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Human SLCO1B1 and ApoE gene polymorphism detection kit, and preparation method and application of same

A detection kit and gene technology, applied in the fields of biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, can solve the problems of long experiment time, low sensitivity and poor specificity of PCR-sequencing method, and prevent Effects of false negative and false positive results, improved specificity and accuracy, and improved typing efficiency

Active Publication Date: 2018-12-14
WUHAN HEALTHCHART BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The PCR-sequencing method has low sensitivity and takes a long time for the experiment, so it is not suitable for clinical promotion; the chip hybridization method has low detection sensitivity and poor specificity, and is prone to false positive results; the high-resolution melting curve method has special requirements for equipment and is not suitable for clinical promotion. Detection sensitivity is not high
The Taqman-qPCR method has high detection sensitivity, but often due to the site sequence, the detection specificity is not high, and it is genotyped by the Genotyping method, which has certain requirements for the number of samples and polymorphism distribution, and is not suitable for detecting mutation frequency too low position

Method used

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  • Human SLCO1B1 and ApoE gene polymorphism detection kit, and preparation method and application of same
  • Human SLCO1B1 and ApoE gene polymorphism detection kit, and preparation method and application of same
  • Human SLCO1B1 and ApoE gene polymorphism detection kit, and preparation method and application of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 prepares SLCO1B1 and ApoE gene detection kit of the present invention

[0055] 1. Primer design and synthesis:

[0056] The 4 sites of SLCO1B1 388, SLCO1B1 521, ApoE 388 and ApoE 526 contain 6 primers in each system; the two upstream primers F1 and F2 of the sense strand, the two downstream primers R1 and R2 of the antisense strand, F1 and R1 are Ordinary primers, F2 and R2 are specific ARMs primers with fluorophore and tag sequences, screened by primers and PCR reaction conditions to ensure that the primer pair F1R1 enriches the template, while F2 and R1, F1 and R2 can be amplified normally Specific genotype PCR products with fluorescence were produced; internal reference primers were added to each gene locus.

[0057] The specific sequence is as follows:

[0058]

[0059]

[0060] The specific combination is as follows:

[0061] Wherein, SEQ ID NO.1 and SEQ ID NO.4 are used to amplify the 388A DNA fragment of the SLCO1B gene locus, and the amplif...

Embodiment 2

[0097] The human SLCO1B1 and ApoE gene polymorphism detection kit prepared in Example 1 was used to detect the samples to be tested. In this example, 100 cases of EDTA anticoagulated venous whole blood samples were collected, genomic DNA was extracted, and human SLCO1B1 and ApoE gene polymorphism detection kits were used to detect the polymorphisms of SLCO1B1 388, SLCO1B1 521, ApoE 388 and ApoE 526 genes. The specific operation The process is as follows:

[0098] (1) Genomic DNA extraction from blood samples: Genomic DNA was extracted using a commercial extraction kit. After extraction, the DNA was eluted with TE buffer and the DNA concentration was measured; the genomic DNA was diluted to 20 ng / μl;

[0099] (2) Fluorescence quantitative detection: Add 30 μl of PCR premixed reaction solution and 20 μl of sample DNA to be tested into the reaction tube; the PCR reaction program is: 95°C for 1 minute pre-denaturation; 15 cycles: 95°C for 5 seconds, 61°C for 32 seconds , do not c...

Embodiment 3

[0120] Use the kit prepared in Example 1 to detect and verify the minimum detection line of this kit, specifically including the following steps:

[0121](1) Use the samples of known SLCO1B1 and ApoE corresponding site genotypes in Example 2, select one case each of the wild type genome, mutant genome and heterozygous genome samples for each site, and dilute the above samples to 10ng / μL, 1ng / μL, 0.5ng / μL, 0.25ng / μL, 0.1ng / μL, 0.05ng / μL, 0.025ng / μL, 0.001ng / μL;

[0122] (2) Fluorescence quantitative detection: Add 30 μl of PCR premixed reaction solution and 20 μl of sample DNA to be tested into the reaction tube; the PCR reaction program is: 95°C for 1 minute pre-denaturation; 15 cycles: 95°C for 5 seconds, 61°C for 32 seconds , do not collect fluorescence; 30 cycles: 95°C for 5 seconds, 61°C for 32 seconds, collect fluorescence; each concentration gradient of each sample is repeated 20 times;

[0123] (3) After the PCR reaction is completed, perform result interpretation acc...

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Abstract

The invention belongs to the field of biotechnologies, and particularly relates to a human SLCO1B1 and ApoE gene polymorphism detection kit, and a preparation method and an application of same. The kit is composed of: a PCR premix reaction solution respectively used for detecting the rs2306283 loca of the SLCO1B1 gene, the rs4149056 loca of the SLCO1B1 gene, the rs429358 loca of the ApoE gene andthe rs7412 loca of the of the ApoE gene, and a positive reference substance and a negative reference substance. The PCR premix reaction solution includes specific primer sequence groups, probe groupsand a PCR reaction solution, which are used for amplifying the mentioned loci. The specific primer sequence groups are composed of a regular outer primer and specific ARMs primers having fluorescencetags. The kit is used for detecting the polymorphism of human SLCO1B1 and ApoE gene, is high in sensitivity and specificity, is easy to use and has reliable results, can complete the detection withinone hour, and is simple and objective in result interpretation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a human SLCO1B1 and ApoE gene polymorphism detection kit and a preparation method and application thereof. Background technique [0002] The human SLCO1B1 gene is located on chromosome 12 and encodes the organic anion transporting polypeptide OATP1B1. OATP1B1 is a transmembrane transport protein, which is mainly distributed in the liver. It has the physiological function of mediating the transport of endogenous and exogenous substances in the liver cell membrane, metabolizing and eliminating them, and plays an important regulatory role in the transport of statins. Studies have shown that the SLCO1B1 gene has genetic polymorphisms, among which 388A>G and 521T>C are two common single nucleotide polymorphisms, which can form four haplotypes SLCO1B1*1a (388A-521T), SLCO1B1*1b (388G-521T), SLCO1B1*5(388A-521C) and SLCO1B1*15(388G-521C). The mutated SLCO1B1 gene weakens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2600/156C12Q2535/137C12Q2537/143
Inventor 王宁付金玲李倩李雪梅叶伦程弘夏陈刚
Owner WUHAN HEALTHCHART BIOLOGICAL TECH
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