Primer set for detecting copy number variation of smn1 gene and smn2 gene and design method thereof

Abstract

This application discloses a primer set and its design method for detecting copy number variations in the SMN1 and SMN2 genes. The primer set includes a first primer for detecting exon 7 of the SMN1 gene, a second primer for detecting exon 8 of the SMN1 gene, a third primer for detecting exon 7 of the SMN2 gene, and a fourth primer for detecting exon 8 of the SMN2 gene. The 3′ ends of each of the first to fourth primers independently meet the following conditions: A1) The first base is absolutely conserved and is specifically complementary to the sequences of exons 7, 8, 7, and 8 of the SMN1, SMN1, and SMN2 genes, respectively; and A2) At least one mismatched base is introduced at positions 2 to 4. Using this primer set to detect copy number variations in the SMN1 and SMN2 genes ensures both specificity and amplification efficiency, as well as minimal dosage.

Description

Technical Field

[0001] This application relates to the field of gene detection technology, specifically to primer sets and their design methods for detecting copy number variations of the SMN1 and SMN2 genes, and further to primer sets and their design methods for detecting copy number variations of the SMN1 and SMN2 genes, kits thereof, and their applications. Background Technology

[0002] Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disease caused by degeneration of the anterior horn motor neurons of the spinal cord and the lower brainstem motor nuclei, resulting in progressive, symmetrical muscle weakness and atrophy.

[0003] Mutations in the SMN1 gene (Survival of Motor Neuron 1) determine the occurrence of the disease; over 95% of SMA cases are caused by homozygous deletions or mutations in the SMN1 gene. Its homolog, SMN2, influences disease severity and progression. Due to a key splicing mutation (c.840C>T), only a small amount of full-length functional protein is produced, but its copy number is negatively correlated with disease severity, serving as an important prognostic and treatment guideline. Therefore, the genetic diagnosis of SMA differs from most single-gene genetic diseases, and many clinical guidelines recommend simultaneous testing of SMN1 and SMN2 copy numbers. The pathogenic gene SMN1 and the modifying gene SMN2 share >99.9% sequence homology, differing by only 5 bases. The full-length transcripts of SMN1 and SMN2 are typically compared at exon 7 (c.840C / T) and exon 8 (c.840C / T). The differential base sites (239G / A) serve as the main reference sites for distinguishing between the two loci, and these two sites are also the most commonly used detection sites in clinical typing.

[0004] Currently, detection technologies for SMN genes are mainly divided into several categories, each with its own applicable scope and inherent limitations. Multiplex linkage-dependent probe amplification (MLPA) is one of the gold standard methods used in clinical laboratories both domestically and internationally for SMA diagnosis and SMN2 copy number analysis, but it is complex to operate, costly, and time-consuming (48-72 hours), making it difficult to implement widely. qPCR has a single detection target, making it difficult to distinguish complex variants and unable to accurately quantify multiple copies; NGS copy number quantification is inaccurate, and in complex repetitive regions, the accuracy of CNV analysis algorithms based on read depth decreases significantly, making it difficult to accurately distinguish between adjacent states such as 2 copies and 3 copies; new technologies based on long-read sequencing (such as Oxford Nanopore ONT and PacBio HiFi), although capable of directly observing heterozygous genes and gene conversion events and providing complete haplotype information, still have higher instrument and single-session costs than traditional methods, complex data analysis, and require specially developed bioinformatics analysis workflows, which is not conducive to large-scale screening.

[0005] Fluorescent PCR-capillary electrophoresis is a high-throughput molecular detection technique that combines multiplex fluorescent PCR amplification with capillary electrophoretic separation and detection. Currently, Amplification Refractory Mutation System PCR (ARMS-PCR) is the most commonly used method for SMN1 and SMN2 detection. ARMS detection is currently mainly focused on SMN1, and due to inherent limitations, it cannot accurately quantify and effectively distinguish multiple copies (≥2) of the SMN1 gene. Although there are studies on the simultaneous detection of SMN1 and SMN2 genes using ARMS-PCR, its application remains relatively limited. For example, patent CN 119685475 A introduces a mismatch at the penultimate base of the primer and combines it with competitive primer PCR (COP PCR) and the TaqMan probe method. Exons 7 and 8 need to be detected independently in two separate tubes, achieving the differentiation of SMN1 and SMN2 genes at the 7th and 8th exon sites. However, it still cannot accurately quantify and effectively differentiate multiple copies (≥2). This is due to the inherent limitations of the ARMS real-time PCR method.

[0006] Capillary electrophoresis (CE) works by using a high-voltage electric field to drive the sample to separate within a capillary. DNA fragments of different lengths migrate at different speeds, achieving high separation efficiency and rapid analysis. Capillary electrophoresis combined with ARMS can effectively overcome the limitation of ARMS-PCR alone in accurately quantifying multiple copies (≥2). However, given the many insertion and deletion mutations in genomic sequences, these mutations, when amplified by PCR, exhibit double peaks on capillary electrophoresis, increasing the complexity of subsequent analysis. Especially when primer specificity is compromised, multiple sets of peaks of very similar size may appear on the electrophoresis pattern, posing significant challenges to further analysis. Therefore, unlike conventional quantitative ARMS-PCR, capillary electrophoresis-based ARMS-PCR primer design requires higher standards. This necessitates that the amplification region avoids insertion and deletion sites as much as possible during primer design, while ensuring the specificity of the amplified products. However, finding a suitable location on the genome for primer design on a capillary electrophoresis platform is quite difficult and requires a thorough evaluation based on comprehensive data from various databases and actual sample testing. For simultaneous single-tube detection of SMN1 and SMN2, some researchers use reverse sequences with higher GC content. While this reduces the difficulty of primer design, small insertion / deletion sites near the detection site in the reverse sequence (such as chr5:70951910 (GRCh38), DELINS, T>-, TT) can cause bimodal distributions on the capillary electrophoresis platform, increasing the complexity of result analysis.

[0007] In summary, the current field of genetic diagnosis of SMA faces a core contradiction: on the one hand, clinical requirements for the accuracy of SMN genotyping are increasing, requiring not only knowledge of copy number but also the presence of heterozygous genes, sequence variations, and their haplotype background in order to achieve true precision medicine; on the other hand, existing mainstream clinical testing technologies (MLPA, qPCR) are insufficient in resolving this complexity, while long-read sequencing technologies that can resolve complexity face limitations such as cost, throughput, and operational complexity, making them difficult to popularize in routine clinical laboratories. Summary of the Invention

[0008] Based on this, this application provides at least one primer set for detecting copy number variations of the SMN1 and SMN2 genes and a method for designing the primer set thereon.

[0009] For example, by introducing a universal sequence and combining it with amplification arrest mutation system PCR (ARMS-PCR) to introduce mismatched bases at positions 2-4 of the detection site, coupled with fluorescent dye-labeled primers and a capillary electrophoresis platform with a resolution of 1 base, the differences between the SMN1 and SMN2 genes at two different sites in exons 7 and 8 can be effectively distinguished.

[0010] In addition, the kit, combined with a stable PCR amplification reaction system and copy number analysis method, can effectively distinguish and accurately quantify the copy numbers of SMN1 and SMN2, thereby providing a basis for the analysis of SMA patients' condition, medication guidance and prognosis assessment.

[0011] In a first aspect of this application, a method is provided for designing a primer set for detecting copy number variations of the SMN1 and SMN2 genes. The primer set designed by the method includes a first primer for detecting exon 7 of the SMN1 gene, a second primer for detecting exon 8 of the SMN1 gene, a third primer for detecting exon 7 of the SMN2 gene, and a fourth primer for detecting exon 8 of the SMN2 gene.

[0012] The 3′ ends of the first primer to the fourth primer each independently conform to:

[0013] A1) The first base is absolutely conserved and is sequence-specifically complementary to exon 7 of the SMN1 gene, exon 8 of the SMN1 gene, exon 7 of the SMN2 gene, and exon 8 of the SMN1 gene, respectively; and,

[0014] A2) Introduce a mismatched base at least one of the bases at positions 2 to 4.

[0015] In a second aspect of this application, a primer set is provided that is designed using the method described in the first aspect for designing primer sets for detecting copy number variations of the SMN1 and SMN2 genes.

[0016] The primer set includes one or more of the first to fourth primers.

[0017] In a third aspect of this application, a kit for detecting copy number variations in the SMN1 and SMN2 genes is provided, comprising the primer set as described in the second aspect.

[0018] In a fourth aspect of this application, a PCR premix for detecting copy number variations in the SMN1 and SMN2 genes is provided, the PCR premix comprising the primer set as described in the second aspect; wherein:

[0019] The concentrations of the first to the fourth primers in the PCR premix are each independently 0.05 μM to 0.1 μM.

[0020] In a fifth aspect of this application, a method for detecting copy number variations of the SMN1 and SMN2 genes based on a capillary electrophoresis platform is provided, the method comprising the following steps:

[0021] S1. Mix genomic DNA, nucleic acid polymerase mixture and primer set as described in the second aspect, perform PCR amplification, and obtain PCR product;

[0022] S2. The PCR product is subjected to denaturation and capillary electrophoresis;

[0023] S3. Export the data and analyze it, then calculate the R value;

[0024] S4. Determine the copy number based on the R value. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0026] Figure 1 This is a schematic diagram of the specific primer structure in one embodiment of this application.

[0027] Figure 2 This is a capillary electrophoresis pattern of sample number 1 detected in one embodiment of this application.

[0028] Figure 3 This is a capillary electrophoresis pattern of sample number 2 detected in one embodiment of this application.

[0029] Figure 4 This is a capillary electrophoresis pattern of sample number 13 detected in one embodiment of this application.

[0030] Figure 5 This is a capillary electrophoresis pattern of sample number 15 detected in one embodiment of this application.

[0031] Figure 6 This is a capillary electrophoresis pattern of sample number 36 detected in one embodiment of this application.

[0032] Figure 7 This is a capillary electrophoresis pattern of sample number 42 detected in one embodiment of this application.

[0033] Figure 8 This is a capillary electrophoresis pattern of sample number 46 detected in one embodiment of this application.

[0034] Figure 9This is a capillary electrophoresis pattern of sample number 54 detected in one embodiment of this application. Detailed Implementation

[0035] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0036] 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. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0037] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0038] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0039] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0040] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0041] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0042] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0043] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0044] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0045] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0046] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0047] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0048] This application creatively achieves the requirement of multiplex detection of exons 7 and 8 of SMN1 and SMN2 in a single tube by introducing only one differential base at positions 2-4 of the 3' end of a specific primer, ensuring both specificity and amplification efficiency while reducing the amount of material used. Its structural features include: for each exon, the first base at the 3' end of the primer is absolutely conserved and specifically complementary to the SMN1 and SMN2 gene sequences, respectively; one mismatched base is introduced at positions 2-4 of the 3' end of each primer, strictly adhering to the cooperative asymmetric mismatch of adjacent or spacer positions.

[0049] One aspect of this application provides a method for designing a primer set for detecting copy number variations of the SMN1 and SMN2 genes, wherein the primer set designed by the method includes a first primer for detecting exon 7 of the SMN1 gene, a second primer for detecting exon 8 of the SMN1 gene, a third primer for detecting exon 7 of the SMN2 gene, and a fourth primer for detecting exon 8 of the SMN2 gene.

[0050] The 3′ ends of the first primer to the fourth primer each independently conform to:

[0051] A1) The first base is absolutely conserved and is sequence-specifically complementary to exon 7 of the SMN1 gene, exon 8 of the SMN1 gene, exon 7 of the SMN2 gene, and exon 8 of the SMN1 gene, respectively; and,

[0052] A2) Introduce a mismatched base at least one of the bases at positions 2 to 4.

[0053] For example, the last four digits of the 3' terminal can be any of the following sequences:

[0054] For the first primer (which specifically binds to Exon7 SMN1), the mismatched bases are mainly concentrated at positions 2-4, with TTNC, THTC, or HTTC being exemplary sequences.

[0055] For the third primer (which specifically binds to Exon7 SMN2), the mismatched bases are mainly concentrated at positions 2-3, with TTDT or TBTT being exemplary sequences.

[0056] For the second primer (which specifically binds to Exon8 SMN1), the mismatched bases are mainly concentrated at positions 2-3, with exemplary sequences being CTBG or CNGG;

[0057] For the fourth primer (which specifically binds to Exon8 SMN2), the mismatched bases are mainly concentrated at positions 2-4, with CTNA, CNGA, or NTGA being exemplary sequences.

[0058] Wherein, N = A, G, T, or C; H = A, C, or T; D = A, G, or T; B = G, C, or T; A is adenine in DNA bases; T is thymine in DNA bases; C is cytosine in DNA bases; and G is guanine in DNA bases.

[0059] In some implementations, the primer combination arrangement is shown in Table A.

[0060] Table A

[0061]

[0062] It should be understood that the “original sequence” in the table refers to the last four positions of the 3′ end without mismatched bases. The genomic sequences of the heterologous binding of the four original sequences are GAAA, AAAA, CCAG and TCAG, respectively.

[0063] In some implementations, each of the first to fourth primers independently includes a second portion, namely a universal sequence located at the 5' end.

[0064] In some embodiments, each of the first to fourth primers independently includes a third portion, namely a padding sequence located between the universal sequence and the 3′ end.

[0065] In some embodiments, the length of the second portion is 18 to 34 bases.

[0066] In some embodiments, the sequence length of the third part is 3 to 7 bases.

[0067] In another aspect of this application, a primer set designed using the method described above for designing primer sets for detecting copy number variations of the SMN1 and SMN2 genes is provided;

[0068] The primer set includes one or more of the first to fourth primers.

[0069] For example, the primer set includes the first to fourth primers as described above.

[0070] In some embodiments, the first primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO: 1; exemplaryly, the sequence of the first primer is shown in SEQ ID NO: 1.

[0071] In some embodiments, the third primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO: 2; exemplaryly, the sequence of the third primer is shown in SEQ ID NO: 2.

[0072] In some embodiments, the second primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO: 4; exemplaryly, the sequence of the second primer is shown in SEQ ID NO: 4.

[0073] In some embodiments, the fourth primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO: 5; exemplaryly, the sequence of the fourth primer is shown in SEQ ID NO: 5.

[0074] In some embodiments, the primer set further includes a first common downstream primer for detecting exon 7 of the SMN1 gene and exon 7 of the SMN2 gene and / or a second common downstream primer for detecting exon 8 of the SMN gene and exon 8 of the SMN2 gene.

[0075] In some embodiments, the first common downstream primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO: 3; and the second common downstream primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO: 6.

[0076] In some embodiments, the nucleotide sequences of one upstream primer and one shared downstream primer used to detect the EXON7 site of the SMN1 and SMN2 genes are shown in SEQ ID No: 1-3, respectively.

[0077] In some embodiments, the nucleotide sequences of one upstream primer and one shared downstream primer used to detect the EXON8 site of the SMN1 and SMN2 genes are shown in SEQ ID No: 4-6, respectively.

[0078] In some embodiments, the primer set further includes a first primer pair for detecting internal control genes.

[0079] In some embodiments, the internal control gene includes Albumin (ALB); exemplaryly, the nucleotide sequences of an upstream primer and a downstream primer for detecting the internal control gene ALB are shown in SEQ ID No: 7 and 8, respectively.

[0080] In some embodiments, the primer set further includes a second primer pair for fluorescent labeling.

[0081] For example, the second primer pair is used for PCR signal amplification and fluorescence detection.

[0082] In some embodiments, the nucleotide sequences of an upstream primer and a downstream fluorescently labeled primer of the second primer pair are shown in SEQ ID No: 9 and 10, respectively.

[0083] In some embodiments, the second primer pair contains at least one fluorescent group selected from 6-FAM, VIC, HEX, NED, PET, ROX, and LIZ.

[0084] In another aspect of this application, a kit is provided for detecting the copy number of human motor neuron survival genes SMN1 and SMN2 based on a capillary electrophoresis platform;

[0085] The kit includes the primer set described above.

[0086] In some embodiments, the kit also includes a nucleic acid polymerase mixture.

[0087] In some embodiments, the kit also includes three normal controls.

[0088] In another aspect of this application, a PCR premix for detecting copy number variations of the SMN1 and SMN2 genes based on a capillary electrophoresis platform is also provided, comprising the primer set as described above.

[0089] In some embodiments, the concentrations of the first to fourth primers in the PCR premix are each independently 0.05 μM to 0.1 μM.

[0090] In some embodiments, the concentration of the first primer pair in the PCR premix is ​​0.05 μM to 0.1 μM.

[0091] In some embodiments, the concentration of the second primer pair in the PCR premix is ​​0.2 μM to 0.4 μM.

[0092] As an example of the present invention, the concentration of primers is shown in Table B.

[0093] Table B

[0094]

[0095] In another aspect of this application, a method for detecting the copy number of human motor neuron survival genes SMN1 and SMN2 is also provided, employing the following technical solution:

[0096] S1. Mix genomic DNA, nucleic acid polymerase mixture and primer set as described above for PCR amplification to prepare PCR product;

[0097] S2. The PCR product is denatured and subjected to capillary electrophoresis;

[0098] S3. Export the data and analyze it, then calculate the R value;

[0099] S4. Determine the copy number based on the R value.

[0100] In some implementations, step S1, the procedure for performing PCR amplification includes:

[0101] Pre-denaturation: 95℃, 2 min to 5 min;

[0102] Denaturation: 95℃, 15 s~30 s;

[0103] Annealing: 56℃, 25 s~30 s;

[0104] Extension: 72℃, 25 s~35 s;

[0105] Final extension: 72℃, 5 min to 10 min;

[0106] The process involved 25 cycles of denaturation to extension.

[0107] In some implementations, the procedure for performing PCR amplification includes:

[0108] Pre-denaturation: 95℃, 2 min;

[0109] Denaturation: 95℃, 15 s;

[0110] Annealing: 56℃, 30 s;

[0111] Extension: 72℃, 30s;

[0112] Final extension: 72℃, 5 min;

[0113] The process involved 25 cycles of denaturation to extension.

[0114] The following are some examples.

[0115] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0116] Example 1: Establishment and application of a multiplex detection system for exons 7 and 8 of the SMN1 and SMN2 genes.

[0117] This invention relates to a method for detecting exons 7 and 8 of the SMN1 and SMN2 genes, including primer design, screening, PCR amplification and capillary electrophoresis analysis, for accurate copy number determination.

[0118] 1 Primer Design and Screening

[0119] 1.1 Design Strategy

[0120] To avoid bimodal interference from high-frequency insertion / deletion sites in the genome during capillary electrophoresis, a reference sequence with low GC content (AT enrichment) was selected, which effectively avoids the direction of variation. Primer design was based on the sequences of exons 7 and 8 of the human SMN1 and SMN2 genes and the internal control gene ALB from the NCBI database, with specific primers designed at functional site base differences.

[0121] 1.2 Primer Structure

[0122] The primers for the internal control ALB gene consist of two parts: a specific recognition sequence and a universal tag sequence.

[0123] The SMN1 exon 7 / 8 primers have similar structures to the ALB primers, containing specific sequences and universal tags.

[0124] The SMN2 exon 7 / 8 primers introduce 3–5 non-homologous nucleotide filler sequences (such as “ACTTC”) between the specific sequence and the universal tag to distinguish the length of the SMN1 amplification product, facilitating capillary electrophoresis analysis.

[0125] The above describes specific primers (see schematic diagram of specific primer structure). Figure 1 ).

[0126] The universal tag sequence is also used as a PCR primer. At least one of the two PCR primers (PCR-F and PCR-R) is fluorescently labeled at its 5′ end for signal amplification, thus avoiding the waste of labeling multiple specific primers individually.

[0127] 1.3 Mismatched base design

[0128] Based on the ARMS-PCR principle, artificial mismatched bases were introduced at positions 2–4 of the 3′ end of the primers to enhance specificity. The design strategy combined strong and weak mismatch combinations and was optimized through two rounds of screening: the first round eliminated non-specific or poorly amplified primers, and the second round validated the qualified primers. Exon 7 and exon 8 primer sets were screened separately, and the results are summarized in tables (see Tables 1 and 2), categorized as "qualified" or "unqualified" based on amplification specificity and effectiveness.

[0129] Table 1. Screening of mismatch primers for Exon7 on SMN1 and SMN2 genes

[0130]

[0131] Note: Qualified - specific, with no or slight primer dimers, and good amplification effect; Unqualified - non-specific amplification or cross-amplification between two genes.

[0132] Table 2. Screening of mismatch primers for Exon8 on SMN1 and SMN2 genes

[0133]

[0134] Note: Qualified - specific, with no or slight primer dimers, and good amplification effect; Unqualified - non-specific amplification or cross-amplification between two genes.

[0135] 1.4 Multiple Primer Set Screening

[0136] The selected primer combinations for exon 7 and exon 8 were combined for multiplex detection, and further optimized by introducing the internal control ALB gene. One primer combination was selected to achieve simultaneous detection of exons 7 and 8 of SMN1 and SMN2 in a single tube. The composition of the selected primer mixes is shown in Table 3 (E7-combination 8 and E8-combination 9).

[0137] Table 3. Primer set final concentration information

[0138]

[0139] 2. PCR amplification

[0140] 2.1 Sample DNA Extraction

[0141] Genomic DNA was extracted from EDTA-anticoagulated whole blood (using the IVD3101-TL-06 kit from MGI Genomics), with an OD260 / 280 ratio of 1.6–2.0.

[0142] 2.2 Amplification reaction system (total volume 15 μL)

[0143] Table 4 Amplification System

[0144]

[0145] 2.3 PCR reaction procedure

[0146] Table 5 Amplification Reaction Procedure

[0147]

[0148] 3. Capillary electrophoresis detection

[0149] • Prepare the denaturation system: Mix 8.7 μL of Hi-Di Formamide and 0.3 μL of Size Standard NS500, aliquot the mixture, and add 1 μL of PCR product to each tube.

[0150] • Denature at 95°C for 3 min, then in an ice bath for 3 min before loading onto an ABI 3500Dx gene analyzer.

[0151] Electrophoresis parameters: injection voltage 1.6 kV, injection time 8 s, running voltage 17 kV, running time 1330 s.

[0152] 4. Data Analysis and Copy Number Interpretation

[0153] 4.1 Calculation of Peak Height Ratio

[0154] • Calculate the ratio B of the peak height of the SMN1 or SMN2 exon 7 / 8 amplification product to the peak height of the internal control ALB in the sample to be tested.

[0155] • Select three normal control samples and calculate their target / internal control peak height ratios A1, A2, and A3 respectively. Take the mean A0 = (A1 + A2 + A3) / 3.

[0156] The final ratio is R = B / A0.

[0157] 4.2 Copy number determination:

[0158] The copy number status of the SMN1 or SMN2 gene is determined based on the R value, see Table 6.

[0159] Table 6 Copy Count Interpretation Table

[0160]

[0161] Example 2 Primer and System Optimization

[0162] Primer concentrations in the experimental reaction system are crucial for genotyping and detection stability. This study involved a single-tube multiplex reaction, making it more complex than conventional single-detection systems. The concentrations of individual primers and the relative ratios between specific and PCR primers in the multiplex system are more sensitive to the experiment, thus requiring further screening and testing. After testing, test group 4 was ultimately determined to be the most suitable primer concentration. Furthermore, the annealing temperature in the reaction program settings plays a significant role in product amplification efficiency and specificity. Deviations may exist between theoretical and actual annealing temperatures during primer design, requiring careful screening and confirmation through testing. After testing and optimization, the final annealing temperature for the reaction system was determined to be 56℃.

[0163] The primer sequences used are shown in Table 7.

[0164] Table 7 Primer sequence information used

[0165]

[0166] The concentrations of each primer are screened as shown in Table 8.

[0167] Table 8 Primer Concentration Adjustment and Screening

[0168]

[0169] The results of tests at different annealing temperatures are shown in Table 9.

[0170] Table 9 Tests at different annealing temperatures

[0171]

[0172] Example 3: Sample Detection and Reference Reagent Consistency Verification

[0173] 1. Ninety-six EDTA-anticoagulated peripheral blood samples were collected from clinical screening. The consistency of copy number detection results was verified using the SALSA MLPA Probemix P060 SMA Carrier (gold standard kit for SMA detection) from MRC-Holland, Netherlands, as a reference method.

[0174] 2. Nucleic acid extraction: Refer to the steps in Example 2.

[0175] 3. PCR amplification and result analysis were performed following the steps in Example 1. The copy number detection results were consistent with the MLPA results, achieving 100% consistency. The detection results are shown in Table 10 (capillary electrophoresis patterns of some samples are shown in Table 10). Figure 2-9 ).

[0176] Table 10 Consistency between this application and MLPA results

[0177]

[0178]

[0179]

[0180] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0181] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method of designing a primer set for detecting copy number variations of the SMN1 gene and the SMN2 gene, characterized in that, The primer set designed by the method includes a first primer for detecting exon 7 of the SMN1 gene, a second primer for detecting exon 8 of the SMN1 gene, a third primer for detecting exon 7 of the SMN2 gene, and a fourth primer for detecting exon 8 of the SMN2 gene. The 3′ ends of the first primer to the fourth primer each independently conform to: A1) The first base is absolutely conserved and is sequence-specifically complementary to exon 7 of the SMN1 gene, exon 8 of the SMN1 gene, exon 7 of the SMN2 gene, and exon 8 of the SMN1 gene, respectively; and, A2) Introduce a mismatched base at least one of the bases at positions 2 to 4.

2. The method of claim 1, wherein the primer set designed to detect copy number variation of the SMN1 gene and the SMN2 gene is characterized in that, The genome sequence specifically bound to the last four positions of the 3' end of the first primer is GAAA, the genome sequence specifically bound to the last four positions of the 3' end of the third primer is AAAA, the genome sequence specifically bound to the last four positions of the 3' end of the second primer is CCAG, and the genome sequence specifically bound to the last four positions of the 3' end of the fourth primer is TCAG. The sequence of the last four positions at the 3′ end of the primer set meets one or more of the following conditions: B1) First primer: TTNC, THTC, or HTTC; B2) Second primer: CTBG or CNGG; B3) Third primer: TTDT or TBTT; B4) Fourth primer: CTNA, CNGA, or NTGA; Where N is A, G, T or C, H is A, C or T, D is A, G or T, and B is G, C or T.

3. The method of claim 2, wherein the primer set is designed to detect copy number variation of the SMN1 gene and the SMN2 gene. The sequence of the last four positions at the 3′ end of the primer set meets one or more of the following conditions: C1) First primer: TTCC, and third primer: TTGT; C2) First primer: TTCC, and third primer: TTAT; C3) First primer: TTCC, and third primer: TGTT; C4) First primer: TTCC, and third primer: TCTT; C5) First primer: TTGC, and third primer: TGTT; C6) First primer: TTGC, and third primer: TCTT; C7) First primer: TATC, and third primer: TTGT; C8) First primer: TCTC, and third primer: TTAT; C9) First primer: ATTC, and third primer: TTGT; C10) First primer: ATTC, and third primer: TTAT; C11) First primer: ATTC, and third primer: TGTT; C12) First primer: ATTC, and third primer: TCTT; C13) First primer: CTTC, and third primer: TTGT; C14) First primer: CTTC, and third primer: TTAT; C15) First primer: CTTC, and third primer: TGTT; C16) Second primer: CTTG, and fourth primer: CTAA; C17) Second primer: CTTG, and fourth primer: CAGA; C18) Second primer: CTTG, and fourth primer: CGGA; C19) Second primer: CTTG, and fourth primer: CCGA; C20) Second primer: CTTG, and fourth primer: GTGA; C21) Second primer: CTTG, and fourth primer: ATGA; C22) Second primer: CTCG, and fourth primer: CAGA; C23) Second primer: CTCG, and fourth primer: CGGA; C24) Second primer: CTCG, and fourth primer: CCGA; C25) Second primer: CTCG, and fourth primer: GTGA; C26) Second primer: CTCG, and fourth primer: TTGA; C27) Second primer: CTCG, and fourth primer: ATGA; C28) Second primer: CAGG, and fourth primer: CTTA; C29) Second primer: CAGG, and fourth primer: GTGA; C30) Second primer: CAGG, and fourth primer: TTGA; C31) Second primer: CAGG, and fourth primer: ATGA; C32) Second primer: CCGG, and fourth primer: CTCA; C33) Second primer: CCGG, and fourth primer: CTTA; C34) Second primer: CCGG, and fourth primer: GTGA; C35) Second primer: CCGG, and fourth primer: TTGA; C36) Second primer: CCGG, and fourth primer: ATGA; C37) Second primer: CGGG, and fourth primer: CTCA; C38) Second primer: CGGG, and fourth primer: CTTA; C39) Second primer: CGGG, and fourth primer: CTAA; C40) Second primer: CGGG, and fourth primer: GTGA; C41) Second primer: CGGG, and fourth primer: TTGA; C42) Second primer: CGGG, and fourth primer: ATGA; C43) Second primer: ATGG, and fourth primer: CTCA; C44) Second primer: ATGG, and fourth primer: CTTA; C45) Second primer: ATGG, and fourth primer: CTAA; C46) Second primer: ATGG, and fourth primer: CAGA; The second primer for C47 is ATGG, and the fourth primer is CCGA.

4. The method of designing a primer set for detecting copy number variations of the SMN1 gene and the SMN2 gene according to any one of claims 1 to 3, wherein Each of the first to the fourth primers independently includes a universal sequence at the 5′ end; and a padding sequence between the universal sequence and the 3′ end; Optionally, the length of the universal sequence is 18 to 34 bases; Optionally, the length of the filling sequence is 3 to 7 bases.

5. Primers designed using the method described in any one of claims 1 to 4 for designing primer sets for detecting copy number variations in the SMN1 and SMN2 genes; The primer set includes one or more of the first to fourth primers.

6. The primer set of claim 5, wherein It meets one or more of the following conditions: D1) The first primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO: 1; D2) The third primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO: 2; D3) The second primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO: 4; D4) The fourth primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO: 5; Optionally, the primer set further includes a first common downstream primer for detecting exon 7 of the SMN1 gene and exon 7 of the SMN2 gene and / or a second common downstream primer for detecting exon 8 of the SMN gene and exon 8 of the SMN2 gene; The first shared downstream primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO: 3; The second common downstream primer comprises a nucleic acid fragment with the sequence shown in SEQ ID NO:

6.

7. The primer set according to claim 5 or 6, wherein The primer set also includes a first primer pair for detecting internal control genes and / or a second primer pair for fluorescent labeling; Optionally, the internal control gene includes Albamin (ALB); the sequences of the first primer pair are optionally shown as SEQ ID NO: 7 and 8, respectively; Optionally, the sequences of the fluorescently labeled primer pairs are shown in SEQ ID NO: 9 and 10, respectively.

8. A kit for detecting copy number variations of the SMN1 gene and the SMN2 gene, characterized by, It includes the primer set as described in any one of claims 5 to 7; Optionally, the kit may also include a nucleic acid polymerase mixture.

9. A PCR master mix for detecting copy number variations of the SMN1 gene and the SMN2 gene, characterized in that, The PCR premix comprises the primer set as described in any one of claims 5 to 7; wherein: The concentrations of the first to fourth primers in the PCR premix were each independently 0.05 μM to 0.1 μM; Optionally, the concentration of the first primer pair in the PCR premix is ​​0.05 μM to 0.1 μM; Optionally, the concentration of the second primer pair in the PCR premix is ​​0.2 μM to 0.4 μM.

10. A method for detecting SMN1 and SMN2 gene copy number variations based on a capillary electrophoresis platform, characterized in that, Includes the following steps: S1. Mix genomic DNA, nucleic acid polymerase mixture and primer set as described in any one of claims 5 to 7, perform PCR amplification, and obtain PCR product; S2. The PCR product is subjected to denaturation and capillary electrophoresis; S3. Export the data and analyze it, then calculate the R value; S4. Determine the copy number based on the R value; Optionally, in step S1, the procedure for PCR amplification includes: Pre-denaturation: 95℃, 2 min to 5 min; Denaturation: 95℃, 15 s~30 s; Annealing: 56℃, 25 s~30 s; Extension: 72℃, 25 s~35 s; Final extension: 72℃, 5 min to 10 min; The process involved 25 cycles of denaturation to extension.

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