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Nucleic acid composition and reagent kit for detecting cardiovascular disease medication related genes based on nucleic acid mass-spectrometric technique, and detection method

A nucleic acid composition and technical detection technology, applied in the field of genetic detection, can solve the problems of inconvenient identification of peak images, poor specificity, low sensitivity, etc., and achieve the reduction of non-specific amplification problems, clear and identifiable results, and rational drug use guidance Effect

Active Publication Date: 2020-05-08
WUHAN YZY MEDICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] At present, nucleic acid mass spectrometry-based detection of a variety of cardiovascular disease drug-related genes still has some shortcomings, such as poor specificity, low efficiency, low sensitivity, and the peak map is not easy to identify, etc.

Method used

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  • Nucleic acid composition and reagent kit for detecting cardiovascular disease medication related genes based on nucleic acid mass-spectrometric technique, and detection method
  • Nucleic acid composition and reagent kit for detecting cardiovascular disease medication related genes based on nucleic acid mass-spectrometric technique, and detection method
  • Nucleic acid composition and reagent kit for detecting cardiovascular disease medication related genes based on nucleic acid mass-spectrometric technique, and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] The nucleic acid composition provided in this example for detecting cardiovascular disease drug-related genes based on nucleic acid mass spectrometry technology includes an amplification primer set for multiplex PCR amplification of cardiovascular disease drug-related genes and a single base extension reaction The extension primer set;

[0114] Wherein, the amplification primer set includes the following primer pairs:

[0115] The first primer pair: it includes the primers shown in SEQ ID NO.1-2;

[0116] The second primer pair: it includes the primers shown in SEQ ID NO.3-4;

[0117] The third primer pair: it includes the primers shown in SEQ ID NO.5-6;

[0118] The fourth primer pair: it includes the primers shown in SEQ ID NO.7-8;

[0119] The fifth primer pair: it includes the primers shown in SEQ ID NO.9-10;

[0120] The sixth primer pair: it includes the primers shown in SEQ ID NO.11-12;

[0121] The seventh primer pair: it includes the primers shown in SEQ I...

Embodiment 2

[0140] This embodiment provides a method for detecting cardiovascular disease medication-related genes based on nucleic acid mass spectrometry using the nucleic acid composition provided in Example 1, including the following steps:

[0141] (1) Sample preparation

[0142] Specimen collection: Depending on the actual situation, different sample types can be collected, such as whole blood, oral exfoliated cells, dried blood films, saliva and other sample tissues containing intact cells.

[0143] Genomic DNA extraction: The absorbance OD260 / 280 of DNA after extraction is between 1.7-2.0, and then the sample is diluted to 10ng / μL for the next step of PCR reaction.

[0144] (2) Primer dilution

[0145] The dry powder of primers amplified by multiplex PCR was dissolved in ultrapure water to prepare a 100 μM stock solution, and mixed so that the final concentration of each amplification primer was 0.5-2 μM.

[0146]The dry powder of the extension primers was dissolved in ultrapure ...

Embodiment 3

[0166] The kit for the detection of cardiovascular disease drug-related genes based on nucleic acid mass spectrometry provided in this example is designed with a reaction master mix, including multiplex PCR amplification, SAP digestion, and iPLEX extension three-tube reaction master mix, as well as a positive control and a blank control There are 5 tubes in total, and the specific composition is shown in Table 3 below.

[0167] table 3

[0168]

[0169] The kit provided in this example is designed in the form of a premixed solution, which is convenient for users to operate, and the sample can be amplified directly after DNA extraction, shortening the processing time, improving work efficiency, and enabling high-throughput detection. The premix not only simply mixes the components, but also adds a PCR reaction enhancer and stabilizer such as DMSO (of course, in other embodiments, DMSO may not be added), which is fast, easy, sensitive, specific It has the advantages of stron...

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Abstract

The invention discloses a nucleic acid composition and reagent kit for detecting cardiovascular disease medication related genes based on a nucleic acid mass-spectrometric technique, and a detection method, and relates to the technical field of gene detection. The nucleic acid composition disclosed by the invention comprises an amplification primer set for performing multiplex PCR amplification oncardiovascular disease medication related genes, and an extension primer set for performing a single-base extension reaction. When the nucleic acid composition, the reagent kit or the detection method provided by the invention is used, 16-site genotypes of the cardiovascular disease medication related genes can be detected through the nucleic acid mass-spectrometric technique, the characteristicsthat the sensitivity is high, the specificity is high, the result is easy to recognize, the accuracy is increased and the operations are convenient are obtained, a more reliable detection means is provided for detection of the cardiovascular disease medication related genes, and more reasonable medication guidance is also provided for individualization medication of cardiovascular diseases.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a nucleic acid composition, kit and detection method for detecting cardiovascular disease drug-related genes based on nucleic acid mass spectrometry technology. Background technique [0002] The iPLEX technology based on the MassARRAY platform uses single-base extension technology and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology to achieve multiple amplification reaction genotype detection. The main feature is to perform single-tube multiplex PCR, and add sequence-specific extension primers to the PCR amplification products. The extension primers are designed to be located one base before the mutation site, and can be extended by one base at the mutation site. The prepared samples were then co-crystallized with the chip matrix with the analytes, the crystals were placed in the vacuum tube of the mass spectrometer, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2533/101C12Q2565/627
Inventor 方闪闪梁瑶李丽琼张喆蔡从利
Owner WUHAN YZY MEDICAL SCI & TECH
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