Detection method and application of personalized children's drug analysis system based on SNP (single nucleotide polymorphisms) sites

A detection method and site technology, which is applied in the detection field of children's personalized drug analysis, can solve the problems of high cost and low detection efficiency, and achieve the effects of low cost, low detection cost and high throughput

Active Publication Date: 2019-01-04

深圳一道医学检验实验室

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[0009] At present, the research on the polymorphism of these gene loci is mainly based on the Sanger sequencing method...

Abstract

The invention discloses a detection method personalized children's drug analysis system based on SNP (single nucleotide polymorphisms) sites. SNP site combination for common anti-infection children'sdrug risk evaluation comprise 67 SNP site combinations and specific primers for detecting drug toxicity, drug metabolism and medicine reactions. Sample DNA is subjected to PCR amplification through site specific primers, the difference between a melting curve and a standard melting curve is analyzed, the Tm values of PCR amplified products are compared, and corresponding genotyping results can beobtained. The detection method can be applied to quick screening of applicability of common drugs to children, provides guide for reasonable selection of children's drugs and has the advantages of being simple to operate, high in efficiency, high in specificity, economical and the like.

Application Domain

Microbiological testing/measurement

Technology Topic

PersonalizationGenotyping +11

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[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0031] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0032] The term "high resolution melting curve method" as used herein mainly refers to the difference based on the physical properties of nucleic acid molecules. The fragment length, GC content, and GC distribution of different nucleic acid molecules are different, so any double-stranded DNA molecule will have its own shape and position of the melting curve when heated and denatured. The basic principle of high-resolution melting curve technology is to distinguish samples according to different melting curves.

[0033] The design mainly follows: use software for comparison to ensure that the annealing temperature difference between the primer pairs is not large, preferably within 3 degrees. The absolute value of ΔG of the primer-dimer formed between the primer pair and the primer pair is less than 5. The length of the combined primer is 20-40bp, so when synthesizing the primer, a higher purification standard should be selected.

[0034] In a preferred embodiment, the "combined primers" of the present invention can be used to prepare a kit, which can be used for the detection of gene loci related to children's drug use.

[0035] Another aspect of the present invention provides a method for detecting genetic loci associated with drug use in children in one or more samples. The method includes the steps of amplifying DNA from a plurality of samples using the "combined primers" described above, followed by sequencing to obtain the sequences of the samples.

[0036] The invention provides a high-resolution melting curve method based on multiplex PCR technology, which can simultaneously detect 58 mutation sites of common children's drug-related genes.

[0037] Note: In order to ensure that the sample is not contaminated by food or drink, do not eat or drink within 30 minutes before sampling.

[0038] 1) Genomic DNA is extracted from the tester's oral swab sample;

[0039] 2) Different combined primers are mixed in equal proportions, and the mixed primers are used as a primer pool. Use a 96-well plate or 384-well plate, configure the reaction system according to the requirements of the kit, stick the film on the plate, and centrifuge briefly to collect the solution on the tube wall to the bottom of the tube; , so that the membrane adheres to the PCR plate.)

[0040] The system is as follows:

[0041]

[0042] Roche Light Cycler 480Ⅱ real-time fluorescent quantitative PCR system, start up;

[0043] Use this primer to amplify according to the method of fluorescent quantitative PCR. The amplification conditions are as follows: pre-denaturation at 95°C for 10 minutes; then denaturation at 95°C for 10 seconds, annealing at 65-55°C (decrease by 0.5°C per cycle) for 10 seconds, and extension at 72°C for 30 seconds The procedure is carried out for 45 cycles; the amplified DNA fragment is the amplified library;

[0044] 3) The DNA fragment in step 2) is subjected to a high-resolution melting curve process according to the high-resolution melting curve conditions. The melting step of the amplified product is carried out immediately after the PCR cycle is completed. 40°C for 1min, then raise the temperature to 65°C for 1s. Fluorescence collection was performed during continuous heating from 65°C to 95°C (25 times/°C). Finally, the temperature was lowered to 40°C.

[0045] Table 1: Design sequence of primers for SNP sites related to drug toxicity:

[0046]

[0047]

[0048] Table 2: SNP site primer design sequence related to drug metabolism:

[0049]

[0050]

[0051] Table 3: Design sequence of primers for drug response-related SNP sites:

[0052]

[0053]

[0054]

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Detection method and application of personalized children's drug analysis system based on SNP (single nucleotide polymorphisms) sites

A detection method and site technology, which is applied in the detection field of children's personalized drug analysis, can solve the problems of high cost and low detection efficiency, and achieve the effects of low cost, low detection cost and high throughput

AI-Extracted Technical Summary

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