CN111073975B

CN111073975B - Probe system for gene SNP locus detection and application thereof

Info

Publication number: CN111073975B
Application number: CN201911413660.1A
Authority: CN
Inventors: 何庆; 王益民; 高堂杰; 周巧; 曹叶
Original assignee: Genetalks Bio Tech Changsha Co ltd
Current assignee: Genetalks Bio Tech Changsha Co ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2023-07-07
Anticipated expiration: 2039-12-31
Also published as: CN111073975A

Abstract

The invention discloses a probe system for detecting SNP loci of genes and application thereof, wherein the probe system at least comprises 2 probe pairs; each probe pair comprising a 5 'fluorescent group modified oligonucleotide sequence and a corresponding complementarily incorporated 3' quencher group modified oligonucleotide sequence; the fluorophores of each probe pair are different; the Tm value of the 5 'fluorescent group modified oligonucleotide sequence does not exceed the Tm value of the 3' quencher group modified oligonucleotide sequence. The invention also provides a reaction system for detecting gene SNP loci, a method for detecting gene SNP loci and application of the probe system in detecting deaf gene SNP loci, wherein the method is used for detecting the deaf gene related SNP loci, and the detection accuracy rate of the probe system on 16 deaf gene related SNP loci is up to 100%.

Description

Probe system for gene SNP locus detection and application thereof

Technical Field

The invention relates to the field of biotechnology and medicine, in particular to a probe system for detecting gene SNP loci and application thereof.

Background

The single nucleotide polymorphism (SNP, single nucleotide polymorphism) site refers to a difference between one base from person to person, and is a DNA sequence polymorphism caused by variation at the genomic nucleotide level, and the genomic sequence difference includes single base transitions, transversions, single base insertions and deletions, and the like. More than 99.9% of the human and human gene sequences are identical, with only 0.1% of the sequences differing, i.e., 1 in 1000 bases, and the human genome has 30 hundred million bases, thus 300 ten thousand bases in the entire human genome. Usually, the 300 ten thousand different bases are called SNP, which is determined by the genetic background of individuals, and in the current research, SNP loci can be used as clinically significant diagnostic markers (markers), which have great significance in clinic and can be used for the discovery of high-risk groups, the identification of disease-related genes, the design and test of medicines, the basic research of biology and the like.

The current method for detecting SNP loci mainly comprises a sequencing method, a chip method and a Taqman method. The sequencing method is very accurate, but the price is very high, so that the method is not suitable for detection of a large sample size; the chip method is suitable for super multi-site analysis, but the chip design cost is high, and due to the complexity of the DNA sample, some SNP can not be detected, so the accuracy is lower; the Taqman method has high accuracy, is suitable for large samples, but has a relatively high price. Therefore, the development of a low-cost, high-efficiency and large-sample-detection-applicable gene SNP locus detection method has very important significance.

According to the latest statistics of the investigation of the disabled people in 2006, the hearing speech disabled people in China reach 2780 ten thousand, about 3 ten thousand of the deaf children are born each year, and 6 to 8 ten thousand of the sick children suffering from delayed deafness are found, wherein most of the sick children are severe or extremely severe sensorineural deafness. This type of deafness severely affects the communication and cognitive abilities of the patient, creating a huge burden on individuals, families and society.

With the completion of the human genome project, the etiology study of hereditary hearing loss has progressed greatly, and it is presumed that 2/3 of congenital sensorineural hearing loss is caused by genetic factors. Non-syndrome deafness accounts for 70% of all hereditary hearing, 80% of which are autosomal recessive inheritance, 15% are autosomal dominant inheritance, and the other 5% are mitochondrial or X-linked inheritance. Although there are many genes involved in deafness, the vast majority of deafness patients are caused by the defect of a few single genes. The molecular diagnosis center for deafness in the general hospital of the liberation army conducts national wide molecular epidemiological investigation of deafness since 2004, determines that GJB2, SLC26A4 and mitochondrial genes mtDNA (A1555G and C1494T mutation) are 3 most common deafness genes which cause most hereditary deafness in China, and finds that 21 percent of deafness patients have GJB2 mutation; 14.5% of patients with SLC26A4 mutation and 3.4% and 0.6% of patients with mtDNA A1555G and C1494T mutation, respectively. Mutations in GJB2 mainly lead to congenital non-syndrome type deafness, which is an autosomal recessive genetic pattern. It is mainly mutated to 235delC. Through the definite GJB2 deafness of gene diagnosis, on the basis of definite etiology, other nervous system diseases can be eliminated, the integrity of cochlear nerves and auditory centers is prompted, and a plurality of researches show that the hearing language rehabilitation effect of the sick children after receiving artificial cochlea implantation is good, so that the method is an ideal effect prediction inspection before the artificial cochlea operation. The SLC26A4 mutation can cause two clinical manifestations, one being Pendred's syndrome, manifested as thyromegaly and deafness; another, most common in china, the infant only presents with deafness, and CT examination can find that the vestibular aqueduct enlarges, known as large vestibular aqueduct syndrome (en-larged vestibular aqueduct syndrome, EVAS). EVAS in China accounts for 20% -28% of the incidence rate of deafness in childhood. The cause of the disease is that the channel connecting the cranial cavity and the inner ear is abnormally enlarged, and the hearing of the EVAS infant can be reduced due to factors causing intracranial pressure change such as mild head collision, cold and the like. In EVAS deafness patients in China, the detection rate of SLC26A4 gene mutation reaches about 97%, and the main mutation is c.919A > G. Deafness caused by mutation of mitochondrial genes mtDNA A1555G and C1494T is mainly related to aminoglycoside drug use. The mutation carrier is extremely sensitive to aminoglycoside drugs, and tinnitus and even serious hearing loss can occur in low-dose use. Since it follows the maternal genetic pattern, i.e., the inheritance of such mutant genes is only passed directly to offspring by females, if there are 1 in the family, it can be deduced that at least 10 in the family carry such mutant genes. If the mother carries the mutant gene, the mother and the offspring can be reminded that the use of aminoglycoside medicines is forbidden, and the occurrence of deafness is avoided; meanwhile, 1 case of diagnosis in families can be used as a drug administration guide of the whole family, thereby blocking the continuous occurrence of deafness in families.

Therefore, through SNP screening of the deafness gene, the screened mtDNA A1555G and C1494T mutation carriers can effectively avoid the occurrence of drug-induced deafness by prohibiting the use of aminoglycoside antibiotics; the GJB2 deaf patient prompts the artificial cochlea to have good prognosis and can directly enter a hearing recovery program. SLC26A4 deafness patient prompts an EVAS infant, and effective hearing of the infant can be maintained as much as possible by taking strict protective measures. And the deafness gene diagnosis can provide scientific and accurate genetic information and guidance for the deafness patients and the mating fertility of the deafness family.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a probe system for detecting gene SNP loci, which can efficiently detect the gene SNP loci for genotyping.

The invention also provides a reaction system for detecting the SNP locus of the gene.

The invention also provides a method for detecting the SNP locus of the gene.

The invention also provides application of the detection method of the gene SNP locus.

According to an embodiment of the first aspect of the present invention, a probe system for detecting SNP loci of genes comprises at least 2 probe pairs; each probe pair comprising a 5 'fluorescent group modified oligonucleotide sequence and a corresponding complementarily incorporated 3' quencher group modified oligonucleotide sequence; the fluorophores of each probe pair are different; the Tm value of the 5 'fluorescent group modified oligonucleotide sequence does not exceed the Tm value of the 3' quencher group modified oligonucleotide sequence.

The probe system according to the embodiment of the first aspect of the invention has at least the following beneficial effects: the same set of probe system can be used for different SNP loci in the invention, and compared with the Taqman method, the detection cost, especially the research and development cost, is greatly saved.

According to some embodiments of the invention, the probe pair is selected from the following 4 probe pairs, probe pair 1 comprising an oligonucleotide sequence modified with a 5 'end fluorescent group having a nucleotide sequence of SEQ ID No.1 and an oligonucleotide sequence modified with a 3' end quencher group having a nucleotide sequence of SEQ ID No. 2;

the probe pair 2 comprises an oligonucleotide sequence with a nucleotide sequence of SEQ ID NO.3 and a oligonucleotide sequence with a nucleotide sequence of SEQ ID NO.4 and a 3' -end quenching group;

the probe pair 3 comprises an oligonucleotide sequence with a nucleotide sequence of SEQ ID NO.5 and modified by a 5 '-end fluorescent group and an oligonucleotide sequence with a nucleotide sequence of SEQ ID NO.6 and modified by a 3' -end quenching group;

the probe pair 4 comprises an oligonucleotide sequence with a nucleotide sequence of SEQ ID NO.7 and an oligonucleotide sequence with a nucleotide sequence of SEQ ID NO.8 and a 3' -end quenching group.

According to some embodiments of the invention, the fluorophore modification comprises at least one of FAM, TET, JOE, HEX, CY3, CY5, ROX, and TAMRA; the quencher group modification includes at least one of BHQ-1 and BHQ-2.

A reaction system for gene SNP site detection according to an embodiment of the second aspect of the invention, the reaction system comprising the probe system described above and a primer set designed for SNP sites; each primer group comprises 1 wild type primer sequence and 1 mutant primer sequence; the wild type primer and the mutant primer both contain oligonucleotide sequences corresponding to probes with fluorescent groups in the probe pair.

Preferably, the oligonucleotide sequences of the primer set are composed of an oligonucleotide sequence corresponding to a 5' -end fluorescent group-modified probe and a SNP site-specific primer sequence.

The reaction system according to the embodiment of the second aspect of the present invention has at least the following advantageous effects: the probe system contained in the reaction system can be suitable for detecting different SNP loci, has the advantages of low price and high efficiency, and is high in detection accuracy and convenient to use when used for detecting SNP loci; the invention adopts the high-fidelity DNA polymerase with 3' to 5' exonuclease activity and the primer modified by 3' end sulfuryl phosphate, thereby greatly reducing non-specific amplification and further accurately typing SNP loci.

According to some embodiments of the invention, the 3' terminal base or adjacent base of the wild-type primer sequence and the mutant primer sequence is modified with phosphorothioate; the vulcanized modified base has a certain tolerance to exonuclease digestion.

According to some embodiments of the invention, the reaction system further comprises a high-fidelity DNA polymerase; primers not modified by phosphorothioate are not tolerant to exonuclease digestion, and when a mismatch occurs, the high-fidelity DNA polymerase can cleave off the mismatched base, and extension is continued; the primer modified by the 3' -end phosphorothioate can resist exonuclease digestion, and when the mismatch occurs, mismatched bases cannot be excised in time, so that the polymerization reaction is stopped, and the extension cannot be performed. Thus, efficient extension can only be performed when the primer and template are perfectly matched, thereby reducing non-specific amplification.

Preferably, the high-fidelity polymerase includes at least one of Pfu polymerase and Deep Vent polymerase.

Preferably, the reaction system comprises the following components in percentage by weight: the concentration of each primer in the primer group is 0.05-0.25 mu M; in the probe system, the concentration of the oligonucleotide sequence modified by the 5 '-end fluorescent group is 0.1 mu M, and the concentration of the oligonucleotide sequence modified by the 3' -end quenching group is 0.15 mu M.

According to a third aspect of the present invention, a method for detecting a gene SNP site comprises the steps of:

s1, constructing the reaction system aiming at each SNP locus;

s2, performing PCR reaction to obtain a detection result.

According to the detection method of the embodiment of the third aspect of the invention, at least the following beneficial effects are achieved: according to the invention, 2 SNP loci can be detected simultaneously by adopting 4 channels, and compared with the conventional detection of 1 SNP locus by adopting 2 channels, the throughput is doubled.

According to some embodiments of the invention, the PCR reaction is programmed to: 94 ℃ for 15min;94℃for 20s,60℃for 60s,10cycles; collecting fluorescence at 94 ℃ for 20s and 55 ℃ for 60s, and collecting fluorescence at 35cycles; fluorescence was collected at 30℃for 60 s.

According to an embodiment of the fourth aspect of the present invention, the above method is applied to the detection of deafness gene-related SNP sites.

The application of the embodiment according to the fourth aspect of the invention has at least the following advantageous effects: the 16 SNP loci of the deafness related susceptibility genes selected by the invention basically cover the main SNP loci of the deafness susceptibility genes in Chinese people, and the detection method provided by the invention can be used for accurately typing the SNP related to the deafness genes, and has the advantages of low price, high use efficiency and high accuracy.

According to some embodiments of the invention, the deafness gene-related SNP sites include 16 SNP sites associated with four deafness genes located in GJB2, GJB3, SLC26A4 and MT-RNR 1; the 16 SNP loci include c.176-191del16b, c.35del G, c.299-300del AT, c.235del C, 512insAACG on the GJB2 gene, c.533C > T on the GJB3 gene, c.919-2A > G on the SLC26A4 (PDS) gene, c.2027T > A, c.2168A > G, c.1174A > T, c.1226G > A, c.1229C > T, 1707+5G > A, c.1975G > C, m.1555A > G, m.1494C > T on the MT-RNR1 (12S rRNA) gene.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a schematic diagram of the detection in example 1 of the present invention, in which F represents a fluorescent group FAM, H represents a fluorescent group HEX, and B represents a quenching group BHQ2;

FIG. 2 shows the result of GJB2-c.176-191del16bp typing in example 2 of the present invention;

FIG. 3 shows the results of GJB2-c.35delG typing in example 2 of the present invention;

FIG. 4 shows the typing results of GJB3-c.538C > T in example 2 of the present invention;

FIG. 5 shows the results of typing SLC26A4-c.919-2A > G in example 2 of the present invention;

FIG. 6 shows the results of typing SLC26A4-c.2027T > A in example 2 of the present invention;

FIG. 7 shows the results of typing SLC26A4-c.2168A > G in example 2 of the present invention;

FIG. 8 shows the result of GJB2-c.235delC typing in example 3 of the present invention;

FIG. 9 shows the results of typing of SLC26A4-c.919-2A > G in example 3 of the present invention.

Detailed Description

In order to describe the technical contents, the achieved objects and effects of the present invention in detail, the following description will be made with reference to the embodiments in conjunction with the accompanying drawings.

Example 1: design of universal probe and primer for detecting deafness gene locus

1. Design thinking

Designing a probe system: a total of 4 probe pairs were designed, each probe pair comprising a 5 'end fluorophore modified oligonucleotide sequence and a corresponding complementarily incorporated 3' end quencher modified oligonucleotide sequence, the fluorophores of each probe pair being different.

The design method of the probe and the primer comprises the following steps:

(1) Each probe pair comprises a 5 'fluorescent group modified oligonucleotide sequence and a 3' quencher group modified oligonucleotide sequence, which are capable of partially or fully complementarily binding to form a luminescence-quenching probe system.

(2) The 5' fluorescent group-modified oligonucleotide sequence and the 3' quenching group-modified oligonucleotide sequence of each probe pair may have different Tm values, the Tm value of the 5' fluorescent group-modified oligonucleotide sequence being the same as the Tm value of the 3' quenching group-modified oligonucleotide sequence, or lower than the Tm value of the 3' quenching group-modified oligonucleotide sequence.

(3) The oligonucleotide sequences of the primer set consist of an oligonucleotide sequence modified by a 5' -end fluorescent group and an SNP site-specific primer sequence. The Tm value of the SNP site specific primer sequence is higher than that of the oligonucleotide sequence modified by the 5' -end fluorescent group.

Designing a related SNP locus detection primer set: the oligonucleotide sequences of the primer set consist of an oligonucleotide sequence modified by a 5' -end fluorescent group and an SNP site-specific primer sequence. The primer set includes a wild primer sequence and a mutant primer sequence. When the wild-type primer or mutant primer is fully paired with the template, a polymerization reaction is performed to produce a product paired with the 5 'fluorophore-modified oligonucleotide sequence, which when bound to the 5' fluorophore-modified oligonucleotide sequence emits a corresponding detectable fluorescent signal. The 3' terminal base or adjacent base of the wild primer sequence and the mutant primer sequence is modified by phosphorothioate, and the base after the sulfuration modification has a certain tolerance to exonuclease digestion.

The primer is modified by phosphorothioate during oligonucleotide synthesis, and can achieve the effects of resisting exonuclease digestion and prolonging the half-life of the oligonucleotide. Zhang Jia and the like find that when the high-fidelity DNA polymerase is in mismatch, the high-fidelity DNA polymerase cannot be repaired or cannot be repaired in time, the premature termination of the polymerization reaction can be caused, and the extension cannot be carried out by comparing the extension reaction of the 3' incompletely paired primer by the high-fidelity DNA polymerase containing 3' to 5' exonuclease activity and the low-fidelity DNA polymerase not containing 3' to 5' exonuclease activity. By utilizing the characteristic of the high-fidelity enzyme, a wild primer and a mutant primer with modified terminal phosphorothioate are designed, and the single nucleotide polymorphism of the corresponding site can be detected by a luminescence-quenching probe system.

The detection principle of the scheme is shown in figure 1, and in the system, when the wild type primer or the mutant primer is completely matched with the template, polymerization reaction is carried out to generate a product matched with the oligonucleotide sequence modified by the 5 'end fluorescent group, and when the product is combined with the oligonucleotide sequence modified by the 5' end fluorescent group, a corresponding detectable fluorescent signal is emitted.

2. Designed probes and primers

The probe and primer sequences designed are shown in Table 1 below:

TABLE 1 primer and probe sequence listing

Example 2: dual system test

And (3) performing fluorescent quantitative PCR reaction on wild type leucocytes, plasmids and corresponding leucocytes and plasmid mixed samples of the leucocytes and plasmids of which the deafness genes are GJB2-c.176-191del16bp, GJB2-c.35del G, GJB3-c.538C > T, SLC A4-c.919-2A > G, SLC A4-c.2027T > A, SLC A4-c.2168A > G6 by adopting a CY5/ROX dual probe.

1. Test sample:

a: human leukocyte DNA;

b: GJB2 mutant plasmid;

c: GJB3 mutant plasmid;

d: SLC26A4 mutant plasmid 1;

e: SLC26A4 mutant plasmid 2;

g: a mixture of human leukocyte DNA and GJB2 mutant plasmids;

h: a mixture of human leukocyte DNA and GJB3 mutant plasmids;

i: a mixture of human leukocyte DNA and SLC26A4 mutant plasmid 1;

j: a mixture of human leukocyte DNA and SLC26A4 mutant plasmid 2;

k: a mixture of human leukocyte DNA and SLC26A4 mutant plasmid 3;

the sample was diluted to 5 ng/. Mu.L after the concentration was measured.

2. The reaction system:

the primers and the common probes for the pre-mixed enzyme buffer and specific site amplification required for the reaction were formulated into a reaction system (in. Mu.L each) as follows in Table 2:

TABLE 2 double reaction System

DNA	2
		2×buffer	5
WF	0.1
		MF	0.1
R	0.25
		CY5-P1	0.1
P2-BHQ2	0.15
		ROX-P3	0.1
P4-BHQ2	0.15
		ddH ₂ O	2.05
Total	10

In Table 2, WF is a wild-type primer, MF is a mutant primer, and R is a common primer, corresponding to the primer and probe sequences in example 1, respectively.

The sample was placed in a fluorescence quantitative reaction apparatus (Bo-day LineGene 9600) and reacted according to the following procedure.

3. The reaction procedure:

94 ℃ for 15min; {94℃20s,60℃60s,10cycles }; {94℃20s,55℃60s, fluorescence was collected, 30cycles }; fluorescence was collected at 30℃for 60 s.

4. Analysis of results:

the GJB2-c.176-191del16bp typing result is shown in figure 2;

the GJB2-c.35delG typing result is shown in figure 3;

the GJB3-c.538C > T typing results are shown in FIG. 4;

the SLC26A4-c.919-2A > G typing results are shown in FIG. 5;

the SLC26A4-c.2027T > A typing results are shown in FIG. 6;

the SLC26A4-c.2168A > G typing results are shown in FIG. 7;

in FIGS. 2 to 7, allele 1 represents a wild type, allele 2 represents a mutant type, and heterozygous represents a heterozygous type.

From the analysis of the figure, the two systems of the deafness genes GJB2-c.176-191del16bp, GJB2-c.35del G, GJB 3-c.533C > T, SLC A4-c.919-2A > G, SLC A4-c.2027T > A, SLC A4-c.2168A > G6 sites carry out fluorescence quantitative reaction on the wild type white blood cells, plasmids and the corresponding white blood cells and plasmid mixed samples, and the typing is correct.

Example 3: quadruple system testing

Fluorescence quantitative PCR reaction is carried out on samples of the deafness genes GJB2-c.235delC and SLC26A4-c.919-2A > G, which are determined by sequencing by using CY5/ROX/FAM/HEX quadruple probes.

1. Test sample:

a is white blood cell DNA;

c.235delC heterozygous samples;

c.235delC pure mutant sample;

c.919-2A > G heterozygous samples;

samples a to D above were diluted to 5ng/uL after concentration determination.

2. The reaction system:

the primers and the common probes for the pre-mixed enzyme buffer and specific site amplification required for the reaction were prepared into a reaction system (in. Mu.L each) as follows in Table 3:

TABLE 3 quadruple reaction system

DNA	2
		2×buffer	5
WF1	0.1
		MF1	0.1
R1	0.25
		WF2	0.1
MF2	0.1
		R2	0.25
CY5-P1	0.1
		P2-BHQ2	0.15
ROX-P3	0.1
		P4-BHQ2	0.15
FAM-P5	0.1
		P6-BHQ2	0.15
HEX-P7	0.1
		P8-BHQ2	0.15
ddH ₂ O	1.1
		Total	10

In Table 3, WF is a wild-type primer, MF is a mutant primer, and R is a common primer, corresponding to the primer and probe sequences of example 1, respectively.

3. The reaction procedure:

94 ℃ for 15min; {94℃20s,60℃60s,10cycles }; {94℃20s,55℃60s, fluorescence was collected, 35cycles }; fluorescence was collected at 30℃for 60 s.

4. Analysis of results:

the GJB2-c.235delC typing result is shown in figure 8;

the SLC26A4-c.919-2A > G typing results are shown in FIG. 9;

in fig. 8 to 9, allele 1 represents a wild type, allele 2 represents a mutant type, and heterozygous represents a heterozygous type.

The analysis of the figure shows that the deafness genes GJB2-c.235delC and SLC26A4-c.919-2A > G quadruple system are used for typing wild white blood cells, c.235delC heterozygous samples, c.235delC pure mutant samples and c.919-2A > G heterozygous samples, and the detection results are correct.

Example 4: deafness clinical sample detection

12 cases of deafness gene pure mutation or mutation carrier with SNP type determined by sequencing are detected, and the specific cases are shown in the following table 4:

TABLE 4 sample genotype conditions

Sample numbering	Gene	Mutation site	Sequencing genotypes
				1	GJB2	c.176delG	Heterozygosity
2	GJB2	c.235delC	Heterozygosity
				3	GJB2	c.299delAT	Heterozygosity
4	GJB2	c.235delC	Homozygous mutation
				5	GJB2	c.299delAT	Homozygous mutation
6	GJB2	c.35delG	Heterozygosity
				7	GJB2	c.35delG	Heterozygosity
8	SLC26A4	c.1174A>T	Heterozygosity
				9	SLC26A4	c.919-2A>G	Homozygous mutation
10	SLC26A4	c.919-2A>G	Heterozygosity
				11	SLC26A4	c.919-2A>G	Heterozygosity
12	SLC26A4	c.919-2A>G	Heterozygosity

1. Sample extraction:

human venous blood was collected and collected with EDTA anticoagulant tubes. 200. Mu.L of peripheral blood was taken, genomic DNA was extracted using DNeasy Blood and Tissue kit of QIAGEN, and the extracted DNA was quantified using Qubit and normalized to 5 ng/. Mu.L.

2. Fluorescent quantitative PCR detection:

the quadruple detection system of example 2 is adopted, the probes and primers designed in example 1 are used for respectively detecting 16 deafness gene mutation sites of DNA extracted from 12 samples, and the corresponding detection sites comprise GJB2-c.176-191del16b, GJB2-c.35delG, GJB2-c.299-300delAT, GJB2-c.235delC, GJB2-512insAACG, GJB 3-c.528C > T, SLC26A4-c.919-2A > G, SLC A4-c.2027T > A, SLC A4-c.2168A > G, SLC A4-c.1174A > T, SLC A4-c.1226G > A, SLC A4-c.1229C > T, SLC A4-1707+5G > A, SLC A4-c.1975G > C, MT-m.1555A > G and-m.144T.

3. Analysis of results:

the results of the 12 deafness samples are shown in Table 5 below, with the white in the table indicating the wild type:

TABLE 5.12 deafness sample test results

From the results of the SNP locus detection of the deafness gene shown in Table 5, the detection accuracy of the SNP locus related to the deafness gene is up to 100%. A significant portion of the chinese population is suffering from hereditary hearing loss caused by 16 high-frequency mutation sites of the 4 common genes GJB2, GJB3, SLC26A4 (PDS), MT-RNR1 (12S rRNA), which have very high rates of portability in normal and dyshearing populations. The invention has the detection accuracy reaching 100 percent, can be effectively applied to the detection of the deafness genes of newborns, and ensures that mutant patients can be intervened and treated in time or deafness-causing medicaments are avoided; meanwhile, the detection system can be used for detecting genes before wedding and pregnancies, provides scientific judgment basis for prenatal and postnatal care, and performs timely intervention and treatment for possible risks.

The invention uses the SNP detection of the deafness related susceptibility gene to carry out detection, but the method provided by the invention is not limited to the application of the deafness susceptibility gene, and other SNP typing detection extended by the method belongs to the protection scope of the patent.

The sample adopted by the implementation of the invention is white blood cell DNA, but the invention is not limited to the white blood cell DNA, and any other nucleic acid sample related to SNP typing detection related to the invention is included in the protection scope of the patent.

In summary, the beneficial effects of the invention are as follows:

1. compared with the Taqman method, the invention reduces the use of probes (the synthesis of the probes is expensive), and greatly saves the detection cost, especially the research and development cost.

2. According to the invention, 2 SNP loci can be detected simultaneously by adopting 4 channels, and compared with the conventional detection of 1 SNP locus by adopting 2 channels, the throughput is doubled.

3. The invention adopts the high-fidelity DNA polymerase with 3' to 5' exonuclease activity and the primer modified by 3' end sulfuryl phosphate, thereby greatly reducing non-specific amplification and further accurately typing SNP loci.

4. The primer system of the invention contains 16 SNP loci of susceptibility genes related to deafness, which basically covers main SNP loci of susceptibility genes of deafness in Chinese people, and the detection method provided by the invention can be used for accurately typing the SNP related to the deafness genes.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent changes made by the specification and drawings of the present invention, or direct or indirect application in the relevant art, are included in the scope of the present invention.

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Claims

1. A method for detecting SNP loci of genes for the purpose of diagnosis or treatment of non-disease is characterized by comprising the steps of: the method comprises the following steps:

s1, constructing a reaction system aiming at each SNP locus;

s2, performing PCR reaction to obtain a detection result;

the SNP loci are deafness gene related SNP loci, and the deafness gene related SNP loci comprise 16 SNP loci related to four deafness genes, namely GJB2, GJB3, SLC26A4 and MT-RNR 1; the 16 SNP loci comprise GJB2-c.176-191del16b, GJB2-c.35del G, GJB2-c.299-300del AT, GJB2-c.235del C, GJB2-512insAACG, GJB3-c.538C > T, SLC A4-c.919-2A > G, SLC A4-c.2027T > A, SLC A4-c.2168A > G, SLC A4-c.1174A > T, SLC A4-c.1226G > A, SLC A4-c.9C > T, SLC A4-1707+5G > A, SLC A4-c.1975G > C, MT-m.1555A > G and MT-m.1494C > T;

the reaction system comprises probe pairs, wherein the probe pairs are selected from the following 4 probe pairs, and the probe pair 1 comprises an oligonucleotide sequence modified by a 5 '-end fluorescent group with a nucleotide sequence of SEQ ID NO.1 and an oligonucleotide sequence modified by a 3' -end quenching group with a nucleotide sequence of SEQ ID NO. 2;

2. The method for detecting a gene SNP site according to claim 1, wherein: the fluorophore modification comprises at least one of FAM, TET, JOE, HEX, CY, CY5, ROX, and TAMRA; the quencher group modification includes at least one of BHQ-1 and BHQ-2.

3. The method for detecting a gene SNP site according to claim 2, wherein: the PCR reaction comprises the following steps: 94 ℃ for 15min;94℃for 20s,60℃for 60s,10cycles; collecting fluorescence at 94 ℃ for 20s and 55 ℃ for 60s, and collecting fluorescence at 35cycles; fluorescence was collected at 30℃for 60 s.

4. A reaction system for detecting SNP loci of genes is characterized in that: the reaction system comprises the probe pair as in claim 1 and a primer set designed for SNP sites; each primer group comprises 1 wild type primer sequence and 1 mutant primer sequence; the wild type primer and the mutant primer both contain oligonucleotide sequences corresponding to probes with fluorescent groups in the probe pair.

5. The reaction system for gene SNP locus detection according to claim 4, wherein: the 3' -terminal base or adjacent base of the wild type primer sequence and the mutant primer sequence is modified by phosphorothioate.

6. The reaction system for gene SNP locus detection according to claim 4, wherein: the reaction system also contains high-fidelity DNA polymerase.

7. The reaction system for gene SNP locus detection according to claim 6, wherein: the high-fidelity DNA polymerase comprises at least one of Pfu polymerase and Deep Vent polymerase.