CN111705122B - Genetic deafness screening method based on MassArray nucleic acid mass spectrometry platform and application thereof - Google Patents
Genetic deafness screening method based on MassArray nucleic acid mass spectrometry platform and application thereof Download PDFInfo
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Abstract
The invention provides a primer group for genetic deafness screening based on a MassArray nucleic acid mass spectrum platform, a method and application thereof. The primer group for detecting the genetic deafness related SNP sites and the detection system thereof are designed based on the MassArray nucleic acid mass spectrum platform, so that the mutation types such as point mutation, deletion mutation, mitochondrial point mutation and the like related to the genetic deafness can be accurately typed, and the rapid and effective detection of the genetic deafness is realized.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a genetic deafness screening method based on a MassArray nucleic acid mass spectrum platform and application thereof.
Background
According to estimation, the total incidence rate of birth defects in China is about 5.6%, and birth defects seriously affect the survival and life quality of children and bring great pain and economic burden to children patients and families.
According to the data in Chinese birth defect prevention and treatment report 2012, hearing impairment is the second largest birth defect disease in China, and accounts for 24.97% of congenital disabilities. 2054 thousands of people with hearing disabilities in China exist, wherein the number of children aged 0-6 exceeds 80 thousands of people, 3 thousands of deaf children are newly added every year, and the number of newly-added hearing-disability children exceeds 6 thousands of cases in total. In the aspect of treatment, severe deafness cannot be treated except for expensive artificial cochlear implants. Therefore, early diagnosis of deafness and prevention of deafness are of great importance.
In China, the proportion of the common population carrying the high-risk gene mutation is 4.37%, which means that almost 1 in every 20 people carries the high-risk deafness gene mutation. Assuming that 15 hundred million people exist in China at present, the carrying population is about 6000 plus 7500 million people. This data is similar to the situation of the mouth of a Chinese hearing impaired person inferred by audiologists.
In 2018, the national health and health committee office issues a national birth defect comprehensive prevention and treatment scheme, and requires comprehensive development of phenylketonuria, congenital hypothyroidism and hearing screening (two screens and listening screen), and prenatal screening, neonatal genetic metabolic disease screening and neonatal hearing screening should be developed at county level. At the local level there are in principle at least 1 neonatal hearing screening center.
Deafness caused by genetic factors such as gene mutation can be divided into three types of congenital deafness, delayed deafness and drug-sensitive deafness according to pathogenic modes and attack time. The congenital deafness patients can be effectively screened by two methods, namely an otoacoustic emission method (OAE) and a brainstem auditory evoked potential method (BAEP), for routine hearing screening of newborn in China, but can be screened only by a deafness gene detection method for two types of delayed deafness and drug-sensitive deafness. Therefore, the combined screening is more helpful for comprehensively evaluating the risk of diseases and taking targeted prevention and treatment measures.
At present, the detection means are numerous and distinctive, and the microarray chip method is simple, convenient and quick to operate, but has limited flux and general sensitivity. The high-flux gene detection technology solves the flux limitation, but has high cost, long detection period and high technical requirements for experiment operation and data analysis personnel, so the high-flux gene detection technology is not widely applied to clinical detection.
In summary, under the condition that the hearing disabilities of the neonates in China account for a large proportion of the congenital disabilities, the risk of the neonates suffering from the deafness is evaluated more comprehensively. Therefore, an economical, rapid and easy-to-operate detection method is needed to meet the requirements of precise medication and treatment. The early discovery and early intervention have important significance for improving the health quality of the children patients.
The invention is provided in view of the above.
Disclosure of Invention
The technical problem to be solved by the present invention is to find a screening method suitable for hereditary deafness, and therefore the object of the present invention is as follows:
the first purpose of the invention is to provide a primer group for detecting SNP sites related to hereditary hearing loss.
The second purpose of the invention is to provide an application of the primer group for detecting the SNP locus related to the hereditary hearing loss in the hereditary hearing loss screening.
The third purpose of the invention is to provide a screening method for hereditary hearing loss.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a primer group for detecting SNP sites related to hereditary hearing loss, wherein the hereditary hearing loss comprises the following components: congenital deafness, delayed deafness, syndromic deafness, and drug-sensitive deafness.
Further, the primer group is used for detecting and typing point mutation, deletion mutation and mitochondrial point mutation of USH2A, SLC26A4, GJB2, CDH23, MT-ND1, KCNQ4, COCH and MYO6 genes.
Further, the SNP sites targeted by the primer sets comprise: rs111033367, rs372347027, rs375668376, rs 3973518039, rs369522997, rs111033199, rs111033242, rs111033220, rs111033313, rs145254330, rs786204730, rs104894408, rs72474224, rs80338942, rs80338943, rs104894396, rs35887622, rs80338939, rs267606617, rs267606618, rs267606619, rs28937588, rs 9566801 and rs 938175, rs876657754, rs777465132, rs201895089 and rs 727504567.
Preferably, the primer sequences are shown in SEQ ID NO.1-56, respectively, or have at least 85% identity with SEQ ID NO. 1-56.
Preferably, the primer set further comprises an extension primer having a sequence as set forth in SEQ ID NO.57-84, or having at least 85% identity with SEQ ID NO. 57-84.
Further, the primer sets can be divided into the following two groups:
the first set comprises primers for rs 9566801, rs80338942, rs80338943, rs876657754, rs375668376, rs111033367, rs267606618, rs 39518039, rs267606619, rs111033242, rs777465132, rs111033199, rs72474224, rs372347027, rs28937588, rs267606617 and rs 104894408.
The second set comprises primers for rs28938175, rs111033220, rs145254330, rs201895089, rs80338939, rs104894396, rs35887622, rs727504567, rs369522997, rs786204730 and rs 111033313.
The invention also provides application of the primer group in preparation of products for screening hereditary hearing loss.
The invention also provides application of the primer group in nucleic acid mass spectrometry detection.
The invention also provides a composition/product/kit for screening of hereditary hearing loss, which is characterized in that the composition/product/kit comprises the primer set as claimed in the claim.
Further, the composition/product/kit further comprises reagents and/or software for detecting SNP sites; more preferably, the software comprises MassARRAY software.
The invention also provides a method for screening the SNP locus related to the hereditary hearing loss or a method for screening the hereditary hearing loss, which is characterized by comprising the step of detecting the nucleotide sequence of the SNP locus in the genome of a sample to be detected by applying the primer set.
Further, the screening is based on the MassARRAY platform.
Furthermore, the primer group for screening the SNP sites related to the mental and neurological diseases is used for carrying out PCR amplification and single base extension reaction on the genome of the object to be detected, and then a MassARRAY platform is used for screening the reaction product to determine the genotype of the SNP sites in the genome of the object to be detected.
The invention has the beneficial technical effects that:
1) the primer group of the genetic deafness related SNP locus is obtained by large sample test and screening, can realize accurate typing of three mutation type samples of point mutation, deletion mutation and mitochondrial point mutation, simultaneously meets the requirements of mass spectrum detection technology, and realizes quick and effective screening of genetic deafness of a sample to be detected by applying a MassARRAY system.
The primer group of the genetic deafness related SNP locus provided by the invention covers 4 kinds of common genetic deafness mutation loci such as congenital deafness, delayed deafness, syndrome deafness, drug-sensitive deafness and the like, and can more effectively screen the genetic deafness.
The product for screening the hereditary hearing loss disease provided by the invention can realize specific detection on the hereditary related site mutation, and compared with the detection method on the market at present, the product has the advantages of strong accuracy, high sensitivity, good repeatability, low cost, short detection period, intuitive result, no need of biological communication intervention and the like. The Chinese medicinal composition is put on the market at present, has obvious market inspection effect, obtains great commercial return, and has great clinical application value and wider market popularization.
The invention can be applied to the detection of 8 genetic deafness related genes and 28 polymorphic sites, greatly improves the detection efficiency and is particularly suitable for batch detection. In addition to non-syndromic deafness, which exhibits hearing impairment as the only phenotype, more than 400 syndromic deafness can lead to multisystemic abnormalities in newborns. However, the targets of common detection are non-syndromic deafness, and the design of syndromic deafness causing multi-system diseases is less. The locus to be detected comprises a hot spot locus of common syndromic deafness, not only focuses on the hearing development condition, but also provides a targeted suggestion for the development of each system. Example (c): usher syndrome can cause congenital hearing impairment in 3% -6% of children, and also causes visual dysplasia, which is prone to progressive visual impairment after the onset of puberty. Therefore, the main pathogenic gene USH2A has detection significance.
The invention overcomes the defect of less SNP site detection at one time in the prior art, has low cost and is suitable for wide popularization; the adaptability of the required sample is strong, and peripheral blood and dry blood spots can be well detected; the invention is suitable for newborns, children and people who need to have a child, can not only provide the living attention items and aminoglycoside drug risk prompts of the examined people and relatives, but also help the examined people with suspected family history to screen pathogenic sites, evaluate pathogenic effects and assist reproduction; the high-risk carrier can take targeted measures as soon as possible, thereby improving the treatment effect and avoiding presbycusis. Provides effective scientific reference for preventing and treating hereditary hearing loss.
Drawings
FIG. 1 is a cluster map of sites before optimization of rs35887622 site provided in embodiment 2 of the present invention
FIG. 2 is a cluster map of sites optimized at the rs35887622 site provided in embodiment 2 of the present invention
FIG. 3 is a peak diagram of the site before optimization of the rs786204730 site provided in embodiment 2 of the present invention
FIG. 4 is a peak diagram of the optimized locus of the rs786204730 locus provided in embodiment 2 of the present invention
FIG. 5 is a cluster map of the rs267606617 locus after merging provided in example 2 of the present invention
FIG. 6 is a cluster map of the site rs267606618 after merging provided in embodiment 2 of the present invention
FIG. 7 is a cluster map of the rs267606619 locus after merging provided in embodiment 2 of the present invention
Fig. 8 is a cluster map of the rs 9566801 locus provided in embodiment 3 of the present invention;
fig. 9 is a cluster map of the rs80338943 site provided in embodiment 3 of the present invention;
FIG. 10 is a cluster map of the rs72474224 site provided in example 4 of the present invention
FIG. 11 is a cluster map of the rs35887622 site provided in example 4 of the present invention
FIG. 12 is a cluster map of rs104894408 sites provided in embodiment 4 of the present invention
FIG. 13 is a cluster map of the rs80338939 site provided in embodiment 4 of the present invention
FIG. 14 is a cluster map of the rs80338942 site provided in embodiment 4 of the present invention
FIG. 15 is a cluster map of site rs104894396 provided in example 4 of the present invention
FIG. 16 is a cluster map of the rs80338943 site provided in embodiment 4 of the present invention
FIG. 17 is a cluster map of the rs201895089 locus provided in example 4 of the present invention
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.
Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.
The term "about" in the present invention denotes an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The following is provided merely to aid in understanding the invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.
The primer group for detecting the SNP loci related to hereditary hearing loss is designed aiming at the main mutation loci related to hereditary hearing loss, can realize specific detection on the mutation loci related to hereditary hearing loss, has high accuracy, covers the mutation loci of common 4 types of hereditary hearing loss, such as congenital hearing loss, delayed hearing loss, syndrome hearing loss, drug-sensitive hearing loss and the like, can greatly shorten the detection period, and simultaneously reduces the detection cost. In addition, the primer group provided by the invention is obtained by changing optimization and large sample test screening, all primer sequences can accurately classify samples, and simultaneously can meet the requirements of a mass spectrometry detection technology, so that rapid and effective detection of hereditary hearing loss by applying a MassARRAY platform is realized.
In some preferred embodiments, the SNP sites corresponding to genetic deafness include: rs111033367, rs372347027, rs375668376, rs 3973518039, rs369522997, rs111033199, rs111033242, rs111033220, rs111033313, rs145254330, rs786204730, rs104894408, rs72474224, rs80338942, rs80338943, rs104894396, rs35887622, rs80338939, rs267606617, rs267606618, rs267606619, rs28937588, rs 9566801 and rs 938175, rs876657754, rs777465132, rs201895089 and rs 727504567.
In some preferred embodiments, the primer set sequences are as set forth in SEQ ID NO.1-56, or are at least 85% identical to SEQ ID NO. 1-56.
It is understood that the primer pairs described in the present invention are PCR primer pairs.
It should be noted that the term "identity" refers to the similarity of sequences. "identity" includes nucleotide sequences having at least 85% (e.g., can be, but is not limited to, 85%, 90%, 95% or more) identity to the single-stranded DNA represented by SEQ ID NO.1 to SEQ ID NO.56 as described herein.
In some preferred embodiments, when extension primers are required for practical use, the primer set further comprises 28 extension primers shown in SEQ ID NO.57-84, or extension primers having at least 85% identity to SEQ ID NO. 57-84.
It is understood that, in the present invention, the primer pair and the extension primer correspond to each other, and the corresponding primer pair and the extension primer are used for detecting nucleotides at the same site. For example, the first primer pair includes an upstream primer of the nucleotide sequence shown in SEQ ID NO.1, a downstream primer of the nucleotide sequence shown in SEQ ID NO.2, and an extension primer of the nucleotide sequence shown in SEQ ID NO.57, which are used for detecting the nucleotide at the same site; the second primer pair comprises an upstream primer of the nucleotide sequence shown as SEQ _ ID NO.3, a downstream primer of the nucleotide sequence shown as SEQ _ ID NO.4 and an extension primer of the nucleotide sequence shown as SEQ _ ID NO.58, wherein the primers are used for detecting the nucleotide at the same site; the third primer pair comprises an upstream primer of the nucleotide sequence shown as SEQ _ ID NO.5, a downstream primer of the nucleotide sequence shown as SEQ _ ID NO.6 and an extension primer of the nucleotide sequence shown as SEQ _ ID NO.59, wherein the primers are used for detecting the nucleotide and the like of the same site.
It is understood that the numbers of the primer pair and the extension primer correspond to the sequence of the SNP sites, i.e., the first primer pair and the first extension primer correspond to the detection site rs 9566801, the second primer pair and the second extension primer correspond to the detection site rs80338942, and the third primer pair and the third extension primer correspond to the detection site rs 80338943.
According to a second aspect of the invention, the invention also provides application of the primer group for detecting related SNP sites of hereditary hearing loss in preparation of a product for detecting hereditary hearing loss.
According to a third aspect of the present invention, there is provided a composition/product/kit for genetic deafness detection, said composition/product/kit comprising the primer set described above.
The composition/product/kit can realize specific detection on site mutation related to detection of hereditary hearing loss, has low detection cost, short period, simple operation and high accuracy, and has great clinical application value and wide market popularization prospect.
In some preferred embodiments, the composition/product/kit for detecting genetic deafness further comprises reagents and/or equipment for detecting SNP sites.
It is to be understood that the reagent and/or apparatus for detecting a SNP site may be a reagent and/or apparatus commonly available in the art for detecting a nucleotide at a SNP site, for example, a reagent or kit for detecting a nucleotide at a SNP site, or a kit for detecting a nucleotide at a SNP site including a reagent.
In some preferred embodiments, the reagents include 10 XPCR Buffer, dNTP Mix, MgCl2Primer Mix, PCR Enzyme, ultrapure water, and the like;
preferably, the apparatus comprises an apparatus for detecting the nucleotide of the SNP site using MassARRAY, preferably the apparatus comprises MassARRAY CPM.
In addition, the invention also provides a method for detecting the SNP locus related to the hereditary hearing loss, which comprises the step of detecting the nucleotide of the SNP locus in the genome of a sample to be detected by applying the primer group.
The method can be used for detecting 28 SNP loci of 8 genetic deafness related genes related to 4 general types of genetic deafness such as congenital deafness, delayed deafness, syndrome deafness, drug-sensitive deafness and the like by applying the primer group provided by the invention, and has the characteristics of strong accuracy, high sensitivity, good repeatability, low cost, short detection period, intuitive result, no need of letter generation intervention and the like.
It can be understood that the invention can detect the whole genome of a sample to be detected, and can also screen specific genes for detection (for example, rs111033367, rs372347027, rs375668376, rs 3973518039, rs369522997, rs111033199, rs111033242, rs111033220, rs111033313, rs145254330, rs786204730, rs104894408, rs 724724, rs80338942, rs80338943, rs104894396, rs35887622, rs80338939, rs 2676617, rs267606618, rs267606619, rs28937588, rs 9566801 and rs28938175, rs876657754, rs777465132, rs201895089 and rs 504567 specific genes), and when detecting specific genes, the interference is smaller, and the detection result is more accurate.
In some preferred embodiments, the primer set is used for performing PCR amplification and base extension reaction on a genome of a sample to be tested, and then a MassARRAY system is used for detecting a product obtained by the reaction, so as to determine the nucleotide of the SNP site in the genome of the sample to be tested.
The MassARRAY gene analysis technology is based on the MALDT-TOF flight time mass spectrum technology, a target sequence is amplified through PCR, then SNP sequence specific extension primers are added according to needs, and extension of a single base is carried out on an SNP locus. The technology applies the characteristic of high sensitivity of mass spectrometry to the quality, and can effectively distinguish two gene sequences with only one different base, thereby further inferring SNP typing.
In the preferred embodiment of the invention, the MassARRAY system is adopted for detection, so that the method for detecting multiple gene loci by using a single platform is realized, the detection efficiency is greatly improved, the method is particularly suitable for batch detection, and a reference is provided for screening of hereditary hearing loss. In addition, the adaptability of the required sample is strong, and peripheral blood and dry blood spots can be well detected.
It will be appreciated in the art that the method may also include a step of dephosphorylating the PCR product prior to the base extension reaction. Specifically, the PCR product was dephosphorylated using shrimp alkaline phosphatase.
Preferably, the method further comprises a step of purifying the reaction product after the base extension reaction, and then detecting the reaction product using MassARRAY. Specifically, the reaction product can be purified by desalting the resin.
In some preferred embodiments, the primer components are grouped into the following 2 groups:
the first set comprises primer sets for detecting rs 9566801, rs80338942, rs80338943, rs876657754, rs375668376, rs111033367, rs267606618, rs 39518039, rs267606619, rs111033242, rs777465132, rs111033199, rs72474224, rs372347027, rs28937588, rs267606617 and rs 104894408.
The second group comprises primer groups for detecting rs28938175, rs111033220, rs145254330, rs201895089, rs80338939, rs104894396, rs35887622, rs727504567, rs369522997, rs786204730 and rs 111033313;
the grouping comprehensively considers factors such as the size of the extension primer and the like, and ensures that the primers in each group are not interfered with each other.
The invention is further described by the accompanying drawings and the following examples, which are intended to illustrate specific embodiments of the invention and are not to be construed as limiting the scope of the invention in any way.
The main reagent information used in the examples of the present invention is as follows:
the main instrument information used in the embodiment of the invention is as follows:
example 1 demonstration and screening of assay sites
Although sites relevant to deafness detection have been disclosed in the prior art, it is not easy to select or combine sites that are truly clinically meaningful and effective for screening. The selection of the site to be detected of the invention needs to have the following functions: 1) mutants of the site may produce a defined pathogenic effect; 2) the mutant has the detection significance of Chinese people and is carried in high frequency in Chinese deaf patients. The invention refers to databases of ACMG, ExAC, 1000Genomes, dbSNP, ClinVar, HGMD and the like, and preliminarily screens the pathogenic sites carried by the patients with Chinese deafness at high frequency.
According to the invention, through medical verification, the site covering the clinically approved pathogenicity and suspected pathogenicity is determined, and the mitochondrial site with higher drug-induced deafness evidence grade is supplemented, so that the site setting has more screening and detecting significance and clinical effectiveness, and positive detection has more prompting effect on prognosis, clinical treatment and life guidance. For example: KCNQ4 is a dominant pathogenic gene related to autosomal dominant deafness type 2A, and mutation carriers can screen hearing at birth normally and can develop full-frequency severe-extremely severe hearing loss after adults with age. At present, the correlation between the hot spot of the gene and diseases is verified, and the pathogenic evidence is sufficient, so that the gene has strong detection significance. Meanwhile, partial pathogenicity of genes in common panel is proved to support insufficient evidence, and if long-term clinical detection is carried out, the clinical pathogenic effect evidential force of the GJB3 gene in common detection is not fully verified, and the method has no wide screening significance.
Meanwhile, the effectiveness of detection and screening is considered, and the site capable of accurately typing three mutation type samples, namely point mutation, deletion mutation and mitochondrial point mutation, is finally obtained through large sample testing and screening. These loci cover 4 kinds of common hereditary deafness mutation loci such as congenital deafness, delayed deafness, syndromic deafness, drug-sensitive deafness and the like, can detect 6 kinds of hereditary deafness diseases, including Usher syndrome type 2A, large vestibular aqueduct syndrome, autosomal recessive deafness type 1A, aminoglycoside drug-induced deafness, autosomal dominant deafness type 2A and autosomal dominant deafness type 9, and provide scientific reference for hereditary deafness screening. Specific genes and sites are shown in the following table:
TABLE 1 genotype correspondence table for each site
| Site/variant | Gene/gene | Genotype(s) | Site/variant | Gene/gene | Genotype(s) |
| rs111033367 | USH2A | CT/Del | rs72474224 | GJB2 | G/T/A |
| rs372347027 | USH2A | A/G | rs80338942 | GJB2 | T/Del |
| rs375668376 | USH2A | G/A | rs80338943 | GJB2 | C/Del |
| rs397518039 | USH2A | A/G | rs104894396 | GJB2 | G/A |
| rs369522997 | USH2A | A/C | rs35887622 | GJB2 | T/C/G |
| rs111033199 | SLC26A4 | G/T | rs80338939 | GJB2 | G/Del |
| rs111033242 | SLC26A4 | C/G | rs267606617 | MT-ND1 | A/G |
| rs111033220 | SLC26A4 | C/T | rs267606618 | MT-ND1 | T/C |
| rs111033313 | SLC26A4 | A/G | rs267606619 | MT-ND1 | C/T |
| rs145254330 | SLC26A4 | C/T | rs28937588 | KCNQ4 | G/T/A |
| rs786204730 | SLC26A4 | Del/T | rs956666801 | KCNQ4 | G/C |
| rs104894408 | GJB2 | C/G/T | rs28938175 | COCH | C/T |
| rs876657754 | CDH23 | A/G | rs201895089 | GJB2 | T/G |
| rs777465132 | USH2A | G/T | rs727504567 | MYO6 | C/T |
The corresponding relationship between the detection sites and the hereditary hearing loss diseases is shown in the following table 2:
TABLE 2 detection sites and corresponding hereditary deafness
Example 2 design of primers and establishment of reaction System
Since the MassARRAY detection is a reaction based on multiplex PCR amplification, the primer combination must avoid the problems of cross amplification, preferential amplification and non-specificity (D.van den Boom et al/International Journal of Mass Spectrometry, 238 (2004); 173-) -188), and thus the primer design of the reaction system needs to be optimized to a certain extent.
Firstly, related parameters are adjusted through MassARRAY website primer Design software (Assay Design Suite), PCR of 28 sites and preliminary Design of UEP primers are completed, designed primers and various parameter files are exported, and the primers are synthesized. And preparing an amplification primer MIX and an extension primer MIX according to a primer configuration table, and finely adjusting the extension primer MIX until the requirements are met. Primer testing and optimization were then performed. The method comprises the following specific steps:
genomic DNA samples were diluted to 10 ng/. mu.L and PCR reaction MIX (hereinafter, single sample size) was prepared as follows
TABLE 3 PCR reaction System
Membrane sealed, 2272g centrifuged for 1 min, and the plate was placed on a PCR instrument for the following thermal cycling:
TABLE 4
(1) Shrimp alkaline phosphatase digestion (SAP)
The PCR plate was removed, centrifuged at 2272g for 1 min, and the SAP reaction was prepared as follows (single sample size:
TABLE 5 SAP reaction System
TABLE 6
| Temperature (. degree.C.) | Time |
| 37 | 40min |
| 85 | |
| 10 | Heat preservation |
(2) Single base Extension (EXT)
Taking out the PCR plate, centrifuging for 1 minute at 2272g, and preparing an EXT reaction system according to the following table, wherein the extended Primer Mix is a mixture of two groups of extension primers with different sites (the following is a single sample amount):
TABLE 7 EXT reaction System
TABLE 8
(3) Resin desalination
Paving clean Resin (Resin) on a sample plate corresponding hole, and air-drying for at least 10 minutes;
taking out the sample plate, and centrifuging for 1 minute by using a plate centrifuge 2272 g;
adding 16 mu L of water into each hole with the sample of the sample plate, and closing the plate;
lightly overturning the sample plate in a volley manner, putting the sample plate on the sample plate with the resin, and then overturning the sample plate together with the sample plate (the two quick plates cannot move horizontally in the process) to allow the resin to fall into the holes;
seventhly, 2272g and centrifuging for 5 minutes.
(4) Dispensing spotting
Samples were spotted using MassARRAY CPM onto the corresponding SpectroCHIP (chip).
(5)MALDI-TOF
Data were obtained using MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometer.
It should be noted that the specificity of the MassARRAY assay is that a fragment containing a target SNP site is amplified by PCR reaction, then the base of the SNP site is extended by the extension primer, and the SNP site information is judged by the molecular weight of the product. The invention can detect information of a plurality of SNP sites in a reaction system, which requires that PCR reaction and extension reaction of each SNP site can not have obvious interference.
Target region adjustment, UEP primer orientation adjustment: primers for rs35887622 and rs786204730 sites were optimized as examples:
no call appears at the rs35887622 site, and after a PCR primer (the sequence of an upstream primer before modification is ACGTTGGATGACAAAGTCGGCCTGCTCATC, and the sequence of a downstream primer before modification is ACGTTGGATGGCATTGGAAAGATCTGGCTC) and a UEP primer (the sequence of the UEP primer before modification is gCCTCTTCATTTTTCGCATTA) are redesigned, the modified PCR primer and the UEP primer are tested according to the steps, so that the test effect is better and the no call phenomenon is improved. No call appears in the 40 samples, the specific clustering peak graph before the primer is changed is shown in figure 1, and the specific clustering peak graph after the primer is changed is shown in figure 2. It can be seen from the figure that the clusters before changing the primers are all no calls, and the clusters after changing are normal and all successfully report the locus genotypes, which indicates that the primers after changing are superior to the primers before changing and meet the detection requirements of the project.
The rs786204730 site shows lower peak and has biased peak, and the PCR primer (the sequence of the upstream primer before the change is shown in the specification) is redesigned to ACGTTGGATGGAGATATGTCTTGATGTTCC, and the sequence of the downstream primer before the change is shown in the specification: ACGTTGGATGAGTTCCTGTCGGATATGGTC) and the altered UEP primer orientation (UEP sequence before alteration: GGTCTCTACTCTGCTTTTTT), testing according to the steps, finding that the modified PCR primer and UEP primer have better testing effect and improved peak appearance. No peak is shown in the test of 40 samples, the peak pattern of the site before optimization is shown in FIG. 3, and the peak pattern of the site after primer optimization is changed is shown in FIG. 4. From the figure, it can be seen that the peak appearance of the site before the primer is changed is low and has a biased peak phenomenon, and the changed cluster is normal and successfully reports the site genotype, which indicates that the changed primer is superior to the primer before the change and meets the detection requirement of the project.
Inter-primer and well alignment: the rs267606617, rs267606618 and rs267606619 site optimized primers are taken as examples.
rs267606617, rs267606618 and rs267606619 locus before hole merging, rs267606619 and rs267606618 in well1 and rs267606617 in well 2; after optimization and adjustment, the detection of aminoglycoside drug-induced deafness sites in a single hole is realized in well 1. By clustering 40 samples, the rs267606617 locus clustering chart 5, the rs267606618 locus clustering chart 6 and the rs267606619 locus clustering chart 7 can be seen to meet the requirements of the project after merging.
In summary, optimal PCR amplification primers and single base extension (UEP) primers were obtained, and the specific primer sequences are shown in tables 7 and 8.
TABLE 9 PCR primer sequences
TABLE 10 UEP primer sequences
Example 3 validation of the reaction System
After the optimal reaction system is confirmed, a series of verification experiments are carried out, including accuracy, precision, personnel comparison and comparison experiments among different batches of primers. The specific verification scheme is as follows:
(1) accuracy experiment verification scheme: one sample was selected for Sanger sequencing at each of the 28 sites and compared to the results of Sanger sequencing and MassARRAY.
Because deafness mutation is a type of mutation which is very rare in clinic, and clinical samples are extremely difficult to collect, plasmids of two types, namely mitochondrial mutation and genome mutation, of the synthetic plasmid respectively comprise sites: rs267606617, rs267606618, rs267606619, and rs 9566801. The fish sperm DNA and two types of plasmids are mixed, 9 mixed plasmid samples are subjected to comparison verification (the plasmid synthesis is verified by Sanger sequencing), and the results of comparing Sanger sequencing with MassARRAY are passed if the consistency is more than 95%.
(2) The precision experiment verification scheme is as follows: 3 samples of the same patient with 3 exceptions of the peripheral blood samples and 3 samples of the dried blood spots are picked, each sample is repeatedly tested for 3 times to carry out one batch of test, 5 batches of test are tested, 15 times of test results of 6 samples have 100% consistency, and the consistency of the inter-batch precision and the intra-batch precision is more than 95%, and the test is passed.
(3) Personnel alignment and reagent alignment experimental validation protocol: two batches of primers (batch A and batch B) were prepared in (2), operator A detected batch 1 and batch 2 using primer batch A, batch 4 using primer batch B, operator B detected batch 3 using primer batch A, and batch 5 using primer batch B, and the results of comparison of the results between the human and the reagents gave a pass if the identity was greater than 95%.
The specific verification process is as follows: first, two batches of amplification primers MIX and extension primers MIX are prepared according to the system addition table provided in example 1 of the present invention, and named as batch A and batch B, respectively. Then, the results were analyzed by PCR amplification, shrimp alkaline phosphatase consumption, single-base extension, resin desalting, and MassARRAY CPM spotting, according to the procedures described in example 1. The accuracy and precision results are shown in the following table.
TABLE 11 verification of accuracy 1
TABLE 12 verification of accuracy 2
The comparison between the MassARRAY result and the Sanger result of 28 samples and 9 mixed plasmids shows that the accuracy of the verification experiment of the system is 100%. In addition, because the detection sites are more, 5 sites rs267606617, rs267606618, rs267606619, rs 9566801 and rs80338943 which are sequenced by Sanger and comprise point mutation, deletion mutation and mitochondrial point mutation are selected for display, and the clustering charts of the 5 sites are respectively shown in a figure 5, a figure 6, a figure 7, a figure 8 and a figure 9. The accuracy of the present invention is 100% as can also be derived from the results of the above figures.
TABLE 13 results of the verification of precision
By comparing the detection results of 3 samples of the peripheral blood and 5 batches of 3 samples of the dry blood spots, the consistency of the batch precision, the personnel comparison and the reagent comparison of the system of the invention is 100 percent.
Example 4 evaluation of Chamber
The invention selects partial sites to participate in European Union indoor evaluation, and specifically participates in 8 site detection report of hereditary hearing loss GJB2 gene of EMQN in 2020, wherein the site names are as follows: rs72474224, rs35887622, rs104894408, rs80338939, rs80338942, rs104894396, rs80338943 and rs201895089, the clustering charts of the above 8 sites are respectively shown in fig. 10, fig. 11, fig. 12, fig. 13, fig. 14, fig. 15, fig. 16 and fig. 17, and the results pass the evaluation of the interventricular septum of the European Union, and the effectiveness, accuracy and authority of the invention are proved.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> Xiansu medical diagnosis Co., Ltd
Jiangsu Xiansheng Medical Devices Co.,Ltd.
NANJING XIANSHENG MEDICAL TESTING Co.,Ltd.
<120> genetic deafness screening method based on MassArray nucleic acid mass spectrum platform and application thereof
<160> 84
<170> SIPOSequenceListing 1.0
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<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
acgttggatg actccgactt ctcctcctac 30
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
acgttggatg tgagtcagga gtcacgatgg 30
<210> 3
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acgttggatg tcttctcatg tctccggtag g 31
<210> 4
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
acgttggatg gagaagtctc cctgttctgt cc 32
<210> 5
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acgttggatg gagaagtctc cctgttctgt cc 32
<210> 6
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
acgttggatg tcttctcatg tctccggtag g 31
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acgttggatg atttctggac ccctagggtg 30
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
acgttggatg ggctgattgt gaaatgtggg 30
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
acgttggatg aaaatgtagt gctgcagagg 30
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
acgttggatg tgccaagtta acgacagcac 30
<210> 11
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
acgttggatg ttgccttgaa gctgttgcac 30
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
acgttggatg actggtcaca caaccaactg 30
<210> 13
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
acgttggatg tctggcgagc agttttgttg 30
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
acgttggatg gctaaaactc acctgagttg 30
<210> 15
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
acgttggatg cacgcatata tcacacgcac 30
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
acgttggatg gctgctggat tttacgtctc 30
<210> 17
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
acgttggatg agtgtaagtt gggtgctttg 30
<210> 18
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
acgttggatg tgaacagggc cctgaagcgc 30
<210> 19
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
acgttggatg gagcaagaag caacacttac 30
<210> 20
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
acgttggatg ttggcagatc ctttggttgg 30
<210> 21
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
acgttggatg gcatggcctt accacaattc 30
<210> 22
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
acgttggatg tcttctggtg ttgacatagg 30
<210> 23
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
acgttggatg gcaccaacct aatagaggta 30
<210> 24
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
acgttggatg actagctgca gttcctgtcg 30
<210> 25
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
acgttggatg gagaagtctc cctgttctgt cc 32
<210> 26
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
acgttggatg tcttctcatg tctccggtag g 31
<210> 27
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
acgttggatg gggtggaagg aaatcctgta 30
<210> 28
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
acgttggatg cactgttgtg tacgaagagc 30
<210> 29
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
acgttggatg ctgcctgtaa cctgtttgtg 30
<210> 30
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
acgttggatg gaaagagatg cccagtaagg 30
<210> 31
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
acgttggatg tgaacagggc cctgaagcgc 30
<210> 32
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
acgttggatg agtgtaagtt gggtgctttg 30
<210> 33
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
acgttggatg ggtgagccag atctttccaa 30
<210> 34
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
acgttggatg tgattcctgt gttgtgtgca t 31
<210> 35
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
acgttggatg cccacatatg ctcgatacag 30
<210> 36
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
acgttggatg gttttaccag aggcttggac 30
<210> 37
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
acgttggatg aaggctgttg ttcctacctg 30
<210> 38
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
acgttggatg cctttgggat cagcaacatc 30
<210> 39
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
acgttggatg gcaccaacct aatagaggta 30
<210> 40
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
acgttggatg actagctgca gttcctgtcg 30
<210> 41
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
acgttggatg tcttctcatg tctccggtag g 31
<210> 42
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
acgttggatg gagaagtctc cctgttctgt cc 32
<210> 43
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
acgttggatg tcttctcatg tctccggtag g 31
<210> 44
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
acgttggatg gagaagtctc cctgttctgt cc 32
<210> 45
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
acgttggatg tcttctcatg tctccggtag g 31
<210> 46
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
acgttggatg gagaagtctc cctgttctgt cc 32
<210> 47
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
acgttggatg tcttctcatg tctccggtag g 31
<210> 48
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
acgttggatg gagaagtctc cctgttctgt cc 32
<210> 49
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
acgttggatg gacaaaatca aagagtacca c 31
<210> 50
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
acgttggatg aaatgaagcc acactgctcc 30
<210> 51
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
acgttggatg gtcactcctt gattaagctg 30
<210> 52
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
acgttggatg agagtgtcac ggttgaatcc 30
<210> 53
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
acgttggatg actagctgca gttcctgtcg 30
<210> 54
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
acgttggatg gcaccaacct aatagaggta 30
<210> 55
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
acgttggatg ccatatgaaa tggcagtagc 30
<210> 56
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
acgttggatg agacacaaaa tcccagtccc 30
<210> 57
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
acgcaccgtc cccca 15
<210> 58
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
<210> 59
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
<210> 60
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
<210> 61
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
aacatccttc atgcaac 17
<210> 62
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
<210> 63
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
tcagataccc cactatgct 19
<210> 64
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
cgtctcagaa gctcatatc 19
<210> 65
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
<210> 66
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
<210> 67
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
tccagttttg acttctaact t 21
<210> 68
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
ggaacatcaa gacatatctc a 21
<210> 69
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
ccaacctcct ttgcagccac aa 22
<210> 70
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
cataggttgt aaacctaaaa tg 22
<210> 71
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
gtggtgtgtc ttgtcaccat agc 23
<210> 72
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
ctccccttac catgttacga cttg 24
<210> 73
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
ggcacgctgc agacgatcct gggg 24
<210> 74
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
<210> 75
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
<210> 76
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
<210> 77
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
gtgcattcgt cttttcc 17
<210> 78
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
gaagacgatc ctggggg 17
<210> 79
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
<210> 80
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
<210> 81
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
ctgctccata atatcaaagt t 21
<210> 82
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
agtccatcct ctctcttttg tc 22
<210> 83
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
gtatgtcagg atagggaaaa aa 22
<210> 84
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
gtttttaaca tcttttgttt tatttc 26
Claims (6)
1. A primer group for detecting SNP sites related to hereditary hearing loss, which is characterized by aiming at the following SNP sites: rs111033367, rs372347027, rs375668376, rs 3973518039, rs369522997, rs111033199, rs111033242, rs111033220, rs111033313, rs145254330, rs786204730, rs104894408, rs72474224, rs80338942, rs80338943, rs104894396, rs35887622, rs80338939, rs267606617, rs267606618, rs267606619, rs28937588, rs 9566801, rs 938175, rs876657754, rs777465132, rs201895089 and rs 727504567; the primer sequences are respectively shown as SEQ _ ID NO. 1-84.
2. The primer set of claim 1, wherein the primer set is further divided into two groups:
the first set comprises primers for rs 9566801, rs80338942, rs80338943, rs876657754, rs375668376, rs111033367, rs267606618, rs 39518039, rs267606619, rs111033242, rs777465132, rs111033199, rs72474224, rs372347027, rs28937588, rs267606617 and rs 104894408;
the second set comprises primers for rs28938175, rs111033220, rs145254330, rs201895089, rs80338939, rs104894396, rs35887622, rs727504567, rs369522997, rs786204730 and rs 111033313.
3. Use of the primer set of any one of claims 1-2 for the preparation of a product for screening genetic deafness.
4. A composition for screening for genetic deafness, said composition comprising the primer set of any one of claims 1-2.
5. A product for screening for genetic deafness, said product comprising the primer set of any one of claims 1-2.
6. A kit for genetic deafness screening, comprising the primer set of any one of claims 1-2.
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| CN112410420B (en) * | 2020-12-19 | 2022-12-06 | 中国人民解放军联勤保障部队第九八四医院 | Special primer for simultaneously and accurately detecting SNP (single nucleotide polymorphism) loci of related genes of 'one palm induced deafness' and 'one needle induced deafness' and application |
| CN115678976B (en) * | 2022-10-31 | 2023-07-14 | 广州凯普医药科技有限公司 | Kit for simultaneously detecting 26 deafness susceptibility gene mutation sites based on time-of-flight mass spectrum and application thereof |
| CN116555417A (en) * | 2023-06-20 | 2023-08-08 | 广州中景医学检验实验室有限公司 | Site for detecting congenital deafness before embryo implantation, primer combination and application thereof |
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| CN105255999A (en) * | 2015-07-22 | 2016-01-20 | 广州市达瑞生物技术股份有限公司 | Method for detecting 20 mutation sites of deaf genes |
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