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Primers for detecting polymorphism of CYP3A4, CYP3A5 and MDR1 genes with ARMs-PCR (amplification refractory mutation system-polymerase chain reaction) method and prepared kit

A CYP3A5 and CYP3A4 technology, applied in the field of molecular biology, can solve the problems of high false positive rate, high annealing temperature, complicated operation procedures, expensive equipment and other problems of the hybridization chip detection method, and achieve accurate and reliable detection results, fast and convenient operation, and time-consuming detection. short effect

Inactive Publication Date: 2015-07-29
UNION STEMCELL & GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the prior art, direct sequencing is used to detect CYP3A4, CYP3A5, and MDR1 gene polymorphisms, which is costly, complicated, and time-consuming; the hybridization chip detection method has the problems of high false positive rate, accurate annealing temperature, and low specificity; Taqman method probe preparation and supporting equipment are expensive and the accuracy is not high
The limitations of the above methods limit the clinical application of CYP3A4, CYP3A5, and MDR1 gene polymorphism detection, and a fast and accurate detection method that does not require expensive equipment is needed for clinical application

Method used

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  • Primers for detecting polymorphism of CYP3A4, CYP3A5 and MDR1 genes with ARMs-PCR (amplification refractory mutation system-polymerase chain reaction) method and prepared kit
  • Primers for detecting polymorphism of CYP3A4, CYP3A5 and MDR1 genes with ARMs-PCR (amplification refractory mutation system-polymerase chain reaction) method and prepared kit
  • Primers for detecting polymorphism of CYP3A4, CYP3A5 and MDR1 genes with ARMs-PCR (amplification refractory mutation system-polymerase chain reaction) method and prepared kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 A human CYP 3A4, CYP 3A5, ABCB1 genotyping detection kit based on the ARMs-PCR method

[0055] The kit contains PCR tubes pre-packed with primers and amplification reaction solution.

[0056] The amplification reaction solution is 44ul, including PCR buffer, dNTP, high-density reagents and hot-start Taq polymerase.

[0057] There are 7 PCR reaction tubes for each person. Tubes 1 to 6 are specific reaction tubes, and tube 7 is a negative control tube. Each tube is pre-dispensed with 7ul of mixed solution, including tracer dye and 3×10 each. -3 nMol primers, the components of each tube are as follows:

[0058]

Embodiment 2

[0059] Example 2 The method and operation steps for detecting human CYP 3A4, CYP 3A5, and ABCB1 genotyping with the use of the kit.

[0060] 1 Extract DNA from the whole blood of the subject and dilute it into a DNA solution of 20-50ng / ul;

[0061] 2 Take out the amplification reaction solution, melt and mix well, take 8ul and add it to the No. 7 reaction tube;

[0062] 3 Add 20ul DNA solution to the remaining amplification reaction solution, mix well and add 8ul each into the 1 to 6 reaction tubes;

[0063] 4 Amplify on the PCR machine, the amplification procedure is:

[0064]

[0065]

[0066] 5 After the reaction is over, take 8ul PCR product from each well and add it to the sample well of 2% agarose gel (containing 0.5μg / mL EB or 0.01% GoodView II) at a voltage of 10-15V / cm gel length Electrophoresis

[0067] 6 Take pictures in the gel imaging system to obtain the electrophoresis results (see figure 1 , The left 1~3 rows are the 3rd typing results of No. 28 specimen, the genotyping...

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Abstract

The invention discloses primers for detecting polymorphism of CYP3A4, CYP3A5 and MDR1 genes with an ARMs-PCR (amplification refractory mutation system-polymerase chain reaction) method and a prepared kit. Primer sequences are shown in SEQ 1-SEQ 18. The primers can perform typing testing on CYP3A4, CYP3A5 and MDR1 genes of humans quickly and accurately through specific amplification after primer combination. Genetic typing or preparation of the typing kit can be performed according to the primers and the primers are used for detecting metabolic capacity of Chinese people for related drugs such as tacrolimus, cyclosporine A and the like, so that a reference basis can be provided for special doctors who determine optimal drug dosage and individual drug use, and drug use risks are reduced.

Description

Technical field [0001] The present invention relates to the field of molecular biology, in particular to a set of primers for detecting CYP3A4, CYP3A5, and MDR1 gene polymorphisms based on the ARMs-PCR method and a kit prepared. Background technique [0002] Drugs such as tacrolimus, cyclosporine A, and rapamycin are commonly used clinically to avoid rejection of organ transplantation, but the optimal dose range of these drugs is narrow, and insufficient doses in clinical applications will lead to immune rejection of transplants Lead to transplantation failure; too high a dose will cause severe liver and kidney toxicity and other adverse reactions. The current clinical routine administration method is to first give different patients the same dose of the drug, and then adjust the dose by real-time detection of the patient’s blood concentration after taking the drug, but this solution still cannot solve the problem of determining the dose based on individual patient differences be...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/106C12Q2600/156
Inventor 韩俊领杜宏伟崔丽娟
Owner UNION STEMCELL & GENE ENG
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