Detection primer pair, kit and detection method for chd7 mutation

Abstract

The application belongs to the technical field of gene detection, and particularly relates to a primer pair, a kit and a detection method for CHD7 mutation. The application can detect 10 potential CHD7 pathogenic mutation sites in a sample to be detected in one PCR reaction by aiming at 10 frequently-occurring CHD7 pathogenic mutation sites selected in a specific manner, and has the characteristic of wide coverage. In addition, the primer pair of the application can further combine with high-resolution melting curve analysis to omit steps such as product recovery after amplification, plasmid construction, sequencing detection and the like in traditional technology, greatly accelerating the detection process and reducing the detection cost, and improving the timeliness of subsequent medical intervention. On the other hand, the primer pair of the application can simultaneously detect 10 sites, and the single detection throughput is several times that of traditional technology, greatly reducing the sample requirement and the number of repeated experiments.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene detection, and particularly relates to a CHD7 mutation detection primer pair, a kit and a detection method. BACKGROUND

[0002] CHARGE syndrome, which can be translated as "coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, ear anomalies / deafness", 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 (a chromatin remodeling protein dependent on adenosine triphosphate), and in a small number of cases, it 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 about 10.4 kb in size, encodes 2997 amino acids, and the encoded protein plays an important role in embryonic development and organ formation. Gene mutation refers to changes in the base pair composition or arrangement order of the structure of the gene. The gene is a DNA fragment with genetic effects, and the DNA molecule is composed of four deoxyribonucleotides. The arrangement order of these nucleotides represents genetic information, and when the arrangement order changes, it means that the gene has mutated.

[0004] There are many types of gene mutations, including point mutations, insertion mutations, deletion mutations, duplication mutations, inversion mutations, etc. CHD7 mutation is usually autosomal dominant inheritance, and the inheritance probability of offspring is 50%. For example, if one of the parents carries the CHD7 mutation gene, their children have a certain risk of developing diseases related to CHD7 mutation. Through gene detection, it can be determined whether the patient carries the CHD7 mutation gene. If one or both of the spouses carry the CHD7 mutation gene, they can choose to screen out embryos that do not carry the gene for implantation through assisted reproductive technology such as in vitro fertilization (IVF) combined with preimplantation genetic diagnosis (PGD) to reduce the risk of offspring.

[0005] Although the traditional technology has recorded a method for detecting CHD7 gene related mutants, it has the related defects of small detection throughput, complex operation, and inability to directly report the detection results after amplification when detecting. SUMMARY

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

[0007] One aspect of the present application provides a CHD7 mutation detection primer pair, which 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 of primer pair 1 to primer pair 10:

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

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

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

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

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

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

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

[0017] primer pair 8 comprises a forward primer F8 with a sequence as shown in SEQ ID NO. 15 and a reverse primer R8 with a sequence as shown in SEQ ID NO. 16;

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

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

[0020] In some embodiments, the primer pair further comprises a primer pair for detecting an internal control.

[0021] In some embodiments, the primer pair for detecting an internal control comprises a forward primer F11 with a sequence as shown in SEQ ID NO. 21 and a reverse primer R11 with a sequence as shown in SEQ ID NO. 22.

[0022] In some embodiments, the primer pair further comprises an internal control probe with a sequence as shown in SEQ ID NO. 23.

[0023] Another aspect of the present application provides a detection kit, which comprises the primer pair for detecting the CHD7 mutation.

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

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

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

[0027] In some embodiments, the detection kit further comprises one or more of a positive control and a negative control.

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

[0029] In some embodiments, the negative control comprises one or more of a 1x TE buffer solution and / or water.

[0030] Another aspect of the present application provides a method for detecting a CHD7 mutant gene, the method comprising the following steps:

[0031] The kit is provided; the PCR reaction solution is mixed with the detection primer pair, centrifuged, and a PCR reaction premix is prepared.

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

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

[0034] Based on high-resolution melting curve analysis, the amplification product of the sample to be tested is detected and compared with the wild-type sample, and the Tm values of the wild-type sample and the sample to be tested are obtained, respectively, Tm1 and Tm2, and whether Tm1 and Tm2 meet the target interval is determined to judge the mutation of the CHD7 gene of the sample to be tested.

[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 of the CHD7 gene of the sample to be tested comprises:

[0037] According to whether Tm1 and Tm2 meet the target interval, it is determined 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, determining whether the CHD7 gene of the sample to be tested contains a mutation comprises: if the mutant Tm2 value meets the target interval, it is considered to have the mutation.

[0039] The target interval is as follows:

[0040] For c.3082A>G, the Tm1 value of the wild type is 54.55-54.95℃, and the Tm2 value of the mutant is 55.40-55.80℃.

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

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

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

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

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

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

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

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

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

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

[0051] 94-96°C, pre-denaturation for 1-3 min;

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

[0053] 24-26°C, 4-6 min;

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

[0055] 94-96°C, pre-denaturation for 2-4 min;

[0056] 38-42°C, 2-4 min;

[0057] 38-42°C to 88-92°C at a rate of 0.008-0.012°C / s, and collect fluorescence; and

[0058] 24-26°C, 0.8-1.2 min.

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

[0060] In order to more clearly illustrate the technical solutions in the embodiments of the application, more completely understand the application and the beneficial effects thereof, the drawings needed to be used in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0061] Figure 1 is a wild-type melting curve peak graph;

[0062] Figure 2 is a mutation site: 1717+33547C>T mutation detection melting curve peak graph;

[0063] Figure 3 is a mutation site: 1716+35339C>T mutation detection melting curve peak graph;

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

[0065] The application will be further described below in conjunction with the embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the application and not to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosed content of the application more thorough and comprehensive. It should also be understood that the application can be realized in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the spirit of the application, and the equivalent forms obtained thereby also fall within the protection scope of the application. In addition, in the following description, a large number of specific details are given in order to provide a more complete 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] Definitions

[0068] The terms or phrases used herein have the following meanings unless otherwise stated or contradicted by context:

[0069] The term "and / or", "or / and", "and / or" used herein in the alternative includes any one of two or more of the associated listed items, and also includes any and all combinations of the associated listed items, including any two of the associated listed items, any more of the associated listed items, or all of the associated listed items. It is to be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it is to be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B, and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C, and D, i.e., includes the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (i.e., the technical solution connected by "logical and").

[0070] In the present application, "multiple", "various", "multiple times", "multiple", etc. are used without specific limitation, which means greater than or equal to two in number. For example, "one or more" means one or greater than or equal to two.

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

[0072] As used herein, "suitable combination manner", "suitable manner", "any suitable manner", etc. are described as "suitable" in the sense of being able to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0073] In the present application, "further", "still further", "in particular", etc. are used for description purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present application.

[0074] In the present application, "optionally", "optional", "optional" means optional, i.e., selected from two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, each "optional" is independent unless otherwise stated, contradicted, or mutually restricted.

[0075] In the present application, the technical features described in an open way include both the closed technical solutions consisting of the listed features and the open technical solutions containing the listed features.

[0076] In the present application, when referring to a numerical interval (i.e. a numerical range), unless otherwise specified, the optional numerical distribution within the numerical interval is considered to be continuous, and includes both numerical end points (i.e. the minimum value and the maximum value) of the numerical range, and every numerical value between the two numerical end points. When a numerical interval refers only to integers within the numerical interval, unless otherwise specified, including both end point integers of the numerical range, and every integer between the two end points, in this document, it is equivalent to directly listing each integer, for example, t is an integer selected from 1-10, which means that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0077] In the present application, unless otherwise specified, the temperature parameter allows both constant temperature treatment and variation 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. It is allowed to fluctuate within the range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C.

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

[0079] All documents mentioned in the present application are incorporated by reference in the present application as if each document was individually incorporated by reference. Unless and to the extent that the cited documents conflict with the purpose and / or technical solutions of the present application, the cited documents are incorporated by reference in their entirety. When the present application refers to the cited documents, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited documents are also incorporated by reference. When the present application refers to the cited documents, the examples and preferred modes of the relevant technical features cited are also incorporated by reference into the present application, but are limited to the implementation of the present application. It should be understood that when the cited content conflicts with the description in the present application, the present application is given priority or is modified adaptively according to the description in the present application.

[0080] The term "mutation site" refers to a specific position in a DNA or RNA sequence that has been altered. This alteration can be a substitution, insertion, or deletion of a single base.

[0081] The term "high-resolution melting" (HRM) is a new technique of gene analysis based on the different melting temperature of single nucleotide to form different melting curve, which has high sensitivity to detect single base difference, and low cost, high throughput, fast speed, accurate result, no limitation of detection site, and realizes the real closed tube operation. HRM analysis technology plays an important role in mutation scanning, single nucleotide polymorphism analysis, methylation research, genotyping, sequence matching and the like.

[0082] Double-stranded DNA has an inherent property-melting temperature (Tm), which refers to the temperature at which half of the double-stranded DNA in the reaction system is denatured, and the temperature is related to the length of double-stranded DNA, base composition and matching degree. In qPCR, fluorescent dyes can be non-specifically combined into the minor groove of double-stranded DNA, and the fluorescence emitted in the free state is weak, and 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 product. When the PCR amplification is completed, the reaction system is detected by heating, and during the temperature rising process, the double-stranded DNA is gradually denatured, and the fluorescent dye embedded in the double-stranded DNA is also dropped, and the fluorescence signal is weakened. When a certain temperature is reached, the double-stranded DNA is rapidly denatured, and the fluorescence signal is suddenly reduced. At this temperature, half of the double-stranded DNA is denatured. Subsequently, the temperature continues to rise until the double-stranded DNA is completely denatured, and the fluorescence signal is weakened to the minimum.

[0083] Therefore, real-time detection of the fluorescence signal in this process can obtain the original graph of the change of fluorescence signal intensity with temperature, and then the fluorescence signal is converted by negative derivative, and the graph of the change of fluorescence signal with temperature is drawn. The temperature corresponding to the characteristic peak appearing in the graph is the Tm value. Based on the difference of Tm value, different PCR products can be distinguished. The high-resolution melting curve analysis technology HRM can accurately show the subtle difference between the melting curves by using saturated fluorescent dyes with stronger DNA binding capacity and PCR instruments with higher temperature control and fluorescence detection precision, so as to realize the detection and distinction of single base mutation.

[0084] In one aspect, the application provides a primer pair for detecting mutations of CHD7, a pathogenic gene of CHARGE syndrome, and the primer pair is selected from one or more pairs of primer pairs for detecting 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 a sample to be tested in one PCR reaction by targeting 10 frequently-occurring CHD7 pathogenic mutation sites selected, and has a wide coverage.

[0087] In addition, the primer pair of the present application can further combine with high-resolution melting curve analysis to eliminate the steps of product recovery after amplification, plasmid construction, sequencing detection and the like in the traditional technology, greatly speeding up the detection process and reducing the detection cost, and improving 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, greatly reducing the sample demand and the number of repeated experiments.

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

[0089] Primer pair 1 comprises: 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 comprises: 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 comprises: 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 comprises: 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 comprises: 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 comprises: a forward primer F6 having a sequence as set forth in SEQ ID NO. 11, and a reverse primer R6 having a sequence as set forth in SEQ ID NO. 12.

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

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

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

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

[0099] In some embodiments, the kit further comprises an internal control detection primer pair.

[0100] In some embodiments, the internal control detection primer pair comprises: a forward primer F11 having a sequence as set forth in SEQ ID NO. 21, and a reverse primer R11 having a sequence as set forth in SEQ ID NO. 22.

[0101] Optionally, the kit further comprises an internal control probe having a sequence as set forth in SEQ ID NO. 23.

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

[0103] Table 1

[0104]

[0105]

[0106] Another aspect of the present application provides a kit for detecting mutations of a CHARGE syndrome pathogenic gene CHD7, the kit comprising: the detection primer pair described above.

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

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

[0109] Optionally, the fluorescent dye comprises 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 a housekeeping gene.

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

[0113] Another aspect of the present application provides a method for detecting a CHD7 mutant gene, which is for diagnostic or non-diagnostic purposes. It is understood that the expression of CHD7 gene has a high correlation with CHARGE syndrome, and thus the method provided by the present application has a high test value in clinical practice.

[0114] In addition, CHD7 gene has high polymorphism, and thus can also be used for research in the field of sociology (such as ethnic distribution, etc.).

[0115] The detection method comprises the following steps: providing the kit, mixing the PCR reaction solution with the detection primer pair, centrifuging, and preparing a PCR reaction premix;

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

[0117] Based on the high-resolution melting curve analysis method, the amplification product of the sample to be tested is detected and compared with a wild-type sample, and the Tm values of the wild-type sample and the sample to be tested are obtained, respectively, denoted as Tm1 and Tm2. According to whether Tm1 and Tm2 meet the target interval, the mutation of the CHD7 gene of the sample to be tested is determined.

[0118] Through a large number of tests on plasmids, quality control products, and real samples using the HRM technology, the present application can obtain the accurate range of Tm values of each test site of the wild-type CHD7. The same test method can also obtain the accurate range of new Tm values generated in the same PCR reaction system after each site produces a corresponding pathogenic mutation. Both data are drawn into a table. In actual operation, by comparing the actual Tm value with 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 comprises one or more of a blood sample, a urine sample, an amniotic fluid sample, and a tissue sample.

[0120] In some embodiments, the mutation of CHD7 gene in the sample to be tested is determined according to the values of Tm1 and Tm2, including:

[0121] According to the values of Tm1 and Tm2, it is determined 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 is 54.55-54.95℃, and the Tm2 value of the mutant is 55.40-55.80℃;

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

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

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

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

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

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

[0129] For c.1716+35339C>T, the Tm1 value of the wild type is 75.40-75.80℃, and the Tm2 value of the mutant is 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 is 78.40-78.80℃; and,

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

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

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

[0134] 94-96°C, denaturation for 14-16 s, 58-62°C annealing and extension for 28-32 s, 44-46 cycles; for example, a temperature of 94°C, 95°C, or 96°C, and any value in between, for a denaturation time of 14 s, 15 s, or 16 s, and any value in between. A temperature of 58°C, 59°C, 60°C, 61°C, or 62°C. Annealing and extension for 28 s, 29 s, 30 s, 31 s, or 32 s; 44, 45, or 46 cycles.

[0135] 24-26°C, 4-6 min; for example, a temperature of 24°C, 25°C, or 26°C, for a time of 4 min, 5 min, or 6 min.

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

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

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

[0139] 38-42°C to 88-92°C at a ramp rate of 0.008-0.012°C / s, collecting fluorescence; for example, a temperature of 38°C, 39°C, 40°C, 41°C, or 42°C, to 88°C, 89°C, 90°C, 91°C, or 92°C, at a ramp 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°C, 0.8-1.2 min. For example, a temperature of 24°C, 25°C, or 26°C, for a time of 0.8 min, 0.9 min, 1.0 min, 1.1 min, 1.2 min.

[0141] The embodiments of the present application will be described in detail below with examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples without specific conditions are preferred to refer to the instructions given in the present application, and can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the experimental methods known in the art.

[0142] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range, unless otherwise specified. With respect to temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0143] It should be understood that the size of the sequence number of each process described above in various embodiments of the present application does not mean the order of execution, and the execution order of each process should be determined according to 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] The present example provides a detection kit for CHARGE syndrome pathogenic gene CHD7 mutation.

[0146] I. The kit comprises detection primers and internal reference primers as shown in Table 1 above, such as SEQ ID NO. 1-SEQ ID NO. 23.

[0147] II. The kit further comprises PCR reaction solution as shown in Table 2:

[0148] Table 2

[0149]

[0150] Example 2

[0151] The present example provides a detection method for CHARGE syndrome pathogenic gene CHD7 mutation.

[0152] I. Urine sample pretreatment

[0153] First, prepare the crude urine extract consisting of 50mM Tris HCl, pH 8.0, 1% (v / v) Triton X-100, 2mM EDTA, 50mM NaOH, 10% (v / v) glycerol and the balance of purified water. Take the above urine sample and add 100 μL of crude urine extract, and place it at 90°C for 5 min for subsequent PCR amplification template.

[0154] II. PCR-HRM melting curve experiment

[0155] 1. Preparation of PCR reaction solution and primer probe mixture, the specific components are shown in Table 3:

[0156] Table 3

[0157] Components Per serving addition (μL) Tris-HCl PCR Buffer 2.5 dNTPs (10 mM) 2 Mg 2+ (5mM)]]> 2 Taq DNA polymerase 1 Saturation 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 Per serving addition (μL) 10 mutation site mixed forward primer-F (100 μM) 0.03 10 mutation site mixed reverse primer-R (100 μM) 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. Sample loading

[0162] 5 μL of negative quality control, positive quality control and clinical sample template in step 1 were added to the above prepared system respectively. PCR amplification reaction was carried out.

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

[0164] step 1: 95℃ pre-denaturation for 2 min;

[0165] step 2: 95℃ denaturation for 15 s, 60℃ annealing and extension for 30 s, 45 cycles;

[0166] step 3: 25℃, 5 min;

[0167] 5. HRM melting curve conditions

[0168] step 1: 95℃ pre-denaturation for 3 min;

[0169] step 2: 40℃, 3 min;

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

[0171] step 4: 25℃, 1 min.

[0172] The wild type melting curve peak diagram is shown in Figure 1 The mutation detection melting curve peak diagram of mutation site 1717+3354 7C>T is shown in Figure 2 The mutation detection of mutation site: 1716-3533 9C>T is shown in Figure 3 The mutation detection melting curve peak diagram of mutation site 2442+5G>C is shown in Figure 4

[0173] 6. The judgment threshold of whether each site occurs mutation is 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 results of the test are shown in Table 6:

[0177] Table 6

[0178]

[0179]

[0180] The above-described embodiments merely express several embodiments of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the scope of patent protection. It should be noted that, for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application. In addition, it should be understood that, after reading the above teaching content of the present application, those skilled in the art can make various modifications or modifications to the present application, and the equivalent forms obtained are also within the scope of protection of the present application. It should also be understood that, on the basis of the technical solutions provided by the present application, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, which are within the scope of protection of the appended claims of the present application. Therefore, the scope of protection of the patent of the present application should be based on the content of the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A primer pair for detecting CHD7 mutations, characterized in that, The detection primer pairs include primer pairs that detect 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; The detection primer pairs are primer pairs 1 to 10 as follows: Primer pair 1 includes: forward primer F1 with the sequence shown in SEQ ID NO.1, and reverse primer R1 with the sequence shown in SEQ ID NO.2; Primer pair 2 includes: forward primer F2 with the sequence shown in SEQ ID NO.3, and reverse primer R2 with the sequence shown in SEQ ID NO.4; Primer pair 3 includes: forward primer F3 with the sequence shown in SEQ ID NO.5, and reverse primer R3 with the sequence shown in SEQ ID NO.6; Primer pair 4 includes: forward primer F4 with the sequence shown in SEQ ID NO.7, and reverse primer R4 with the sequence shown in SEQ ID NO.8; Primer pair 5 includes: forward primer F5 with the sequence shown in SEQ ID NO.9, and reverse primer R5 with the sequence shown in SEQ ID NO.10; Primer pair 6 includes: forward primer F6 with the sequence shown in SEQ ID NO.11, and reverse primer R6 with the sequence shown in SEQ ID NO.12; Primer pair 7 includes: forward primer F7 with the sequence shown in SEQ ID NO.13, and reverse primer R7 with the sequence shown in SEQ ID NO.14; Primer pair 8 includes: forward primer F8 with the sequence shown in SEQ ID NO.15, and reverse primer R8 with the sequence shown in SEQ ID NO.16; Primer pair 9 comprises: forward primer F9 with the sequence shown in SEQ ID NO. 17, reverse primer R9 with the sequence shown in SEQ ID NO. 18; and, Primer pair 10 includes: forward primer F10 with the sequence shown in SEQ ID NO.19, and reverse primer R10 with the sequence shown in SEQ ID NO.

20.

2. The detection primer pair for CHD7 mutation according to claim 1, characterized in that, It also includes internal reference detection primer pairs.

3. The detection primer pair for CHD7 mutation according to claim 2, characterized in that, The internal reference detection primer pair includes the forward primer F11 with the sequence shown in SEQ ID NO.21 and the reverse primer R11 with the sequence shown in SEQ ID NO.

22.

4. The detection primer pair for CHD7 mutation according to claim 3, characterized in that, It also includes an internal reference probe, the sequence of which is shown in SEQ ID NO.

23.

5. A test kit, characterized in that, The kit comprises: the detection primer pair for CHD7 mutation as described in any one of claims 1 to 4.

6. The detection kit according to claim 5, characterized in that, The kit also includes PCR reaction solution.

7. The detection kit according to claim 6, characterized in that, The PCR reaction solution includes: PCR buffer, Mg 2 + One or more of dNTPs, Taq DNA polymerase, and fluorescent dyes.

8. The detection kit according to claim 7, characterized in that, The fluorescent dyes include one or more of LC Green, LCGreen Plus, SYTO 9, and Eva Green.

9. The detection kit according to any one of claims 5 to 8, characterized in that, The test kit also includes one or more of positive and negative controls.

10. The test kit according to claim 9, characterized in that, The positive control sample includes a DNA fragment of the internal reference gene.

11. The test kit according to claim 9, characterized in that, The negative control includes one or more of 1×TE buffer solution and / or water.

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