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Mononucleotide polymorphism detection method based on melting curves and kit thereof

A single nucleotide polymorphism and melting curve technology, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve problems such as complex operation, high price, and application limitations

Inactive Publication Date: 2014-10-01
徐高连 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

5. DNA sequencing method: DNA Sanger sequencing can accurately and directly reflect sequence differences, but in terms of operation cycle, this method takes at least 3-4 days from sample processing to reporting, and the price is relatively expensive
However, currently on the market, most of the detection methods for single-base point mutations at multiple gene sites in multiple gene fragments require special analytical instruments, which are not only expensive, but also complicated to operate, so they are subject to certain restrictions in application. limit

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  • Mononucleotide polymorphism detection method based on melting curves and kit thereof
  • Mononucleotide polymorphism detection method based on melting curves and kit thereof
  • Mononucleotide polymorphism detection method based on melting curves and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0336] Application in detection of clarithromycin resistance of Helicobacter pylori

[0337] This example will detect common clarithromycin-resistant loci, such as A2143G, A2142G and A2142C.

[0338] 1. The composition of the test kit, including:

[0339] (1) Positive control solution stored at -20°C;

[0340] (2) Negative control solution stored at -20°C;

[0341] (3) Amplification primer A stored at -20°C:

[0342] A forward primer: 5-CGTCAGTCGCAAGATGAAGCG (SEQ ID NO: 1)

[0343] A reverse primer: 5-CGCATGATATTCCCCATTAGCAG (SEQ ID NO: 2);

[0344](4) Specific primer B stored at -20°C: used to detect A2143G, A2142G and A2142C mutation sites

[0345] Site-specific primer B15-GCGGCAAGACGGGA (SEQ ID NO: 3)

[0346] Site-specific primer B25-GCGGCAAGACGGCA (SEQ ID NO: 4)

[0347] Site-specific primer B35-CGGCAAGACGGAGA (SEQ ID NO:5);

[0348] (5) Reagents required for fluorescent dyes and amplification reaction systems;

[0349] (6) ddH stored at -20°C 2 O.

[0350] 2. ...

Embodiment 2

[0380] Application in detection of quinolone drug resistance of Helicobacter pylori

[0381] This example will detect common clarithromycin-resistant sites, such as Asn87Lys and Asp(GAT)91Gly(GGT) / Tyr(TAT) / Asn(AAT).

[0382] 1. The composition of the test kit, including:

[0383] (1) Positive control solution stored at -20°C;

[0384] (2) Negative control solution stored at -20°C;

[0385] (3) Amplification primer A stored at -20°C:

[0386] A forward primer: 5-TTTAGCTTATTCAATGAGCGT (SEQ ID NO: 6)

[0387] A reverse primer: 5-GCAGACGGCTTGGTAGAATA (SEQ ID NO: 7);

[0388] (4) Specific primer B stored at -20°C: used to detect Asn87Lys and Asp(GAT)91Gly(GGT) / Tyr(TAT) / Asn(AAT) mutation sites

[0389] Site-specific primer B15-CCATGGCGATAAAG (SEQ ID NO: 8)

[0390] Site-specific primer B25-CCATGGCGATAAGG (SEQ ID NO: 9)

[0391] Site-specific primer B35-ACGCGGTTTATTA (SEQ ID NO: 10)

[0392] Site-specific primer B45-ACGCGGTTTATAA (SEQ ID NO: 11)

[0393] Site-specific primer...

Embodiment 3

[0428] Application in detection of mitochondrial DNA G11778A single nucleotide polymorphism in Leber's disease

[0429] Leber's hereditary optic neuropathy (LHON) is a maternally inherited binocular optic nerve disease. In 1988, Wallace et al. first reported that the primary mutation (G→A) at the 11778th nucleotide site of mitochondrial DNA (mtDNA) could cause LHON. In view of the diagnostic significance of the mitochondrial G11778A site typing for Leber's hereditary optic neuropathy, this example detects the polymorphism of the G11778A site.

[0430] 1. The composition of the test kit, including:

[0431] (1) Positive control solution stored at -20°C;

[0432] (2) Negative control solution stored at -20°C;

[0433] (3) Amplification primer A stored at -20°C:

[0434] A forward primer: 5-GCGCAGTCATTCTCATAATCG (SEQ ID NO: 13)

[0435] A reverse primer: 5-GGCGAGGTTAGCGAGGCTTG (SEQ ID NO: 14);

[0436] (4) Specific primer B stored at -20°C: used to detect the G11778A mutati...

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Abstract

The invention relates to a mononucleotide polymorphism detection method based on melting curves and a kit thereof. The detection method comprises the following steps: in a same PCR amplification system, subjecting a locus target template containing SNPs and single-base mutation to PCR amplification, carrying out AS-PCR amplification, which can differentiate the locus specificities according to the SNPs gene type and types of single-base mutation, monitoring the fluorescent dye, which can combine with DNA double strands, in solution, running a product melting curve analysis program, and detecting the polymorphism gene type of mononucleotide in amplification products. The whole process from the extraction of sample nucleic acid to the final detection result only lasts for 2 to 3 hours, at the same time the detection method also has the advantages of simple and clear result, large detection throughput, and low detection cost. The detection method and kit thereof can be used in common molecular labs and hospital laboratory department for corresponding detection, can also be applied to clinical detection, greatly increases the accuracy of clinical diagnosis, shortens the detection time for patients, and reduces the costs of a medicine and healthcare system.

Description

technical field [0001] The invention belongs to the field of detection of single nucleotide polymorphism and single base mutation in genetic engineering, and in particular relates to a detection method of single nucleotide polymorphism based on melting curve and a kit thereof. Background technique [0002] Single nucleotide polymorphisms (SNPs) refer to the existence of two or more different bases at a specific nucleotide position at the genome level, and the frequency of any allele in the population is not less than 1 %. With the completion of human genome sequencing, SNPs screening and detection have become the focus of extensive attention of researchers. Most of the methods currently used for SNPs detection are based on PCR technology, combined with methods such as fluorescence, mass spectrometry, gene chips or direct sequencing, and have entered the high-throughput era of SNPs detection. However, most of these methods are complicated to operate, expensive, and take a l...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
CPCC12Q1/6858C12Q2531/113C12Q2527/107C12Q2563/107
Inventor 徐高连
Owner 徐高连