CN106811533B - Genetic deafness gene detection kit - Google Patents

Abstract

The invention relates to the field of gene detection, in particular to a nucleic acid membrane strip and a kit for genetic deafness gene detection. The genetic deafness gene detection kit comprises PCR reaction liquid, wherein the PCR reaction liquid comprises PCR reaction liquid A, PCR reaction liquid B, PCR reaction liquid C, PCR reaction liquid D and PCR reaction liquid E; the PCR reaction solution comprises corresponding primers and probes. The kit of the invention utilizes sequences of various mutation sites reported in literature to design a LATE-PCR special primer for specific amplification, and adopts ZNA modified by locked nucleic acid^TMAnd a probe for detecting the most sites in the reaction solution in the least tube. The PCR reaction solution A, B, C, D, E in the kit can be tested on the same fluorescent PCR instrument by using the same amplification program, and the requirements of clinical rapid and convenient deafness detection are met.

Description

Genetic deafness gene detection kit

Technical Field

The invention relates to the field of gene detection, in particular to a genetic deafness gene detection kit.

Background

The existing deafness gene detection products in the current market mainly comprise nine genetic deafness related gene detection kits (microarray chip method) and fifteen genetic deafness related gene detection kits (microarray chip method) of Boao; kypr deafness susceptibility gene detection kit (PCR + flow-through hybridization method); four deafness gene detection kits (ARMS-PCR method) controlled by Zhongsheng north; congenital deafness gene detection kit (fluorescent PCR method), drug-induced deafness gene detection kit (fluorescent PCR method), deafness gene GJB 2235 delC detection kit (fluorescent PCR method) and PDS gene mutation detection kit (fluorescent PCR method) of Jinan Yingsheng; a kit (fluorescence PCR method) for detecting drug-induced deafness gene mutation of Zhihai bioengineering Co. Although there are many products for detecting deafness at present, there is no product for directly, rapidly and simultaneously detecting multiple mutation sites.

The detection kit for detecting deafness susceptibility gene mutation and the preparation method and application thereof (patent publication No. 104263848A) related to Xiamen university are the most detection sites in all patents, but only 23 non-syndrome hereditary deafness gene mutation sites are detected, related patent of Wuxi Zhongde American biotech Limited company, namely, a hereditary deafness gene detection kit (patent publication No. 103352080A) only detects 17 non-syndrome hereditary deafness gene mutations, Chengyo Chinese population deafness gene screening kit and application thereof (patent publication No. 103911452A) detects 17 non-syndrome hereditary deafness susceptibility gene mutations, related patent of Beijing Shenjin instrument biotechnology Limited company, namely, a high specificity deafness susceptibility gene detection kit and application thereof (patent publication No. 102534031A) detect 12 deafness susceptibility gene mutations, the detection capabilities of the detection kits are limited, or low throughput, or time and labor consuming, and more importantly, difficult to perform high-throughput detection on multiple mutation sites of different genes simultaneously. The related patent of the company is ' a nucleic acid membrane strip and a kit for genetic deafness gene detection ' (patent application No. 104498609A) ' and the patent applied by the company for the invention does not belong to the same technical platform at present.

At present, the kit for detecting the non-syndrome genetic deafness genes clinically applied in China is mainly developed based on the principle of a liquid-phase chip hybridization method, realizes the detection of a few mutation sites in a plurality of genes in a multi-tube PCR system, for example, fifteen genetic deafness related gene detection kits and deafness susceptible gene detection kits of Boao biotechnology limited company and Kjeep biochemistry limited company in Chaipu, China can respectively realize the detection of 15 and 9 deafness susceptible gene mutation sites, but the methods have high price of required special instruments, low flux or complex operation, time consumption and labor consumption.

In addition to the above-mentioned kits based on liquid chip hybridization, there is also a clinical development based on the principle of fluorescence PCR technology, for example, the kits developed by Zhongsheng Beijing and Jinan Yingsheng biotechnology limited company can only detect a few loci of deafness susceptibility genes, especially, each kit of Jinan Yingsheng can only detect a few loci of one gene, and has few detection loci and low flux, and if it is necessary to simultaneously detect several deafness susceptibility genes of one sample, it is necessary to use three kits at the same time, which is high in cost.

At present, no kit for detecting a plurality of mutation sites of a plurality of deafness susceptibility genes of Chinese people exists in China. Therefore, it is necessary to establish a high-throughput, high-efficiency and low-cost screening method for deafness gene mutation, which has realized clinical rapid detection or large-scale population screening.

Disclosure of Invention

The invention aims to provide a genetic deafness gene detection kit which can simultaneously detect 30 mutation sites in four hot spot genes related to genetic deafness at one time.

The invention provides a genetic deafness gene detection kit, which comprises PCR reaction liquid, wherein the PCR reaction liquid comprises PCR reaction liquid A, PCR reaction liquid B, PCR reaction liquid C, PCR reaction liquid D and PCR reaction liquid E;

the PCR reaction solution A comprises the following primers and probes:

primer G2F 1: 5'-TAGTGATTCCTGTGTTGTGTG-3', respectively;

primer G2R 1: 5'-AGCCTTCGATGCGGACCTTCTG-3', respectively;

internal reference primer GF 1: 5'-CTATGACTTAGTTGCGTTAC-3', respectively;

internal reference primer GR 1: 5'-GAGAAGTGGGGTGGCTTTTAGGATGG-3', respectively;

probe P1: 5' -GTTTGTTCACACCCCCCAGGA-Z4-BHQ1

Probe P2: 5'-CAGCCACAATGAGGATC-3', respectively;

probe P3: 5'-TGCAGACAAAGTCGGCCT-3', respectively;

probe P4: 5'-TAGCACACGTTCTTGCAGCCTGGCTGCAG-3', respectively;

probe P5: 5' -CAGCTGCAGGGCCCATAG-3'；

Probe P6: 5' -CTTCTCGTCTCCGGTAGGC-Z4-3'；

Probe P25: 5'-CGCTCCAACCGACTGCTGTCACCTTCAC-3', respectively;

the PCR reaction solution B comprises the following primers and probes:

primer G2F 2: 5'-GACCTACACAAGCAGCATC-3', respectively;

primer G2R 2: 5'-TTCAGCAGGATGCAAATTCCAGACAC-3', respectively;

primer G3F 1: 5'-AGTTCCTCTTCCTCTACCTG-3', respectively;

primer G3R 1: 5'-CACAGATGGTGAGTACGATGCAGACG-3', respectively;

primer SF 1: 5'-AAATACCGAGTCAAGGAATG-3', respectively;

primer SR 1: 5'-GCCTTGAAGGGTAAGCAACCATCTGTC-3', respectively;

primer SF 2: 5'-GAACACTTTCTCGTATCCAGCAGC-3', respectively;

primer SR 2: 5'-TCCATCTATATTTTACTTGTAAGTTC-3', respectively;

primer SF 3: 5'-CACAGCTAAAGATTGTCCTC-3', respectively;

primer SR 3: 5'-CCATCCCTGGAGCAAGAAGCAACAC-3', respectively;

internal reference primer GF 1: 5'-CTATGACTTAGTTGCGTTAC-3', respectively;

internal reference primer GR 1: 5'-GAGAAGTGGGGTGGCTTTTAGGATGG-3', respectively; probe P7: 5'-AGGCGTTCGTTGCACTTCACCA-3', respectively;

probe P8: 5' -CTTCTTGGTAGGTCGGGCAAT-3'；

Probe P9: 5'-TAGCCCAATACTAACTCCCG-3', respectively;

probe P10: 5 '-CCTGACTCTGCTGGTTGGAAT-Z4-3';

probe P11: 5'-GATAATAGGGAGAACTCCATTGT-3', respectively;

probe P25: 5'-CGCTCCAACCGACTGCTGTCACCTTCAC-3', respectively; the PCR reaction solution C comprises the following primers and probes:

primer SF 4: 5'-CTGGATTGCTCACCATTGTCGTCTG-3', respectively;

primer SR 4: 5'-GTACTAAGAGGAACACCACAC-3', respectively;

primer SF 5: 5'-TCATCCAGTCTCTTCCTTAG-3', respectively;

primer SR 5: 5'-AGCCTTCCTCTGTTGCCATTCCTC-3', respectively;

primer SF 6: 5'-GAGCAATGCGGGTTCTTTGACGAC-3', respectively;

primer SR 6: 5'-TCTTGAGATTTCACTTGGTTC-3', respectively;

internal reference primer GF 1: 5'-CTATGACTTAGTTGCGTTAC-3', respectively;

internal reference primer GR 1: 5'-GAGAAGTGGGGTGGCTTTTAGGATGG-3', respectively; probe P12: 5'-TTTGTTTTATTTCAGACGAT-3', respectively;

probe P13: 5'-CCTGAGAAGATGTTGCTGAT-3', respectively;

probe P14: 5'-GCCGTGCGGGAAAGAGCAGTG-3', respectively;

probe P15: 5' -TTTGATGGTCCATGATGC-Z4-3'；

Probe P25: 5'-CGCTCCAACCGACTGCTGTCACCTTCAC-3', respectively;

the PCR reaction solution D comprises the following primers and probes:

primer SF 7: 5'-AGATTCTTAGATTTTCCAGTCC-3', respectively;

primer SR 7: 5'-AGAGGGTCTAGGGCCTATTCCTGATTG-3', respectively;

primer SF 8: 5'-GTGAACGTTCCCAAAGTGCCAATCC-3', respectively;

primer SR 8: 5'-ATACTGGACAACCCACATC-3', respectively;

primer MF 1: 5'-CGCGGTCACACGATTAACCCAAGTC-3', respectively;

primer MR 1: 5'-TGTTAAGCTACACTCTGGTTC-3', respectively;

internal reference primer GF 1: 5'-CTATGACTTAGTTGCGTTAC-3', respectively;

internal reference primer GR 1: 5'-GAGAAGTGGGGTGGCTTTTAGGATGG-3', respectively;

probe P16: 5'-ATAAAATACTTACTGTGGACTT-3', respectively;

probe P17: 5 '-TCCATAGCCTTCTGCTTGACTGTG-Z4-3';

probe P18: 5'-GAGATCACAGCGGGTGGTAAG-3', respectively;

probe P19: 5' -ATCACCCCCTCCCCAATA-3'；

Probe P20: 5'-CCACTATGCTTAGCCCTA-3', respectively;

probe P25: 5'-CGCTCCAACCGACTGCTGTCACCTTCAC-3', respectively;

the PCR reaction solution E comprises the following primers and probes:

primer MF 1: 5'-CGCGGTCACACGATTAACCCAAGTC-3', respectively;

primer MR 1: 5'-TGTTAAGCTACACTCTGGTTC-3', respectively;

primer MF 2: 5'-AGGCTCATTCATTTCTCTAAC-3', respectively;

primer MR 2: 5'-CATGGGGTTGGCTTGAAACCAGC-3', respectively;

primer MF 3: 5'-CCACAACACAATGGGGCTCACTCAC-3', respectively;

primer MR 3: 5'-AGGGTGGTTATAGTAGTGTG-3', respectively;

internal reference primer GF 1: 5'-CTATGACTTAGTTGCGTTAC-3', respectively;

internal reference primer GR 1: 5'-GAGAAGTGGGGTGGCTTTTAGGATGG-3', respectively;

probe P21: 5' -ACCGCCCGTCACCCTCCTCAA-3'；

Probe P22: 5'-TAGAGGAGACAAGTCGTAACAT-3', respectively;

probe P23: 5' -TTTGCCTAGATTTTATGTATACG-3'；

Probe P24: 5 '-CGACCCCTTATTTACCGA-Z4-3';

probe P25: 5'-CGCTCCAACCGACTGCTGTCACCTTCAC-3', respectively;

wherein, the probe containing Z4 in the probe sequence represents that the probe is ZNA^TMThe underlined bases are located below the probe and are modified by the locked nucleic acid.

Preferably, in the genetic deafness gene detection kit, 5 'of the probe is provided with a fluorescent group, 3' of the probe is provided with a quenching group, and the fluorescent group is one of FAM, TET, CY5, HEX or ROX; the quenching group is one of BHQ1, BHQ2 or BHQ3 groups which can be matched with the fluorescent group.

Preferably, in the above genetic deafness gene detection kit, the PCR reaction solution comprises: MgCl₂PCRBuffer, dNTP mix, Taq enzyme and UNG enzyme.

The invention has the beneficial effects that: the kit can simultaneously detect 30 mutation sites in four hot spot genes related to hereditary hearing loss at one time, and can quickly and stably diagnose the genotypes of the sites. The detection kit can be used for more comprehensively and earlier finding the children with deafness genes, particularly the late hearing-deficient children, reducing the missing rate of the gene defects caused by the conventional deafness gene detection kit on the market, helping to take intervention measures to prevent speech disorders in time and effectively reducing the incidence rate of deafness and dumb, and meanwhile, the product can also be applied to prenatal gene diagnosis and is used as a basis, and the increase of new deafness can be avoided through human intervention.

The kit of the invention utilizes sequences of various mutation sites reported in literature to design a LATE-PCR special primer for specific amplification, so as to apply multiple asymmetric PCR to carry out same-tube detection (total five tubes) on multiple genotypes, and adopts locked nucleic acid modified ZNA^TMThe probes have stronger discrimination capability on SNP, have low fluorescence background and can completely discriminate two probes which are marked by the same fluorescence in the reaction solution of the same tube, so that the reaction solution of the least tube can detect the most sites. The combination of the primer and the probe designed by the invention can detect 30 mutation sites in four hot spot genes related to hereditary hearing loss through one-time experiment, and the PCR reaction solution A, B, C, D, E in the kit can be tested on the same fluorescent PCR instrument by using the same amplification program, thereby meeting the requirements of rapid clinical and convenient hearing loss detection.

Drawings

FIG. 1 is a ZNA of example 1 of an embodiment of the present invention^TMThe structure of the probe is shown schematically;

FIG. 2a illustrates the use of ZNA in example 1 of an embodiment of the present invention^TMA melting curve result chart of the probe;

FIG. 2b illustrates the use of ZNA in example 1 of an embodiment of the present invention^TMA melting curve result chart of the probe;

FIG. 3 is a ZNA of example 1 in accordance with an embodiment of the present invention^TMThe action mechanism diagram of the probe;

FIG. 4 is a schematic diagram of a locked nucleic acid structure in example 1 of the present invention;

FIG. 5 is a graph showing the results of melting curves obtained by the genetic deafness gene detection kit in example 2 according to the embodiment of the present invention;

FIG. 6 is a graph showing the results of melting curves obtained by the genetic deafness gene detection kit in example 2 according to the embodiment of the present invention;

FIG. 7 is a graph showing the results of melting curves obtained by the genetic deafness gene detection kit in example 2 according to the embodiment of the present invention;

FIG. 8 is a graph showing the results of melting curves obtained by the genetic deafness gene detection kit in example 2 according to the embodiment of the present invention;

Detailed Description

In order to explain technical contents, structural features, and objects and effects of the present invention in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

The most key concept of the invention is as follows: 30 mutation sites in four hot spot genes related to hereditary hearing loss can be detected by one-time experiment, and the PCR reaction solution A, B, C, D, E in the kit can be tested on the same fluorescent PCR instrument by using the same amplification program, so that the requirements of clinical rapid and convenient hearing loss detection are met.

Example 1

The kit can simultaneously detect 30 mutation sites, namely GJB2(35delG, 109G > A, 155delTCTG, 167delT, 176-191del16, 235delC, 299-300delAT, 512insAACG), GJB3(538C > T, 547G > A), SLC26A4(281C > T, 589G > A, 749T > C, 754T > C, IVS7-2A > G, 1174A > T, 1226G > A, 1229C > T, 1975G > C, 2027T > A, IVS15+5G > A, 2162C > T, 2168A > G), mtDNA (961T > G, 1095T > C, 1494C > T, 1555A > G, 7444G > A, 7445A > G, 12201T > C) in four hot spot genes related to hereditary hearing loss at one time.

1. Design and screening of primers

Firstly, sequences of various mutation sites reported in literature are utilized to design a LATE-PCR special primer for specific amplification, multiple asymmetric PCR is applied to carry out same-tube detection on multiple genotypes (total five tubes), different primers are randomly combined, annealing temperature gradient is optimized, and finally a batch of primers with good specificity are selected, so that the requirements of rapid and convenient clinical deafness detection can be met.

The LATE-PCR here is an advanced form of asymmetric PCR (asymmetric PCR). It is required that the difference between the Tm values of the two primers is at least 5 ℃ wherein the Tm value is the concentration-corrected Tm value and the Tm value of the restriction primer is the lower concentration primer^LValue is higher than the Tm of the higher concentration primer, i.e., the non-limiting primer^XThe value is high so as to ensure that during the exponential amplification phase, i.e. at higher annealing temperatures, there is a higher valueThe annealing temperature is as low as possible below Tm^LThereby ensuring efficient use of the limiting primer while being sufficiently above Tm^XSo as to reduce the interference caused by the non-limiting primer with high concentration, ensure that the amplification at the stage has higher amplification efficiency, reduce non-specific amplification and obtain more double-stranded DNA which can be used for a next template; the amplification efficiency at this stage, which is increased by using a lower annealing temperature for the non-limiting primer during the linear amplification stage, is increased by the non-limiting primer (Tm)^X) And amplicon (Tm)^A) Determination of annealing temperature, Tm^AAnd Tm^XCannot differ too much, Tm^A－Tm^XLess than or equal to 15 ℃. The preferred combination sequences of the amplification primers for each mutation site are shown in Table 1.

TABLE 1 detection primer sequences for various genotypes of deafness

2. Design and screening of probes

2.1 ZNA^TMProbe needle

ZNA^TMThe probe is composed of an oligonucleotide sequence connected with a repeated cationic spermine unit, and the structure is shown in figure 1. The cationic spermine unit herein may be linked in plural as necessary. ZNA^TMThe probe has the advantages that the Tm value of the probe can be improved, and the fluorescence background signal can be reduced, because the connected cationic spermine unit is positively charged, the target nucleotide is negatively charged due to the phosphate skeleton, and the positive cationic spermine unit can reduce the electrostatic repulsion effect between the cationic spermine unit and the target DNA, so that the probe and the target DNA are combined more firmly, and the Tm value of the probe is improved; ZNA^TMThe lower fluorescence background signal of the probe may be due to the fact that the oligo cation end is folded toward the negative oligonucleotide chain, which is shortened due to the inside of the probeThe distance between the two ends formed by the electrostatic repulsion is the distance between the fluorescent group and the quenching group, so that the fluorescence emitted by the fluorescent group can be completely absorbed by the quenching group.

When a SNP is genotyped, the shorter the probe is, the higher the discrimination ability is, but as the length becomes shorter, the Tm value of the probe becomes lower, and the binding efficiency of the probe during the cycle becomes lower. When a multicolor probe melting curve analysis is used, if two probes are detected by one fluorescence channel, four melting peaks, namely four Tm values, are supposed to appear under the condition of double heterozygote, but the crossing of the Tm values is found in the experimental process in many cases, so that the result cannot be interpreted. Thus, we used two probes of the same fluorescent label in the same tube, one of which used ZNA^TMProbes, such that four Tm values can be completely distinguished, e.g., FIG. 2 shows two detection probes of the same detection channel, and neither probe of FIG. 2a employs ZNA^TMThe probe finds that the Tm values of the two middle peaks only differ by 2.5 ℃, and the Tm values have a fluctuation range when a clinical sample is detected, so that the two peaks can be overlapped, and the interpretation of the result is not facilitated; whereas one of the probes of FIG. 2b employs ZNA^TMThe Tm value of the probe was found to be improved as a whole for the yellow probe, and the Tm value was completely distinguishable from the melting peak of the other probe.

ZNA^TMThe principle of action of the probe is shown in FIG. 3: in the absence of target sequence, ZNA^TMThe probe is free to curl, and at the moment, because the fluorescent group and the quenching group are relatively close to each other, the fluorescence emitted by the fluorescent group is absorbed by the quenching group, and only a weak fluorescence signal can be detected; when complementary target sequences exist in the reaction system, ZNA is at low temperature in the whole melting curve process^TMThe probe is hybridized with a target sequence to form a rigid and stable hybrid with a double-chain structure, the distance between the fluorescent group and the quenching group is relatively long, and the fluorescence emitted by the fluorescent group can not be absorbed by the quenching group, so that a strong fluorescence signal can be detected; as the temperature increases, the double-stranded hybrid gradually melts and when the melting point is reached, fluorescence is observedThe point at which the light rapidly decreases and the fluorescence changes most strongly corresponds to the melting point (Tm) at which the probe and the target sequence form a double-stranded structure. ZNA^TMThe double-stranded structure formed by the hybridization of the probe and different target sequences has different stability, so that the probe has different melting points, and the difference of the target sequences can be judged according to the difference of the melting points.

2.2 locked nucleic acid modified probes

The locked nucleic acid is a bicyclic nucleic acid derivative, wherein the 2 '-O position and the 4' -C position of ribose form an oxymethylene bridge through the action of water and are connected into a ring, the ring bridge locks the N configuration of furanose C3 internal form, the flexibility of the ribose structure is reduced, the stability of the partial structure of the phosphate framework is increased, and the specific structure of the locked nucleic acid is shown in figure 4. Locked nucleic acids have sequence specificity which better distinguishes between correctly and incorrectly paired sequences than conventional nucleic acids, LNA-DNA hybrids containing mismatched bases are more unstable than DNA-DNA hybrids containing mismatched bases, where one mismatched base in the LNA-DNA duplex lowers the Tm by at least 20 ℃ and one mismatched base in the corresponding DNA-DNA duplex lowers the Tm by 4-l6 ℃. The invention adopts locked nucleic acid to modify the probes with weak wild and mutant discrimination ability, some directly modify the detection site, and some modify the detection site and adjacent bases, thereby achieving the capability of identifying single base mismatch.

Table 2 shows the sequences of the probes for detecting various loci of hereditary hearing loss in accordance with the present invention, wherein Z4 is contained therein and represents ZNA as the probe^TMThe underlined bases are located below the probe and are modified by the locked nucleic acid.

TABLE 2

3. Determination of primer concentration, Probe concentration and other Components of the reaction System

The kit for detecting the hereditary hearing loss gene comprises PCR reaction liquid, wherein the PCR reaction liquid comprises PCR reaction liquid A, PCR reaction liquid B, PCR reaction liquid C, PCR reaction liquid D and PCR reaction liquid E; the primer and the probe are the sequences described in the two points 1 and 2, the concentration range of the primer and probe storage solution is 0.1-1 mu mol/L, the final concentration of MgCl2 is 1.5mM-9mM, the final concentration of dNTP of a mixed solution of four kinds of deoxyadenosine triphosphates (dATP, dGTP, dCTP and dTTP) with equal concentration is 100nM-300nM, the final concentration of deoxyuridine triphosphate (dUTP) is 100nM-300nM, the final concentration of DNA polymerase is 1-7.5U/reaction, and by utilizing an orthogonal test method and through a large number of experimental comparisons, the optimal PCR reaction system is finally determined and is shown in a formula of a PCR reaction solution A in Table 3, a formula of a PCR reaction solution B in Table 4, a formula of a PCR reaction solution C in Table 5, a formula of a PCR reaction solution D in Table 6 and a formula of a PCR reaction solution E in Table 7.

TABLE 3

TABLE 4

TABLE 5

TABLE 6

TABLE 7

Note: the amount of DNA added was 3. mu.L, and the total reaction volume was 25. mu.L.

The screening and system optimization of the primers and the probes are the key points of the invention, because the reaction system A, B, C, D, E is a multiple asymmetric PCR and also comprises a plurality of probes, the multiple PCR is easy to generate mutual inhibition of each amplified fragment, and part of genes have high GC content, high homology and complex secondary structure, so that the amplification is unstable, and if complex secondary structures are formed between the probes and the primers, the result of a subsequent melting curve is also influenced, so that no melting peak is directly caused. Through a large number of groping experiments, the five reaction systems are optimized.

Determination of PCR reaction conditions

The genetic deafness gene detection kit adopts a two-section PCR system. The reaction conditions are optimized by the following steps:

through a large number of experimental comparison and optimization, the reaction solution A, B, C, D, E can be tested on the same fluorescent PCR instrument by using the same amplification program, and the optimal reaction conditions are as follows:

the annealing temperature and the annealing time have great influence on the PCR amplification efficiency and the specific amplification, and the condition optimization result shows that a non-specific amplification band can be generated when the annealing temperature is low, so that a false positive result is generated; the amplification efficiency is low when the temperature is higher, and the sensitivity is reduced. The experiment can amplify all genotypes without non-specificity by controlling the annealing temperature and the annealing time, and has good specificity, high amplification efficiency and high sensitivity.

4. The genetic deafness gene detection kit is used for the detection process of non-syndromic genetic deafness genotypes, and sequentially comprises the following steps:

(1) extracting DNA of a sample to be detected: genomic DNA was extracted from peripheral blood at a concentration of 5-200 ng.

(2) Fluorescent PCR amplification: and performing fluorescence PCR amplification by taking the extracted genome DNA as a template to obtain a melting curve result.

(3) And (4) interpretation of results: and judging whether the sample to be detected contains corresponding gene mutation or not and the type of the gene mutation according to the result of the multi-color probe melting curve, namely the change of the Tm difference value of the melting peak of each channel of the sample to be detected and the standard contrast. The standard control is a wild-type plasmid and can be used to correct experimental errors caused by external conditions. The result interpretation step is as follows: firstly, reading the Tm value of a standard control in each detection channel; secondly, reading the Tm values of the sample to be detected in each detection channel; and finally, subtracting the Tm values corresponding to the standard control in each detection channel from the Tm values of the sample to be detected in each detection channel to obtain the delta Tm values of each detection channel, and referring to the table 10 to judge whether the sample to be detected has mutation and the type of the mutation.

5. The kit has the beneficial effects

1) The invention adopts the multiple LATE-PCR technology and the multicolor fluorescence probe melting curve technology to simultaneously detect 8 mutations on the GJB2 gene, 2 mutations on the GJB3 gene, 13 mutations on the SLC26A4 gene and 7 mutations on the mtDNA gene. Compared with the prior similar patents for detecting deafness gene mutation, the invention increases the detection of 155delTCTG, 512insAACG, 281C > T, 589G > A, 1095T > C and 12201T > C, the mutation of 155delTCTG and 512insAACG can cause congenital moderate-intensity sensorineural deafness, the mutation of 281C > T and 589G > A can cause large vestibular aqueduct syndrome and congenital or acquired moderate-intensity sensorineural deafness, the mutation of 1095T > C and 12201T > C can cause maternally inherited delayed deafness, if the detection is not carried out, the detection omission can be caused, the birth probability of deafness children can be increased, and serious burden can be brought to families and society. The existing similar patents for detecting deafness can not realize the detection of the gene mutation of more than 30 deafness. By using the kit, the detection of 30 mutations in four hot spot genes related to hereditary hearing loss can be completed by one-time test, so that the cost and the time are greatly saved;

2) simple and quick, can detect a plurality of gene mutation sites simultaneously, and has short time consumption: the kit for detecting the deafness gene mutation can complete 30 mutation detections on four genes related to hereditary deafness in five-tube PCR reaction solution, and the whole operation can be completed only by 2.5-3 h through simple sample loading and loading, so that the operation steps are few, and the time consumption is short;

3) the detection flux is high: the melting curve method of the multicolor probe adopted by the invention is mainly divided into two parts, namely PCR amplification and melting curve analysis, so that the two parts can be simultaneously completed on a fluorescence PCR instrument and can also be amplified on a common PCR instrument, then the melting curve analysis is carried out on the fluorescence PCR instrument, and the detection is carried out by matching one fluorescence PCR instrument with a plurality of common PCR instruments, thus greatly improving the detection flux;

4) good specificity and accurate detection result: locked nucleic acid modified ZNA used in the invention^TMThe probes have stronger discrimination capability on SNP, have low fluorescence background and can completely discriminate two probes which are marked by the same fluorescence in the reaction solution of the same tube, so that the reaction solution of the least tube can detect the most sites.

5) Homogeneous phase detection, closed tube operation: the invention is a homogeneous phase detection system, PCR amplification and melting curve analysis are completed in the same closed reaction tube after adding the template, PCR post-treatment is not needed, and the probability of PCR product pollution is reduced.

Example 2

The genetic deafness gene detection kit adopts LATE-PCR and multicolor fluorescent probe PCR melting curve technology. Designing a corresponding probe in a mutation region to be detected, designing an upstream primer and a downstream primer at the periphery of the designed probe, carrying out PCR amplification on a segment containing a region to be detected by using the upstream primer and the downstream primer, and carrying out melting curve analysis after the PCR amplification is finished. And subtracting the Tm values corresponding to the standard control in the detection channels from the Tm values of the sample to be detected in the detection channels to obtain the delta Tm values of the detection channels, and judging whether the sample to be detected has mutation or not and the mutation type according to the delta Tm values.

1. The genetic deafness gene detection kit mainly comprises the components shown in the table 8, wherein the primers and the probes are defined by corresponding sequences in the embodiment 1.

TABLE 8

2. Adapted for instruments

The kit is suitable for a real-time PCR amplification instrument with FAM, HEX, ROX and CY5 detection channels and a melting curve analysis function, and has the models of Bio-Rad CFX96 and Roche LightCycler480 II.

3. Storage condition and shelf life

The kit is stored in the dark at the temperature below 18 ℃ below zero, and the effective period is 6 months.

The kit needs to be stored and transported at low temperature, and the time limit in transit is not longer than 5 days.

The kit is not suitable to be stored for more than 5 days at 37 ℃.

The repeated freezing and thawing of the kit is not suitable for more than 10 times.

The reagent kit is stored below 18 ℃ below zero after the bottle is opened, and has stable performance in the expiration date.

Production lot number, production date, expiration date: see label.

4. Sample requirement

1) Applicable samples were: the sample source of the kit is peripheral anticoagulated whole blood, the used anticoagulant is sodium citrate or EDTA, and heparin anticoagulation cannot be used.

2) Collecting samples: 5mL of venous blood is extracted and put into a tube containing anticoagulant, and sample information such as name and number of the sample is marked.

3) Blood sample preservation: the anticoagulated whole blood is placed at room temperature (15-25 ℃) for no more than 24 hours, stored at 2-8 ℃ for no more than one month, stored at-18 ℃ for no more than two years, stored at-70 ℃ for a long time, and repeatedly frozen and thawed during frozen storage.

4) Blood sample transportation: the transportation of anticoagulated whole blood needs to be sealed by a foam box and an ice bag, and the time limit in transit is not longer than 72 hours.

5. Inspection method

1) Extraction of DNA: it is recommended to extract human genomic DNA using "nucleic acid extraction reagent" of the Asian energy company. Before PCR amplification, a nucleic acid quantifier or an ultraviolet spectrophotometer can be adopted to measure the concentration and purity of the template DNA. The kit requires that the concentration of the genomic DNA to be detected is 5-200 ng/mu L, and the purity (A260/A280) is 1.7-2.1.

2) Preparing a reaction solution: all components were taken out of the kit and thawed at room temperature and mixed well with shaking, and centrifuged briefly at 5000 rpm.

The required amount of each tube reaction solution is equal to the number of samples to be detected, N +1 wild type control +1 negative control.

To each well of the eight-manifold or 96-well plate, 22. mu.L of the reaction solution was added using a microsyringe.

3) Sample application

Add 3. mu.L of the sample to be tested, wild type control and negative control to each well separately with a microsyringe, cover the vial lid tightly, and centrifuge briefly at 5000 rpm.

4) PCR amplification

The amplification procedure is as in table 9.

TABLE 9

6. Reference value (reference range)

The melting point (Tm) value ranges for the wild type control in each system and channel are detailed in Table 10 below.

Watch 10

7. Interpretation of test results

And (3) judging whether the sample is mutated or not by comparing the difference of the melting points (delta Tm values) of the total 20 detection channels of the sample to be detected in A, B, C, D and E systems and a wild type control. When the difference (Δ Tm value) between the melting peak of the sample and the melting peak of the wild-type control is within ± 1 ℃, the melting peak is the wild-type peak, and the difference exceeds ± 2 ℃ is the mutant peak, the details are shown in the attached tables 11, 12 and 13, which are the detected mutation types and the Δ Tm values of each mutation type in the corresponding channel, and the specific conditions are as follows:

(1) and if 20 detection channels of the sample to be detected only have wild type peaks, the sample is wild type.

(2) If the sample to be detected has both wild type peak and mutant peak in a certain channel, and the other channel is wild type peak, the sample is heterozygote of the mutation, and the mutation type is judged by calculating the difference (delta Tm value) of the melting points of the corresponding wild type peak and mutant peak.

(3) If the sample to be detected only has one mutant peak in a certain channel and other channels are wild type peaks, the sample is homozygous for the mutation, and the mutation type is judged by calculating the difference (delta Tm value) between the melting points of the corresponding wild type peak and the mutant peak.

(4) If the sample has two mutant peaks in two channels and the other channel is a wild type peak, the sample is a double-heterozygote of the two mutations, and the mutation type is judged by calculating the difference (delta Tm value) of the melting points of the corresponding wild type peak and the mutant peak.

TABLE 11

TABLE 12

Watch 13

8. Limitations of the inspection methods

The kit adopts a probe fluorescence melting curve method, and the method can detect sequence variation in the probe coverage range, so that mutation except 30 sites of the kit can be detected.

Since mitochondrial mutations are heterogeneous, there are varying degrees of mutation levels (0-100%). The detection limit of 7 detection sites of mitochondria should be distinguished by the shape of the corresponding wild-type peak, and is 20% or more.

9. Matters of attention

(1) The kit must be strictly stored according to required storage conditions, and the components are fully melted, uniformly mixed, centrifuged for a short time and then uncapped for use before use, so as to ensure the complete volume of a reaction system and prevent potential pollution.

(2) The experiment is strictly operated in different areas, articles in all the areas are used together, and false positive results can be caused by environmental pollution, reagent pollution and sample cross pollution in a laboratory; improper reagent transport, storage or reagent placement may cause reduced reagent detection performance, false negatives or inaccurate detection results.

(3) In order to ensure the accuracy of the experimental result, the concentration and purity of DNA must be measured before amplification, and the subsequent experiment is carried out after the sample is ensured to meet the detection requirement of the reagent.

(4) The kit is suitable for the sample of peripheral sodium citrate ring EDTA anticoagulation whole blood, and if other types of samples are used, the detection result difference is irrelevant to the quality of the kit.

(5) And (3) avoiding light as much as possible in the operation processes of reaction liquid preparation, subpackaging and sample adding, covering the tube cover as soon as possible after sample adding, and centrifuging for a short time.

(6) Quality control should be performed for each experiment.

(7) The test sample should be considered as having infectious material and the handling and treatment should be in compliance with relevant regulatory requirements.

(8) The suction head used in the experiment is directly driven into a waste liquid tank containing disinfectant, and can be discarded after being sterilized together with other wastes.

(9) The kit is only used for in vitro diagnosis.

10. Results of detecting samples Using the genetic deafness Gene detection kit of the present invention

FIG. 5 is a graph showing the result of melting curve of ROX channel in reaction system E of the kit of the present invention, in which the standard control represents a wild-type peak, and the other peak represents a mutant-type peak, so that the sample to be tested is 1494 heterozygous;

FIG. 6 is a graph showing the result of melting curve of CY5 channel in reaction system C of the kit of the present invention, wherein the standard control represents the wild type peak, and the other peak represents the mutant type peak, so that the sample to be tested is 1174 homozygous mutant;

FIG. 7 is a graph showing the results of the melting curve of the FAM channel in the reaction system A of the kit of the present invention, in which the standard control 1 represents a 109G > A wild-type peak, the standard control 2 represents a 35delG wild-type peak, the sample 1 to be tested represents a 109G > A mutant-type peak, and the sample 2 to be tested represents a 35delG mutant-type peak, so that the sample 1 to be tested is a 109 homozygous mutant type, and the sample 2 to be tested is a 35delG homozygous mutant type;

FIG. 8 is a diagram showing the result of a melting curve of a ROX channel in a reaction system C of the kit of the present invention, in which a standard control represents a wild-type peak, a sample 1 to be tested represents a 1226G > A mutant-type peak, a sample 2 to be tested represents a 1229C > T mutant-type peak, a sample 3 to be tested has both a wild-type peak and a 1226G > A mutant-type peak, and is therefore a 1226G > A heterozygous mutant-type peak, and a sample 4 to be tested has both a wild-type peak and a 1229C > T mutant-type peak, and is therefore a 1229C > T heterozygous mutant-type peak.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

SEQUENCE LISTING

<110> Yaenergetic Biotechnology (Shenzhen) Limited

<120> genetic deafness gene detection kit

<160>55

<170>PatentIn version 3.5

<210>1

<211>21

<212>DNA

<213> Artificial sequence

<400>1

tagtgattcc tgtgttgtgt g 21

<210>2

<211>22

<212>DNA

<213> Artificial sequence

<400>2

agccttcgat gcggaccttc tg 22

<210>3

<211>19

<212>DNA

<213> Artificial sequence

<400>3

gacctacaca agcagcatc 19

<210>4

<211>26

<212>DNA

<213> Artificial sequence

<400>4

ttcagcagga tgcaaattcc agacac 26

<210>5

<211>20

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<213> Artificial sequence

<400>5

agttcctctt cctctacctg 20

<210>6

<211>26

<212>DNA

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<400>6

cacagatggt gagtacgatg cagacg 26

<210>7

<211>20

<212>DNA

<213> Artificial sequence

<400>7

aaataccgag tcaaggaatg 20

<210>8

<211>27

<212>DNA

<213> Artificial sequence

<400>8

gccttgaagg gtaagcaacc atctgtc 27

<210>9

<211>24

<212>DNA

<213> Artificial sequence

<400>9

gaacactttc tcgtatccag cagc 24

<210>10

<211>26

<212>DNA

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<400>10

tccatctata ttttacttgt aagttc 26

<210>11

<211>20

<212>DNA

<213> Artificial sequence

<400>11

cacagctaaa gattgtcctc 20

<210>12

<211>25

<212>DNA

<213> Artificial sequence

<400>12

ccatccctgg agcaagaagc aacac 25

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<211>25

<212>DNA

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<400>13

ctggattgct caccattgtc gtctg 25

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<211>21

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gtactaagag gaacaccaca c 21

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<212>DNA

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tcatccagtc tcttccttag 20

<210>16

<211>24

<212>DNA

<213> Artificial sequence

<400>16

agccttcctc tgttgccatt cctc 24

<210>17

<211>24

<212>DNA

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gagcaatgcg ggttctttga cgac 24

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<212>DNA

<213> Artificial sequence

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tcttgagatt tcacttggtt c 21

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<212>DNA

<213> Artificial sequence

<400>19

agattcttag attttccagt cc 22

<210>20

<211>27

<212>DNA

<213> Artificial sequence

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agagggtcta gggcctattc ctgattg 27

<210>21

<211>25

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<400>21

gtgaacgttc ccaaagtgcc aatcc 25

<210>22

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<213> Artificial sequence

<400>22

atactggaca acccacatc 19

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cgcggtcaca cgattaaccc aagtc 25

<210>24

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tgttaagcta cactctggtt c 21

<210>25

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<213> Artificial sequence

<400>25

aggctcattc atttctctaa c 21

<210>26

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<213> Artificial sequence

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catggggttg gcttgaaacc agc 23

<210>27

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ccacaacaca atggggctca ctcac 25

<210>28

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agggtggtta tagtagtgtg 20

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ctatgactta gttgcgttac 20

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gagaagtggg gtggctttta ggatgg 26

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<211>21

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gtttgttcac accccccagg a 21

<210>32

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<212>DNA

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cagccacaat gaggatc 17

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<211>18

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<213> Artificial sequence

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tgcagacaaa gtcggcct 18

<210>34

<211>29

<212>DNA

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tagcacacgt tcttgcagcc tggctgcag 29

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cagctgcagg gcccatag 18

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cttctcgtct ccggtaggc 19

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aggcgttcgt tgcacttcac ca 22

<210>38

<211>21

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<213> Artificial sequence

<400>38

cttcttggta ggtcgggcaa t 21

<210>39

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<213> Artificial sequence

<400>39

tagcccaata ctaactcccg 20

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<211>21

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<213> Artificial sequence

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cctgactctg ctggttggaa t 21

<210>41

<211>23

<212>DNA

<213> Artificial sequence

<400>41

gataataggg agaactccat tgt 23

<210>42

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<213> Artificial sequence

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tttgttttat ttcagacgat 20

<210>43

<211>20

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<213> Artificial sequence

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cctgagaaga tgttgctgat 20

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<211>21

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<213> Artificial sequence

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gccgtgcggg aaagagcagt g 21

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<213> Artificial sequence

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tttgatggtc catgatgc 18

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<211>22

<212>DNA

<213> Artificial sequence

<400>46

ataaaatact tactgtggac tt 22

<210>47

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<400>47

tccatagcct tctgcttgac tgtg 24

<210>48

<211>21

<212>DNA

<213> Artificial sequence

<400>48

gagatcacag cgggtggtaa g 21

<210>49

<211>18

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<213> Artificial sequence

<400>49

atcaccccct ccccaata 18

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<211>18

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<213> Artificial sequence

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ccactatgct tagcccta 18

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accgcccgtc accctcctca a 21

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tagaggagac aagtcgtaac at 22

<210>53

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tttgcctaga ttttatgtat acg 23

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cgacccctta tttaccga 18

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cgctccaacc gactgctgtc accttcac 28

Claims (2)

1. A genetic deafness gene detection kit is characterized by comprising PCR reaction liquid, wherein the PCR reaction liquid comprises 5 tubes of PCR reaction liquid, and the 5 tubes of PCR reaction liquid are respectively PCR reaction liquid A, PCR reaction liquid B, PCR reaction liquid C, PCR reaction liquid D and PCR reaction liquid E; the 5 tubes of PCR reaction liquid all comprise: MgCl₂PCR Buffer, dNTP mixture, Taq enzyme and UNG enzyme;

the PCR reaction solution A comprises the following primers and probes:

primer G2F 1: 5'-TAGTGATTCCTGTGTTGTGTG-3', respectively;

primer G2R 1: 5'-AGCCTTCGATGCGGACCTTCTG-3', respectively;

internal reference primer GF 1: 5'-CTATGACTTAGTTGCGTTAC-3', respectively;

internal reference primer GR 1: 5'-GAGAAGTGGGGTGGCTTTTAGGATGG-3', respectively;

probe P1: 5' -GTTTGTTCACACCCCCCAGGA-Z4-BHQ1

Probe P2: 5'-CAGCCACAATGAGGATC-3', respectively;

probe P3: 5'-TGCAGACAAAGTCGGCCT-3', respectively;

probe P4: 5'-TAGCACACGTTCTTGCAGCCTGGCTGCAG-3', respectively;

probe P5: 5' -CAGCTGCAGGGCCCATAG-3'；

Probe P6: 5' -CTTCTCGTCTCCGGTAGGC-Z4-3'；

Probe P25: 5'-CGCTCCAACCGACTGCTGTCACCTTCAC-3', respectively;

the PCR reaction solution B comprises the following primers and probes:

primer G2F 2: 5'-GACCTACACAAGCAGCATC-3', respectively;

primer G2R 2: 5'-TTCAGCAGGATGCAAATTCCAGACAC-3', respectively;

primer G3F 1: 5'-AGTTCCTCTTCCTCTACCTG-3', respectively;

primer G3R 1: 5'-CACAGATGGTGAGTACGATGCAGACG-3', respectively;

primer SF 1: 5'-AAATACCGAGTCAAGGAATG-3', respectively;

primer SR 1: 5'-GCCTTGAAGGGTAAGCAACCATCTGTC-3', respectively;

primer SF 2: 5'-GAACACTTTCTCGTATCCAGCAGC-3', respectively;

primer SR 2: 5'-TCCATCTATATTTTACTTGTAAGTTC-3', respectively;

primer SF 3: 5'-CACAGCTAAAGATTGTCCTC-3', respectively;

primer SR 3: 5'-CCATCCCTGGAGCAAGAAGCAACAC-3', respectively;

internal reference primer GF 1: 5'-CTATGACTTAGTTGCGTTAC-3', respectively;

internal reference primer GR 1: 5'-GAGAAGTGGGGTGGCTTTTAGGATGG-3', respectively;

probe P7: 5'-AGGCGTTCGTTGCACTTCACCA-3', respectively;

probe P8: 5' -CTTCTTGGTAGGTCGGGCAAT-3'；

Probe P9: 5'-TAGCCCAATACTAACTCCCG-3', respectively;

probe P10: 5 '-CCTGACTCTGCTGGTTGGAAT-Z4-3';

probe P11: 5'-GATAATAGGGAGAACTCCATTGT-3', respectively;

probe P25: 5'-CGCTCCAACCGACTGCTGTCACCTTCAC-3', respectively;

the PCR reaction solution C comprises the following primers and probes:

primer SF 4: 5'-CTGGATTGCTCACCATTGTCGTCTG-3', respectively;

primer SR 4: 5'-GTACTAAGAGGAACACCACAC-3', respectively;

primer SF 5: 5'-TCATCCAGTCTCTTCCTTAG-3', respectively;

primer SR 5: 5'-AGCCTTCCTCTGTTGCCATTCCTC-3', respectively;

primer SF 6: 5'-GAGCAATGCGGGTTCTTTGACGAC-3', respectively;

primer SR 6: 5'-TCTTGAGATTTCACTTGGTTC-3', respectively;

internal reference primer GF 1: 5'-CTATGACTTAGTTGCGTTAC-3', respectively;

internal reference primer GR 1: 5'-GAGAAGTGGGGTGGCTTTTAGGATGG-3', respectively;

probe P12: 5'-TTTGTTTTATTTCAGACGAT-3', respectively;

probe P13: 5'-CCTGAGAAGATGTTGCTGAT-3', respectively;

probe P14: 5'-GCCGTGCGGGAAAGAGCAGTG-3', respectively;

probe P15: 5' -TTTGATGGTCCATGATGC-Z4-3'；

Probe P25: 5'-CGCTCCAACCGACTGCTGTCACCTTCAC-3', respectively;

the PCR reaction solution D comprises the following primers and probes:

primer SF 7: 5'-AGATTCTTAGATTTTCCAGTCC-3', respectively;

primer SR 7: 5'-AGAGGGTCTAGGGCCTATTCCTGATTG-3', respectively;

primer SF 8: 5'-GTGAACGTTCCCAAAGTGCCAATCC-3', respectively;

primer SR 8: 5'-ATACTGGACAACCCACATC-3', respectively;

primer MF 1: 5'-CGCGGTCACACGATTAACCCAAGTC-3', respectively;

primer MR 1: 5'-TGTTAAGCTACACTCTGGTTC-3', respectively;

internal reference primer GF 1: 5'-CTATGACTTAGTTGCGTTAC-3', respectively;

internal reference primer GR 1: 5'-GAGAAGTGGGGTGGCTTTTAGGATGG-3', respectively;

probe P16: 5'-ATAAAATACTTACTGTGGACTT-3', respectively;

probe P17: 5 '-TCCATAGCCTTCTGCTTGACTGTG-Z4-3';

probe P18: 5'-GAGATCACAGCGGGTGGTAAG-3', respectively;

probe P19: 5' -ATCACCCCCTCCCCAATA-3'；

Probe P20: 5'-CCACTATGCTTAGCCCTA-3', respectively;

probe P25: 5'-CGCTCCAACCGACTGCTGTCACCTTCAC-3', respectively;

the PCR reaction solution E comprises the following primers and probes:

primer MF 1: 5'-CGCGGTCACACGATTAACCCAAGTC-3', respectively;

primer MR 1: 5'-TGTTAAGCTACACTCTGGTTC-3', respectively;

primer MF 2: 5'-AGGCTCATTCATTTCTCTAAC-3', respectively;

primer MR 2: 5'-CATGGGGTTGGCTTGAAACCAGC-3', respectively;

primer MF 3: 5'-CCACAACACAATGGGGCTCACTCAC-3', respectively;

primer MR 3: 5'-AGGGTGGTTATAGTAGTGTG-3', respectively;

internal reference primer GF 1: 5'-CTATGACTTAGTTGCGTTAC-3', respectively;

internal reference primer GR 1: 5'-GAGAAGTGGGGTGGCTTTTAGGATGG-3', respectively;

probe P21: 5' -ACCGCCCGTCACCCTCCTCAA-3'；

Probe P22: 5'-TAGAGGAGACAAGTCGTAACAT-3', respectively;

probe P23: 5' -TTTGCCTAGATTTTATGTATACG-3'；

Probe P24: 5 '-CGACCCCTTATTTACCGA-Z4-3';

probe P25: 5'-CGCTCCAACCGACTGCTGTCACCTTCAC-3', respectively;

wherein, the probe containing Z4 in the probe sequence represents that the probe is ZNA^TMThe underlined bases are located below the probe and are modified by the locked nucleic acid.

2. The genetic deafness gene detection kit of claim 1, wherein 5 'of the probe is provided with a fluorophore, 3' is provided with a quencher, and the fluorophore is one of FAM, TET, CY5, HEX or ROX; the quenching group is one of BHQ1, BHQ2 or BHQ3 groups which can be matched with the fluorescent group.

Images