Personalized medication gene detection kit and application

A gene detection and kit technology, applied in the field of individualized drug gene detection kits, can solve the problems of poor amplification uniformity, difficult amplification depth, and difficulty in ensuring detection accuracy, achieve less non-specific amplification, overcome mutual Interfering, specific effects

Active Publication Date: 2018-10-26
CAPITALBIO GENOMICS
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0004] The use of multiplex PCR capture and next-generation sequencing technology for gene detection has obvious advantages such as simple operation, low cost, and a large number of enriched capture regions in a short time, which can greatly shorten the detection process and detection time; however, due to drug metabolism, efficacy and toxicity related There are as many as hundreds of polymorphic sites in the gene, and the use of multiplex PCR capture technology often leads to poor amplification uniformity, large differences in GC content in the amplified region, and the large interaction between multiplex PCR primers, which makes it difficult to achieve one-tube operation. As a result, it is difficult to guarantee the acc

Method used

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  • Personalized medication gene detection kit and application
  • Personalized medication gene detection kit and application
  • Personalized medication gene detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, a kind of personalized drug gene detection primer set

[0056] A primer set for individualized drug gene detection, including a primer set for amplifying 110 drug gene polymorphic sites in Table 1, the primer sequences of which are shown in SEQ ID NO: 1 to SEQ ID NO: 186 in Table 1 Show.

[0057] The present invention refers to the Genetic Detection Technical Guidelines of the Health and Family Planning Commission, the FDA drug label of the U.S. Food and Drug Administration and the NIH pharmGKB pharmacogenomics database, and screens out 110 drug gene polymorphism sites related to drug metabolism, curative effect and toxicity (such as Shown in Table 1), the 110 polymorphic sites are distributed on the 54 genes shown in Table 2; the inventor creatively designs, screens, and optimizes the selected 110 polymorphic sites , and finally obtain the multiplex PCR primers shown in Table 1, which have strong specificity, less non-specific amplification, and can well...

Embodiment 2

[0067] Embodiment 2, a kind of individualized drug gene detection kit

[0068] A gene detection kit for individualized medicine, comprising:

[0069] (1) A primer set for amplifying 110 drug gene polymorphic sites in Table 1, its primer sequence is shown in SEQID NO:1~SEQ ID NO:186 in Table 1, the 5' of each primer sequence There is also a universal sequence connected to the end, and the universal sequence is: 5'-AAATGGGCGGTAGGCTTG-3' (SEQ ID NO: 193); when the kit is used, each primer is mixed and matched as shown in the pooling coefficient of each primer in Table 1;

[0070] (2) Linker: 5'-CCTCTCTATGGGCAGTCGGTGATAAATGGGCGGTAGGCTTG-3' (SEQ ID NO: 194);

[0071] (3) Tag linker: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNGATAAATGGGCGGTAGGCTTG-3' (SEQ ID NO: 195), N in the sequence represents any base of AGCT, and NNNNNNNN is used to identify libraries constructed from different samples;

[0072] (4) Multiple PCR buffers, high concentration multiple PCR buffers such as 2X, 5X an...

Embodiment 3

[0076] Example 3. A method for constructing a nucleic acid sequencing library for gene detection of individualized medication

[0077] A method for constructing a nucleic acid sequencing library for gene detection of individualized medicine, comprising the following steps:

[0078] (1) One-step multiplex PCR amplification

[0079] Using the kit in Example 2, configure a 25 μL system for one-step multiplex PCR amplification, including: 200 ng of sample DNA, 0.6 μL of primer set, 0.25 μL of adapter, 0.25 μL of index adapter, 12.5 μL of multiplex PCR buffer (2X), DNA polymerization Enzyme 1U / μL, DMSO 5% (V / V), nuclease-free water as the balance, wherein, the mixing ratio of each primer in the primer set is shown in Table 5; mix the system thoroughly, centrifuge briefly, and put On the PCR instrument, run the PCR reaction program: 95°C for 3min; 35 cycles of amplification (95°C for 10s, 58°C for 1.5min, 72°C for 30s), 72°C for 1min, 16°C Hold.

[0080] Table 5. Primer pooling co...

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Abstract

The invention discloses a personalized medication gene detection kit and application. The personalized medication gene detection kit is suitable for multiple kinds of sample types including dried blood spots, whole blood and buccal swabs, information of all medicine gene polymorphic sites can be acquired at once, the sequencing effective rate is 50% or above, the sequencing cover degree is 100%, the average sequencing depth exceeds 3000 X, the library building success rate is larger than or equal to 98.5%, and the medicine gene polymorphic site detection accuracy is larger than or equal to 99.9%; a primer group is high in specificity and little in nonspecific amplification, interference among primers can be well overcome, under the situation of up to 186 primer sequences, one-tube multiplePCR reaction is achieved, moreover, amplicon is high in uniformity, and under the situation of large amplification area GC content difference, overall effective amplification can be conducted. The detection kit, the primer group and a library building method can be applied to personalized medication gene detection, and guidance is provided for personalized medication.

Description

technical field [0001] The invention belongs to the field of gene sequencing, and in particular relates to a gene detection kit for individualized medicine and its application. Background technique [0002] According to the data of the United Nations World Health Organization (WHO), one-third of the global death patients died from irrational drug use, not from natural diseases themselves. This is related to irreversible damage to organs caused by adverse reactions caused by improper use of drugs. Cause death, especially the incidence of adverse drug reactions in children's medication is about 13%, and the incidence of newborns is as high as 24.4%. In addition, the effectiveness of clinical medication is also insufficient, especially in the treatment of chronic diseases such as hypertension, hyperlipidemia, and diabetes. For the same drug, there will be differences among ineffective groups, safe and effective groups, and toxic response groups. Fast-metabolizing subjects may ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11C12Q1/6806C40B50/06
Inventor 糜庆丰向书芹钟婉平郭怿盈吴春求黄铨飞刘丽菲
Owner CAPITALBIO GENOMICS
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