Deafness genetic gene mutation detection kit, reagent combination, probe and method for detecting gene mutation

Abstract

The present invention discloses a deafness genetic gene mutation detection kit, a reagent combination, a probe and a method for detecting gene mutation. The kit includes a pre-treatment solution, an amplification reaction solution and a mutation detection liquid group, and the mutation detection liquid group is selected from at least one of mutation detection liquid A, mutation detection liquid B, mutation detection liquid C and mutation detection liquid D; the pre-treatment solution includes Tween20, NP‑40, Triton‑X100, PVP and NaOH; the amplification reaction solution includes hot start Taq enzyme, UNG enzyme, dNTP, dUTP, 5×PCR buffer, and the 5×PCR buffer includes Tris‑HCl, MgCl2, BSA, mannitol, (NH4)2SO4, Tween20 and trehalose. The kit is treated in one step to a trace blood sample (3ul blood or 3mm dry blood spot), and the treatment solution directly performs subsequent amplification detection, and does not need to carry out cumbersome steps such as genomic DNA purification, saves cost and time, and is suitable for screening and detecting dry blood spots of newborns.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a kit for detecting genetic mutation of deafness, a reagent combination, a probe and a method for detecting genetic mutation. Background Art

[0002] Neonatal disease screening, screening and diagnosis of diseases in newborns, is an important measure for the three-level prevention and control of birth defects, achieving early detection, early diagnosis, and early treatment, significantly improving the prognosis and quality of life of children. Deafness is a major birth defect, and genetic factors are the main cause of deafness. A large number of molecular epidemiological studies have shown that GJB2, SLC26A4, mitochondrial 12S rRNA, and GJB3 genes are the most common genetic pathogenic genes for deafness in the Chinese population. The main 20 sites in these four genes cover more than 90% of deafness pathogenic mutations. Therefore, clinically, deafness gene testing is usually performed by detecting these hotspot mutations. The significance of genetic testing for deafness in newborns is particularly significant, which advances the time point for deafness intervention and avoids more deafness. Neonatal disease screening is managed and implemented by the neonatal disease screening centers in various regions. Heel blood is usually collected on the third day after the birth of the newborn, and dried blood spots are made and delivered to the neonatal disease screening center for testing. Therefore, the realization of the newborn disease screening kit must be able to achieve detection through dried blood spots.

[0003] The main methods for detecting deafness genes include restriction fragment length polymorphism analysis (RFLP), Taqman fluorescence quantification, denaturing high-performance liquid chromatography (DHPLC) technology, allele-specific PCR, SNaP shot sequencing technology, high-throughput sequencing technology, gene chips and DNA direct sequencing. These methods are generally used more in clinical diagnostic laboratories. Due to the cumbersome detection steps, expensive instruments, and detection throughput limitations, their clinical application and promotion are greatly restricted.

[0004] At present, the most clinically used deafness gene detection kits include the 15-item hereditary deafness-related gene detection kit (microarray chip method) of Beijing Bio-Tech Co., Ltd. and the deafness susceptibility gene detection kit (PCR+flow-through hybridization method) of Chaozhou Keyu Biotechnology. The technologies of these two kits are mainly based on the principle of liquid chip hybridization, which can realize multi-tube PCR amplification to detect 15 mutation hotspots. However, these methods require special instruments, which are expensive, have many and complicated operation steps, are time-consuming and labor-intensive, and require the lid to be opened to handle the PCR products, which is extremely prone to PCR contamination.

[0005] The deafness gene detection kit developed by BGI is based on high-throughput sequencing technology. Although it can detect 20 mutation hotspots, high-throughput sequencing technology has high detection costs, cumbersome operations, and requires a large sample size. It is not suitable for newborn screening tests for large populations.

[0006] Xiamen University's related patents "A Deafness Susceptibility Gene Mutation Detection Kit and Its Preparation Method and Application" (Patent No. CN104263848B) and Shenzhen Yaneng Bio's "A Hereditary Deafness Gene Detection Kit" (Patent No. CN106811533B) both use fluorescent quantitative PCR platforms and can simultaneously detect more than 20 mutation hotspots through the fluorescent probe melting curve method. However, both require the use of DNA purified from anticoagulant blood, while newborn screening can only collect limited dried blood spots and cannot provide anticoagulant blood. Therefore, these two kits are not suitable for newborn deafness gene screening.

[0007] At present, there is still a lack of a test kit suitable for neonatal deafness gene screening for newborn dry blood spot samples, which is simple to operate, low in cost, has multiple detection sites and high throughput in China. This has greatly restricted the promotion and popularization of neonatal deafness gene screening. The most meaningful detection of deafness gene mutation sites is newborn screening, so it is very necessary to develop a test kit with higher sensitivity directly for dry blood spot sample detection. With the increase in the number of newborn screening diseases, the demand for the number of dry blood spots produced by blood sampling has also increased accordingly. Therefore, it is very important to develop a micro-dry blood spot sample detection technology, without increasing the number of newborn dry blood spots collected, to complete the detection on the existing blood spots, which is very important for the popularization of newborn deafness gene mutation site screening detection. Summary of the invention

[0008] One of the purposes of the present invention is to provide a deafness susceptibility gene mutation detection kit based on multicolor probe melting curve technology that can detect trace blood samples, has rapid processing, is simple and quick, has high sensitivity, strong reliability and / or low cost, in order to address the shortcomings of the prior art, such as the need for more blood samples, complicated pre-treatment and detection operations, long time consumption, high detection cost, easy contamination and low throughput.

[0009] The present invention provides a deafness genetic gene mutation detection kit, comprising a pretreatment solution, an amplification reaction solution and a mutation detection liquid group, wherein the mutation detection liquid group is selected from at least one of mutation detection liquid A, mutation detection liquid B, mutation detection liquid C and mutation detection liquid D;

[0010] The pre-treatment solution includes Tween20, NP-40, Triton-X100, PVP and NaOH;

[0011] The amplification reaction solution includes hot start Taq enzyme, UNG enzyme, dNTP, dUTP, and 5×PCR buffer, and the 5×PCR buffer includes Tris-HCl, MgCl2, BSA, mannitol, (NH4)2SO4, Tween20, and trehalose;

[0012] The mutation detection solution A includes amplification primers for specifically amplifying a target fragment containing a mutation site combination A and a probe specific to the target fragment, wherein the mutation site combination A includes at least one selected from the group consisting of GJB2 gene site 35 (c.35delG), GJB2 gene site 167 (c.167delT), GJB2 gene site 176_191 (c.176_191del 16), GJB2 gene site 299_300 (c.299_300del AT), GJB2 gene site 235 (c.235delC) and SLC26A4 gene site 1707+5 (c.1707+5G>A);

[0013] The mutation detection solution B includes amplification primers for specifically amplifying a target fragment containing a mutation site combination B and a probe specific to the target fragment, wherein the mutation site combination B includes at least one selected from the group consisting of site 919 of the SLC26A4 gene (c.919-2A>G), site 1174 of the SLC26A4 gene (c.1174A>T), site 1226 of the SLC26A4 gene (c.1226G>A), site 1229 of the SLC26A4 gene (c.1229C>T), site 538 of the GJB3 gene (c.538C>T) and site 547 of the GJB3 gene (c.547G>A);

[0014] The mutation detection solution C includes amplification primers for specifically amplifying a target fragment containing a mutation site combination C and a probe specific to the target fragment, wherein the mutation site combination C includes at least one selected from the group consisting of mitochondrial 12S rRNA gene 1494 site (c.1494C>T), mitochondrial 12S rRNA gene 1555 site (c.1555A>G), SLC26A4 gene 2162 site (c.2162C>T), SLC26A4 gene 2168 site (c.2168A>G), SLC26A4 gene 1975 site (c.1975G>C) and SLC26A4 gene 2027 site (c.2027T>A);

[0015] The mutation detection liquid D includes amplification primers for specifically amplifying a target fragment containing a mutation site combination D and a probe specific to the target fragment, wherein the mutation site combination D includes at least one selected from the group consisting of SLC26A4 gene 917 site (c.917insG), SLC26A4 gene 281 site (c.281C>T), SLC26A4 gene 589 site (c.589G>A), GJB2 gene 427 site (c.427C>T) and GJB2 gene 512 site (c.512513insAACG).

[0016] Optionally, the probe type is a dual-probe quenching system.

[0017] The present invention uses the independently developed Multiplex adjacent probes melting curve analysis (MapMCA) technology to design an adjacent quenching dual probe system (i.e., dual probe quenching system) for melting curve analysis. The principle is as follows: Figure 1 , the adjacent quenching dual probe system is used in conjunction with two probes, one mutation detection probe is labeled with a fluorescent group, and the matching anchor probe is labeled with a quenching group. The mutation detection probe and the anchor probe are adjacent to the binding position of the target DNA. After asymmetric PCR amplification produces a single-stranded target product DNA, during the melting curve process, the two probes anneal and hybridize on the target DNA sequence, the fluorescent group and the quenching group are close, the fluorescence of the fluorescent group is quenched, and the fluorescence signal is low. As the temperature rises, the hybrid double strands slowly melt, the fluorescently labeled mutation detection probe separates from the anchor probe, the distance between the fluorescent group and the quenching group increases, the quenching effect gradually weakens, and the fluorescence signal becomes stronger. The temperature at which the fluorescence signal changes fastest is the melting point (Tm) value of the probe detection. Figure 2 As shown, the mutation detection probe covers the mutation site to be detected. Due to the existence of the mutation site, the mutation detection probe cannot completely bind to the mutant site in the target, resulting in a decrease in the stability of the double-stranded structure. Therefore, in the melting curve analysis, Tm decreases, thereby realizing the function of detecting genetic gene mutation sites.

[0018] The adjacent quenching dual probe system is different from the self-quenching probe principle commonly used in the market (such as Figure 1 The self-quenching probe is different from the self-quenching probe, which is a probe with a fluorescent group and a quenching group labeled at both ends. When there is no target, the probe curls naturally, the fluorescent group and the quenching group are close to each other, and the fluorescence of the fluorescent group is quenched. When there is a target, the probe anneals and hybridizes with the target, the distance between the fluorescent group and the quenching group is pulled apart, and a fluorescent signal appears.

[0019] The sensitivity and effect of fluorescent probe melting curve in detecting targets depend on the signal difference generated when the fluorescent group dissociates from the quenching group. When the self-quenching probe is in a naturally curled state, the fluorescent group and the quenching group still have a large distance between them. The adjacent quenching double probes are hybridized at adjacent positions of the target DNA. The fluorescent group and the quenching group are close together through double-stranded hybridization. The quenching group is closer to the fluorescent group and the quenching effect is better. The intensity of the fluorescent signal difference generated after the quenching group separates is better than that of the self-quenching probe melting curve, that is, the Tm peak is high, thereby improving the detection effect and realizing the detection of smaller amounts of dry blood spot samples.

[0020] The above-mentioned deafness genetic gene mutation detection kit may also include a standard control solution and / or a negative control solution. The standard control solution contains normal human genomic DNA or normal human dried blood spots, and the normal human genomic DNA is human genomic DNA that does not contain the above-mentioned mutation site mutation. The negative control solution does not contain the above-mentioned target fragment, for example, the negative control solution can be deionized water.

[0021] Optionally, according to the above-mentioned deafness genetic gene mutation detection kit, the pretreatment solution includes 0.5%-2.5% (volume percentage) Tween20, 0.02%-1% (volume percentage) NP-40, 0.05%-0.2% (volume percentage) Triton-X100, 0.02%-1% (mass percentage) PVP, 0.02-0.4M NaOH; 6.55ul of the amplification reaction solution includes 0.5-2U hot start Taq enzyme, 0.025-0.25U UNG enzyme, 5-10nmol dNTP, 5-10nmol dUTP, 5×PCR buffer; the 5×PCR buffer in the amplification reaction solution includes 50-500mM Tris-HCl (pH 8.8), 15-30mM MgCl2, 0.25%-10% (mass percentage) BSA, 0%-15% (mass percentage) mannitol, 50-200mM (NH4)2SO4, 0.025%-0.5% (volume percentage) Tween20 and 5-30% (mass percentage) trehalose.

[0022] Optionally, according to the above-mentioned deafness genetic gene mutation detection kit, the probe is composed of a mutation detection probe that specifically binds to the target fragment and an anchor probe that specifically binds to the target fragment, the mutation detection probe specifically binds to the mutation site, and of the two probes, the mutation detection probe and the anchor probe, one probe is labeled with a fluorescent group at one end, and the other probe is labeled with a corresponding quencher group at one end, and after the mutation detection probe and the anchor probe bind to the target fragment, the quencher group interacts with the fluorescent group and does not produce fluorescence.

[0023] The fluorescent group can be selected from at least one of AMCA, Alexa Fluor 488, Alexa Fluor 594, Atto 425, ATTO590, Cy3, Cy5, CY5.5, Cy7, 6-FAM, HEX, JOE, NED, ROX, Pacific Blue, Quasar 570, Quasar670, TAMRA, TET, Texas Red and VIC. The quenching group can be selected from at least one of DABCYL, BHQ quenchers, ECLIPSE, MGB and TAMRA.

[0024] Optionally, according to the above-mentioned deafness genetic gene mutation detection kit,

[0025] The probe of the hybridization mutation site combination A includes at least one of the FP1-Q1 probe, the FP2-Q2 probe, the FP3-Q3 probe, the FP4-Q4 probe and the FP5-Q5 probe,

[0026] The FP1-Q1 probe consists of a mutation detection probe with a sequence of SEQ ID No.23 and an anchor probe with a sequence of SEQ ID No.24.

[0027] The FP2-Q2 probe consists of a mutation detection probe with the sequence of SEQ ID No.25 and an anchor probe with the sequence of SEQ ID No.26.

[0028] The FP3-Q3 probe consists of a mutation detection probe with the sequence of SEQ ID No.27 and an anchor probe with the sequence of SEQ ID No.28.

[0029] The FP4-Q4 probe consists of a mutation detection probe with a sequence of SEQ ID No.29 and an anchor probe with a sequence of SEQ ID No.30.

[0030] The FP5-Q5 probe consists of a mutation detection probe with the sequence of SEQ ID No. 31 and an anchor probe with the sequence of SEQ ID No. 32;

[0031] The probe of the hybridization mutation site combination B includes at least one of the FP6-Q6 probe, the FP7-Q7 probe, the FP8-Q7 probe, and the FP9-Q8 probe.

[0032] The FP6-Q6 probe consists of a mutation detection probe with the sequence of SEQ ID No.33 and an anchor probe with the sequence of SEQ ID No.34.

[0033] The FP7-Q7 probe consists of a mutation detection probe with the sequence of SEQ ID No.35 and an anchor probe with the sequence of SEQ ID No.36.

[0034] The FP8-Q7 probe consists of a mutation detection probe with a sequence of SEQ ID No.37 and an anchor probe with a sequence of SEQ ID No.36.

[0035] The FP9-Q8 probe consists of a mutation detection probe with the sequence SEQ ID No.38 and an anchor probe with the sequence SEQ ID No.39.

[0036] The probe of the hybridization mutation site combination C includes at least one of the FP10-Q9 probe, the FP11-Q10 probe, the FP12-Q11 probe, the FP13-Q12 probe, and the FP14-Q12 probe,

[0037] The FP10-Q9 probe consists of a mutation detection probe with a sequence of SEQ ID No.40 and an anchor probe with a sequence of SEQ ID No.41.

[0038] The FP11-Q10 probe consists of a mutation detection probe with a sequence of SEQ ID No.42 and an anchor probe with a sequence of SEQ ID No.43.

[0039] The FP12-Q11 probe consists of a mutation detection probe with a sequence of SEQ ID No.44 and an anchor probe with a sequence of SEQ ID No.45.

[0040] The FP13-Q12 probe consists of a mutation detection probe with a sequence of SEQ ID No.46 and an anchor probe with a sequence of SEQ ID No.47.

[0041] The FP14-Q12 probe consists of a mutation detection probe with a sequence of SEQ ID No.48 and an anchor probe with a sequence of SEQ ID No.47.

[0042] The probe of the hybridization mutation site combination D includes at least one of the FP15-Q13 probe, the FP16-Q14 probe, the FP17-Q15 probe, the FP18-Q16 probe, and the FP19-Q17 probe.

[0043] The FP15-Q13 probe consists of a mutation detection probe with a sequence of SEQ ID No.49 and an anchor probe with a sequence of SEQ ID No.50.

[0044] The FP16-Q14 probe consists of a mutation detection probe with a sequence of SEQ ID No.51 and an anchor probe with a sequence of SEQ ID No.52.

[0045] The FP17-Q15 probe consists of a mutation detection probe with a sequence of SEQ ID No.53 and an anchor probe with a sequence of SEQ ID No.54.

[0046] The FP18-Q16 probe consists of a mutation detection probe with a sequence of SEQ ID No.55 and an anchor probe with a sequence of SEQ ID No.56.

[0047] The FP1P19-Q17 probe consists of a mutation detection probe with the sequence of SEQ ID No.57 and an anchor probe with the sequence of SEQ ID No.58.

[0048] Optionally, the 3' end of the mutation detection probe is labeled with a fluorescent group, and the 5' end of the anchor probe is labeled with a quencher group, or the 5' end of the mutation detection probe is labeled with a fluorescent group, and the 3' end of the anchor probe is labeled with a quencher group. Optionally, the 3' end of the unlabeled fluorescent group or the quencher group is modified with a C3 Spacer.

[0049] Optionally, according to the above-mentioned deafness genetic gene mutation detection kit,

[0050] The amplification primers for specifically amplifying the target fragment containing the mutation site combination A are at least one primer pair in the A primer pair composition, and the A primer pair composition consists of an F1-R1 primer pair and an F2-R2 primer pair. The sequences of the two single-stranded DNAs of the F1-R1 primer pair are shown in SEQ ID No. 1-2, and the sequences of the two single-stranded DNAs of the F2-R2 primer pair are shown in SEQ ID No. 3-4.

[0051] The amplification primers for specifically amplifying the target fragment containing the mutation site combination B are at least one primer pair in the B primer pair composition, and the B primer pair composition consists of a F3-R3 primer pair, a F4-R5 primer pair and a F5-R5 primer pair. The sequences of the two single-stranded DNAs of the F3-R3 primer pair are shown in SEQ ID Nos. 5-6, the sequences of the two single-stranded DNAs of the F4-R4 primer pair are shown in SEQ ID Nos. 7-8, and the sequences of the two single-stranded DNAs of the F5-R5 primer pair are shown in SEQ ID Nos. 9-10.

[0052] The amplification primers for specifically amplifying the target fragment containing the mutation site combination C are at least one primer pair in the C primer pair composition, and the C primer pair composition consists of a F6-R6 primer pair, a F7-R7 primer pair, and a F8-R8 primer pair. The sequences of the two single-stranded DNAs of the F6-R6 primer pair are shown in SEQ ID Nos. 11-12, the sequences of the two single-stranded DNAs of the F7-R7 primer pair are shown in SEQ ID Nos. 13-14, and the sequences of the two single-stranded DNAs of the F8-R8 primer pair are shown in SEQ ID Nos. 15-16.

[0053] The amplification primers for specifically amplifying the target fragment containing the mutation site combination D are at least one primer pair in the D primer pair composition, and the D primer pair composition consists of an F3-R3 primer pair, an F9-R9 primer pair, an F10-R10 primer pair, and an F11-R11 primer pair. The sequences of the two single-stranded DNAs of the F9-R9 primer pair are shown in SEQ ID Nos. 17-18, the sequences of the two single-stranded DNAs of the F10-R10 primer pair are shown in SEQ ID Nos. 19-20, and the sequences of the two single-stranded DNAs of the F11-R11 primer pair are shown in SEQ ID Nos. 21-22.

[0054] Each primer pair consists of two single-stranded DNA fragments.

[0055] The present invention also provides a reagent composition for use in melting curve analysis, wherein the reagent composition comprises the above-mentioned pre-treatment solution and the above-mentioned amplification reaction solution.

[0056] The present invention also provides a reagent composition for extracting DNA, and the reagent composition comprises the above-mentioned pre-treatment solution.

[0057] The present invention also provides a probe composition for detecting a genetic mutation of deafness, comprising at least one of the above-mentioned probes.

[0058] The present invention also provides a probe-primer composition for detecting a genetic mutation of deafness, comprising at least one of the above-mentioned probes and at least one of the above-mentioned primer pairs.

[0059] The application of the above-mentioned deafness genetic gene mutation detection kit, the above-mentioned reagent composition, the above-mentioned probe composition or the above-mentioned probe primer composition in any of the following also falls within the protection scope of the present invention. The specific application may be:

[0060] (1) Genetic screening for hereditary deafness, such as genetic screening for hereditary deafness in newborns;

[0061] (2) Identification of deafness-causing gene types, such as identification of deafness-causing gene types in newborns;

[0062] (3) Prevent the occurrence of deafness;

[0063] (4) Preparation of genetic screening products for hereditary deafness;

[0064] (5) Prepare products for identifying deafness-causing gene types;

[0065] (6) Prepare products for preventing deafness.

[0066] The present invention also provides a method for detecting gene mutation, comprising

[0067] (a1) amplifying a nucleic acid fragment containing a mutation site of a gene to be detected by asymmetric PCR, wherein a PCR amplification primer is relatively excessive in the reaction mixture, and the chain generated by its extension binds to a probe, and the probe is added to the amplification reaction before amplification, wherein the probe is composed of a mutation detection probe and an anchor probe, and the mutation detection probe specifically binds to the mutation site of the gene to be detected. Of the two probes, one probe is labeled with a fluorescent group at one end, and the other probe is labeled with a corresponding quenching group at one end. After the mutation detection probe and the anchor probe bind to the nucleic acid fragment, the quenching group interacts with the fluorescent group and does not generate fluorescence;

[0068] (a2) After the amplification is completed, a melting curve analysis is performed on the amplification product containing the probe obtained in step (a1) to obtain a fluorescent signal of the nucleic acid fragment; and whether the nucleic acid fragment has a corresponding gene mutation is determined based on the fluorescent signal of the nucleic acid fragment.

[0069] The nucleic acid fragment without gene mutation is named as wild-type gene. The nucleic acid fragment with gene mutation is named as mutant gene.

[0070] Optionally, in the above method, determining whether the nucleic acid fragment has a corresponding gene mutation according to the fluorescence signal of the nucleic acid fragment includes determining whether the corresponding gene mutation exists according to the difference in fluorescence signals between the nucleic acid fragment and a standard control.

[0071] The standard control is a nucleic acid fragment containing the gene mutation site to be tested, and the gene mutation site to be tested does not have a gene mutation (i.e., a wild-type gene). The fluorescence signal of the standard control is obtained by the following steps:

[0072] (b1) amplifying the standard control by asymmetric PCR, wherein the PCR amplification primer is in a relatively excessive amount in the reaction mixture, the chain generated by its extension binds to the probe, and the probe is added to the amplification reaction before amplification;

[0073] (b2) After the amplification is completed, a melting curve analysis is performed on the amplification product containing the probe obtained in step (b1) to obtain the fluorescence signal of the standard control.

[0074] Optionally, one end of the mutation detection probe is labeled with a fluorescent group, and one end of the anchor probe is labeled with a corresponding quencher group.

[0075] The specific binding of the mutation detection probe to the mutation site of the gene to be detected may be the specific binding of the mutation detection probe to the wild-type gene, or the specific binding of the mutation detection probe to the mutant gene.

[0076] The fluorescent group can be selected from at least one of AMCA, Alexa Fluor 488, Alexa Fluor 594, Atto 425, ATTO590, Cy3, Cy5, CY5.5, Cy7, 6-FAM, HEX, JOE, NED, ROX, Pacific Blue, Quasar 570, Quasar670, TAMRA, TET, Texas Red and VIC. The quenching group can be selected from at least one of DABCYL, BHQ quenchers, ECLIPSE, MGB and TAMRA.

[0077] The above-mentioned method for detecting gene mutations can be, for example, a method for detecting gene mutations in multiple regions. In this method, the PCR amplification primers are multiple pairs of PCR amplification primers corresponding to nucleic acid fragments in different regions, and the probes are multiple probes corresponding to nucleic acid fragments in different regions.

[0078] The above-mentioned method for detecting gene mutation can specifically use the genomic DNA of the sample to be tested as a template, and use the above-mentioned deafness genetic gene mutation kit or the above-mentioned reagent combination to perform PCR amplification and melting curve analysis;

[0079] The PCR amplification program includes 50°C for 2 min, 95°C for 10 min; 95°C for 10 s, 62°C for 10 s, 72°C for 20 s, 15 cycles; 95°C for 10 s, 56°C for 15 s, 72°C for 20 s, 50 cycles;

[0080] The melting curve analysis program includes 95°C for 1 min, 37°C for 3 min, and heating from 40°C to 85°C at a rate of 0.04°C / s for melting curve analysis.

[0081] The above-mentioned method for detecting gene mutations may also include mixing the pretreatment solution and the sample to be tested, 95°C, 10 minutes to obtain a mixed solution; centrifuging the mixed solution, 2500rpm (500g), 5 minutes to obtain an extract; mixing the amplification reaction solution, a mutation detection solution in the mutation detection solution group and the extract to obtain a detection reaction system; performing the PCR amplification on the detection reaction system to obtain a PCR amplification product; performing the melting curve analysis on the PCR amplification product to obtain a fluorescent signal; and determining whether the sample to be tested has a corresponding gene mutation based on the fluorescent signal.

[0082] The above-mentioned method for detecting gene mutation may also include: using the standard control solution as a sample, using the above-mentioned deafness genetic gene mutation kit or the above-mentioned reagent combination to perform PCR amplification and melting curve analysis to obtain a standard control fluorescent signal, and determining Tm according to the standard control fluorescent signal. 标准对照Then, determining whether the sample to be tested has a corresponding gene mutation according to the fluorescence signal includes: determining the Tm value of the melting curve according to the fluorescence signal, according to ATm=Tm-Tm 标准对照 Obtain the ΔTm value, and determine whether the sample to be tested has a corresponding gene mutation according to the ΔTm value. Use a standard control as a calibration to reduce the melting point error caused by different instruments and personnel operations.

[0083] Optionally, determining whether the sample to be tested has a corresponding gene mutation according to the ΔTm value is specifically determining whether the sample to be tested has a corresponding gene mutation by referring to Tables 9 to 12 in the following embodiments.

[0084] The sample to be tested may be blood or a dried blood spot, such as a trace amount of blood or a trace amount of dried blood spot.

[0085] In view of the shortcomings of existing deafness gene detection reagents, such as requiring a large amount of blood samples and purifying genomic DNA, and being unsuitable for application in newborn dry blood spot screening and detection, the kit provided by the present invention processes trace blood samples (3ul blood or 3mm dry blood spots) in one step, and the treated liquid is directly used for subsequent amplification detection, without the need for tedious steps such as genomic DNA purification, thus saving cost and time, and being suitable for newborn dry blood spot screening and detection.

[0086] The kit, primer-probe combination and detection method provided by the present invention can enhance the detection signal through the adjacent quenching double probe system, with a sensitivity of 1ng, and realize the detection of multiple deafness gene mutation sites with trace blood samples (dried blood spots 3mm, whole blood 3u1), which is beneficial for newborn screening detection without adding additional blood samples.

[0087] The kit and detection method provided by the present invention use a reaction system containing an amplification optimizer to achieve PCR amplification of genomic DNA from trace blood samples (3 mm dried blood spots, 3 ul whole blood) without purification. The pre-treatment is completed in 15 minutes and the entire detection is completed in about 2.5 hours, saving time and purification costs. The kit is suitable for high-throughput screening and detection of deafness gene mutation sites in dried blood spots of newborns.

[0088] The kit provided by the present invention is efficient and convenient, and has multiple detection sites. One reaction detects 23 common deafness gene mutation sites through 4 reaction systems. Hot-start Taq enzyme is used to reduce nonspecific amplification, and UDG enzyme is used for anti-pollution treatment. There is no need to open the PCR product, thus avoiding aerosol contamination of the PCR product.

[0089] The kit provided by the present invention can not only detect homogeneous mutations of mitochondrial gene mutation sites c.1494C>T and c.1555A>G, but also detect heterogeneous mutations (mutation detection ratio of more than 10%).

[0090] The probe sequence of the kit provided by the present invention is optimized to increase the difference in Tm values ​​between wild-type and mutant sites, which is beneficial for interpretation and resolution, and the detection results of sample mutation sites are automatically analyzed in combination with the positive sample curve. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 It is the principle of self-quenching probe and adjacent quenching double probe.

[0092] Figure 2 It is the principle of dual probe detection to distinguish between wild type and mutant types of genes.

[0093] Figure 3 The comparison of melting peaks detected by the dual-probe system and the self-quenching probe of the present invention is shown in FIG. Figure 3 In A, 1-5 are melting peaks detected by the dual probe system of the present invention, 6-10 are melting peaks detected by the self-quenching probe, 1 and 10 are c.1707+5G>A sites, 2 and 6 are c.235delC sites, 3 and 9 are c.299-300delAT sites, 4 and 7 are c.35delG sites, 5 and 8 are c.176-191del16 sites, Figure 3 In B, 1-4 are melting peaks detected by the dual probe system of the present invention, 5-8 are melting peaks detected by the self-quenching probe, 1 and 5 are c.919-2A>G, 2 and 6 are c.1226G>A / c.1229C>T sites, 3 and 8 are c.1174A>T sites, 4 and 7 are c.538C>T sites, Figure 3 In C, 1-5 are melting peaks detected by the dual probe system of the present invention, 6-10 are melting peaks detected by the self-quenching probe, 1 and 7 are c.1975G>C site, 2 and 9 are c.1555A>G site, 3 and 6 are c.2168A>G site, 4 and 8 are c.2027T>A site, 5 and 10 are c.1494C>T site, Figure 3 In D, 1-5 are melting peaks detected by the dual-probe system of the present invention, 6-10 are melting peaks detected by self-quenching probes, 1 and 6 are c.917ins G sites, 2 and 8 are c.427C>T sites, 3 and 7 are c.589G>A sites, 4 and 9 are c.281C>T sites, and 5 and 10 are c.512513insAACG sites.

[0094] Figure 4 Comparison between dual-probe system and self-quenching probe detection.

[0095] Figure 5 Comparison of direct extraction from dried blood spots and blood genome purification detection.

[0096] Figure 6 The comparison between the amplification optimizer buffer system of the present invention and the enzyme universal buffer is shown in the figure. Figure 6 A detection reaction system A signal comparison, Figure 6 B is the signal comparison of detection reaction system D.

[0097] Figure 7 To detect the plasmid at each site in the detection reaction system A.

[0098] Figure 8 To detect the plasmid at each site in the detection reaction system B.

[0099] Fig. 9 To detect the plasmid at each site in the detection reaction system C.

[0100] Fig.10 To detect the plasmid at each site in the detection reaction system D.

[0101] Fig.11 Optimized for c.2162C>T and c.2168A>G site probes. Fig.11 A is the mutation detection probe FP12 to detect c.2162C>T and c.2168A>G sites, Fig.11 B is the mutation detection probe FP12b detecting c.2162C>T and c.2168A>G sites.

[0102] Fig.12 Optimized for c.1226G>A and c.1229C>T loci probes. Fig.12 A is the mutation detection probe FP8 to detect c.1226G>A and c.1229C>T sites, Fig.12 B is the mutation detection probe FP8b detecting c.1226G>A and c.1229C>T sites.

[0103] Fig.13 For the detection of different proportions of mitochondrial c.1555A>G and c.1494C>T mutations, Fig.13 A is the detection of different proportions of c.1555A>G mutation type, Fig.13 B is the detection of different proportions of c.1494C>T mutation.

[0104] Fig.14 For clinical sample detection reaction system A mid-site mutation detection.

[0105] Fig.15 For clinical sample detection reaction system B mid-site mutation detection.

[0106] Fig.16 For clinical sample detection reaction system C midpoint mutation detection.

[0107] Fig.17 For clinical sample detection reaction system D mid-site mutation detection.

[0108] Fig.18It is a site mutation detection in the clinical dried blood spot sample detection reaction system A.

[0109] Fig.19 It is a site mutation detection in the clinical dried blood spot sample detection reaction system B.

[0110] Fig. 20 It is a clinical dried blood spot sample detection reaction system C midpoint mutation detection.

[0111] Fig.21 It is the mid-point detection of the clinical dried blood spot sample detection reaction system D. DETAILED DESCRIPTION

[0112] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0113] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0114] SPSS11.5 statistical software was used to process the data. The experimental results were expressed as mean ± standard deviation and tested by One-way ANOVA.

[0115] The nucleotide sequence of the GJB2 gene in the normal human genomic DNA described in the present invention is from positions 20187470 to 20192938 of genbank accession number NC_000013.11, November 22, 2021; the nucleotide sequence of the GJB3 gene is from positions 34781214 to 34786364 of genbank accession number NC_000001.11, November 22, 2021; the nucleotide sequence of the SLC26A4 gene is from positions 107660828 to 107717809 of genbank accession number NC_000007.14, November 22, 2021; the nucleotide sequence of the mitochondrial 12S rRNA gene is from positions 648 to 1601 of genbank accession number NC_012920.1, September 2, 2020.

[0116] Hot start Taq enzyme: Sangon Biotechnology (Shanghai) Co., Ltd., catalog number: B110003-2500.

[0117] UNG enzyme: Shanghai Sangon Biotechnology Co., Ltd., catalog number: B110042-0200.

[0118] Example 1: Preparation and use of a kit for detecting genetic mutations in the deafness gene

[0119] This kit can simultaneously detect mutations at 23 sites of 4 hereditary deafness genes, including c.35del G, c.176_191del 16, c.167delT, c.235del C, c.299_300del AT, c.427C>T, c.512_513insAACG on GJB2, c.538C>T, c.547G>A on GJB3, c.281C>T, c.589G>A, c.2162C>T, c.2168A>G, c.919-2A>G (IVS7-2A>G), c.917insAACG on SLC26A4. G, c.1174A>T, c.1226G>A, c.1229C>T, c.1707+5G>A(IVS15+5G>A), c.1975G>C, c.2027T>A, c.1494C>T, c.1555A>G on mitochondrial 12SrRNA.

[0120] 1. Primer Preparation

[0121] Multiple pairs of primers were designed for the sequences of the 23 mutation sites. Through screening and combination, a group of primers with good specificity and effect were finally selected. The amplification primers and preferred combination sequences of each mutation site are shown in Table 1. Primers were synthesized according to the primer sequences in Table 1 below.

[0122] Table 1 Primer related information

[0123]

[0124]

[0125] 2. Preparation of probe

[0126] Probes were prepared according to the information in Table 2 below, wherein FP1-FP19 were mutation detection probes and Q1-Q17 were anchor probes.

[0127] The fluorescent group is one of FAM, HEX, CY5, and ROX, the quenching group is one of BHQ1, BHQ2, or BHQ3 quenching groups that can match the fluorescent group, part of the anchor probe is labeled with MGB quenching group to increase the Tm value, and C3 Spacer is a blocking group that prevents the probe from extending as a primer at the 3 end of the probe DNA sequence. The mutation detection probe and anchor probe are used in combination, the mutation detection probe is 5' labeled with a fluorescent group and 3' modified with C3 Spacer, and the matching anchor probe is 3' labeled with a quenching group; or the mutation detection probe is 3' labeled with a fluorescent group, and the matching anchor probe is 5' labeled with a quenching group and 3' modified with C3 Spacer.

[0128] Among them, because some mutation sites are close to each other, c.1174A>T, c.1226G>A and c.1229C>T sites share a quencher probe Q7, and c.1975G>C and c.2027T>A share a quencher probe Q12.

[0129] Table 2 Probe related information

[0130]

[0131]

[0132] 3. Preparation of a Deafness Gene Mutation Detection Kit

[0133] The deafness genetic gene mutation detection kit (100 servings) consists of 15 ml of pretreatment solution, 700 μl of amplification reaction solution, 660 μl of mutation detection solution A, 660 μl of mutation detection solution B, 660 μl of mutation detection solution C, 660 μl of mutation detection solution D, 100 μl of standard control solution, and 500 μl of negative control solution.

[0134] The pretreatment solution is composed of a solute and a solvent, the solvent is water, and the solute and its concentration are: 1.5% (volume percentage) Tween20, 0.2% (volume percentage) NP-40, 0.1% (volume percentage) Tiiton-X100, 0.2% (mass percentage) PVP, 0.2M NaOH. The above reagents were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0135] Each amplification reaction solution is composed of a solvent and a solute, the solvent is water, and the solute and its concentration are: in 6.55ul amplification reaction solution, 2U hot start Taq enzyme, 0.25U UNG enzyme, 10nmol dNTP, 10nmol dUTP, 5ul 5×PCR buffer and water are composed, wherein the 5×PCR buffer contains an amplification optimizer, which is composed of 375mM Tris-HCl (PH 8.8), 20mM MgCl2, 2.5% (mass percentage) BSA, 15% (mass percentage) mannitol, 80mM (NH4)2SO4, 0.05% (volume percentage) Tween20, 25% (mass percentage) trehalose and water. The above reagents (except BSA) were purchased from Sangon Biotechnology (Shanghai) Co., Ltd., and BSA was purchased from Sigma.

[0136] The amount of amplification reaction solution (for one amplification reaction) is shown in Table 3.

[0137] Table 3 Amplification reaction solution dosage

[0138] Reagents 1 serving (μl) 5xPCR Buffer 5 5U / ul Hot-Taq enzyme (Shanghai Biotechnology Co., Ltd.) 0.4 10mM dNTP (Shanghai Biotech) 1 100mM dUTP (Shanghai Biotech) 0.1 5U / ul UDG enzyme (Shanghai Biotech) 0.05 total 6.55

[0139] Mutation detection solution A is prepared by mixing the primer storage solution, probe storage solution and deionized water shown in Table 4.

[0140] Table 4 Mutation Detection Solution A Formula

[0141]

[0142]

[0143] Mutation detection solution B is prepared by mixing the primer storage solution, probe storage solution and deionized water shown in Table 5.

[0144] Table 5 Mutation Detection Solution B Formula

[0145]

[0146] Mutation detection solution C is prepared by mixing the primer storage solution, probe storage solution and deionized water shown in Table 6.

[0147] Table 6 Mutation Detection Solution C Formula

[0148]

[0149]

[0150] Mutation detection solution D is prepared by mixing the primer storage solution, probe storage solution and deionized water shown in Table 7.

[0151] Table 7 Mutation Detection Solution D Formula

[0152]

[0153]

[0154] The standard control is a normal human dried blood spot, that is, a wild-type dried blood spot.

[0155] The negative control solution was deionized water.

[0156] IV. Detection method using the deafness genetic gene mutation detection kit

[0157] 1. Pre-treatment of samples to be tested

[0158] Add 100ul of pretreatment solution to the sample to be tested (3mm dried blood spot or 3ul whole blood), heat it at 95°C for 10min to obtain a mixed solution, centrifuge the mixed solution at 2500rpm for 5min to obtain an extract.

[0159] 2. Configure the detection reaction system

[0160] Mix 6.55ul of the amplification reaction solution and 2ul of the detection genome template (test sample extract, plasmid or purified genomic DNA) with 6ul of mutation detection solution A, B, C, and D respectively, make up to 25ul with deionized water, and centrifuge to remove bubbles to obtain detection reaction system A, detection reaction system B, detection reaction system C, and detection reaction system D.

[0161] Mix 6.55ul of the amplification reaction solution and 2ul of the standard control blood spot sample extract with 6ul of mutation detection solutions A, B, C, and D, respectively, and make up to 25ul with deionized water to obtain standard control detection reaction system A, standard control detection reaction system B, standard control detection reaction system C, and standard control detection reaction system D.

[0162] Mix 6.55ul of the amplification reaction solution and 2ul of the negative control with 6ul of mutation detection solutions A, B, C, and D, respectively, and make up to 25ul with deionized water to obtain negative control detection reaction system A, negative control detection reaction system B, negative control detection reaction system C, and negative control detection reaction system D.

[0163] 3. Detection

[0164] Fluorescence quantitative PCR instrument (Shanghai Hongshi SLAN-96S) was used to perform PCR amplification and melting curve analysis on the above detection reaction system.

[0165] PCR amplification procedure:

[0166] 50℃2min, 95℃10min;

[0167] 95℃10s; 62℃10s; 72℃20s; 15 cycles

[0168] 95℃10s; 56℃15s; 72℃20s; 50 cycles

[0169] Melting curve analysis procedure:

[0170] The temperature was increased from 40°C to 80°C at a rate of 0.04°C / s for melting curve analysis, and the fluorescence signals of FAM, HEX, ROX, and CY5 were collected simultaneously.

[0171] 4. Results Analysis

[0172] The Tm value of each channel of the reaction detection system is obtained according to the fluorescence signal by the configuration software of the fluorescence quantitative PCR instrument.

[0173] The Tm value is based on the value automatically judged by the instrument. However, when the instrument cannot give a Tm value, the Tm value can be obtained by adjusting the baseline or manual judgment.

[0174] The FAM / HEX / CY5 / ROX channels in the standard control reaction systems A / B / C / D all have melting peaks with Tm within the reference value range (as shown in Table 8), and the FAM / HEX / CY5 / ROX channels in the negative control reaction system have no clear melting peaks, which means that the detection amplification system is valid.

[0175] If the melting peak is missing in the FAM / HEX / CY5 / ROX channel in the detection reaction system A / B / C / D, it means that the corresponding site amplification has failed, the test result is invalid, and the sample needs to be re-extracted for testing.

[0176] If a melting peak appears in the fluorescence channel of the negative control detection reaction system, it means that the experimental results are invalid and it is recommended to repeat the experiment.

[0177] There are no melting peaks in each channel of the standard control detection reaction system, indicating that there is a problem with the experiment. It is recommended to check whether the reagents are within the validity period or whether the PCR reaction program is set correctly.

[0178] Table 8 Reference value range of standard control melting peak Tm

[0179]

[0180] The detection principle of this method is to compare the melting peak appearing in the FAM / HEX / CY5 / ROX channel in the detection reaction system A / B / C / D with the wild-type melting peak (i.e., the melting peak appearing in the FAM / HEX / CY5 / ROX channel in the standard control reaction system A / B / C / D), and determine whether a mutation occurs based on the Tm difference. Among the melting peaks appearing in the FAM / HEX / CY5 / ROX channel in the detection reaction system A / B / C / D, if there is only a wild-type peak, the corresponding gene locus of the sample to be tested is wild-type; if there is only a mutant peak, the corresponding gene locus of the sample to be tested is a homozygous mutant; if both the wild-type peak and the mutant peak appear, the corresponding gene locus of the sample to be tested is a heterozygous mutant.

[0181] The specific judgment methods and standards are described as follows.

[0182] According to the changes in the Tm values ​​of the melting peaks of the sample to be tested and the standard control in each channel in the multicolor probe melting curve analysis results, it is judged whether the sample to be tested contains the corresponding gene mutation and the type of gene mutation. The Tm value of each channel of the detection reaction system is recorded as Tm, and the Tm value of each channel of the corresponding standard control detection reaction system is recorded as Tm 标准对照 , through the formula ΔTm=Tm-Tm 标准对照 Obtain the ΔTm value, and determine whether the sample to be tested has the corresponding gene mutation and the mutation type according to the ΔTm value with reference to Tables 9 to 12. This involves reading the Tm value of the sample to be tested and the standard control (automatically obtained by the configuration software of the fluorescent PCR instrument), and the standard control is used as a correction to reduce the melting point error caused by different instruments and personnel operations.

[0183] In Tables 9 and 11, the ROX channel has two melting peaks, the one with a low Tm value is ROX1, and the one with a high Tm value is ROX2. In Table 12, the FAM channel has two melting peaks, the one with a low Tm value is FAM1, and the one with a high Tm value is FAM2.

[0184] Table 9 Mutation judgment criteria for reaction system A

[0185]

[0186]

[0187] Table 10 Mutation judgment criteria for reaction system B

[0188]

[0189] Table 11 Mutation judgment criteria for reaction system C

[0190]

[0191] Table 12 Mutation judgment criteria for reaction system D

[0192]

[0193] Example 2: Comparative detection of adjacent quenching dual probes and self-quenching probes

[0194] The sample to be tested in this embodiment is a normal dried blood spot.

[0195] The deafness genetic gene mutation detection kit prepared in Example 1 was used to detect the sample to be tested, and the detection method was the same as that in Example 1.

[0196] In order to compare the detection effect of adjacent quenched double probes and self-quenched probes, self-quenched probes of common major mutation sites of deafness genes were synthesized (Shanghai Sangon Biotechnology Co., Ltd.), the sequence of which is the same as the DNA sequence of the detection mutation probes of each detection site in the kit of the present invention, FAM / HEX / CY5 / ROX are labeled fluorescent groups, BHQ1 / BHQ2 are labeled quenching groups, and the specific structure of the probe is shown in Table 13.

[0197] Table 13 Self-quenching probes for comparison of main mutation sites and dual probe detection systems

[0198]

[0199] The preparation of the self-quenching detection reaction system, the primer concentration and the fluorescence detection probe concentration were consistent with those in Example 1 of the present invention.

[0200] The specific preparation method of self-quenching reaction system A is shown in Table 14.

[0201] Table 14 Self-quenching reaction system A formula

[0202]

[0203] The specific preparation method of self-quenching reaction system B is shown in Table 15.

[0204] Table 15 Self-quenching reaction system B formula

[0205]

[0206] The specific preparation method of self-quenching reaction system C is shown in Table 16.

[0207] Table 16 Self-quenching reaction system C formula

[0208]

[0209]

[0210] The specific preparation method of the self-quenching reaction system D is shown in Table 17.

[0211] Table 17 Self-quenching reaction system D formula

[0212]

[0213] The PCR amplification system was prepared according to the method in Example 1: 6.55ul of the amplification reaction solution was added with 2ul of the same dried blood extract solution, 6ul of the mutation detection reaction system A / B / C / D of the present invention and 6ul of the self-quenching detection reaction system A / B / C / D in Example 1 were used respectively, and deionized water was added to 25ul, and PCR amplification and analysis were performed according to the method in Example 1.

[0214] The results showed that the adjacent quenching double probes of the present invention detected the melting peaks of the major deafness gene mutation sites, and the two sites c.176-191del16 and c.538C>T were similar to the self-quenching probe detection, and the other sites were significantly stronger than the self-quenching probe to varying degrees. The comparison of the melting peak detection effect is shown in Figure 3 The dual probe system of the present invention and the self-quenching probe detect the melting peaks, and the data analysis is shown in Figure 4 Comparison between the dual-probe system and the self-quenching probe detection. Specific data are shown in Table 18. Detection 1-8 represents a repeated detection.

[0215] Table 18 Detection results of dual probe system and self-quenching probe

[0216]

[0217]

[0218] Example 3: Sensitivity test

[0219] The reaction system was prepared as in Example 1, and blood DNA was purified using the Tiangen genome purification kit and graded diluted. 20ng, 10ng, 5ng, 2.5ng, 1.25ng, 0.625ng, 0.312ng, and 0.156ng of genomic DNA were added to each reaction system, respectively. The detection method refers to "IV. Detection method using the deafness genetic gene mutation detection kit" in Example 1.

[0220] The results showed that most sites had good signals at 0.156ng, a few sites had significantly weakened signals at 0.312ng, and all sites had good signals at 0.625ng genomic DNA template, so the detection sensitivity was set to 1ng / 25ul reaction system to ensure the quality of the detection signal. 1ng of genomic DNA was diluted and added to each reaction, and 20 repeated reactions were tested, and the detection signals were all good.

[0221] Example 4, DNA extraction experiment

[0222] The sample in this embodiment is a normal dried blood spot sample.

[0223] The deafness genetic gene mutation detection kit prepared in Example 1 was used to directly extract DNA from the sample, and the extraction method was the same as "IV. Detection method using the deafness genetic gene mutation detection kit 1. Pretreatment of the sample to be tested" in Example 1.

[0224] A genomic extraction kit was used to extract DNA from the same dried blood spot sample mentioned above. The genomic extraction kit was a dried blood spot genomic DNA extraction kit DP334-2 (Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0225] The extracted DNA was tested. The detection method was as described in Example 1, "IV. Detection method using a deafness genetic gene mutation detection kit". 6.55ul of the amplification reaction solution and 2ul (2ng / ul) of the DNA sample extracted by the genome purification kit were mixed with mutation detection solutions A, B, C, and D respectively, and the mixture was made up to 25ul with deionized water. The bubbles were removed by centrifugation to obtain detection reaction system A, detection reaction system B, detection reaction system C, and detection reaction system D. The analysis of PCR amplification and signal detection results was as described in Example 1, "IV. Detection method using a deafness genetic gene mutation detection kit".

[0226] The results show that Figure 5 , see Table 19 for specific data, and tests 1-6 represent repeated tests. 2ul of dried blood spot treatment solution was added, and the detection signal was good. The direct treatment solution of dried blood spots and the purified blood genomic DNA detection results were consistent. The direct treatment of dried blood spot samples by this kit and the detection of purified genomes obtained by using a commercial genome extraction kit were consistent.

[0227] Table 19 Results of direct extraction of dried blood spots and blood purification genome testing

[0228]

[0229]

[0230] Example 5, Optimizer Effect

[0231] The dried blood spot whole blood sample has not been purified for nucleic acid and contains many interfering components. In order to realize the direct extraction of the dried blood spot sample for detection and analysis, the reaction system of the present invention adds an amplification optimizer to improve the anti-inhibition ability of blood inhibitors. The kit undergoes a series of optimization screening and finally determines the amplification reaction buffer containing the amplification optimizer, and conducts a comparative test with the reaction buffer provided by the ordinary Taq enzyme (Shanghai Sangon Biotechnology Co., Ltd.).

[0232] The sample in this embodiment is a normal dried blood spot sample.

[0233] The experiment was divided into 2 groups, the experimental group and the control group.

[0234] The experimental group used the deafness genetic gene mutation detection kit prepared in Example 1 to detect the samples, and the detection method was the same as "IV. Detection method using the deafness genetic gene mutation detection kit" in Example 1.

[0235] The treatment methods of the control group and the experimental group were similar, except that the 5×PCR buffer used the reaction buffer provided by the common Taq enzyme (Sangon Biotech (Shanghai) Co., Ltd.).

[0236] The results are as follows Figure 6 As shown, for the direct extract of the blood spot sample, the amplification reaction buffer containing the amplification optimizer of the present invention has good signals for all the tests, while the reaction system using the reaction buffer provided by the ordinary Taq enzyme has no detected signals at some sites (c.35del G, c.176_191del 16, c.235del C, c.299_300del AT of the GJB2 gene in reaction system A and c.427C>T, 512_513insAACG of the GJB2 gene in reaction system D), and the signal intensity of other sites is also lower than that of the amplification reaction system of the present invention.

[0237] Example 6: Detection of plasmid

[0238] The sample of this embodiment is a plasmid (relevant information is shown in Table 20), which contains the wild-type or mutant gene sequence of the gene site to be detected (the preparation was commissioned to Sangon Biotechnology (Shanghai) Co., Ltd., and the sequence of each gene site was synthesized by gene synthesis and cloned into the PUC-57 plasmid).

[0239] Table 20 Gene synthesis containing plasmids of each deafness gene locus sequence

[0240]

[0241]

[0242] Plasmid Z1-Z30 was diluted to 0.1 pg / ul, and plasmid Z31-Z33 was diluted to 10 pg / ul. 2 ul was taken as an amplification template for amplification detection. The deafness genetic gene mutation detection kit prepared in Example 1 was used to detect the sample to be tested. The detection method was the same as that in Example 1.

[0243] Test results, such as Figure 7-Figure 10 This kit can effectively detect the wild type and homozygous mutant types of the gene loci to be tested.

[0244] Example 7: Early stage probe optimization

[0245] The present invention uses an adjacent quenching double probe system to detect gene loci, the probe is complementary to the detection target, and the difference between the wild type and mutant type of the gene loci is used to cause the difference in melting Tm value, so as to achieve the purpose of detecting gene loci. Because the c.2162C>T and c.2168A>G and c.1226G>A and c.1229C>T sites are close, the same mutation detection probe is used during design, and the sequence in the probe covers two sites, which is complementary to the wild type of one site and the mutant type of the other site. In this way, the two sites can be analyzed at the same time, and the Tm value of the mutant type of one site will decrease, and the Tm value of the mutant type of the other site will increase. Because the probe sequence will affect the difference in Tm between the wild type and the mutant type, the difference in Tm value is too small, and the melting peak is difficult to distinguish, which affects the detection effect.

[0246] 1. Optimization of probes for c.2162C>T and c.2168A>G sites

[0247] The samples to be tested are c.2162C>T / c.2168A>G wild-type plasmid Z18, c.2168A>G homozygous mutant plasmid Z19, and c.2162C>T homozygous mutant plasmid Z20, which are diluted to 0.1 pg / ul and set aside.

[0248] The amplification reaction solution was prepared as in Example 1. The amplification primers for c.2162C>T and c.2168A>G were upstream primer F7 and downstream primer R7.

[0249] The sequence of c.2162C>T, c.2168A>G mutation detection probe 1 (FP12) is:

[0250] CY5-TTTTGATGGTCCATGATGCT-C3 Spacer,

[0251] The sequence of c.2162C>T, c.2168A>G mutation detection probe 2 (FP12b) is:

[0252] CY5-TCTTTTGATGGTCCATGATGC-C3 Spacer,

[0253] The sequence of the anchor probe (Q11) is: TTGACGACAACATTAGAAAGGACACATT-BHQ2.

[0254] The detection reaction system is:

[0255] Amplification reaction solution: 6.55ul

[0256] Upstream primer F7 (20uM) 0.1ul

[0257] Downstream primer R7 (20uM) 1ul

[0258] Mutation detection probe 1 (FP12) or mutation detection probe 2 (FP12b) (10uM) 0.7ul

[0259] Anchor probe Q11: (10uM) 0.7ul

[0260] Sample to be tested (0.1pg / ul plasmid Z18, Z19, Z20): 1ul

[0261] Deionized water: 14.95ul, total volume 25ul.

[0262] PCR amplification and melting analysis were performed using the method in Example 1. The results are as follows: Fig.11 , the detection results of c.2162C>T, c.2168A>G mutation detection probe 1 (FP12) showed that the wild-type Tm value of c.2162C>T, c.2168A>G was 58.5℃, the mutant Tm value of c.2168A>G was 51℃, and the mutant Tm value of c.2162C>T was 62℃, while the detection results of mutation detection probe 2 (FP12b) showed that the wild-type Tm value of c.2162C>T, c.2168A>G was 64.5℃, c.21 The Tm value of the 68A>G mutant is 58℃, and the Tm value of the c.2162C>T mutant is 66.5℃. The Tm difference between the c.2162C>T and wild-type mutation detection probe 2 (FP12b) is only 2℃, which makes it difficult to distinguish the heterozygous state. The Tm difference between the c.2162C>T and wild-type mutation detection probe 1 (FP12) is 3.5℃, which has a better distinction effect. Therefore, the c.2162C>T and c.2168A>G sites are preferably the mutation detection probe 1 (FP12). 2. Optimization of probes for c.1226G>A and c.1229C>T sites

[0263] The samples to be tested are normal wild-type samples and c.1226G>A, c.1229C>T heterozygous mutation samples. The sample type is anticoagulated blood purified DNA, diluted to 2ng / ul, and the samples to be tested are ready for use.

[0264] The amplification primers for c.1226G>A and c.1229C>T were upstream primer F4 and downstream primer R4.

[0265] The sequence of c.1226G>A, c.1229C>T mutation detection probe 1 (FP8) is:

[0266] ROX-TTTCCCACACGGCCGTCCA-C3 Spacer

[0267] The anchor probe sequence is: Q7: BHQ 1-TTCTTCTCTTGTTTTGTGGCCACCACTGCT-BHQ2

[0268] The sequence of c.1226G>A, c.1229C>T mutation detection probe 2 (FP8b) is: CY5-ctctttcccacacggccgtcca-C3 Spacer,

[0269] The anchor probe sequence is: Q7b:ttcttctcttgttttgtggccaccac-BHQ2,

[0270] Detection reaction system 25ul:

[0271] Amplification reaction solution: 6.55ul

[0272] Upstream primer F4 (20uM) 0.1ul

[0273] Downstream primer R4 (20uM) 1ul

[0274] Mutation detection probe 1 (FP8) (10uM) 0.7ul and anchor probe Q7 (10uM) 0.7ul

[0275] Or mutation detection probe 1 (FP8b) (10uM) 0.7ul and anchor probe Q7b (10uM) 0.7ul

[0276] Sample to be tested: 1ul

[0277] Deionized water: 14.95ul

[0278] PCR amplification and melting analysis were performed using the method in Example 1. The results are as follows: Fig.12 The detection results of mutation detection probe 1 (FP8) showed that the wild-type Tm value of c.1226G>A and c.1229C>T was 62.5℃, the mutant Tm value of c.1229C>T was 50.2℃, and the mutant Tm value of c.1226G>A was 69.2℃. The detection results of mutation detection probe 2 (FP8b) showed that the wild-type Tm value of c.1226G>A and c.1229C>T was 66℃, the mutant Tm value of c.1229C>T was 57.5℃, and the mutant Tm value of c.1226G>A was 71℃. In mutation detection probe 1 (FP8), the Tm difference between c.1226G>A and the wild-type was 6.7℃, and the discrimination effect was better. Therefore, mutation detection probe 1 (FP8) is preferred for c.1226G>A and c.1229C>T sites.

[0279] Example 8: Detection of mitochondrial site mutation ratio

[0280] Because mitochondria have heterogeneous mutations, the mutant types account for different proportions. In this example, the kit of the present invention is used to detect the mutation ratio of mitochondrial loci (c.1494C>T and c.1555A>G). The synthesized wild-type and mutant plasmids Z31-Z33 with c.1494C>T and c.1555A>G are diluted to 10pg / ul, and the mutant type: wild type are mixed in proportion. The mutant types account for 0%, 5%, 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, and 100%. 2ul is taken as an amplification template for amplification detection. The deafness genetic gene mutation detection kit prepared in Example 1 is used to detect the sample to be tested, and the detection method is the same as that in Example 1.

[0281] Some test results are as follows Fig.13 As shown, the results show that the kit of the present invention can effectively detect the mutants of c.1494C>T and c.1555A>G when the mutant ratio is 10% or more.

[0282] Example 9: Clinical application

[0283] The samples to be tested in this embodiment are 31 clinical deaf patients' anticoagulated blood purified genomes, diluted to 2ng / ul, and 1ul is taken as a template for amplification detection. The deafness genetic gene mutation detection kit prepared in Example 1 is used to detect the samples to be tested, and the detection method is the same as that in Example 1. Sanger sequencing verification is used as a control.

[0284] Some test results can be found in Figure 14-17 The test kit of the present invention has a 100% success rate and accuracy rate for clinical positive samples, which is consistent with the Sanger sequencing verification results. Specific data are shown in Table 21.

[0285] Table 21 Clinical application test results of Example 9

[0286]

[0287]

[0288] Example 10: Clinical application

[0289] The samples to be tested in this embodiment are 405 dried blood spot samples of newborns, all of which are made by collecting heel blood from newborns 72 hours after birth.

[0290] The deafness genetic gene mutation detection kit prepared in Example 1 was used to detect the sample to be tested, and the detection method was the same as that in Example 1. The 15-item deafness gene kit (Cat. No.: 300068-01) of Biobio was used as a control.

[0291] Some test results are as follows Figure 18-Figure 21, 405 newborn clinical samples were tested, 152 samples carried mutations, and 1 sample was a homozygous mutation. For specific data, please see Table 22. After comparison and testing with BGI's 15 deafness gene test kits, the compliance rate was 100%.

[0292] Table 22 Clinical application test results of Example 10

[0293]

[0294]

[0295] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. SEQUENCE LISTING <110> Suzhou Ganjiang Biotechnology Co., Ltd. <120> Deafness genetic gene mutation detection kit, reagent combination, probe and method for detecting gene mutation <130> GNCYZ220963 <160> 58 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> DNA <213> Artificial sequence <400> 1 cagagcaaac cgcccagagt agaag 25 <210> 2 <211> twenty three <212> DNA <213> Artificial sequence <400> 2 gagccttcga tgcggacctt ctg 23 <210> 3 <211> 27 <212> DNA <213> Artificial sequence <400> 3 tctgagcaac tgtgacttga ctccttg 27 <210> 4 <211> twenty one <212> DNA <213> Artificial sequence <400> 4 ggtctagggc ctattcctga t 21 <210> 5 <211> 28 <212> DNA <213> Artificial sequence <400> 5 agacacaaaa tcccagtccc tattccta 28 <210> 6 <211> 27 <212> DNA <213> Artificial sequence <400> 6 gtactaagag gaacaccaca ctcaccc 27 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <400> 7 accaccacgc agagtaggca t 21 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <400> 8 cctctgttgc cattcctcga ct 22 <210> 9 <211> 19 <212> DNA <213> Artificial sequence <400> 9 ctgccccaac atcgtggac 19 <210> 10 <211> twenty three <212> DNA <213> Artificial sequence <400> 10 cagatgaggt agcagagctc aca 23 <210> 11 <211> 25 <212> DNA <213> Artificial sequence <400> 11 accccagaaa actacgatagccctt 25 <210> 12 <211> twenty three <212> DNA <213> Artificial sequence <400> 12 agctacactc tggttcgtcc aag 23 <210> 13 <211> 26 <212> DNA <213> Artificial sequence <400> 13 cagcctgggc aatagaatga gactct 26 <210> 14 <211> 29 <212> DNA <213> Artificial sequence <400> 14 gaaccttgac cctcttgaga tttcacttg 29 <210> 15 <211> 31 <212> DNA <213> Artificial sequence <400> 15 tgacagtgtt ttcttcgttt agaatggcat c 31 <210> 16 <211> 31 <212> DNA <213> Artificial sequence <400> 16 aacccacatc attttactat tgccaaagct c 31 <210> 17 <211> twenty four <212> DNA <213> Artificial sequence <400> 17 gctccccaaa taccgagtca agga 24 <210> 18 <211> twenty one <212> DNA <213> Artificial sequence <400> 18 tctgccaaca tcttaccttg c 21 <210> 19 <211> twenty four <212> DNA <213> Artificial sequence <400> 19 cagctagagt cctgattgcc agtg 24 <210> 20 <211> twenty three <212> DNA <213> Artificial sequence <400> 20 agcacctgac ctaaaacaac gta 23 <210> twenty one <211> twenty three <212> DNA <213> Artificial sequence <400> twenty one cagaaggtcc gcatcgaagg ctc 23 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <400> twenty two ggcaatgcgt taaactggct 20 <210> twenty three <211> twenty one <212> DNA <213> Artificial sequence <400> twenty three gtttgttcac accccccagg a 21 <210> twenty four <211> twenty three <212> DNA <213> Artificial sequence <400> twenty four cgtctgcagc gtgccccaat cca 23 <210> 25 <211> twenty two <212> DNA <213> Artificial sequence <400> 25 tcgtagctgg ctgcagggtg tt 22 <210> 26 <211> 30 <212> DNA <213> Artificial sequence <400> 26 cagacaaagt cggcctgctc atctccccac 30 <210> 27 <211> twenty one <212> DNA <213> Artificial sequence <400> 27 atcagctgca gggcccatag c 21 <210> 28 <211> twenty four <212> DNA <213> Artificial sequence <400> 28 ggatgtggga gatggggaag tagt 24 <210> 29 <211> 33 <212> DNA <213> Artificial sequence <400> 29 ttcctcttct tctcatgtct ccggtaggcc acg 33 <210> 30 <211> 32 <212> DNA <213> Artificial sequence <400> 30 gcatggccac taggagcgct ggcgtggaca cg 32 <210> 31 <211> twenty four <212> DNA <213> Artificial sequence <400> 31 ctagggataa aatacttact gtgg 24 <210> 32 <211> 30 <212> DNA <213> Artificial sequence <400> 32 gaagtctcaa aagaggttag aaaacaaatt 30 <210> 33 <211> 27 <212> DNA <213> Artificial sequence <400> 33 ttgttttattcagacgata attgcta 27 <210> 34 <211> 29 <212> DNA <213> Artificial sequence <400> 34 tgccatttca tatggagcca acctggaaa 29 <210> 35 <211> twenty four <212> DNA <213> Artificial sequence <400> 35 tgggatcagc aacatcttct cagg 24 <210> 36 <211> 30 <212> DNA <213> Artificial sequence <400> 36 ttcttctctt gttttgtggc caccactgct 30 <210> 37 <211> 19 <212> DNA <213> Artificial sequence <400> 37 tttcccacac ggccgtcca 19 <210> 38 <211> twenty two <212> DNA <213> Artificial sequence <400> 38 acattgcccg acctaccaag aa 22 <210> 39 <211> 33 <212> DNA <213> Artificial sequence <400> 39 aaaatcttca cctacttcat ggtgggcgcc tcc 33 <210> 40 <211> twenty four <212> DNA <213> Artificial sequence <400> 40 caccgcccgt caccctcctc aagt 24 <210> 41 <211> 26 <212> DNA <213> Artificial sequence <400> 41 gttgaacagg gccctgaagc gcgtac 26 <210> 42 <211> twenty two <212> DNA <213> Artificial sequence <400> 42 tatatagagg agacaagtcg ta 22 <210> 43 <211> 28 <212> DNA <213> Artificial sequence <400> 43 aggacattta actaaaaccc ctacgcat 28 <210> 44 <211> 20 <212> DNA <213> Artificial sequence <400> 44 ttttgatggt ccatgatgct 20 <210> 45 <211> 28 <212> DNA <213> Artificial sequence <400> 45 ttgacgacaa cattagaaag gacacatt 28 <210> 46 <211> twenty two <212> DNA <213> Artificial sequence <400> 46 atccatagcc ttgtgcttga ct 22 <210> 47 <211> 31 <212> DNA <213> Artificial sequence <400> 47 tggagctata tctttcctgg acgttgttgg a 31 <210> 48 <211> 20 <212> DNA <213> Artificial sequence <400> 48 tgagatcact gcgggtggta 20 <210> 49 <211> twenty four <212> DNA <213> Artificial sequence <400> 49 gaagtaattg tggtaagtag aata 24 <210> 50 <211> 32 <212> DNA <213> Artificial sequence <400> 50 gtagttagaa agttcagcat tatttggttg ac 32 <210> 51 <211> twenty one <212> DNA <213> Artificial sequence <400> 51 agttagtact gggctagtgg c 21 <210> 52 <211> twenty four <212> DNA <213> Artificial sequence <400> 52 ggctgcttag tgacgtcatt tcgg 24 <210> 53 <211> twenty one <212> DNA <213> Artificial sequence <400> 53 tgtataattc caaccagcag a 21 <210> 54 <211> 26 <212> DNA <213> Artificial sequence <400> 54 ctatatttta cttgtaagtt cattac 26 <210> 55 <211> 20 <212> DNA <213> Artificial sequence <400> 55 catcttcttc cgggtcatct 20 <210> 56 <211> 29 <212> DNA <213> Artificial sequence <400> 56 cgaagccgcc ttcatgtacg tcttctatg 29 <210> 57 <211> 29 <212> DNA <213> Artificial sequence <400> 57 gtgaagtgca acgcctggcc ttgtcccaa 29 <210> 58 <211> twenty four <212> DNA <213> Artificial sequence <400> 58 cgacggcttc tccatgcagc ggct 24

Claims

1. A kit for detecting genetic mutation of deafness, characterized in that: It includes a pre-treatment solution, an amplification reaction solution and a mutation detection solution group, wherein the mutation detection solution group is mutation detection solution A; The pre-treatment solution includes Tween20, NP-40, Triton-X100, PVP and NaOH; The amplification reaction solution includes hot start Taq enzyme, UNG enzyme, dNTP, dUTP, and 5×PCR buffer, and the 5×PCR buffer includes Tris-HCl, MgCl2, BSA, mannitol, (NH4)2SO4, Tween20, and trehalose; The mutation detection solution A includes amplification primers for specifically amplifying a target fragment containing mutation site combination A and a probe specific to the target fragment; The probe is composed of a mutation detection probe that specifically binds to the target fragment and an anchor probe that specifically binds to the target fragment. The mutation detection probe specifically binds to the mutation site. Of the two probes, one probe is labeled with a fluorescent group at one end, and the other probe is labeled with a corresponding quenching group at one end. After the mutation detection probe and the anchor probe bind to the target fragment, the quenching group interacts with the fluorescent group and does not generate fluorescence. The probes hybridizing the mutation site combination A consist of FP1-Q1 probe, FP2-Q2 probe, FP3-Q3 probe, FP4-Q4 probe and FP5-Q5 probe; The FP1-Q1 probe consists of a mutation detection probe with the sequence SEQ ID No. 23 and an anchor probe with the sequence SEQ ID No.

24. The FP2-Q2 probe consists of a mutation detection probe with a sequence of SEQ ID No. 25 and an anchor probe with a sequence of SEQ ID No.

26. The FP3-Q3 probe consists of a mutation detection probe with the sequence SEQ ID No. 27 and an anchor probe with the sequence SEQ ID No.

28. The FP4-Q4 probe consists of a mutation detection probe with the sequence SEQ ID No. 29 and an anchor probe with the sequence SEQ ID No.

30. The FP5-Q5 probe consists of a mutation detection probe with the sequence SEQ ID No. 31 and an anchor probe with the sequence SEQ ID No. 32; The amplification primers used to specifically amplify the target fragment containing the mutation site combination A are an A primer pair composition, and the A primer pair composition consists of an F1-R1 primer pair and an F2-R2 primer pair. The sequences of the two single-stranded DNAs of the F1-R1 primer pair are shown in SEQIDNo.1-2, and the sequences of the two single-stranded DNAs of the F2-R2 primer pair are shown in SEQIDNo.3-4.

2. A kit for detecting genetic mutation of deafness, characterized in that: It includes a pre-treatment solution, an amplification reaction solution and a mutation detection solution group, wherein the mutation detection solution group consists of mutation detection solution A, mutation detection solution B, mutation detection solution C and mutation detection solution D; The pre-treatment solution includes Tween20, NP-40, Triton-X100, PVP and NaOH; The amplification reaction solution includes hot start Taq enzyme, UNG enzyme, dNTP, dUTP, and 5×PCR buffer, and the 5×PCR buffer includes Tris-HCl, MgCl2, BSA, mannitol, (NH4)2SO4, Tween20, and trehalose; The mutation detection solution A includes amplification primers for specifically amplifying a target fragment containing mutation site combination A and a probe specific to the target fragment; The mutation detection solution B includes amplification primers for specifically amplifying a target fragment containing mutation site combination B and a probe specific to the target fragment; The mutation detection solution C includes amplification primers for specifically amplifying a target fragment containing mutation site combination C and a probe specific to the target fragment; The mutation detection solution D includes amplification primers for specifically amplifying a target fragment containing the mutation site combination D and a probe specific to the target fragment; The probe is composed of a mutation detection probe that specifically binds to the target fragment and an anchor probe that specifically binds to the target fragment. The mutation detection probe specifically binds to the mutation site. Of the two probes, one probe is labeled with a fluorescent group at one end, and the other probe is labeled with a corresponding quenching group at one end. After the mutation detection probe and the anchor probe bind to the target fragment, the quenching group interacts with the fluorescent group and does not generate fluorescence. The probes hybridizing the mutation site combination A consist of FP1-Q1 probe, FP2-Q2 probe, FP3-Q3 probe, FP4-Q4 probe and FP5-Q5 probe, The FP1-Q1 probe consists of a mutation detection probe with the sequence SEQ ID No. 23 and an anchor probe with the sequence SEQ ID No.

24. The FP2-Q2 probe consists of a mutation detection probe with a sequence of SEQ ID No. 25 and an anchor probe with a sequence of SEQ ID No.

26. The FP3-Q3 probe consists of a mutation detection probe with the sequence SEQ ID No. 27 and an anchor probe with the sequence SEQ ID No.

28. The FP4-Q4 probe consists of a mutation detection probe with the sequence SEQ ID No. 29 and an anchor probe with the sequence SEQ ID No.

30. The FP5-Q5 probe consists of a mutation detection probe with the sequence SEQ ID No. 31 and an anchor probe with the sequence SEQ ID No. 32; The probes for hybridizing the mutation site combination B consist of FP6-Q6 probe, FP7-Q7 probe, FP8-Q7 probe, and FP9-Q8 probe, The FP6-Q6 probe consists of a mutation detection probe with the sequence SEQ ID No. 33 and an anchor probe with the sequence SEQ ID No.

34. The FP7-Q7 probe consists of a mutation detection probe with the sequence SEQ ID No. 35 and an anchor probe with the sequence SEQ ID No.

36. The FP8-Q7 probe consists of a mutation detection probe with a sequence of SEQ ID No. 37 and an anchor probe with a sequence of SEQ ID No.

36. The FP9-Q8 probe consists of a mutation detection probe with the sequence SEQ ID No. 38 and an anchor probe with the sequence SEQ ID No.

39. The probes for hybridizing the mutation site combination C consist of FP10-Q9 probe, FP11-Q10 probe, FP12-Q11 probe, FP13-Q12 probe, and FP14-Q12 probe, The FP10-Q9 probe consists of a mutation detection probe with the sequence SEQ ID No. 40 and an anchor probe with the sequence SEQ ID No.

41. The FP11-Q10 probe consists of a mutation detection probe with the sequence SEQ ID No. 42 and an anchor probe with the sequence SEQ ID No.

43. The FP12-Q11 probe consists of a mutation detection probe with a sequence of SEQ ID No. 44 and an anchor probe with a sequence of SEQ ID No.

45. The FP13-Q12 probe consists of a mutation detection probe with a sequence of SEQ ID No. 46 and an anchor probe with a sequence of SEQ ID No.

47. The FP14-Q12 probe consists of a mutation detection probe with a sequence of SEQ ID No. 48 and an anchor probe with a sequence of SEQ ID No.

47. The probes for hybridizing the mutation site combination D consist of FP15-Q13 probe, FP16-Q14 probe, FP17-Q15 probe, FP18-Q16 probe, and FP19-Q17 probe, The FP15-Q13 probe consists of a mutation detection probe with a sequence of SEQ ID No. 49 and an anchor probe with a sequence of SEQ ID No.

50. The FP16-Q14 probe consists of a mutation detection probe with the sequence SEQ ID No. 51 and an anchor probe with the sequence SEQ ID No.

52. The FP17-Q15 probe consists of a mutation detection probe with a sequence of SEQ ID No. 53 and an anchor probe with a sequence of SEQ ID No.

54. The FP18-Q16 probe consists of a mutation detection probe with a sequence of SEQ ID No. 55 and an anchor probe with a sequence of SEQ ID No.

56. The FP19-Q17 probe consists of a mutation detection probe with the sequence SEQ ID No. 57 and an anchor probe with the sequence SEQ ID No. 58; The amplification primers used for specifically amplifying the target fragment containing the mutation site combination A are a primer pair composition A, and the primer pair composition A is composed of a primer pair F1-R1 and a primer pair F2-R2, and the sequences of the two single-stranded DNAs of the F1-R1 primer pair are shown in SEQ ID No. 1-2, and the sequences of the two single-stranded DNAs of the F2-R2 primer pair are shown in SEQ ID No. 3-4; The amplification primers for specifically amplifying the target fragment containing the mutation site combination B are a B primer pair composition, which is composed of a F3-R3 primer pair, a F4-R5 primer pair and a F5-R5 primer pair. The sequences of the two single-stranded DNAs of the F3-R3 primer pair are shown in SEQ ID Nos. 5-6, the sequences of the two single-stranded DNAs of the F4-R4 primer pair are shown in SEQ ID Nos. 7-8, and the sequences of the two single-stranded DNAs of the F5-R5 primer pair are shown in SEQ ID Nos. 9-10. The amplification primers for specifically amplifying the target fragment containing the mutation site combination C are a C primer pair composition, which is composed of a F6-R6 primer pair, a F7-R7 primer pair and a F8-R8 primer pair. The sequences of the two single-stranded DNAs of the F6-R6 primer pair are shown in SEQ ID Nos. 11-12, the sequences of the two single-stranded DNAs of the F7-R7 primer pair are shown in SEQ ID Nos. 13-14, and the sequences of the two single-stranded DNAs of the F8-R8 primer pair are shown in SEQ ID Nos. 15-16. The amplification primers for specifically amplifying the target fragment containing the mutation site combination D are in the D primer pair composition, and the D primer pair composition consists of a F3-R3 primer pair, a F9-R9 primer pair, a F10-R10 primer pair and a F11-R11 primer pair, the sequences of the two single-stranded DNAs of the F3-R3 primer pair are shown in SEQ ID Nos. 5-6, the sequences of the two single-stranded DNAs of the F9-R9 primer pair are shown in SEQ ID Nos. 17-18, the sequences of the two single-stranded DNAs of the F10-R10 primer pair are shown in SEQ ID Nos. 19-20, and the sequences of the two single-stranded DNAs of the F11-R11 primer pair are shown in SEQ ID Nos. 21-22, Each primer pair consists of two single-stranded DNA fragments.

3. The deafness genetic gene mutation detection kit according to claim 1 or 2, characterized in that: The pretreatment solution includes 0.5%-2.5% Tween20, 0.02%-1% NP-40, 0.05%-0.2% Triton-X100, 0.02%-1% PVP, and 0.02-0.4M NaOH; the amplification reaction solution includes 0.5-2U hot start Taq enzyme, 0.025-0.25U UNG enzyme, 5-10nm old NTP, 5-10nm old UTP, and 5× PCR buffer; the 5× PCR buffer in the amplification reaction solution includes 50-500mM Tris-HCl pH8.8, 15-30mM MgCl2, 0.25%-10% BSA by mass, 0%-15% mannitol by mass, 50-200mM (NH4)2SO4, 0.025%-0.5% Tween20 by volume, and 5-30% trehalose by mass.

4. A probe primer composition for detecting a deafness genetic gene mutation, characterized in that: The probe primer composition for detecting genetic mutations of deafness comprises the amplification primers for specifically amplifying the target fragment containing mutation site combination A as described in claim 1 and a probe specific to the target fragment.

5. The probe primer composition according to claim 4, characterized in that: The probe primer composition also includes the amplification primers for specifically amplifying the target fragment containing the mutation site combination B and the probe specific to the target fragment as described in claim 2, the amplification primers for specifically amplifying the target fragment containing the mutation site combination C and the probe specific to the target fragment as described in claim 2, and the amplification primers for specifically amplifying the target fragment containing the mutation site combination D and the probe specific to the target fragment as described in claim 2.

6. Use of the deafness genetic gene mutation kit according to any one of claims 1 to 3 or the probe primer combination according to claim 4 or 5 in any of the following: (1) Preparation of genetic screening products for hereditary deafness; (2) Prepare products for identifying deafness-causing gene types.

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