Primer for detecting deafness gene SLC26A4 point mutation and application thereof

Abstract

The invention belongs to the field of medical detection, and particularly relates to a primer for detecting deafness gene SLC26A4 point mutation and application of the primer, an LNA nucleoside monomer modification technology is creatively introduced to a deafness gene locus 2162, and high-sensitivity specific detection of the deafness gene locus 2162 is achieved in combination with a multiple PCR technology, a single base extension technology and a time-of-flight mass spectrometry technology. In addition, the invention further discloses a deafness gene multi-site detection kit which can be used for simultaneously detecting hotspot mutation of a plurality of deafness genes. The primer is high in extension efficiency, rapid in diagnosis, simple to operate and low in cost, and provides reference for clinical detection of the deafness gene.

Description

technical field

[0001] The invention belongs to the field of medical detection, in particular to primers for detecting point mutations of deafness gene SLC26A4 and applications thereof Background technique

[0002] Deafness is one of the most common clinical genetic diseases. At present, the traditional physical hearing defect screening method is generally used in the screening of deafness. Deafness) defects, resulting in the continuous occurrence of deaf tragedies. Genetic detection of deafness can change the occurrence of deafness from traditional passive treatment to active prevention, so as to achieve early detection and early intervention, so as to avoid the occurrence of deafness. Therefore, the detection of deafness susceptibility genes is of great significance.

[0003] At present, there are many gene detection methods on the market, such as: fluorescent quantitative PCR, sequencing and mass spectrometry detection, etc. Fluorescent quantitative PCR is widely used be...

Examples

Embodiment 12162

[0076] Example 1 2162C>T detection site primer design

[0077] A. Locked nucleic acid spacer modification

[0078] Table 4 2162C>T detection site primers

[0079]

[0080]

[0081] Bases underlined are bases modified by locked nucleic acids.

[0082] The result is as Figure 1-9 As shown, the specificity of the primer (SEQ-ID-NO.1) designed by the conventional method is poor, and under the normal dosage situation, the extension efficiency is low, and the residual level of the primer is high (characterized by the peak intensity of the primer / peak intensity of the product). )( figure 1 ). Different numbers of locked nucleic acid modifications were sequentially designed on the bases spaced between the 5' and 3' ends of the primer of SEQ-ID-NO.1, and the changes in elongation efficiency were investigated.

[0083] There was no significant change in elongation efficiency when 1 or 2 locked nucleic acid modifications were added at the 5' end (e.g. figure 2 / 3); when add...

Embodiment 2

[0090] Embodiment 2 A kind of test kit for detecting deafness gene polymorphism

[0091] The kit disclosed by the invention comprises multiple PCR reaction reagents, phosphatase digestion reagents, single base extension reagents, desalting resin and chip components which are independent of each other. The multiple PCR reaction reagents include multiple PCR reaction solutions and amplification enzymes; the phosphatase digestion reagents include phosphatase digestion reaction solutions and phosphatase; the single base extension reagents include single base extension reaction solutions and elongation enzymes . In this method, a purification column can be used to replace the resin, and the purpose of purifying the product can also be achieved; the chip in this method can be any substrate suitable for forming nucleic acid-matrix co-crystals, such as glass sheets, silicon sheets, stainless steel, etc.

[0092] The polymorphic sites selected by the present invention all have high po...

Embodiment 3

[0105] Embodiment 3 clinical experiment

[0106]Use the locked nucleic acid modified primer SEQ-ID-NO.4 to detect the 2162C>T site, and use other 19 detection sites to analyze the 20 SNP site gene detection results of 4 clinical samples, combined with deafness Interpretation of the significance of the SNP loci of related susceptibility genes, and analysis of the risk of deafness in the tested samples.

[0107] Table 8 Correspondence between deafness-related susceptibility genes and SNPs

[0108]

[0109] The SNP typing results of deafness-related susceptibility genes are shown in Table 9 below and Figure 16-19 :

[0110] Table 9 SNP typing of deafness-related susceptibility genes

[0111]

[0112] It can be seen from the above table that the genotypes of the 20 loci can be detected correctly, and the genotype distribution conforms to the genotype distribution frequency of Chinese Han people. Clinical sample C has a mutation at one site in the SLC26A4 gene, and there...

technical field

[0001] The invention belongs to the field of medical detection, in particular to primers for detecting point mutations of deafness gene SLC26A4 and applications thereof Background technique

[0002] Deafness is one of the most common clinical genetic diseases. At present, the traditional physical hearing defect screening method is generally used in the screening of deafness. Deafness) defects, resulting in the continuous occurrence of deaf tragedies. Genetic detection of deafness can change the occurrence of deafness from traditional passive treatment to active prevention, so as to achieve early detection and early intervention, so as to avoid the occurrence of deafness. Therefore, the detection of deafness susceptibility genes is of great significance.

[0003] At present, there are many gene detection methods on the market, such as: fluorescent quantitative PCR, sequencing and mass spectrometry detection, etc. Fluorescent quantitative PCR is widely used be...

Examples

Embodiment 12162

[0076] Example 1 2162C>T detection site primer design

[0077] A. Locked nucleic acid spacer modification

[0078] Table 4 2162C>T detection site primers

[0079]

[0080]

[0081] Bases underlined are bases modified by locked nucleic acids.

[0082] The result is as Figure 1-9 As shown, the specificity of the primer (SEQ-ID-NO.1) designed by the conventional method is poor, and under the normal dosage situation, the extension efficiency is low, and the residual level of the primer is high (characterized by the peak intensity of the primer / peak intensity of the product). )( figure 1 ). Different numbers of locked nucleic acid modifications were sequentially designed on the bases spaced between the 5' and 3' ends of the primer of SEQ-ID-NO.1, and the changes in elongation efficiency were investigated.

[0083] There was no significant change in elongation efficiency when 1 or 2 locked nucleic acid modifications were added at the 5' end (e.g. figure 2 / 3); when add...

Embodiment 2

[0090] Embodiment 2 A kind of test kit for detecting deafness gene polymorphism

[0091] The kit disclosed by the invention comprises multiple PCR reaction reagents, phosphatase digestion reagents, single base extension reagents, desalting resin and chip components which are independent of each other. The multiple PCR reaction reagents include multiple PCR reaction solutions and amplification enzymes; the phosphatase digestion reagents include phosphatase digestion reaction solutions and phosphatase; the single base extension reagents include single base extension reaction solutions and elongation enzymes . In this method, a purification column can be used to replace the resin, and the purpose of purifying the product can also be achieved; the chip in this method can be any substrate suitable for forming nucleic acid-matrix co-crystals, such as glass sheets, silicon sheets, stainless steel, etc.

[0092] The polymorphic sites selected by the present invention all have high po...

Embodiment 3

[0105] Embodiment 3 clinical experiment

[0106]Use the locked nucleic acid modified primer SEQ-ID-NO.4 to detect the 2162C>T site, and use other 19 detection sites to analyze the 20 SNP site gene detection results of 4 clinical samples, combined with deafness Interpretation of the significance of the SNP loci of related susceptibility genes, and analysis of the risk of deafness in the tested samples.

[0107] Table 8 Correspondence between deafness-related susceptibility genes and SNPs

[0108]

[0109] The SNP typing results of deafness-related susceptibility genes are shown in Table 9 below and Figure 16-19 :

[0110] Table 9 SNP typing of deafness-related susceptibility genes

[0111]

[0112] It can be seen from the above table that the genotypes of the 20 loci can be detected correctly, and the genotype distribution conforms to the genotype distribution frequency of Chinese Han people. Clinical sample C has a mutation at one site in the SLC26A4 gene, and there...

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