comprises the following steps:

[0059] a) Take 1 μL of the saliva (or blood sample) sample to be tested and add directly to PCR reaction tube Ⅰ containing 20 μL of ApoE 526C>T and ApoE A388T>C combined detection reaction solution and 20 μL of SLCO1B1 388A>G and SLCO1B1521T>C combined detection In the PCR reaction tube II of the reaction solution, after mixing and centrifuging, put it into the fluorescence quantitative PCR instrument and carry out the PCR amplification reaction at the same time: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 10 seconds; 60°C annealing for 30 seconds ;40 loops;

[0060] b) Perform fluorescence detection on the amplified product, and directly interpret the polymorphism of the test sample according to the fluorescence curve and Ct value:

[0061] b1) Perform fluorescence detection on the PCR reaction product of PCR reaction tube I, an...

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Abstract

The invention discloses a kit and method used for detecting polymorphisms of an apolipoprotein E (ApoE) gene and a solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene. The kit comprises an ApoE 526C>T and ApoE A388T>C joint detection reaction solution and an SLCO1B1 388A>G and SLCO1B1 521T>C joint detection reaction solution. According to the kit, experiment prove that primers and probes in the joint detection reaction solutions have high specificity, and saliva / blood samples can be directly employed to perform polymerase chain reaction (PCR) amplification and analysis;moreover, typing detection of two single nucleotide polymorphisms (SNPs) can be realized in a reaction tube, and thus, not only is the detection efficiency obviously increased, but also the detectionperiod is obviously shortened; and moreover, the kit and the method have the advantages of being high in sensibility, high in specificity, accurate and reliable, convenient and fast to operate, and the like.

Description

technical field [0001] The invention relates to a kit and method for detecting ApoE and SLCO1B1 gene polymorphisms, belonging to the technical field of in vitro gene detection. Background technique [0002] Hyperlipidemia refers to a disease in which cholesterol and triglycerides in serum are elevated. Blood lipids are mainly composed of four substances: total cholesterol, triglycerides, low-density lipoprotein and high-density lipoprotein. As long as any one of the three items of total cholesterol, triglycerides and low-density lipoprotein exceeds the standard, it is hyperlipidemia. Cholesterol in the blood combines with arterial elastin, the blood vessels harden and become brittle, and lose their original elastic activity, resulting in insufficient arterial blood supply to vital organs, leading to coronary heart disease, stroke, and limb necrosis. Not only that, hyperlipidemia can also cause various diseases such as high blood pressure, diabetes, Alzheimer's, liver cirrh...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6851
CPCC12Q1/6883C12Q1/6851C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 朱田李春雨章泽林
Owner 上海未凡生物科技有限公司