CN115851905A

CN115851905A - Primer group and method for detecting 108 polymorphic site mutations of deafness-related gene

Info

Publication number: CN115851905A
Application number: CN202211203338.8A
Authority: CN
Inventors: 郭慈琳; 胡昌明; 王华文; 甘立佳; 孙明明; 唐驻景
Original assignee: Guangzhou Kingmed Diagnostics Group Co ltd; Guangzhou Kingmed Diagnostics Central Co Ltd
Current assignee: Guangzhou Kingmed Diagnostics Group Co ltd; Guangzhou Kingmed Diagnostics Central Co Ltd
Priority date: 2022-09-29
Filing date: 2022-09-29
Publication date: 2023-03-28

Abstract

The invention discloses a primer group and a method for detecting 108 polymorphic site mutations of deafness related genes. Designing a primer group aiming at gene segments where 108 mutation sites of 18 genes related to hereditary hearing loss are located, adopting an amplicon capture sequencing method, firstly utilizing the primer group to carry out multiplex PCR amplification to construct a sequencing library, then combining a new generation high-throughput sequencing platform Illumina miniseq to carry out detection, and realizing the relative quantification of different bases of each site of a DNA sequence and the sequence judgment of the DNA segment by analyzing all sequencing signals. The method provided by the invention is simple and convenient to operate, high in flux and low in cost, and is suitable for large-scale population diagnosis and screening of hereditary hearing loss.

Description

Primer group and method for detecting 108 polymorphic site mutations of deafness-related gene

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a primer group and a method for detecting 108 polymorphic site mutations of deafness related genes.

Background

Deafness is a general term for the organic or functional lesion of auditory conduction pathway, resulting in hearing impairment of different degrees, and is the most common sensory defect in human beings. According to WHO statistics, the total number of patients with disabled hearing loss is 4.66 hundred million, which accounts for 5 percent of the total number of people, wherein the number of children accounts for 3400 ten thousand. The clinical manifestations are mainly hearing impairment of different degrees, some types of deafness can be recovered, and some types of deafness are permanent. The etiology of deafness is complex, and it can be related to ear diseases, genetic factors, drugs, environment, etc., and the genetic factors account for more than 60% of the etiology, and are the more common types of deafness. Hearing impairment can have a non-negligible effect on speech development, leading to developmental disturbances or speech function retardation that in turn affects the patient's mental, intellectual and social abilities to interact.

Deafness is one of the most common birth defects, and the pathogenic causes are genetic and various environmental factors, wherein at least 50% of preschool deafness is caused by genetic factors. The current research shows that the hereditary hearing loss genetic mode mainly comprises classical autosomal dominant inheritance, normally-dyed recessive inheritance, X-linked dominant inheritance, X-linked recessive inheritance and Y-linked inheritance, and also comprises mitochondrial maternal inheritance and the like. The most common 3 genes for most genetic deafness are GJB2 (c.235delC homozygous mutation), SLC26A4 (c.2168A > G/IVS 7-2A > G), mitochondrial mtDNA (A155G and C1494T mutation), respectively. By screening the deafness genes of the couple before childbirth, the fetus and the newborn, the couple can find that the couple carries the same deafness pathogenic gene, drug deafness sensitive individuals and late hereditary deafness individuals so as to make correct childbirth guidance, reduce and even prevent the deafness patients and realize the secondary prevention of hereditary deafness.

At present, deafness gene screening work aiming at target people such as newborns, pregnant women and the like is widely carried out in China, and most hospitals introduce deafness hot spot screening related items. According to incomplete statistics, more than 900 million neonates and pre-pregnancy adult deafness gene screens and nearly 26 million pregnancy women deafness gene screens are completed in the whole country. If the newborn infant fails to treat the hearing disorder timely and effectively, the newborn infant may seriously threaten the normal growth and development of the infant and even negatively affect the family and society of the sick child along with the development of the illness state.

The molecular mechanism of hereditary hearing loss is relatively clear, and the detection of hearing loss gene is an effective measure for preventing and controlling hereditary hearing loss. However, due to the diversity of variation types of deafness genes, including point variation, small segment insertion loss (Indel), copy Number Variation (CNV), structural Variation (SV), etc., a single detection technology is difficult to work, and the current methods for genetic deafness diagnosis mainly include gene chip, gene Sanger sequencing, targeted capture sequencing of target deafness genes based on Next Generation Sequencing (NGS) technology, sequencing of Whole Exome (WES), sequencing of Whole Genome (WGS), etc.

For clinical or transformation studies, the detection is not only accurate, but also rapid and economical. The traditional detection method of gene Sanger sequencing can only detect individual and partial loci at one time, and can detect few mutation types, while target deafness gene target capture sequencing, whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS) based on Next Generation Sequencing (NGS) technology have complicated and tedious hybridization capture experimental process, too long manual operation time, uncontrollable influence on experimental results possibly caused by too many manual intervention processes, high cost and extremely fatal clinical application. In addition, the detection methods have the defects of false positive mutation detection, high detection rechecking rate and unstable process.

Amplicon targeted sequencing is very useful as a complementary technique to whole genome sequencing, which greatly simplifies the experimental flow and analysis goals. This technique has proven to be a fast, efficient technique and has played a unique part in the next generation of high throughput sequencing, has generated many exciting new discoveries, and the application field is becoming more and more extensive. Meanwhile, the simplification of the experimental process of the amplicon capturing technology greatly reduces the professional threshold of operators, so that more people can complete the experiment, and the risk of insufficient hands is thoroughly liberated.

Therefore, the primer is designed according to the target region of the deafness related gene aiming at the amplicon target sequencing of the deafness, so that the primer can cover the common gene mutation sites of hereditary deafness and has very important significance.

Disclosure of Invention

Based on this, one of the purposes of the present invention is to provide a primer set for detecting 108 polymorphic site mutations of deafness-related genes, wherein the primer set can be used for amplifying 108 polymorphic site regions of 18 deafness-related genes, and can cover gene mutation sites common to hereditary deafness.

The specific technical scheme for realizing the aim of the invention comprises the following steps:

a primer group for detecting 108 polymorphic site mutations of deafness related genes comprises:

a primer pair for detecting the site mutation of the GJB3 gene, wherein the primer pair comprises a forward primer with a sequence shown as SEQ ID NO.1 and a reverse primer with a sequence shown as SEQ ID NO. 2;

a primer pair for detecting site mutation of the DSPP gene, wherein the primer pair comprises a forward primer with a sequence shown as SEQ ID NO.3 and a reverse primer with a sequence shown as SEQ ID NO. 4;

a primer pair for detecting the site mutation of the GPR98 gene, wherein the primer pair comprises a forward primer with a sequence shown as SEQ ID NO.5 and a reverse primer with a sequence shown as SEQ ID NO. 6;

a primer pair for detecting site mutation of the DFNA5 gene, wherein the primer pair comprises a forward primer with a sequence shown as SEQ ID NO.7 and a reverse primer with a sequence shown as SEQ ID NO. 8;

a primer pair for detecting a site mutation of the SLC26A4 gene, said primer pair comprising: a forward primer with a sequence shown as SEQ ID NO.9 and a reverse primer with a sequence shown as SEQ ID NO. 10; a forward primer with a sequence shown as SEQ ID NO.11 and a reverse primer with a sequence shown as SEQ ID NO. 12; a forward primer with a sequence shown as SEQ ID NO.13 and a reverse primer with a sequence shown as SEQ ID NO. 14; a forward primer with a sequence shown as SEQ ID NO.15 and a reverse primer with a sequence shown as SEQ ID NO. 16; a forward primer with a sequence shown as SEQ ID NO.17 and a reverse primer with a sequence shown as SEQ ID NO. 18; a forward primer with a sequence shown as SEQ ID NO.19 and a reverse primer with a sequence shown as SEQ ID NO. 20; a forward primer with a sequence shown as SEQ ID NO.21 and a reverse primer with a sequence shown as SEQ ID NO. 22; a forward primer with a sequence shown as SEQ ID NO.23 and a reverse primer with a sequence shown as SEQ ID NO. 24; a forward primer with a sequence shown as SEQ ID NO.25 and a reverse primer with a sequence shown as SEQ ID NO. 26; a forward primer with a sequence shown as SEQ ID NO.27 and a reverse primer with a sequence shown as SEQ ID NO. 28; a forward primer with a sequence shown as SEQ ID NO.29 and a reverse primer with a sequence shown as SEQ ID NO. 30; a forward primer with a sequence shown as SEQ ID NO.31 and a reverse primer with a sequence shown as SEQ ID NO. 32; a forward primer with a sequence shown as SEQ ID NO.33 and a reverse primer with a sequence shown as SEQ ID NO. 34; a forward primer with a sequence shown as SEQ ID NO.35 and a reverse primer with a sequence shown as SEQ ID NO. 36; a forward primer with a sequence shown as SEQ ID NO.37 and a reverse primer with a sequence shown as SEQ ID NO. 38; a forward primer with a sequence shown as SEQ ID NO.39 and a reverse primer with a sequence shown as SEQ ID NO. 40;

a primer pair for detecting site mutation of TMC1 gene, the primer pair comprising: a forward primer with a sequence shown as SEQ ID NO.41 and a reverse primer with a sequence shown as SEQ ID NO. 42; a forward primer with a sequence shown as SEQ ID NO.43 and a reverse primer with a sequence shown as SEQ ID NO. 44;

a primer pair for detecting the site mutation of the MYO7A gene, wherein the primer pair comprises a forward primer with a sequence shown as SEQ ID NO.45 and a reverse primer with a sequence shown as SEQ ID NO. 46;

a primer pair for detecting site mutation of the TECTA gene, wherein the primer pair comprises a forward primer with a sequence shown as SEQ ID NO.47 and a reverse primer with a sequence shown as SEQ ID NO. 48;

a primer pair for detecting a site mutation of the DIABLO gene, the primer pair comprising a forward primer having a sequence shown in SEQ ID No.49 and a reverse primer having a sequence shown in SEQ ID No. 50;

the primer pair for detecting the locus mutation of the GJB2 gene comprises: a forward primer with a sequence shown as SEQ ID NO.51 and a reverse primer with a sequence shown as SEQ ID NO. 52; a forward primer with a sequence shown as SEQ ID NO.53 and a reverse primer with a sequence shown as SEQ ID NO. 54; a forward primer with a sequence shown as SEQ ID NO.55 and a reverse primer with a sequence shown as SEQ ID NO. 56;

a primer pair for detecting the site mutation of the COCH gene, wherein the primer pair comprises a forward primer with a sequence shown as SEQ ID NO.57 and a reverse primer with a sequence shown as SEQ ID NO. 58;

a primer pair for detecting the site mutation of the MYO15A gene, wherein the primer pair comprises a forward primer with a sequence shown as SEQ ID NO.59 and a reverse primer with a sequence shown as SEQ ID NO. 60;

a primer pair for detecting a site mutation of a PRPS1 gene, the primer pair comprising: a forward primer with a sequence shown as SEQ ID NO.61 and a reverse primer with a sequence shown as SEQ ID NO. 62; a forward primer with a sequence shown as SEQ ID NO.63 and a reverse primer with a sequence shown as SEQ ID NO. 64;

a primer pair for detecting the site mutation of the MITO related genes, wherein the MITO related genes comprise an MT-RNR1 gene, an MT-CO1 gene, an MT-TL1 gene, an MT-TS1 gene and an MT-TH gene; the primer pair comprises: a forward primer with a sequence shown as SEQ ID NO.65 and a reverse primer with a sequence shown as SEQ ID NO. 66; a forward primer with a sequence shown as SEQ ID NO.67 and a reverse primer with a sequence shown as SEQ ID NO. 68; a forward primer with a sequence shown as SEQ ID NO.69 and a reverse primer with a sequence shown as SEQ ID NO. 70; a forward primer with a sequence shown as SEQ ID NO.71 and a reverse primer with a sequence shown as SEQ ID NO. 72.

In some of these embodiments, the 108 polymorphic sites comprise:

IVS7-2A >G, c.2164A > G, c.1229C > T, c.1174A > T, c.1975G > C, c.2027T > A, c.2162C > T, c.589G > A, c.1226G > A, c.281C > T, IVS15+5G > A, c.2086C > T, c.754T > C, c.107C > T, c.259G > T, c.1343C > T, c.92C > T, c.1919G1540 > A, c.2000T > C, c.679G > C, IVS14-2A 397G, c.169019A, c.920C > T, c.0T > A, c.1340C > T, c.1161 _3 del3T, TCT, c.131C > 131C.131C > C. c.1555_1556delAA, c.1586T > G, c.1594A > C, c.1634T > C, c.1673A > T, c.1717G > T, c.1746delG, c.2054G > T, c.2082delA, c.2107C > G, c.227C > T, c.230A > T, c.269C > T, c.334C > T, c.34delC, c.387delC, c.404A > G, c.4343A > G, c.697G > C, c.812A > G, c.241G > T, IVS13+ C > -G, IVS14+ G >, IVS 14-G19A, IVS16-6G > A, c.1105A > G, c.706T > C.10T, c.10T > C.14G > T, c.159C > T, c.14A > C.14T > C > T;

c.538C > T, c.547G > A, c.423delATT, c.497A > G and c.421A > G on GJB3 gene;

m.1555A > G and m.1494C > T on the MT-RNR1 gene;

m.7444G > A on MT-CO1 gene;

m.3243A > G on the MT-TL1 gene;

m.7445A > G, m.7505T > C and m.7511T > C on MT-TS1 gene;

m.12201T > C on the MT-TH gene;

c.52G > T on the DSPP gene;

c.10088 — 10091delTAAG in GPR98 gene;

IVS8+4A > G on DFNA5 gene;

c.150delT and c.1334G > A on TMC1 gene;

c.652g > a and c.731g > C on the MYO7A gene;

c.4525t > G on the TECTA gene;

c.377c > T on the DIABLO gene;

c.1535t > C and c.1625g > a on the COCH gene;

c.8767C > T on MYO15A gene;

c.193G > A, c.259G > A, c.869T > C and c.916G > A on the PRPS1 gene;

c.235delC, c.299_300delAT, c.35delG, c.176_191del16, c.167delT, c.512insAACG, c.231G > A, c.218A > G, c.427C > T, c.416G > A, c.257C > G, c.253T > C, c.107T > C, c.99delT, c.94C > T, c.250G > T, c.238C > T, c.230G > A, c.223C > T and c.35insG on the GJB2 gene.

The invention also provides application of the primer group in preparation of products for detecting 108 polymorphic site mutations of deafness related genes.

In some of these embodiments, the product is a kit.

The invention also provides a kit for detecting 108 polymorphic site mutations of the deafness related gene, which comprises the primer group for detecting the 108 polymorphic site mutations of the deafness related gene.

In some of these embodiments, the primer pairs SEQ ID NO. 1-2, SEQ ID NO. 5-6 each have a working concentration of 0.06uM; the working concentrations of the primer pair SEQ ID NO. 3-4, SEQ ID NO. 41-42 and SEQ ID NO. 49-50 are respectively 0.0715uM; the working concentrations of the primer pairs SEQ ID NO. 7-8, SEQ ID NO. 47-48 and SEQ ID NO. 61-62 are respectively 0.078uM; the working concentration of the primer pair SEQ ID NO. 9-10 is 0.075uM respectively; the working concentration of the primer pair SEQ ID NO. 11-12 is 0.0585uM respectively; the working concentrations of the primer pair SEQ ID NO. 13-14, SEQ ID NO. 15-16 and SEQ ID NO. 55-56 are respectively 0.039uM; the working concentrations of the primer pair SEQ ID NO. 17-18, SEQ ID NO. 19-20, SEQ ID NO. 33-34 and SEQ ID NO. 35-36 are respectively 0.0325uM; the working concentrations of the primer pairs SEQ ID NO. 21-22 and SEQ ID NO. 23-24 are respectively 0.09uM; the working concentrations of the primer pair SEQ ID NO. 25-26, SEQ ID NO. 27-28, SEQ ID NO. 29-30, SEQ ID NO. 31-32 and SEQ ID NO. 39-40 are respectively 0.052uM; the working concentrations of the primer pairs SEQ ID NO. 37-38, SEQ ID NO. 43-44, SEQ ID NO. 57-58 and SEQ ID NO. 63-64 are respectively 0.0845uM; the working concentrations of the primer pairs SEQ ID NO. 45-46, SEQ ID NO. 51-52 and SEQ ID NO. 53-54 are respectively 0.0455uM; the working concentration of the primer pair SEQ ID NO. 59-60 is 0.104uM respectively; the working concentration of the primer pair SEQ ID NO. 65-66 is 0.02uM respectively; the working concentration of the primer pair SEQ ID NO. 67-68 is 0.01uM respectively; the working concentration of the primer pair SEQ ID NO. 69-70 is 0.04uM respectively; the working concentrations of the primer pairs SEQ ID NO. 71-72 are respectively 0.03uM.

The invention also provides a method for detecting 108 polymorphic site mutations of deafness related genes, which comprises the following steps: and (3) taking the DNA of a sample to be detected as a template, performing multiple PCR amplification by using the kit, constructing a library, and sequencing.

In some of these embodiments, the sequencing is performed based on an illumina sequencing platform.

In some embodiments, the multiplex PCR amplification comprises a first PCR amplification and a second PCR amplification, and the reaction procedure of the first PCR amplification is: 95 ℃ for 5min;95 ℃ for 10s; 30s at 60 ℃;72 ℃ for 15s;20times;72 ℃ for 1min; at 15 ℃, oc; the reaction procedure of the second PCR amplification is as follows: at 95 ℃ for 10min; at 95 ℃ for 30s; 90s at 65 ℃;10times; at 15 ℃ and oc.

In some embodiments, the reaction system for the first PCR amplification comprises: SKa Multiplex PCR Master Mix, DMSO, primer Mix, H ₂ O; the reaction system for the second PCR amplification comprises: SKa Multiplex PCR Master Mix, first round PCR amplification product, linker.

Compared with the prior art, the invention has the following beneficial effects:

the method for detecting 108 polymorphic site mutations of deafness related genes comprises the steps of designing a primer group aiming at gene segments where 108 mutation sites of 18 genes related to hereditary deafness are located, adopting an amplicon capture sequencing method, firstly carrying out multiplex PCR (polymerase chain reaction) amplification by using the primer group to construct a sequencing library, then combining a new generation high-throughput sequencing platform Illumina miniseq for detection, and realizing the relative quantification of different bases of each site of a DNA sequence and the sequence judgment of the DNA segment by analyzing all sequencing signals; and the sequencing data is subjected to sequence reading and result judgment by combining a bioinformatics analysis method, so that comprehensive and high-value reference information is finally provided for clinical accurate medication, molecular typing and curative effect recurrence monitoring, and doctors are assisted to make more complete treatment schemes. The method provided by the invention is simple and convenient to operate, high in flux and low in cost, and is suitable for large-scale population diagnosis and screening of hereditary hearing loss.

Drawings

FIG. 1 is a flowchart of the method for detecting 108 polymorphic site mutations of deafness-related gene in example 2 of the present invention.

FIG. 2 is a schematic diagram of a method for detecting 108 polymorphic site mutations of a deafness-related gene in example 2 of the present invention.

FIG. 3 is a flowchart of bioinformatic analysis in example 2 of the present invention.

FIG. 4 is a schematic diagram of the determination of the size of the library fragments using a 2100 bioanalyzer in example 2 of the present invention.

FIG. 5 is a partial comparison of the results of bioinformatics analysis in example 2 of the present invention.

FIG. 6 shows the results of sequencing tests on the primer pairs before and after optimization in test example 1 of the present invention.

FIG. 7 is a graph of IGV depth after using 1.

FIG. 8 shows the statistical results of the primers and depth in the test process in test example 2 of the present invention.

FIG. 9 shows the results of the detection with reagent C in test example 3 of the present invention.

FIG. 10 shows the results of the assay using reagent A in test example 3 of the present invention.

FIG. 11 shows the results of detection using reagent B in test example 3 of the present invention.

FIG. 12 shows the results of the in-batch precision in test example 4 of the present invention.

FIG. 13 shows the results of the inter-lot precision in test example 4 of the present invention.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples were commercially available unless otherwise specified.

According to the genetic deafness related genes GJB3, DSPP, GPR98, DFNA5, SLC26A4, TMC1, MYO7A, TECTA, DIABLO, GJB2, COCH, MYO15A, PRPS1 and MITO mitochondrial related genes (including MT-RNR1, MT-CO1, MT-TL1, MT-TS1 and MT-TH), 18 genes are used as target genes, 108 polymorphic site mutation areas are used as target amplification areas, a set of primer groups suitable for genetic deafness related gene detection is obtained through design and screening, the primer groups comprise 36 pairs, and the concentration of each pair of primers in the primer groups is further optimized. The primer group of the 36 pairs of primers is used for carrying out multiple PCR amplification, and the amplification uniformity of each pair of primers achieves an ideal effect by combining the optimization of amplification conditions, so that the constructed library covers the detection of common gene mutation of hereditary hearing loss, and meanwhile, the operation is simple and convenient, the flux is high, the cost is low by high-throughput sequencing, and the method is suitable for large-scale population diagnosis and screening of hereditary hearing loss.

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1 specific primers for detecting 108 polymorphic site mutations of deafness-related genes

In this embodiment, a set of primer sets suitable for genetic deafness related gene detection is designed and screened according to 18 genes in total of genetic deafness related genes GJB3, DSPP, GPR98, DFNA5, SLC26A4, TMC1, MYO7A, TECTA, DIABLO, GJB2, COCH, MYO15A, PRPS1 and MITO mitochondria related genes (MT-RNR 1, MT-CO1, MT-TL1, MT-TS1 and MT-TH) as target genes and 108 polymorphic site mutation regions as target amplification regions, and the detected genes and site information are specifically shown in Table 1. Specific primer sequences are shown in table 2.

TABLE 1 detection Gene and site information (deafness 108 sites)

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TABLE 2 primer sequences and detection ranges

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Example 2 method for detecting 108 polymorphic site mutations of deafness-related genes

Please refer to fig. 1, which is a flowchart of the method for detecting 108 polymorphic site mutations of deafness-related gene according to the present invention. The primer pair shown in the table 2 of the embodiment 1 is utilized to detect 108 polymorphic site mutations of deafness related genes of 384 samples, and the method specifically comprises the following steps:

1. DNA extraction

Genomic DNA was extracted from 384 samples of peripheral blood and blood filter disc samples using a commercial nucleic acid extraction and purification kit (Boo Muwa).

2. First round PCR amplification and purification

(1) PCR amplification

Melting 2 Xmultiplex PCR buffer solution and enzyme, placing on an ice plate, preparing a reaction system (except a DNA template) according to the table 3, subpackaging into a 96-hole half skirt PCR plate, adding 10 mu L of the DNA template in the step (1) by using a semi-automatic sample adding instrument BenchSmart 96, and carrying out a first round of PCR reaction. The concentrations of the primers in the Primer Mix are shown in table 4, and the reaction procedure is shown in table 5.

TABLE 3 first round PCR reaction System

Components	Volume (mu L)
		SKaMultiplexPCRMasterMix	25
DMSO	4
		PrimerMix	5
H ₂ O	6
		DNA template	10

TABLE 4 primer concentrations

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TABLE 5 first round PCR reaction procedure

(2) Purifying the mixture

a. Adding 50 mu LVAHTS DNA Clean Beads (Novozan biotechnology, 1.0 magnetic bead ratio, balancing to room temperature, fully shaking before use) into each sample, blowing, uniformly mixing, and standing at room temperature for 5min;

b. placing in a magnetic frame for 2min, clarifying the solution, and removing the supernatant;

c. adding 200 μ L of freshly prepared 80% ethanol, standing for 30s, clarifying the solution, and removing the supernatant by suction; repeating the steps once; standing at room temperature for 5-10min to volatilize alcohol;

d. add 23.5. Mu.L of ddH ₂ And O, resuspending the magnetic beads, uniformly mixing the magnetic beads by blowing, standing the mixture at room temperature for 5min, placing the mixture on a magnetic frame, and carefully transferring 21.5 mu L of supernatant into a second round of PCR amplification system after the solution is clarified.

The purification operation can be performed through an Agilent automation instrument, the manual operation time can be shortened under the condition of large sample size, and the method is quicker and more convenient. Firstly, preparing reagents and consumables required by library purification according to a table 6, then detecting the sample amount according to the current day, selecting a VWorks workshop PCR base _2 \ u plate \ Illumina1.Add beads and binding for Adaptor AMPure XP.pro program on a VWorks operation interface, clicking START on a program operation interface, placing the reagents and consumables according to the program prompt, and operating the program to finish the whole purification operation.

TABLE 6 library purification reagent consumables

3. Second round PCR amplification and purification

(1) PCR amplification

Melting 2x multiplex PCR buffer solution and enzyme, placing on an ice plate, preparing a reaction system (except for a first round amplification product and a linker) according to table 7, subpackaging into 4 pieces of 96-well PCR plates, respectively adding 21.5 μ L of the purified first round PCR amplification product obtained in the step 2, then adding 2.5 μ L of a corresponding linker (with the concentration of 2.5 μ M, linker sequences SEQ ID No. 73-SEQ ID No.456, specifically shown in table 8) to each sample by using a semi-automatic sample adding instrument BenchSmart 96 to distinguish the samples, and carrying out a second round PCR reaction. The reaction sequence is shown in table 9.

TABLE 7 second round PCR reaction System

Components	Volume (μ L)
		SKaMultiplexPCRMasterMix	25
First round PCR amplification product	22.5
		Joint (indexPrimer (2.5P))	2.5
Total	50

TABLE 8 linker sequences

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Note: from Tag001 to Tag384, the sequences are named SEQ ID No.73 to SEQ ID No.456.

TABLE 9 second round PCR reaction procedure

(2) Purifying the mixture

a. Transferring the product after the second round of amplification to a new tube according to 10 muL per hole, fully shaking and mixing uniformly, sucking 100 muL of mixed sample to a new EP tube, adding 100 muL of VAHTS DNA Clean Beads (Novozan bioscience, 1.0 magnetic bead ratio, balancing to room temperature, fully shaking before use), blowing and mixing uniformly, and standing for 5min at room temperature;

c. adding 200 μ L of freshly prepared 80% ethanol, rotating the EP tube for several times, clarifying the solution, and then sucking off the supernatant; repeating the steps once; standing at room temperature for 5-10min to volatilize alcohol;

d. add 27. Mu.L of ddH ₂ O resuspending the magnetic beads, blowing and mixing uniformly, standing at room temperature for 5min, placing on a magnetic rack, carefully transferring 25 mu L of supernatant into a new EP tube after the solution is clarified, marking as a library tube, and then storing at 4 ℃.

3. Library concentration determination and quality control

Determining the concentration of the library by using the Qubit, wherein the concentration of the library is required to be more than or equal to 1ng/uL; the size of the library fragments was determined using an Agilent 2100 bioanalyzer (DNA 1000 Kit). FIG. 2 is a diagram showing the determination of the size of the library fragments, which is 300-500 bp, corresponding to the expected fragment size.

4. Sequencing on computer

Using a high throughput sequencer Miniseq ^TM The DX-CN platform was used for sequencing. Miniseq selected by the detection method of the invention ^TM The DX-CN platform adopts SBS sequencing technology and is matched with an amplicon capture method for detection. The SBS sequencing technology can effectively reduce sequencing errors of homopolymers and repeated sequences through continuously perfecting updated sequencing reagents and detection systems, and has high base coverage and accuracy. Meanwhile, the simplification of the experiment process of the amplicon capturing technology greatly reduces the professional threshold of operators, so that more people can complete the experiment, and the risk of insufficient hands is thoroughly liberated. Amplicon capture sequencing is very useful as a complementary technique to whole genome sequencing, greatly simplifying the experimental flow and the analysis objective. This technique has proven to be a fast, efficient technique and has played a unique part in the next generation of high throughput sequencing, has generated many exciting new findings and is becoming more and more widespread.

The principle of detecting 108 polymorphic site mutations of deafness-related genes based on the Illumina platform is shown in figure 3.

(5) Bioinformatics analysis

Please refer to fig. 4 for the bioinformatics analysis flow.

Transmitting a Raw Data (bcl) file generated by sequencing to a computer server special for a laboratory, generating a fastq file through special software, analyzing and processing sequencing Data, wherein the fastq file comprises low-quality Data in the filtering sequencing, removing joints and primers, comparing the low-quality Data to a position corresponding to reference genome Data, counting sequencing mapping information, sequence number, target region coverage size, average sequencing depth, sequencing uniformity and the like, selecting and testing corresponding software or Selfscript for mutation analysis according to various mutation types, determining annotations such as gene, coordinate, mRNA locus, amino acid change, SNP function (missense mutation/nonsense mutation/variable shearing locus), protein function prediction and the like of a mutation locus by combining annotation software and a database, and finally outputting a visual table result. And when the average sequencing depth of the target area of the detection sample is more than or equal to 20X, the analysis requirement of the clinical sample is met.

Mitochondrial variation and chromosome variation are screened by two algorithms of somatic variation and germ line variation respectively to obtain the specific variation condition of the known deafness variation site, and the screening of the lowest depth and frequency is carried out. Has faster analysis speed and can sensitively capture the variation information of specific genes.

FIG. 5 is a diagram showing the alignment results of SNV GJB2: c.109G > A. The sample is sequenced by the method, then data is compared with a human genome reference sequence, and the GJB gene of the sample is found to generate a c.109G > A variation (namely, G base is replaced by A base). The G base is labeled blue, the A base is labeled red, and if identical to the reference sequence, the color is uniformly gray.

Test example 1 optimization of primer sequences

Finding out a fragment file in which 108 site mutations of 18 genes are located according to NCBI, obtaining a proper primer through Blast, selecting a primer with proper parameters such as TM, GC, length and the like, synthesizing the primer, and then performing sequencing detection. The primer pair KHL015. For detecting the site mutation of SLC26A4 gene is taken as an example for explanation.

Sequencing detection is carried out on the primer pairs before and after optimization, and the result is shown in figure 6. As can be seen from FIG. 6, the primer pair before optimization only sequences to 74 reads, and the primer pair after optimization sequences to 271 reads, which meets the requirements of further detection.

Test example 2 optimization of primer concentration

According to the test example 1, after a primer group for detecting 108 polymorphic site mutations of deafness related genes is screened, the optimal primer concentration is searched, and the method specifically comprises the following steps:

1. the primers were mixed according to 1;

2. adjusting the concentration of each pair of primers according to the sequencing depth of off-line data analysis;

as shown in fig. 7, which is a sequencing depth map of a sample tested by using the primer mixture of 1; the sequencing depth of the PRPS1 site is obviously higher than that of other sites, so that the concentration of the primer of the PRPS1 can be properly reduced.

The statistical results of the primers and depth during the test are shown in fig. 8 and table 10 after 5 rounds of adjustment.

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3. After multiple adjustment tests, the final primer concentrations were obtained as shown in Table 4 in example 1.

Test example 3 optimization of amplification conditions for detection

Due to the detection requirement, the enzyme with the shortest amplification time and up-to-standard performance needs to be selected. Therefore, this test example will be described with respect to KHL015.SLC26A4 as the primer. The same samples were tested using different Taq enzymes and different reaction protocols (first round PCR) (other conditions were the same as in example 2), as shown in Table 11.

TABLE 11 reaction enzymes and reaction procedure testing

The results of the amplification using the reagents and procedures in Table 11 are shown in FIGS. 9-11, and it can be seen from the results that the detection using the reagent C and the corresponding procedure has the shortest time and the performance reaching the standard, and each primer can be successfully amplified. And the reagent A and the corresponding reaction program, and the reagent B and the corresponding reaction program are adopted for detection, so the effect is not ideal.

Test example 4 methodological validation of the method of the invention

The method of embodiment 2 is adopted to detect 1032 samples, so that the performances of the method of the invention, such as accuracy, sensitivity and the like, are verified.

Wherein, the sample is detected by a Boolmuhua gene deafness detection platform (a commercial kit, which is considered to have 100 percent of accuracy): 303 samples with detected variation (275 SNV positive sites and 57 Indel samples); 672 samples of which no variation is detected at 108 sites in the detection region; 57 cases of NTC quality control.

1. Margin of detection

In order to confirm the blank detection limit of the detection method of the present invention, the analysis data for detecting NTC quality control is counted (13 batches of 57 NTC data are counted), and the statistical results are shown in table 12.

TABLE 12NTC quality control analysis data

According to the results in Table 12, when NTC Mapped Reads > 1007 and mean Depth > 20, it is determined that the sample is contaminated and the cause needs to be checked, and the process is out of control.

2. Frequency range of variation

In order to determine the MAF fluctuation range of the heterozygous and homozygous variation of the detection method, the heterozygous and homozygous variation sites of all samples are counted. The range of the SNV heterozygous and homozygous variation MAF fluctuation is shown in FIG. 12, and when SNV is detected, the variation is heterozygous when the MAF fluctuation range is 30-70%, and is homozygous when the MAF fluctuation range is 79-100%.

The range of Indel heterozygous and homozygous variant MAFs fluctuation is shown in fig. 13. When indels are detected, the variation is heterozygous when the MAF fluctuation range is 25-60%, and homozygous when the MAF fluctuation range is 90-100%.

3. Accuracy of

The accuracy of the detection method is defined as the consistency of the detection result of the sample with other platforms or known results, 975 samples detected by the Boolwood gene deafness detection platform in the test example are counted to obtain the consistency of the detection results of the platform and the Boolwood gene deafness detection platform, and the statistical results are shown in Table 13.

TABLE 13 accuracy verification results

Positive coincidence rate = 315/(315 + 0) × 100% =100%

The negative coincidence rate = 660/(660 + 0) × 100% =100%

Total coincidence rate =315+ 660/(315 +660+ 0) =100%

4. Specificity of&Sensitivity of the probe

The specificity of the detection method is expressed by using a negative predictive value and a coincidence rate; the sensitivity of the detection method is expressed by using a positive predictive value and a coincidence rate.

1. The test detects samples (comprising 267 positive samples and 708 negative samples, wherein 275 positive sites exist in total, and 105025 negative sites exist in total) with known SNV results detected by a Boolmuwa gene deafness detection platform, and the statistical results are shown in Table 14.

Table 14 specificity & sensitivity validation results

Positive predictive value PPV = TP/(TP + FP) = 275/(275 + 0) =100%;

positive coincidence rate PPA = TP/(TP + FN) = 275/(275 + 0) =100%

Negative predictive value NPV = TN/(TN + FN) = 105025/(105025 + 0) =100%

Negative coincidence rate NPA = TN/(TN + FP) = 105025/(105025 + 0) =100%

2. The test performed on samples (containing 57 positive samples and 918 negative samples) with known Indel results detected by the boohu-warfarin deafness detection platform, and the statistical results are shown in table 15.

Table 15 specificity & sensitivity validation results

Positive predictive value PPV = TP/(TP + FP) = 57/(57 + 0) =100%;

positive coincidence rate PPA = TP/(TP + FN) = 57/(57 + 0) =100%;

negative predictive value NPV = TN/(TN + FN) = 918/(918 + 0) =100%;

negative coincidence rate NPA = TN/(TN + FP) = 918/(918 + 0) =100%

5. Precision degree

The precision of the present assay is defined as the ability to perform repeated assays on a sample with the same result. The verification is carried out for comparison experiments among different persons, different time, different instruments and different holes of the same sample, and the difference between batches is counted.

1. Internal precision: 22 positive clinical samples and 4 negative samples (detected by the Boohu Muhua gene deafness detection platform) were selected for detection, and each sample was subjected to parallel detection for 3 times, with the results shown in Table 16.

TABLE 16 verification of precision in batch

2. Precision between batches: 22 positive clinical samples and 4 negative clinical samples (detected by the Boohu Muhua gene deafness detection platform) were selected for precision detection in batches, the detection was performed once a day in 3 days, and the results are shown in Table 17 by 3 technicians at intervals.

TABLE 17 verification of in-batch precision

The intra and inter-batch results show: all the sample detection qualitative results are consistent, and the precision in batches and between batches meets the requirement.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims

1.A primer group for detecting 108 polymorphic site mutations of deafness-related genes is characterized by comprising:

2. The primer set for detecting 108 polymorphic sites of deafness-related genes according to claim 1, wherein the 108 polymorphic sites comprise:

IVS7-2A >;

c.538C > T, c.547G > A, c.423delATT, c.497A > G and c.421A > G on the GJB3 gene;

m.1555A > G and m.1494C > T on MT-RNR1 gene;

m.7444G > A on MT-CO1 gene;

m.3243A > G on the MT-TL1 gene;

m.7445A > G, m.7505T > C and m.7511T > C on MT-TS1 gene;

m.12201T > C on the MT-TH gene;

c.52G > T on the DSPP gene;

c.10088-10091 delTAAG in GPR98 gene;

IVS8+4A > Gon the DFNA5 gene;

c.150delT and c.1334G > A on TMC1 gene;

c.652g > a and c.731g > C on the MYO7A gene;

c.4525t > G on the TECTA gene;

c.377C > T in the DIABLO gene;

c.1535t > C and c.1625g > a on the COCH gene;

c.8767c > T on the MYO15A gene;

c.193G > A, c.259G > A, c.869T > C and c.916G > A on PRPS1 gene;

3. Use of the primer set of claim 1 or 2in the preparation of a product for detecting 108 polymorphic site mutations of a deafness-associated gene.

4. The use according to claim 3, said product being a kit.

5. A kit for detecting 108 polymorphic site mutations of a deafness-related gene, which is characterized by comprising the primer group for detecting 108 polymorphic site mutations of the deafness-related gene according to claim 1 or 2.

6. The kit for detecting 108 polymorphic site mutations of a deafness-related gene according to claim 5, wherein the working concentrations of the primer pairs SEQ ID No. 1-2 and SEQ ID No. 5-6 are 0.06uM respectively;

the working concentrations of the primer pair SEQ ID NO. 3-4, SEQ ID NO. 41-42 and SEQ ID NO. 49-50 are respectively 0.0715uM;

the working concentrations of the primer pairs SEQ ID NO. 7-8, SEQ ID NO. 47-48 and SEQ ID NO. 61-62 are respectively 0.078uM;

the working concentration of the primer pair SEQ ID NO. 9-10 is 0.075uM respectively;

the working concentration of the primer pair SEQ ID NO. 11-12 is 0.0585uM respectively;

the working concentrations of the primer pair SEQ ID NO. 13-14, SEQ ID NO. 15-16 and SEQ ID NO. 55-56 are respectively 0.039uM;

the working concentrations of the primer pairs SEQ ID NO. 17-18, SEQ ID NO. 19-20, SEQ ID NO. 33-34 and SEQ ID NO. 35-36 are respectively 0.0325uM;

the working concentrations of the primer pairs SEQ ID NO. 21-22 and SEQ ID NO. 23-24 are respectively 0.09uM;

the working concentration of the primer pair SEQ ID NO. 25-26, SEQ ID NO. 27-28, SEQ ID NO. 29-30, SEQ ID NO. 31-32 and SEQ ID NO. 39-40 is 0.052uM respectively;

the working concentrations of the primer pairs SEQ ID NO. 37-38, SEQ ID NO. 43-44, SEQ ID NO. 57-58 and SEQ ID NO. 63-64 are respectively 0.0845uM;

the working concentrations of the primer pairs SEQ ID NO. 45-46, SEQ ID NO. 51-52 and SEQ ID NO. 53-54 are respectively 0.0455uM;

the working concentration of the primer pair SEQ ID NO. 59-60 is 0.104uM respectively;

the working concentration of the primer pair SEQ ID NO. 65-66 is 0.02uM respectively;

the working concentration of the primer pair SEQ ID NO. 67-68 is 0.01uM respectively;

the working concentration of the primer pair SEQ ID NO. 69-70 is 0.04uM respectively;

the working concentrations of the primer pairs SEQ ID NO. 71-72 are respectively 0.03uM.

7. A method for detecting 108 polymorphic site mutations of deafness related genes is characterized by comprising the following steps: performing multiplex PCR amplification by using the DNA of a sample to be tested as a template and the kit of claim 5 or 6, constructing a library, and sequencing.

8. The method for detecting 108 polymorphic site mutations of a deafness-related gene according to claim 7, wherein the sequencing is performed based on an illumina sequencing platform.

9. The method for detecting 108 polymorphic site mutations of a deafness-related gene according to claim 7, wherein said multiplex PCR amplification comprises a first PCR amplification and a second PCR amplification, and the reaction procedure of said first PCR amplification is: 95 ℃ for 5min;95 ℃ for 10s; 30s at 60 ℃;72 ℃ for 15s;20times;72 ℃ for 1min; at 15 ℃, oc; the reaction procedure of the second PCR amplification is as follows: at 95 ℃ for 10min; at 95 ℃ for 30s; 90s at 65 ℃;10times; at 15 ℃ and oc.

10. The method for detecting 108 polymorphic site mutations of a deafness-related gene according to claim 7, wherein the reaction system of the first PCR amplification comprises: SKa Multiplex PCR Master Mix, DMSO, primer Mix, H ₂ O; the reaction system for the second PCR amplification comprises: SKa Multiplex PCR Master Mix, first round PCR amplification product, linker.