Primer set, kit and method for detecting the number of (cta·tag)n and (ctg·cag)n trinucleotide repeats of atxn8os and atxn8 genes

Abstract

The present application relates to a kind of primer group, kit and method for detecting ATXN8OS and ATXN8 gene (CTA·TAG) n And (CTG·CAG) n Trinucleotide repeat number, belong to gene detection technical field.The primer group includes first primer pair and second primer pair;The forward primer sequence of the first primer pair is as shown in SEQ ID NO.1, and the reverse primer sequence is as shown in SEQ ID NO.2;The forward primer sequence of the second primer pair is as shown in SEQ ID NO.1, and the reverse primer sequence is as shown in SEQ ID NO.3.The present application is realized by the design of two primer pairs, complementary verification is realized, detection result is comprehensive and reliable, make up the technical deficiency of one generation sequencing and two generation sequencing in long fragment, high GC content repeat sequence detection, and detection cost is low, detection cycle is short.

Description

Technical Field

[0001] This invention relates to a primer set, kit, and method for detecting the number of trinucleotide repeats (CTA·TAG)n and (CTG·CAG)n in the ATXN8OS and ATXN8 genes, belonging to the field of gene detection technology. Background Technology

[0002] Spinocerebellar ataxia type 8 (SCA8) is an autosomal dominant, chronic, progressive ataxia. Its typical initial clinical manifestation is dysarthria characterized by slowed speech and prolonged articulation, often accompanied by gait instability. Patients may also experience oculomotor abnormalities (such as nystagmus, abnormal visual tracking, and saccades), limb ataxia, limb spasticity, and sensory loss. In severe cases, patients may be unable to walk independently by age 40-60. The age of onset is wide, ranging from infancy to over 70 years of age, and the course of the disease can last for decades. Epidemiological data shows that SCA8 accounts for approximately 2-5% of autosomal dominant ataxia patients, and due to the significant decrease in penetrance, its actual prevalence in the population may be even higher. Currently, there is no specific treatment; treatment mainly focuses on symptomatic support and rehabilitation training.

[0003] Genetic studies have shown that the pathogenesis of SCA8 is associated with abnormal expansion of trinucleotide repeat sequences in the overlapping genes ATXN8OS and ATXN8. Both genes are located on the long arm of human chromosome 13, region 2, band 1 (13q21), with their 3′ ends overlapping in opposite directions. The ATXN8OS gene contains a (CTG)n repeat, while the ATXN8 gene contains a complementary (CAG)n repeat. Since the 5′ end of the CTG or CAG repeat sequence is adjacent to the CTA or TAG repeat sequence, in clinical testing, the sum of the number of (CTA·TAG)n and (CTG·CAG)n repeats is usually used to determine the extent of trinucleotide repeat expansion. The number of repeats in the normal population is 15-50; 51-70 repeats are considered intermediate, with unclear clinical significance; pathogenic repeats range from 71-1300 repeats, exhibiting significant penetrance, and can be seen in both ataxia patients and asymptomatic individuals.

[0004] Accurate detection of the trinucleotide repeat counts (CTA·TAG)n and (CTG·CAG)n in the ATXN8OS and ATXN8 genes is crucial for genetic counseling, family screening, prenatal diagnosis, and preimplantation genetic testing of SCA8. However, this detection faces the following technical challenges:

[0005] First, the repetitive sequence structure is complex and has a high GC content. The repetitive sequence in this region is not a single (CTG·CAG)n repeat, but a mixed repeat of (CTA·TAG)n and (CTG·CAG)n. The sequence is complex and has a high GC content, which makes it easy to form secondary structures, making conventional PCR amplification difficult.

[0006] Second, the range of repetition counts is extremely wide. The repetition counts of normal and pathogenic alleles vary greatly (from 15 to over 1300 times), making the amplification of long allele fragments particularly difficult. This can easily lead to failure in the amplification of large allele fragments, resulting in false negative test results.

[0007] Existing detection methods all have significant shortcomings. The detection methods reported in the literature mainly include the following:

[0008] (1) Conventional fluorescent PCR-capillary electrophoresis: Specific primers are designed for amplification in conserved regions on both sides of the repetitive sequence. This method is simple to operate and low in cost, but when the number of allele repetitions is too long, the PCR amplification efficiency is extremely low or even completely fails due to the high GC content and the excessively long amplified fragment, which makes it impossible to detect long fragment alleles and may misclassify abnormal heterozygotes as normal homozygotes.

[0009] (2) Repeat Primer PCR (TP-PCR): Developed to overcome the difficulties of amplifying long fragments, this method typically uses three primers (one fluorescently labeled upstream specific primer, one universal primer, and one downstream primer consisting of a repeat sequence with a universal primer tail) for amplification. It can produce ladder-like products with a 3 bp difference, enabling qualitative or semi-quantitative detection of ultra-long repeat sequences. However, this technique has the following drawbacks: using three primers, primers are prone to dimerization, resulting in a high risk of non-specific amplification; primer ratios and amplification conditions (such as ion concentration and primer concentration) require fine optimization, leading to poor system stability.

[0010] (3) First-generation sequencing: It has poor sequencing accuracy for long, high-GC-content repetitive sequences, and signal attenuation is prone to occur in repetitive regions, making accurate interpretation impossible.

[0011] (4) Second generation sequencing: Because this region is a repetitive sequence region, second generation sequencing has limitations or cannot detect it.

[0012] (5) Third-generation sequencing: For example, CN 111378653 A discloses a pair of primer sequences for SCA8 detection, and the amplified product is used as a template for third-generation sequencing. Although third-generation sequencing has certain advantages in the detection of repetitive sequences, its application in practical testing is currently limited by the high requirements for DNA sample quality, complex operation procedures, long detection cycle, high cost, and lack of conclusive data on the accuracy of repetitive sequence detection, especially in prenatal diagnosis and preimplantation genetic testing.

[0013] In clinical practice, there is a need for an accurate, rapid, and economical method to detect the (CTA·TAG)n and (CTG·CAG)n triple nucleic acid repeats of the ATXN8OS and ATXN8 genes. Summary of the Invention

[0014] The first objective of this invention is to provide a primer set for detecting the number of trinucleotide repeats (CTA·TAG)n and (CTG·CAG)n in the ATXN8OS and ATXN8 genes.

[0015] To achieve the first objective of this invention, the primer set comprises a first primer pair and a second primer pair;

[0016] The forward primer sequence of the first primer pair is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2;

[0017] The forward primer sequence of the second primer pair is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.3.

[0018] In one specific embodiment, the primer shown in SEQ ID NO.1 is labeled with a fluorescent dye at its 5′ end.

[0019] The fluorescent dyes mentioned can be FAM, HEX, VIC, NED, etc.

[0020] The second objective of this invention is to provide a kit for detecting the number of trinucleotide repeats (CTA·TAG)n and (CTG·CAG)n in the ATXN8OS and ATXN8 genes.

[0021] To achieve the second objective of the present invention, the kit comprises the primer set described above.

[0022] In one specific embodiment, the kit further includes PCR reaction reagents, which include at least one of DNA polymerase, PCR buffer, dNTPs, GC enhancer, and dimethyl sulfoxide.

[0023] A third objective of this invention is to provide a method for detecting the number of trinucleotide repeats (CTA·TAG)n and (CTG·CAG)n in the ATXN8OS and ATXN8 genes for non-diagnostic purposes.

[0024] To achieve the third objective of this invention, the method includes the following steps:

[0025] (1) Provide genomic DNA from the subject's sample;

[0026] (2). Using genomic DNA as a template, PCR amplification was performed using the first primer pair and the second primer pair described above to obtain the amplification products;

[0027] (3) Perform capillary electrophoresis analysis on the amplification products from step (2);

[0028] (4) Based on the capillary electrophoresis results, analyze the length of the amplified fragment and calculate the number of trinucleotide repeats of (CTA·TAG)n and (CTG·CAG)n.

[0029] In one specific embodiment, in step (2), the concentration of the primer is 10 μmol / L.

[0030] In one specific embodiment, in step (2), the DNA concentration of the DNA template is 10–30 ng / uL.

[0031] In one specific embodiment, in step (2), the procedure for PCR amplification using the first primer pair and the second primer pair is landing PCR, which includes:

[0032] (a) High-temperature pre-denaturation step: Heat the reaction system to a temperature sufficient to completely denature the template DNA and maintain the denaturation time for 3 to 10 minutes;

[0033] (b) Landing amplification phase: Perform the first number of cycles; each cycle consists of the following steps: denaturation at a denaturing temperature for 1–2 minutes, annealing at a temperature higher than the primer melting temperature, with the annealing temperature gradually decreasing in each cycle; and extension at a temperature at which the DNA polymerase is catalytically active.

[0034] (c) Conventional amplification phase: Perform the second number of cycles; each cycle consists of the following steps: denaturation at denaturation temperature for 1 to 2 minutes, annealing at a final annealing temperature of 60 to 50°C, and extension at 68 to 72°C.

[0035] (d) Finally, maintain at 68–72°C for 5–10 minutes for the final extension time;

[0036] The first number of cycles is 10 to 15 times; the second number of cycles is 20 to 30 times.

[0037] In one specific embodiment, in step (4), the formula for calculating the number of trinucleotide repeats is: number of repeats = (length of amplified fragment - length of conserved sequence) / 3;

[0038] The conserved sequence length was 231 bp when amplified using the first primer pair and 62 bp when amplified using the second primer pair.

[0039] When multiple ladder-like amplification fragments differing by 3 bp are obtained by using the second primer pair, the longest amplification fragment is selected and substituted into the formula for calculation.

[0040] In one specific implementation, capillary electrophoresis uses GS 1200 LIZ as a fragment length standard.

[0041] GS 1200 LIZ is GeneScan TM GS 1200 LIZ Size Standard.

[0042] Beneficial effects:

[0043] The method of this invention can be used to obtain trinucleotide repeat information of the ATXN8OS and ATXN8 genes in vitro, and has the following advantages compared with the prior art:

[0044] (1) Comprehensive and Reliable Detection Results: This invention achieves precise quantification of normal repeat counts and stable qualitative detection of ultra-long repetitive sequences through the complementary design of two primer pairs. The first primer pair (P1 / P2) amplifies the full-length repetitive region, accurately quantifying the repeat count within the normal range; the second primer pair (P1 / P3) employs an improved dual-primer TP-PCR technique, effectively amplifying ultra-long repetitive sequences and generating ladder-like amplicones, avoiding missed detection of large allele fragments. The results of the two primer pairs can be mutually verified, significantly improving the reliability of the detection.

[0045] (2) Simplified operation and reduced cost: Compared with existing TP-PCR technology, this invention simplifies the three-primer system to a two-primer system, eliminating the need for universal primers, reducing the types of primers, lowering the risk of primer dimer formation, simplifying the system optimization process, and improving experimental stability and reproducibility. Furthermore, the detection method of this invention does not require complex third-generation sequencing equipment; it can be completed based on conventional PCR and capillary electrophoresis, resulting in low detection cost and short cycle time, making it suitable for large-scale clinical screening.

[0046] (3) Overcoming the shortcomings of existing technologies: This invention overcomes the technical deficiencies of first-generation and second-generation sequencing in the detection of long, high-GC-content repetitive sequences, solves the problem of failure in amplification of ultra-long repetitive sequences using conventional PCR methods, and avoids the complexity and instability of the TP-PCR three-primer system. Compared with third-generation sequencing, this invention has no special requirements for DNA sample quality, a simple operation process, a short detection cycle, and low cost, making it more suitable for clinical testing services, especially in prenatal diagnosis and preimplantation genetic testing.

[0047] (4) High specificity for SCA8: The present invention designs primers for the (CTA·TAG)n and (CTG·CAG)n mixed repetitive sequences unique to the ATXN8OS and ATXN8 genes, which fully considers the complexity of the two genes overlapping and bidirectional transcription, and has high target specificity. Attached Figure Description

[0048] Figure 1 This is a schematic diagram illustrating the amplification principle of the first primer pair (P1 / P2) of the present invention.

[0049] Figure 2 This is a schematic diagram of the amplification principle of the second primer pair (P1 / P3) of the present invention (double primer improved TP-PCR).

[0050] Figure 3 This is a schematic diagram of the amplification principle of the existing repeat primer PCR (TP-PCR) method (three-primer system).

[0051] Figure 4 The fragment standard in Example 3 of this invention is GS 600 LIZ. Samples A and B were amplified using the first primer pair (amplifier 1) and then subjected to capillary electrophoresis. A: Sample of the family subject (pathogenic duplication, heterozygous); B: Sample of the mother of the family subject (pathogenic duplication, heterozygous).

[0052] Figure 5 The image shows the results of capillary electrophoresis of samples C and D using the first primer pair (amplifier 1) in Example 3 of this invention, with the fragment standard being GS 600 LIZ; C: Father of the subject (normal / negative control, heterozygous); D: Positive control sample (outside the normal repeat range, heterozygous).

[0053] Figure 6 The fragment standard in Example 3 of this invention is GS 1200 LIZ. Samples A and B were amplified using the first primer pair (amplifier 1) and subjected to capillary electrophoresis. A: Family subject sample (pathogenic duplication, heterozygous); B: Sample of the mother of the family subject (pathogenic duplication, heterozygous).

[0054] Figure 7 The image shows the results of capillary electrophoresis of samples C and D using the first primer pair (amplifier 1) in Example 3 of this invention, with the fragment standard being GS 1200 LIZ; C: Father of the subject (normal / negative control, heterozygous); D: Positive control sample (outside the normal repeat range, heterozygous).

[0055] Figure 8The image shows the results of capillary electrophoresis of samples A and B in Example 3 of this invention using the second primer pair (amplifier 2); A: Family subject sample (pathogenic duplication, heterozygous); B: Sample of the mother of the family subject (pathogenic duplication, heterozygous).

[0056] Figure 9 The image shows the results of capillary electrophoresis of samples C and D in Example 3 of this invention using the second primer pair (amplifier 2); C: Father of the subject (normal / negative control, heterozygous); D: Positive control sample (outside the normal repeat range, heterozygous).

[0057] Figure 10 The diagram shows the repeatability verification of the positive control sample D at two different times in Example 3 of the present invention; Figures A and B are capillary electrophoresis diagrams of amplicon 1 at two different times, and Figures C and D are capillary electrophoresis diagrams of amplicon 2 at two different times.

[0058] Figure 11 This is an agarose gel electrophoresis image of amplicon 1 of samples A, B, and C in an embodiment of the present invention. Detailed Implementation

[0059] To achieve the first objective of the present invention, the primer set for detecting the number of trinucleotide repeats of (CTA·TAG)n and (CTG·CAG)n in the ATXN8OS and ATXN8 genes comprises a first primer pair and a second primer pair.

[0060] The forward primer sequence of the first primer pair is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2;

[0061] The forward primer sequence of the second primer pair is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.3.

[0062] The primer shown in SEQ ID NO.3 consists of 5 CAG trinucleotide repeats and 11 conserved bases on the 3′ flanking side, and its sequence is SEQ ID NO.3.

[0063] In one specific embodiment, the primer shown in SEQ ID NO.1 is labeled with a fluorescent dye at its 5′ end.

[0064] The fluorescent dye can be FAM, HEX, VIC, NED, etc.

[0065] The first primer pair is used to amplify the target fragment containing the (CTA·TAG)n and (CTG·CAG)n trinucleotide repeat regions of the ATXN8OS and ATXN8 genes and their flanking conserved sequences. The forward primer sequence is shown in SEQ ID NO. 1, and the reverse primer sequence is shown in SEQ ID NO. 2. The amplification reference genome location is chr13:70139303_70139608, and the reference genome is GRCh38 / hg38.

[0066] The second primer pair is used to amplify the core regions of the (CTA·TAG)n and (CTG·CAG)n trinucleotide repeat sequences and generate stepwise amplicones differing by 3 bp. Its forward primer sequence is the same as that of the first primer pair, SEQ ID NO. 1, and its reverse primer sequence is shown in SEQ ID NO. 3: consisting of 5 CAG trinucleotide repeats and 11 conserved bases adjacent to the 3′ flanking ends. Its amplification reference genome position is chr13:70139303_70139439, and the reference genome is GRCh38 / hg38.

[0067] To achieve the second objective of the present invention, the kit comprises the above-described primer set.

[0068] In one specific embodiment, the kit further includes PCR reaction reagents, which include at least one of DNA polymerase, PCR buffer, dNTPs, GC enhancer, and dimethyl sulfoxide.

[0069] In one specific implementation, the kit includes:

[0070] First primer pair mixture: primers containing the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, with a concentration of 10 μmol / L for each primer;

[0071] Second primer pair mixture: primers containing the sequences shown in SEQ ID NO.1 and SEQ ID NO.3, each primer concentration is 10 μmol / L; 2×PCR Master Mix (containing DNA polymerase, buffer, dNTPs);

[0072] GC Enhancer; DMSO; Ultrapure Water.

[0073] To achieve the third objective of this invention, the method includes the following steps:

[0074] (1) Extract genomic DNA from the subject's sample;

[0075] (2) Using genomic DNA as a template, PCR amplification was performed using the first primer pair and the second primer pair to obtain amplification products; the first primer pair is the primers shown in SEQ ID NO.1 and SEQ ID NO.2, and the second primer pair is the primers shown in SEQ ID NO.1 and SEQ ID NO.3;

[0076] (3) Perform capillary electrophoresis analysis on the amplification products of step (2);

[0077] (4) Based on the capillary electrophoresis results, analyze the length of the amplified fragment and calculate the number of trinucleotide repeats of (CTA·TAG)n and (CTG·CAG)n.

[0078] The first primer pair is used to detect the number of trinucleotide repeats in (CTA·TAG)n and (CTG·CAG)n. The primer sequences (P1 and P2) are conserved sequences flanking the repeat sequences. PCR amplification of the same template produces amplicons of the same length, allowing for quantitative calculation of the number of trinucleotide repeats. However, repeat sequences that are too long cannot be amplified. The amplification principle is explained in [link to amplification principle]. Figure 1 .

[0079] The second pair of primers is used for the relative quantitative or qualitative detection of longer or very long (CTA·TAG)n and (CTG·CAG)n trinucleotide repeat sequences: (i) such as Figure 2 and Figure 3 As shown, the present invention replaces the three primers (PI, PII, and PIII) required for repeat primer amplification with two primers (P1 and P3); (ii) The advantages of the P3 primer of the present invention are: it can be completely complementary to the target sequence for amplification, and the primer length is shorter than that of PIII for repeat primer PCR, making it easier to bind to the template and amplify; (iii) The P3 primer of the present invention replaces the PII and PIII primers of the repeat primer; (iv) The P3 primer of the present invention consists of 5 CAG trinucleotides plus 11 conserved bases adjacent to the 3′ flanking ends. The amplification principle of the second primer pair of this invention is as follows: PCR amplification is performed using two primers, one of which is a fluorescently labeled upstream primer (P1), and the other is a primer (P3) consisting of 5 trinucleotide repeats (such as CAG) plus more than 10 bases adjacent to the 3′ end of the repeat sequence. The two primers are added to the amplification system in a similar proportion. In each PCR cycle, primers P1 and P3 amplify a set of PCR products with a length difference of 3 bp. The products then become the templates for the next amplification cycle. After 20 to 30 PCR cycles, the requirement for detecting ultra-long repeat sequences is met, and multiple amplicons with a length difference of 3 bp are generated.

[0080] In one specific embodiment, in step (2), the PCR amplification system of the first primer pair is as follows: taking a total PCR reaction system of 25 μL as an example, 10×dNTP 1µL, 10×PCR buffer 2.5µL, DdH2O about 16.0µL (to bring the total volume to 25µL), DMSO 1.5~2.5µL, Taq enzyme 0.5µL, primers P1 / P2 1µL each, template DNA 1µL, and the total system is 25µL.

[0081] The PCR amplification system for the second primer pair was AmpliTaq Gold 360 Master Mix, with 5% GCEnhancer and 5%-10% DMSO added, for a total amplification volume of 25µL.

[0082] In one specific embodiment, in step (2), the concentration of the primer is 10 μmol / L.

[0083] In one specific embodiment, in step (2), the DNA concentration of the DNA template is 10–30 ng / µL.

[0084] In one specific embodiment, in step (2), the PCR amplification procedure using the first primer pair and the second primer pair is both landing PCR, wherein the landing PCR includes:

[0085] (a) High-temperature pre-denaturation step: Heat the reaction system to a temperature sufficient to completely denature the template DNA and maintain the denaturation time for 3 to 10 minutes;

[0086] (b) Landing amplification phase: Perform the first number of cycles; each cycle consists of the following steps: denaturation at a denaturing temperature for 1–2 minutes, annealing at a temperature higher than the primer melting temperature, with the annealing temperature gradually decreasing in each cycle; and extension at a temperature at which the DNA polymerase is catalytically active.

[0087] (c) Conventional amplification phase: Perform the second number of cycles; each cycle consists of the following steps: denaturation at denaturation temperature for 1 to 2 minutes, annealing at a final annealing temperature of 60 to 50°C, and extension at 68 to 72°C.

[0088] (d) Finally, maintain at 68–72°C for 5–10 minutes for the final extension time;

[0089] The first number of cycles is 10 to 15 times; the second number of cycles is 20 to 30 times.

[0090] In one specific embodiment, the landing PCR includes:

[0091] (a) Pre-denaturation at 95℃ for 5 minutes;

[0092] (b) Then, a 10-15 cycle of descent phase is performed, each cycle consisting of denaturation at 95°C for 1 minute, annealing at 68-55°C for 2 minutes, and extension at 72°C for 2-3 minutes, wherein the annealing temperature is decreased in each cycle;

[0093] (c) Then perform 20 to 30 cycles, each cycle consisting of denaturation at 95°C for 1 minute, annealing at 55°C for 2 minutes, and extension at 72°C for 2 to 3 minutes;

[0094] (d) Finally, extend at 72°C for 5 to 10 minutes.

[0095] Number of repetitions = (length of amplified fragment - length of conserved sequence) / 3;

[0096] The conserved sequence length was 231 bp when amplified using the first primer pair and 62 bp when amplified using the second primer pair.

[0097] When multiple ladder-like amplified fragments differing by 3 bp are obtained using the second primer pair, the longest amplified fragment is selected and substituted into the formula for calculation. In one specific implementation, it is recommended to use GS 1200 LIZ as a fragment length standard for capillary electrophoresis.

[0098] The specific embodiments of the present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the embodiments described herein.

[0099] Example 1

[0100] Primer sequence

[0101] The primer sequences used in this invention are shown in Table 1 below. All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0102] Table 1

[0103]

[0104] Example 2

[0105] Except for the primers, all other reagents used in the test are commercially available, and their composition is as follows:

[0106] 1. First primer pair: Primers (P1 / P2) containing the sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2, with a concentration of 10 μM for each primer.

[0107] 2. Second primer pair: Primers (P1 / P3) containing the sequences shown in SEQ ID NO. 1 and SEQ ID NO. 3, with a concentration of 10 μM for each primer.

[0108] 3. AmpliTaq Gold 360 Master Mix

[0109] 4. 10×dNTP

[0110] 5. 10×PCR buffer: contains Mg + Tris-HCl, etc.

[0111] 6. GC Enhancer.

[0112] 7. DMSO.

[0113] 8. Ultrapure water.

[0114] Example 3

[0115] Methods for detecting the number of repeats (CTA·TAG)n and (CTG·CAG)n in the ATXN8OS and ATXN8 genes

[0116] This embodiment provides a detailed explanation using three samples (A, B, and C) from one family and one positive control sample (D).

[0117] I. Genomic DNA Extraction from Samples

[0118] Peripheral blood was collected from each subject, with 0.2 mL of peripheral blood sample taken. DNA was extracted according to the instructions of the blood genomic DNA extraction kit. The extracted DNA sample was measured using a micro-ultraviolet spectrophotometer, and the concentration was diluted to 10-30 ng / μL for later use. This DNA sample served as a template for PCR reactions.

[0119] II. PCR amplification of the (CTA·TAG)n and (CTG·CAG)n trinucleotide repeat regions of the ATXN8OS and ATXN8 genes.

[0120] The design and synthesis of primers for amplifying the (CTA·TAG)n and (CTG·CAG)n trinucleotide repeat regions of the ATXN8OS and ATXN8 genes are shown in Table 2.

[0121] Table 2

[0122]

[0123] The PCR reaction reagents and system configuration for amplicon 1 (P1 / P2) amplification included: 1µL of 10×dNTP, 2.5µL of 10×PCR buffer, 16.5µL of DdH2O, 1.5µL of DMSO, 0.5µL of Taq enzyme, 1µL each of primers P1 and P2, and 1µL of template DNA, for a total system volume of 25µL.

[0124] The PCR reaction reagents and system configuration for amplicon 2 (P1 / P3) amplification include: AmpliTaq Gold TM The total volume of the mixture is 25 µL, consisting of 12.5 µL of 360Master Mix, 1.2 µL of GC Enhancer, 6.3 µL of DdH2O, 1.5 µL of DMSO, 0.5 µL of Taq enzyme, 1 µL each of primers P1 and P3, and 1 µL of template DNA.

[0125] The first primer pair (P1 / P2) and the second primer pair (P1 / P3) were amplified in different reaction systems to obtain amplicon 1 and amplicon 2.

[0126] The PCR amplification parameters for amplicon 1 and amplicon 2 are the same, and are set as follows:

[0127] 95℃ for 5 minutes, 1 cycle;

[0128] Denaturation at 95℃ for 1 min, annealing at 62-55℃ (fall PCR) for 2 min each, extension at 72℃ for 2 min, touch-down cycle number 10;

[0129] Denaturation at 95℃ for 1 min, annealing at 55℃ for 2 min, extension at 72℃ for 2 min, 25 cycles;

[0130] Extend at 72℃ for 7 minutes.

[0131] III. Capillary electrophoresis detection using ABI 3500Dx.

[0132] Take 0.5 μL of PCR product and mix it with 9.5 μL of fragment standard mixture (containing 9.0 μL HIDI and 0.5 μL GS600 LIZ standard or 0.5 μL GS 1200 LIZ standard). Perform capillary electrophoresis using an ABI 3500Dx gene analyzer. Results are shown below. Figures 4 to 11 .in, Figure 4 and 5 The capillary electrophoresis image shows the results after four samples were amplified using the first primer pair (P1 / P2) (i.e., amplicon 1) and the fragment standard GS 600 LIZ was added. Figure 6 and 7 The capillary electrophoresis image shows the results after amplification of four samples using the first primer pair (P1 / P2) (i.e., amplicon 1) with the addition of GS 1200 LIZ fragment standard. Figure 8 and 9 This is a capillary electrophoresis image of four samples after amplification using the second primer pair (P1 / P3) (i.e., amplicon 2); Figure 10The graph shows the results of two tests at different times for positive control sample D, demonstrating the good repeatability of the present invention. Figure 11 Table 5 shows the agarose gel electrophoresis images of amplicon 1 from samples A, B, and C. The first-generation sequencing results of the amplicon 1 products from samples A, B, and C are shown in Table 5. The first-generation sequencing results are consistent with the fragment analysis results (fragment standard: GS 600 LIZ) or differ by 1-2 replicates (fragment standard: GS 1200 LIZ), demonstrating that the detection accuracy is good and within the acceptable range. In the figure, A represents the sample from the family pedigree subject, B represents the sample from the mother of the family pedigree subject, C represents the sample from the father of the family pedigree subject, and D represents the positive control sample.

[0133] IV. Results Analysis

[0134] Fragment analysis was performed on the electrophoresis results using GeneMapper analysis software.

[0135] Results analysis: The number of trinucleotide repeats was calculated based on the difference between the length of the amplified fragment and the length of the conserved sequence in the amplicon reference sequence.

[0136] Calculation formula: Number of repetitions = (Length of amplified fragment - Length of conserved sequence) / 3;

[0137] Calculation reference: The conserved sequence length of amplicon 1 is 231 bp; the conserved sequence length of amplicon 2 is 62 bp. Note: Amplicon 2 consists of multiple fragments differing by 3 bp; the amplified fragment length is calculated based on the longer fragment.

[0138] Figure 4-7 All results are capillary electrophoresis results of PCR products from amplicon 1 of the four samples. The difference is: Figure 4 and 5 The standard for the four sample products was calculated to be GS 600 LIZ when a fragment was added during capillary electrophoresis. Figure 6 and 7 The standard for the four sample products was calculated to be GS 1200 LIZ when the fragment was added during capillary electrophoresis.

[0139] according to Figure 4-7 It is known that for the same sample, a difference of 4-5 bp in fragment length and a difference of 1-2 trinucleotide repeats between different fragment standards will not affect the interpretation of the results. It is recommended to use GS 1200 LIZ standards for capillary electrophoresis, as its maximum standard fragment is 1200 bp, making it relatively accurate for calculating fragment sizes exceeding 700 bp (e.g., the 825.17 bp fragment in sample B). GS 600 LIZ standards have a maximum standard fragment size of 600 bp; for fragments larger than 600 bp, there are no comparable standard fragments, and the detection may be affected by limitations imposed by the electrophoresis parameters (e.g., sample B only shows a 307.77 bp fragment). Figure 4-7 The specific calculation method and results of the number of repeats of the ATXN8OS and ATXN8 genes (CTA·TAG)n and (CTG·CAG)n (written as "(CTA·TAG)n(CTG·CAG)n" in the table) are shown in Table 3.

[0140] Table 3

[0141]

[0142] Note: Because a normal gene has two alleles, heterozygosity amplifies two fragment lengths, namely allele 1 and allele 2. The two columns of each test result represent the test results of allele 1 and allele 2, respectively.

[0143] Figure 8-9 The capillary electrophoresis results of PCR products of amplicon 2 from four samples are shown below. Figure 8-9 It can be seen that the improved repeat primer method (second primer pair amplification) can qualitatively detect ultra-long repeat fragments with good amplification effect, and is an effective supplement to the first primer pair (as can be seen from the peak diagram of sample A, there are long fragments that the first primer pair cannot amplify). The specific calculation process and results of the capillary sample repeat number are shown in Table 4.

[0144] Table 4

[0145]

[0146] Figure 10 The capillary electrophoresis results for positive control sample D at two different time points show a fragment length difference of less than 1 bp. Figures A and B are capillary electrophoresis images of amplicon 1 at two different time points, and figures C and D are capillary electrophoresis images of amplicon 2 at two different time points. This demonstrates the good repeatability and stability of this detection method.

[0147] Figure 11 The agarose gel electrophoresis images of amplicon 1 of samples A, B, and C are shown in Table 5. The purified products with a band of about 300 bp were analyzed by first-generation sequencing. The repeat counts of (CTA·TAG)n and (CTG·CAG)n are shown in Table 5. Compared with the detection method of the present invention (Table 3), the repeat counts of trinucleotides are consistent (fragment standard LIZ600) or differ by 1-2 times (fragment standard LIZ1200), which is within the allowable range. Therefore, the detection method of the present invention has good detection accuracy.

[0148] Table 5

[0149]

Claims

1. A primer set for detecting the number of trinucleotide repeats (CTA·TAG)n and (CTG·CAG)n in the ATXN8OS and ATXN8 genes, characterized in that, It contains a first primer pair and a second primer pair; The forward primer sequence of the first primer pair is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2; The forward primer sequence of the second primer pair is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.

3.

2. The primer set according to claim 1, characterized in that, The primer shown in SEQ ID NO.1 is labeled with a fluorescent dye at its 5′ end.

3. A kit for detecting the number of trinucleotide repeats (CTA·TAG)n and (CTG·CAG)n in the ATXN8OS and ATXN8 genes, characterized in that, It includes the primer set as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, It also includes PCR reaction reagents, which include at least one of DNA polymerase, PCR buffer, dNTPs, GC enhancer, and dimethyl sulfoxide.

5. A method for detecting the number of trinucleotide repeats (CTA·TAG)n and (CTG·CAG)n in the ATXN8OS and ATXN8 genes for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Provide genomic DNA from the subject's sample; (2). Using genomic DNA as a template, PCR amplification is performed using the first primer pair and the second primer pair as described in claim 1 or 2, respectively, to obtain amplification products; (3) Perform capillary electrophoresis analysis on the amplification products from step (2); (4) Based on the capillary electrophoresis results, analyze the length of the amplified fragment and calculate the number of trinucleotide repeats of (CTA·TAG)n and (CTG·CAG)n.

6. The method according to claim 5, characterized in that, In step (2), the concentration of the primer is 10 μmol / L.

7. The method according to claim 5, characterized in that, In step (2), the DNA concentration of the DNA template is 10–30 ng / uL.

8. The method according to claim 5, characterized in that, In step (2), the procedure for PCR amplification using the first primer pair and the second primer pair is landing PCR, which includes: (a) High-temperature pre-denaturation step: Heat the reaction system to a temperature sufficient to completely denature the template DNA and maintain the denaturation time for 3 to 10 minutes; (b) Landing amplification phase: Perform the first number of cycles; each cycle consists of the following steps: denaturation at a denaturing temperature for 1–2 minutes, annealing at a temperature higher than the primer melting temperature, with the annealing temperature gradually decreasing in each cycle; and extension at a temperature at which the DNA polymerase is catalytically active. (c) Conventional amplification phase: Perform the second number of cycles; each cycle consists of the following steps: denaturation at denaturation temperature for 1 to 2 minutes, annealing at a final annealing temperature of 60 to 50°C, and extension at 68 to 72°C. (d) Finally, maintain at 68–72°C for 5–10 minutes for the final extension time; The first number of cycles is 10 to 15 times; the second number of cycles is 20 to 30 times.

9. The method according to claim 5, characterized in that, In step (4), the formula for calculating the number of trinucleotide repeats is: Number of repetitions = (length of amplified fragment - length of conserved sequence) / 3; The conserved sequence length was 231 bp when amplified using the first primer pair and 62 bp when amplified using the second primer pair. When multiple ladder-like amplification fragments differing by 3 bp are obtained by using the second primer pair, the longest amplification fragment is selected and substituted into the formula for calculation.

10. The method according to claim 5, characterized in that, In step (3), GS 1200 LIZ was used as a fragment length standard for capillary electrophoresis.

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