CHD7 mutation detection primer pair, kit and detection method

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a CHD7 mutation detection primer pair, a kit and a detection method. According to the application, 10 potential CHD7 pathogenic mutation sites in the to-be-detected sample can be detected in one tube of PCR reaction by aiming at 10 specifically selected multiple CHD7 pathogenic mutation sites, and the method has the characteristic of wide coverage. In addition, the primer pair is further combined with a high-resolution melting curve analysis method, so that the steps of product recovery after amplification, plasmid construction, sequencing detection and the like in the traditional technology can be omitted, the detection process is greatly accelerated, the detection cost is reduced, and the timeliness of subsequent medical intervention is improved. On the other hand, the primer pair can detect 10 sites at the same time, the single detection flux is several times that of a traditional technology, and the sample demand quantity and the repeated experiment frequency are greatly reduced.

Description

Technical Field

[0001] The present application belongs to the field of gene detection technology, and specifically relates to a detection primer pair, a kit and a detection method for CHD7 mutation. Background Art

[0002] CHARGE syndrome, whose full English name is Coloboma, Heart defect, Atresia choanae, Retarded growth and development, Genital hypoplasia, Ear anomalies / deafness, can be translated as "eye defect, heart malformation, choanal atresia, growth retardation, reproductive and urinary abnormalities, and ear malformation syndrome", is a complex congenital disease with multiple clinical manifestations and genetic characteristics.

[0003] The disease is mainly caused by heterozygous mutations in the CHD7 gene (an adenosine triphosphate-dependent chromatin remodeling protein), and in a few cases may be caused by mutations in the SEMA3E gene. CHD7 mutation refers to abnormal changes in the CHD7 gene on chromosome 8q12.2. The gene contains 38 exons, is approximately 10.4kb in size, and encodes 2997 amino acids. The protein it encodes plays an important role in embryonic development and organ formation. Gene mutation refers to a change in the base pair composition or arrangement order of the gene structure. Genes are DNA fragments with genetic effects, and DNA molecules are composed of four deoxynucleotides. The order of these nucleotides represents genetic information. When this order changes, it means that the gene has mutated.

[0004] There are many types of gene mutations, the most common ones include point mutations, insertion mutations, deletion mutations, duplication mutations, inversion mutations, etc. CHD7 mutations are usually autosomal dominant, with a 50% chance of inheritance in offspring. For example, if one of the parents carries the CHD7 mutation gene, their children have a certain chance of suffering from diseases related to the CHD7 mutation. Genetic testing can determine whether the patient carries the CHD7 mutation gene. If one or both of the couple carries the CHD7 mutation gene, they can choose to use assisted reproductive technology, such as in vitro fertilization (IVF) combined with embryo genetic diagnosis (PGD) to screen out embryos that do not carry the gene for implantation to reduce the risk of disease in offspring.

[0005] Although traditional technologies have documented methods for detecting CHD7 gene-related mutants, they have related defects such as low detection throughput, complex operation, and inability to directly report detection results after amplification. Summary of the invention

[0006] Based on this, an embodiment of the present application provides a primer pair, a kit and a detection method for CHD7 mutation.

[0007] On the one hand, the present application provides a primer pair for detecting a CHD7 mutation, wherein the primer pair comprises a primer pair for detecting one or more of the following target mutation sites:

[0008] c.3082A>G, c.1717-39602A>G, c.5405-17G>A, c.1717-11753G>A, c.3398C>T, c.1717-33547C>T, c.2501C>T, c.1716+35339C>T, c.2442+5G>C, and c.1716+20548G>C.

[0009] In some embodiments, the detection primer pair is selected from one or more pairs of the following primer pairs 1 to 10:

[0010] Primer pair 1, comprising: a forward primer F1 having a sequence as shown in SEQ ID NO.1, and a reverse primer R1 having a sequence as shown in SEQ ID NO.2;

[0011] Primer pair 2, comprising: a forward primer F2 having a sequence as shown in SEQ ID NO.3, and a reverse primer R2 having a sequence as shown in SEQ ID NO.4;

[0012] Primer pair 3, comprising: a forward primer F3 having a sequence as shown in SEQ ID NO.5, and a reverse primer R3 having a sequence as shown in SEQ ID NO.6;

[0013] Primer pair 4, comprising: a forward primer F4 having a sequence as shown in SEQ ID NO.7, and a reverse primer R4 having a sequence as shown in SEQ ID NO.8;

[0014] Primer pair 5, comprising: a forward primer F5 having a sequence as shown in SEQ ID NO.9, and a reverse primer R5 having a sequence as shown in SEQ ID NO.10;

[0015] Primer pair 6, comprising: a forward primer F6 having a sequence as shown in SEQ ID NO.11, and a reverse primer R6 having a sequence as shown in SEQ ID NO.12;

[0016] Primer pair 7, comprising: a forward primer F7 having a sequence as shown in SEQ ID NO.13, and a reverse primer R7 having a sequence as shown in SEQ ID NO.14;

[0017] Primer pair 8, comprising: a forward primer F8 having a sequence as shown in SEQ ID NO.15, and a reverse primer R8 having a sequence as shown in SEQ ID NO.16;

[0018] Primer pair 9, comprising: a forward primer F9 having a sequence as shown in SEQ ID NO.17, a reverse primer R9 having a sequence as shown in SEQ ID NO.18; and,

[0019] The primer pair 10 comprises: a forward primer F10 having a sequence as shown in SEQ ID NO.19, and a reverse primer R10 having a sequence as shown in SEQ ID NO.20.

[0020] In some of these embodiments, an internal reference detection primer pair is also included.

[0021] In some of the embodiments, the internal reference detection primer pair includes a forward primer F11 having a sequence as shown in SEQ ID NO.21 and a reverse primer R11 having a sequence as shown in SEQ ID NO.22.

[0022] In some of the embodiments, an internal reference probe is also included, and the sequence of the internal reference probe is shown in SEQ ID NO.23.

[0023] On the other hand, the present application provides a detection kit, which includes: the detection primer pair for CHD7 mutation.

[0024] In some embodiments, the kit further comprises a PCR reaction solution.

[0025] In some embodiments, the PCR reaction solution includes: PCR buffer, Mg 2+ , dNTPs, Taq DNA polymerase and one or more of fluorescent dyes.

[0026] In some embodiments, the fluorescent dye includes one or more of LC Green, LC Green Plus, SYTO 9 and EvaGreen.

[0027] In some embodiments, the detection kit also includes one or more of a positive quality control product and a negative quality control product.

[0028] In some embodiments, the positive control comprises a DNA fragment of an internal reference gene;

[0029] In some embodiments, the negative control material includes one or more of 1×TE buffer solution and / or water.

[0030] On the other hand, the present application provides a method for detecting a CHD7 mutant gene, the detection method comprising the following steps:

[0031] Providing the kit; mixing and centrifuging the PCR reaction solution and the detection primer pair to prepare a PCR reaction premix;

[0032] The DNA of the sample to be tested is placed in the PCR reaction premix, and PCR amplification is performed to obtain the amplified product of the sample to be tested.

[0033] In some embodiments, the method for detecting the CHD7 mutant gene further comprises:

[0034] Based on the high-resolution melting curve analysis method, the amplified product of the sample to be tested is detected and compared with the wild-type sample control, and the Tm values ​​of the wild-type sample and the sample to be tested are obtained, which are recorded as Tm1 and Tm2 respectively. According to whether Tm1 and Tm2 meet the target interval, the mutation of the CHD7 gene of the sample to be tested is judged;

[0035] In some embodiments, the sample to be tested includes one or more of a blood sample, a urine sample, and an amniotic fluid sample.

[0036] In some embodiments, comparing whether Tm1 and Tm2 meet the target interval to determine the mutation status of the CHD7 gene of the sample to be tested includes:

[0037] According to whether Tm1 and Tm2 meet the target interval, it is judged whether the CHD7 gene of the sample to be tested contains a mutation, and / or the genotype of the mutation is determined.

[0038] In some embodiments, judging whether the CHD7 gene of the sample to be tested contains a mutation includes: if the mutant Tm2 value meets the target interval, it is considered to have the mutation;

[0039] The target intervals are as follows:

[0040] For c.3082A>G, the Tm1 value of the wild type was 54.55°C to 54.95°C, and the Tm2 value of the mutant was 55.40 to 55.80°C;

[0041] For c.1717-39602A>G, the Tm1 value of the wild type was 57.20℃~57.60℃, and the Tm2 value of the mutant was 58.35℃~58.75℃;

[0042] For c.5405-17G>A, the Tm1 value of the wild type was 59.65℃~60.05℃, and the Tm2 value of the mutant was 58.90℃~59.30℃;

[0043] For c.1717-11753G>A, the Tm1 value of the wild type was 62.10℃~62.50℃, and the Tm2 value of the mutant was 60.80℃~61.20℃;

[0044] For c.3398C>T, the Tm1 value of the wild type was 64.80℃~65.20℃, and the Tm2 value of the mutant was 63.65℃~64.05℃;

[0045] For c.1717-33547C>T, the Tm1 value of the wild type was 68.20℃~68.60℃, and the Tm2 value of the mutant was 66.60℃~67.00℃;

[0046] For c.2501C>T, the Tm1 value of the wild type was 71.80℃~72.20℃, and the Tm2 value of the mutant was 70.30℃~70.70℃;

[0047] For c.1716+35339C>T, the Tm1 value of the wild type was 75.40℃~75.80℃, and the Tm2 value of the mutant was 73.95℃~74.35℃;

[0048] For c.2442+5G>C, the Tm1 value of the wild type is 78.00℃~78.40℃, and the Tm2 value of the mutant type is 78.40℃~78.80℃; and,

[0049] For c.1716+20548G>C, the Tm1 value of the wild type was 81.30℃~81.70℃, and the Tm2 value of the mutant type was 81.80℃~82.20℃.

[0050] In some embodiments, the PCR amplification procedure includes:

[0051] 94℃~96℃, pre-denaturation 1min~3min;

[0052] 94°C to 96°C, denaturation for 14s to 16s, annealing and extension at 58°C to 62°C for 28s to 32s, 44 to 46 cycles; and

[0053] 24℃~26℃, 4min~6min;

[0054] Optionally, the conditions based on high resolution melting curve analysis include:

[0055] 94℃~96℃, pre-denaturation 2min~4min;

[0056] 38℃~42℃, 2min~4min;

[0057] 38°C to 42°C to 88°C to 92°C, with a heating rate of 0.008 to 0.012°C / s, and collecting fluorescence; and,

[0058] 24℃~26℃, 0.8min~1.2min.

[0059] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0061] Figure 1 This is the wild-type melting curve peak;

[0062] Figure 2 For mutation site: 1717+33547C>T mutation detection melting curve peak diagram;

[0063] Figure 3 For mutation site: 1716+35339C>T mutation detection melting curve peak diagram;

[0064] Figure 4 This is the mutation site: 2442+5G>C mutation detection melting curve peak diagram. DETAILED DESCRIPTION

[0065] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0067] the term

[0068] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0069] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0070] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0071] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0072] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0073] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0074] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0075] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0076] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0077] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0078] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0079] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0080] The term "mutation site" refers to a specific position where a change occurs in a DNA or RNA sequence. This change can be a single base substitution, insertion or deletion.

[0081] The term "high-resolution melting", in English high-resolution melting, HRM, is a new gene analysis technology based on the formation of different melting curves of different forms due to the different melting temperatures of single nucleotides. It has extremely high sensitivity and can detect the difference of a single base. It is low-cost, high-throughput, fast, accurate, and not limited by the detection site, realizing a true closed-tube operation. HRM analysis technology plays an important role in mutation scanning, single nucleotide polymorphism analysis, methylation research, genotyping, sequence matching, etc.

[0082] Double-stranded DNA has an inherent characteristic - melting temperature (Tm), which refers to the temperature at which half of the double-stranded DNA in the reaction system melts. This temperature is related to the length of the double-stranded DNA, the base composition and the degree of matching. In qPCR, fluorescent dyes can bind non-specifically to the minor groove of double-stranded DNA. In the free state, the fluorescence emitted is weak. Once embedded in double-stranded DNA, the fluorescence signal is greatly enhanced, so it can be used for real-time detection of double-stranded DNA amplification products. After the PCR amplification is completed, the reaction system is heated for detection. During the temperature increase, the double-stranded DNA gradually melts, and the fluorescent dye embedded in the DNA double strand also falls off, and the fluorescence signal weakens. When a certain temperature is reached, the double-stranded DNA melts rapidly, and the fluorescence signal drops sharply. At this temperature, half of the double-stranded DNA melts. Subsequently, the temperature continues to rise until the double-stranded DNA is completely melted and the fluorescence signal weakens to the minimum.

[0083] Therefore, by real-time detection of the fluorescence signal in the process, the original spectrum of the fluorescence signal intensity changing with temperature can be obtained, and then the fluorescence signal is converted into a negative derivative, and a graph of the relationship between the fluorescence signal change and temperature is drawn. The temperature corresponding to the characteristic peak in the graph is the Tm value. Based on the difference in Tm values, different PCR products can be distinguished. High-resolution melting curve analysis technology HRM, by using saturated fluorescent dyes with stronger DNA binding ability and PCR instruments with higher temperature control and fluorescence detection accuracy, can accurately show the subtle differences between melting curves, thereby realizing the detection and distinction of single-base mutations.

[0084] On the one hand, the present application provides a detection primer pair for mutation of CHARGE syndrome pathogenic gene CHD7, wherein the detection primer pair is selected from one or more pairs of the following mutation sites:

[0085] Site 1: c.3082A>G, site 2: c.1717-39602A>G, site 3: c.5405-17G>A, site 4: c.1717-11753G>A, site 5: c.3398C>T, site 6: c.1717-33547C>T, site 7: c.2501C>T, site 8: c.1716+35339C>T, site 9: c.2442+5G>C and site 10: c.1716+20548G>C.

[0086] The present application can detect 10 potential CHD7 pathogenic mutation sites in the sample to be tested in one tube of PCR reaction by targeting 10 specifically selected common CHD7 pathogenic mutation sites, and has the characteristic of wide coverage.

[0087] In addition, the primer pair of the present application is further combined with the high-resolution melting curve analysis method to save the steps of post-amplification product recovery, plasmid construction, sequencing detection, etc. in the traditional technology, which greatly speeds up the detection process and reduces the detection cost, and improves the timeliness of subsequent medical intervention. On the other hand, the primer pair of the application can detect 10 sites at the same time, and the single detection throughput is several times that of the traditional technology, which greatly reduces the sample demand and the number of repeated experiments.

[0088] In some embodiments, the detection primer pair is selected from one or more pairs of the following primer pair 1 to primer pair 10:

[0089] Primer pair 1, comprising: a forward primer F1 having a sequence as shown in SEQ ID NO.1, and a reverse primer R1 having a sequence as shown in SEQ ID NO.2;

[0090] Primer pair 2, comprising: a forward primer F2 having a sequence as shown in SEQ ID NO.3, and a reverse primer R2 having a sequence as shown in SEQ ID NO.4;

[0091] Primer pair 3, comprising: a forward primer F3 having a sequence as shown in SEQ ID NO.5, and a reverse primer R3 having a sequence as shown in SEQ ID NO.6;

[0092] Primer pair 4, comprising: a forward primer F4 having a sequence as shown in SEQ ID NO.7, and a reverse primer R4 having a sequence as shown in SEQ ID NO.8;

[0093] Primer pair 5, comprising: a forward primer F5 having a sequence as shown in SEQ ID NO.9, and a reverse primer R5 having a sequence as shown in SEQ ID NO.10;

[0094] Primer pair 6, comprising: a forward primer F6 having a sequence as shown in SEQ ID NO.11, and a reverse primer R6 having a sequence as shown in SEQ ID NO.12;

[0095] Primer pair 7, comprising: a forward primer F7 having a sequence as shown in SEQ ID NO.13, and a reverse primer R7 having a sequence as shown in SEQ ID NO.14;

[0096] Primer pair 8, comprising: a forward primer F8 having a sequence as shown in SEQ ID NO.15, and a reverse primer R8 having a sequence as shown in SEQ ID NO.16;

[0097] Primer pair 9, comprising: a forward primer F9 having a sequence as shown in SEQ ID NO.17, a reverse primer R9 having a sequence as shown in SEQ ID NO.18; and,

[0098] The primer pair 10 comprises: a forward primer F10 having a sequence as shown in SEQ ID NO.19, and a reverse primer R10 having a sequence as shown in SEQ ID NO.20.

[0099] In some of these embodiments, an internal reference detection primer pair is also included.

[0100] In some of the embodiments, the internal reference detection primer pair includes a forward primer F11 having a sequence as shown in SEQ ID NO.21 and a reverse primer R11 having a sequence as shown in SEQ ID NO.22.

[0101] Optionally, an internal reference probe is also included, and the sequence of the internal reference probe is shown in SEQ ID NO.23.

[0102] The specific primer sequences are shown in Table 1 below:

[0103] Table 1

[0104]

[0105]

[0106] On the other hand, the present application provides a detection kit for mutation of the CHARGE syndrome pathogenic gene CHD7, the kit comprising: the above-mentioned detection primer pair.

[0107] In some embodiments, the kit further comprises a PCR reaction solution.

[0108] In some embodiments, the PCR reaction solution includes: PCR buffer, Mg 2+ , dNTPs, Taq DNA polymerase and one or more of fluorescent dyes.

[0109] Optionally, the fluorescent dye includes one or more of LC Green, LC Green Plus, SYTO 9 and Eva Green.

[0110] In some embodiments, the kit further comprises a positive control and a negative control.

[0111] In some embodiments, the positive control comprises a DNA fragment of an internal reference gene.

[0112] In some embodiments, the negative control comprises 1×TE buffer solution or water.

[0113] On the other hand, the present application provides a method for detecting a CHD7 mutant gene, which is for diagnostic or non-diagnostic purposes. It can be understood that the expression of the CHD7 gene is highly correlated with CHARGE syndrome, and thus the method provided in the present application has a high clinical testing value.

[0114] In addition, the CHD7 gene is highly polymorphic and can therefore also be used for research in fields such as sociology (e.g., ethnic distribution, etc.).

[0115] The detection method comprises the following steps: providing the above-mentioned kit, mixing and centrifuging the PCR reaction solution and the detection primer pair to prepare a PCR reaction premix;

[0116] The DNA of the sample to be tested is placed in a PCR reaction premix, and PCR amplification is performed to obtain an amplified product of the sample to be tested;

[0117] Based on the high-resolution melting curve analysis method, the amplified products of the test samples were detected and compared with the wild-type samples. The corresponding Tm values ​​of the wild-type samples and the test samples were obtained, recorded as Tm1 and Tm2, ​​respectively. The mutation status of the CHD7 gene of the test sample was judged based on whether Tm1 and Tm2 were in the target range.

[0118] This application uses HRM technology to test a large number of plasmids, quality control products and real samples to obtain the precise range of Tm values ​​of each tested site of wild-type CHD7. The same test method can also obtain the precise range of new Tm values ​​generated in the same PCR reaction system after each site produces a corresponding pathogenic mutation, and both data are plotted into a table. In actual operation, by comparing the actual Tm value and the Tm value data in the table, it can be determined whether a pathogenic gene mutation has occurred at each site.

[0119] In some embodiments, the sample to be tested includes one or more of a blood sample, a urine sample, an amniotic fluid sample, and a tissue sample.

[0120] In some embodiments, judging the mutation of the CHD7 gene in the sample to be tested according to the values ​​of Tm1 and Tm2 includes:

[0121] According to the Tm1 value and the Tm2 value, it is judged whether the CHD7 gene of the sample to be tested contains a mutation, and / or the genotype of the mutation is determined.

[0122] For c.3082A>G, the Tm1 value of the wild type was 54.55°C to 54.95°C, and the Tm2 value of the mutant was 55.40 to 55.80°C;

[0123] For c.1717-39602A>G, the Tm1 value of the wild type was 57.20℃~57.60℃, and the Tm2 value of the mutant was 58.35℃~58.75℃;

[0124] For c.5405-17G>A, the Tm1 value of the wild type was 59.65℃~60.05℃, and the Tm2 value of the mutant was 58.90℃~59.30℃;

[0125] For c.1717-11753G>A, the Tm1 value of the wild type was 62.10℃~62.50℃, and the Tm2 value of the mutant was 60.80℃~61.20℃;

[0126] For c.3398C>T, the Tm1 value of the wild type was 64.80℃~65.20℃, and the Tm2 value of the mutant was 63.65℃~64.05℃;

[0127] For c.1717-33547C>T, the Tm1 value of the wild type was 68.20℃~68.60℃, and the Tm2 value of the mutant was 66.60℃~67.00℃;

[0128] For c.2501C>T, the Tm1 value of the wild type was 71.80℃~72.20℃, and the Tm2 value of the mutant was 70.30℃~70.70℃;

[0129] For c.1716+35339C>T, the Tm1 value of the wild type was 75.40℃~75.80℃, and the Tm2 value of the mutant was 73.95℃~74.35℃;

[0130] For c.2442+5G>C, the Tm1 value of the wild type is 78.00℃~78.40℃, and the Tm2 value of the mutant type is 78.40℃~78.80℃; and,

[0131] For c.1716+20548G>C, the Tm1 value of the wild type was 81.30℃~81.70℃, and the Tm2 value of the mutant type was 81.80℃~82.20℃.

[0132] In some embodiments, the PCR amplification procedure includes:

[0133] 94°C to 96°C, pre-denaturation for 1 min to 3 min; for example, the temperature is 94°C, 95°C or 96°C and any value in between, and the pre-denaturation time is 1 min, 2 min or 3 min and any value in between.

[0134] 94°C to 96°C, denaturation for 14s to 16s, annealing and extension at 58°C to 62°C for 28s to 32s, 44 to 46 cycles; for example, the temperature is 94°C, 95°C or 96°C and any value therebetween, the denaturation time is 14s, 15s or 16s and any value therebetween. The temperature is 58°C, 59°C, 60°C, 61°C or 62°C. Annealing and extension for 28s, 29s, 30s, 31s or 32s; 44, 45 or 46 cycles.

[0135] 24°C~26°C, 4min~6min; for example, the temperature is 24°C, 25°C or 26°C, and the time is 4min, 5min or 6min.

[0136] In some embodiments, the high resolution melting curve analysis method comprises:

[0137] 94°C ~ 96°C, pre-denaturation 2min ~ 4min; for example, the temperature is 94°C, 95°C or 96°C and any value in between, and the pre-denaturation time is 2min, 3min or 4min and any value in between.

[0138] 38°C to 42°C, 2min to 4min; for example, the temperature is 38°C, 39°C, 40°C, 41°C or 42°C, and the time is 2min, 3min or 4min and any value in between.

[0139] The temperature is increased from 38°C to 42°C to 88°C to 92°C at a heating rate of 0.008°C / s to 0.012°C / s, and fluorescence is collected; for example, the temperature is 38°C, 39°C, 40°C, 41°C or 42°C, and increased to 88°C, 89°C, 90°C, 91°C or 92°C at a heating rate of 0.008°C / s, 0.009°C / s, 0.010°C / s, 0.011°C / s or 0.012°C / s and any value in between.

[0140] 24℃~26℃, 0.8min~1.2min. For example, the temperature is 24℃, 25℃ or 26℃, and the time is 0.8min, 0.9min, 1.0min, 1.1min, 1.2min.

[0141] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0142] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0143] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0144] Example 1

[0145] This embodiment provides a detection kit for mutation of the CHARGE syndrome pathogenic gene CHD7.

[0146] 1. The kit includes the detection primers and internal reference primers shown in SEQ ID NO.1 to SEQ ID NO.23 as shown in Table 1 above.

[0147] 2. The kit also includes the PCR reaction solution shown in Table 2:

[0148] Table 2

[0149]

[0150] Example 2

[0151] This embodiment provides a method for detecting mutations in the CHARGE syndrome pathogenic gene CHD7.

[0152] 1. Urine sample pretreatment

[0153] First, a crude urine extract was prepared, consisting of 50 mM Tris HCl, pH 8.0, 1% Triton X-100, 2 mM EDTA, 50 mM NaOH, 10% glycerol, and the remainder purified water. 100 μL of the crude urine extract was added to the urine sample, placed at 90°C for 5 minutes, and used as a template for subsequent PCR amplification.

[0154] 2. PCR-HRM melting curve experiment

[0155] 1. Prepare PCR reaction solution and primer probe mixture. The specific components are shown in Table 3:

[0156] Table 3

[0157] Components Amount per person (μL) Tris-HCl PCR Buffer 2.5 dNTPs(10mM) 2 <![CDATA[Mg 2+ (5mM)]]> 2 Taq DNA polymerase 1 Saturated fluorescent dye 0.1 Purified water Make up to 15 μL

[0158] 2. The specific components of the mutation site primer mixture are shown in Table 4:

[0159] Table 4

[0160] Components Amount per person (μL) 10 mutation sites mixed forward primer-F (100μM) 0.03 10 mutation sites mixed negative primer-R (100μM) 0.03 GAPDH gene-F11 (100 μM) 0.03 GAPDH gene-R11 (100 μM) 0.03 GAPDH gene-FP (100 μM) 0.05 Purified water Make up to 5 μL

[0161] 3. Add sample

[0162] Add 5 μL of negative control, positive control and clinical sample template in step 1 to the above prepared system respectively. Perform PCR amplification reaction.

[0163] 4. The amplification procedure is as follows:

[0164] Step 1: pre-denaturation at 95°C for 2 min;

[0165] Step 2: denaturation at 95 °C for 15 s, annealing and extension at 60 °C for 30 s, 45 cycles;

[0166] step3: 25℃, 5min;

[0167] 5. HRM melting curve conditions

[0168] Step 1: pre-denaturation at 95°C for 3 min;

[0169] step2: 40℃, 3min;

[0170] Step 3: 40℃ to 90℃, the heating rate is 0.01℃ / s (collect fluorescence);

[0171] step4: 25℃, 1min.

[0172] The wild type melting curve peak is shown in Figure 1 As shown, taking the mutation site 1717+33547C>T as an example, the peak graph of the mutation detection melting curve is as follows Figure 2 As shown; Taking the mutation site: 1716-35339C>T as an example, the mutation detection of this site is as follows Figure 3 Taking the mutation site 2442+5G>C as an example, the peak graph of the melting curve of this mutation detection is as follows Figure 4 shown.

[0173] 6. The thresholds for determining whether a mutation occurs at each site are shown in Table 5:

[0174] Table 5

[0175] Mutation site Wild-type Tm value Mutant Tm value c.3082A>G 54.75±0.20 55.60±0.20 c.1717-39602A>G 57.40±0.20 58.55±0.20 c.5405-17G>A 59.85±0.20 59.10±0.20 c.1717-11753G>A 62.30±0.20 61.00±0.20 c.3398C>T 65.00±0.20 63.85±0.20 c.1717-33547C>T 68.40±0.20 66.80±0.20 c.2501C>T 72.00±0.20 70.50±0.20 c.1716+35339C>T 75.60±0.20 74.15±0.20 c.2442+5G>C 78.20±0.20 78.60±0.20 c.1716+20548G>C 81.50±0.20 82.00±0.20

[0176] 7. The test results are shown in Table 6:

[0177] Table 6

[0178]

[0179]

[0180] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A primer pair for detecting CHD7 mutation, characterized in that: The detection primer pair includes a primer pair for detecting one or more of the following target mutation sites: c.3082A>G, c.1717-39602A>G, c.5405-17G>A, c.1717-11753G>A, c.3398C>T, c.1717-33547C>T, c.2501C>T, c.1716+35339C>T, c.2442+5G>C, and c.1716+20548G>C.

2. The primer pair for detecting CHD7 mutation according to claim 1, characterized in that: The detection primer pair is selected from one or more pairs of the following primer pairs 1 to 10: Primer pair 1, comprising: a forward primer F1 having a sequence as shown in SEQ ID NO.1, and a reverse primer R1 having a sequence as shown in SEQ ID NO.2; Primer pair 2, comprising: a forward primer F2 having a sequence as shown in SEQ ID NO.3, and a reverse primer R2 having a sequence as shown in SEQ ID NO.4; Primer pair 3, comprising: a forward primer F3 having a sequence as shown in SEQ ID NO.5, and a reverse primer R3 having a sequence as shown in SEQ ID NO.6; Primer pair 4, comprising: a forward primer F4 having a sequence as shown in SEQ ID NO.7, and a reverse primer R4 having a sequence as shown in SEQ ID NO.8; Primer pair 5, comprising: a forward primer F5 having a sequence as shown in SEQ ID NO.9, and a reverse primer R5 having a sequence as shown in SEQ ID NO.10; Primer pair 6, comprising: a forward primer F6 having a sequence as shown in SEQ ID NO.11, and a reverse primer R6 having a sequence as shown in SEQ ID NO.12; Primer pair 7, comprising: a forward primer F7 having a sequence as shown in SEQ ID NO.13, and a reverse primer R7 having a sequence as shown in SEQ ID NO.14; Primer pair 8, comprising: a forward primer F8 having a sequence as shown in SEQ ID NO.15, and a reverse primer R8 having a sequence as shown in SEQ ID NO.16; Primer pair 9, comprising: a forward primer F9 having a sequence as shown in SEQ ID NO.17, a reverse primer R9 having a sequence as shown in SEQ ID NO.18; and, The primer pair 10 includes: a forward primer F10 having a sequence as shown in SEQ ID NO.19, and a reverse primer R10 having a sequence as shown in SEQ ID NO.

20.

3. The primer pair for detecting CHD7 mutation according to claim 1, characterized in that: An internal reference detection primer pair is also included; Optionally, the internal reference detection primer pair includes a forward primer F11 having a sequence as shown in SEQ ID NO.21, and a reverse primer R11 having a sequence as shown in SEQ ID NO.22; Optionally, an internal reference probe is also included, and the sequence of the internal reference probe is shown in SEQ ID NO.

23.

4. A detection kit, characterized in that: The kit comprises: a primer pair for detecting a CHD7 mutation according to any one of claims 1 to 3; Optionally, the kit further comprises a PCR reaction solution; Optionally, the PCR reaction solution includes: PCR buffer, Mg 2+ , one or more of dNTPs, Taq DNA polymerase, and fluorescent dyes; Optionally, the fluorescent dye includes one or more of LC Green, LC Green Plus, SYTO 9 and Eva Green.

5. The detection kit according to claim 4, characterized in that The detection kit also includes one or more of a positive quality control product and a negative quality control product; Optionally, the positive quality control product includes a DNA fragment of an internal reference gene; Optionally, the negative control product includes one or more of 1×TE buffer solution and / or water.

6. A method for detecting a CHD7 mutant gene, characterized in that: The detection method comprises the following steps: Providing a detection kit according to any one of claims 4 to 5; The PCR reaction solution and the detection primer pair are mixed and centrifuged to prepare a PCR reaction premix; The DNA of the sample to be tested is placed in the PCR reaction premix, and PCR amplification is performed to obtain the amplified product of the sample to be tested.

7. The method for detecting a CHD7 mutant gene according to claim 6, characterized in that: Also includes: Based on the high-resolution melting curve analysis method, the amplified product of the sample to be tested is detected and compared with the wild-type sample control, and the Tm values ​​of the wild-type sample and the sample to be tested are obtained, which are recorded as Tm1 and Tm2 respectively. According to whether Tm1 and Tm2 meet the target interval, the mutation of the CHD7 gene of the sample to be tested is judged; Optionally, the sample to be tested includes one or more of a blood sample, a urine sample and an amniotic fluid sample.

8. The method for detecting a CHD7 mutant gene according to claim 6, characterized in that: Comparing whether Tm1 and Tm2 meet the target interval and determining the mutation status of the CHD7 gene of the sample to be tested includes: According to whether Tm1 and Tm2 meet the target interval, it is judged whether the CHD7 gene of the sample to be tested contains a mutation, and / or the genotype of the mutation is determined.

9. The method for detecting a CHD7 mutant gene according to claim 8, characterized in that: Determining whether the CHD7 gene of the sample to be tested contains a mutation includes: if the mutant Tm2 value meets the target interval, it is considered to have the mutation; The target intervals are as follows: For c.3082A>G, the Tm1 value of the wild type was 54.55°C to 54.95°C, and the Tm2 value of the mutant was 55.40 to 55.80°C; For c.1717-39602A>G, the Tm1 value of the wild type was 57.20℃~57.60℃, and the Tm2 value of the mutant was 58.35℃~58.75℃; For c.5405-17G>A, the Tm1 value of the wild type was 59.65℃~60.05℃, and the Tm2 value of the mutant was 58.90℃~59.30℃; For c.1717-11753G>A, the Tm1 value of the wild type was 62.10℃~62.50℃, and the Tm2 value of the mutant was 60.80℃~61.20℃; For c.3398C>T, the Tm1 value of the wild type was 64.80℃~65.20℃, and the Tm2 value of the mutant was 63.65℃~64.05℃; For c.1717-33547C>T, the Tm1 value of the wild type was 68.20℃~68.60℃, and the Tm2 value of the mutant was 66.60℃~67.00℃; For c.2501C>T, the Tm1 value of the wild type was 71.80℃~72.20℃, and the Tm2 value of the mutant was 70.30℃~70.70℃; For c.1716+35339C>T, the Tm1 value of the wild type was 75.40℃~75.80℃, and the Tm2 value of the mutant was 73.95℃~74.35℃; For c.2442+5G>C, the Tm1 value of the wild type is 78.00℃~78.40℃, and the Tm2 value of the mutant type is 78.40℃~78.80℃; and, For c.1716+20548G>C, the Tm1 value of the wild type was 81.30℃~81.70℃, and the Tm2 value of the mutant type was 81.80℃~82.20℃.

10. The method for detecting a CHD7 mutant gene according to any one of claims 6 to 9, characterized in that: The PCR amplification procedure includes: 94℃~96℃, pre-denaturation 1min~3min; 94°C to 96°C, denaturation for 14s to 16s, annealing and extension at 58°C to 62°C for 28s to 32s, 44 to 46 cycles; and 24℃~26℃, 4min~6min; Optionally, the conditions based on high resolution melting curve analysis include: 94℃~96℃, pre-denaturation 2min~4min; 38℃~42℃, 2min~4min; 38°C to 42°C to 88°C to 92°C, with a heating rate of 0.008°C / s to 0.012°C / s, collecting fluorescence; and, 24℃~26℃, 0.8min~1.2min.

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