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Primer group and kit for detecting polymorphic sites of aspirin-resistant related genes, and application thereof

An aspirin and polymorphic site technology is applied in the field of primer sets and kits for detecting aspirin resistance-related gene polymorphic sites, which can solve the problems of easy contamination, time-consuming, complicated operation, etc., achieve easy results and avoid calculation , The signal is clear, accurate and stable

Inactive Publication Date: 2019-03-19
WUHAN YZY MEDICAL SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Sequencing is the gold standard for genetic testing. However, this method has two obvious disadvantages: first, the operation is cumbersome and time-consuming; second, PCR products need to be processed later, which is easy to contaminate
However, this method has obvious defects in the detection of gene polymorphisms. First, the detection throughput is low. To complete the detection of a sample, two tubes of PCR reaction solution are required, one tube is used for amplification and detection of the wild-type template, and the other tube is used to amplify and detect the wild-type template. Amplify and detect mutant templates; second, the result interpretation is not intuitive, and the results need to be judged according to △CT; third, non-specific amplification cannot be completely eliminated

Method used

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  • Primer group and kit for detecting polymorphic sites of aspirin-resistant related genes, and application thereof
  • Primer group and kit for detecting polymorphic sites of aspirin-resistant related genes, and application thereof
  • Primer group and kit for detecting polymorphic sites of aspirin-resistant related genes, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1 primer / probe sequence selection

[0103] According to the sequence characteristics of SNP, 3 sets of primers and 5 sets of probes were designed, and PCR systems were prepared respectively. Use the above system to detect wild-type and mutant genomic DNA samples, and compare the differences in amplification performance between different primer-probe combinations. The results are as follows: figure 1 with figure 2 as shown, figure 1 It is the result graph of wild-type samples amplified by different probe sequences of PAI-1 gene, figure 2 It is the result graph of amplifying mutant samples with different probe sequences of PAI-1 gene. After a large number of screening experiments, the best primer-probe combination (probe combination 2), that is, the combination of SEQ ID NO.25 / SEQ ID NO.26 primer pair and SEQ ID NO.27 / SEQ ID NO.28 probe pair was obtained The composed PCR system has the best performance among all the combinations, showing good specificit...

Embodiment 2

[0104] Embodiment 2 different dosages of probes are selected

[0105] The probe uses FAM fluorescence and VIC fluorescence to monitor the amplification of the target sequence in real time. Probe concentration directly affects the value of the amplified fluorescence signal. When the probe concentration is low, the fluorescence signal value is low. When the probe concentration is high, the fluorescence signal is high, and the non-specific signal is also high.

[0106] On the basis of the optimal primer-probe combination screened above, the system performance was further optimized by adjusting the probe concentration. Set 3 probe concentrations (a: 0.1 μM, b: 0.2 μM, c: 0.3 μM)

[0107] Prepare the PCR reaction system separately. Use the above system to detect wild-type and mutant genomic DNA samples, and compare the difference in amplification performance under different probe concentrations. Test results such as image 3 with Figure 4 As shown, among them, image 3 The r...

Embodiment 3

[0109] Example 3 PCR buffer component optimization

[0110] Usually, repeated sequences (especially >3 G repeats) near the SNP site will reduce the ability of the probe to recognize the target SNP site. Due to the presence of repetitive sequences, the target detection gene PAI-1 (4G>5G) has poor specificity and cannot tolerate high concentration (eg 300ng / μL) genomic samples. The specificity could not be improved by adding conventional dimethyl sulfoxide, so a new sulfur-containing organic compound, tetramethylene sulfoxide, was added to the PCR buffer instead. The performance of tetramethylene sulfoxide in improving PCR specificity is stronger than that of dimethylene sulfoxide, but it is particularly critical to accurately select its optimal concentration. If the amount of tetramethylene sulfoxide is too large, it will easily inhibit PCR, and if the amount is too small, the effect of improving system specificity is limited. Therefore, the optimal concentration of this comp...

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Abstract

The invention relates to the technical field of in-vitro nucleic acid detection and particularly provides a primer group and kit for detecting polymorphic sites of aspirin-resistant related genes, andan application thereof. The primer group and the kit for detecting polymorphic sites of aspirin-resistant related genes and the kit can detect polymorphism of genes such as COX1, COX2, GPIIIa, PEAR1,P2Y1, GPIa and PAI-1; the sensitivity is high, so that genome DNA low to 0.1ng / muL can be accurately detected; the specificity is good, the genome DNA up to 300ng / muL can not generate non-specific amplification; the whole Fluorescence PCR detection process can be finished only within 90 minutes, and the detection result is intuitive and easy in interpretation.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a primer set and kit for detecting polymorphic sites of genes related to aspirin resistance and applications thereof. Background technique [0002] Aspirin, as the first-line antiplatelet drug, is widely used in the secondary prevention of cardiovascular and cerebrovascular diseases. However, there are individual differences in aspirin in different populations, and some patients still have cardiovascular and cerebrovascular ischemic events after taking aspirin, that is, there is aspirin resistance. The incidence of aspirin resistance is 5% to 45%, and its mechanism is related to gene polymorphism. At present, the genes related to aspirin resistance mainly include: cyclooxygenase (COX) gene in thromboxane activation pathway, GPIIb / IIIa receptor gene, platelet endothelial cell aggregation receptor 1 (PEAR1) gene, platelet surface ADP receptor gene P2Y1, pl...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/166C12Q2563/107C12Q2561/00C12Q2545/101C12Q2531/113
Inventor 蔡从利李丽琼刘林
Owner WUHAN YZY MEDICAL SCI & TECH
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