Primer group, kit and method for detecting ATXN3 gene (CAG) n trinucleotide repetition number

Abstract

The invention relates to a primer group, a kit and a method for detecting an ATXN3 gene (CAG) n trinucleotide repetition number, and belongs to the technical field of gene detection. The technical problem to be solved by the invention is to provide the primer group for detecting the n trinucleotide repetition number of the ATXN3 gene (CAG). The primer group comprises a first primer pair and a second primer pair, a forward primer sequence of the first primer pair is as shown in SEQ ID NO. 1, and a reverse primer sequence of the first primer pair is as shown in SEQ ID NO. 2; a forward primer sequence of the second primer pair is as shown in SEQ ID NO. 3, and a reverse primer sequence of the second primer pair is as shown in SEQ ID NO. 4. Through the design of the two pairs of primers, complementary verification is achieved, the detection result is comprehensive and reliable, the technical defects of first-generation sequencing and second-generation sequencing on long-fragment and high-GC-content repetitive sequence detection are overcome, the detection cost is low, the detection period is short, and the primer pair is suitable for clinical detection service.

Description

TECHNICAL FIELD

[0001] The present application relates to a primer set, a kit and a method for detecting the number of (CAG)n trinucleotide repeats of the ATXN3 gene, belonging to the technical field of gene detection. BACKGROUND

[0002] Spinocerebellar ataxia (SCA) is a group of genetic neurological diseases with progressive motor coordination dysfunction and balance disorder as the main clinical manifestations. There are about 50 subtypes, among which SCA3 is the subtype with the highest incidence worldwide, accounting for 20-50%.

[0003] SCA3, also known as Machado-Joseph disease (MJD), is an autosomal dominant progressive neurodegenerative disease, mainly manifested as progressive cerebellar ataxia, including drunken gait, clumsy movement, poetic language, nystagmus, diplopia, pyramidal tract damage, including hyperreflexia, ankle clonus, and positive pathological signs; extrapyramidal symptoms, including Parkinson's syndrome, dystonia, rigidity, etc. Patients have a large difference in age of onset, usually adult onset, gradually progressing, and there is no accurate data on the incidence, which is a rare disease. SCA3 progresses gradually after onset, and the symptoms of ataxia in limbs and gait become obvious, and walking becomes more and more difficult, usually 10-15 years after onset, requiring auxiliary equipment or even being unable to walk; in the late stage of the disease, dysphagia, weak cough, malnutrition or aspiration are the main causes of death, and a study shows that the average survival time after onset is 21 years. At present, there is no specific treatment method, and the main treatment is symptomatic support, intensive care and rehabilitation training. Genetic studies have shown that the expansion of the number of (CAG)n trinucleotide repeats of the ATXN3 gene is the genetic cause of SCA3.

[0004] SCA3 not only causes serious physical pain and mental stress to patients, but also brings heavy economic burden and emotional test to patient families. Therefore, genetic testing of patients or family members with reproductive needs can determine the cause of the disease and provide prognosis; providing prenatal diagnosis or preimplantation genetic testing for couples carrying abnormal variations can provide scientific guidance for the birth of healthy next generation.

[0005] The ATXN3 gene is located on the long arm of human chromosome 14, region 3, band 2, and subband 1 (14q32.12). The gene is approximately 61 kb in length and contains 11 exons (reference genome: GRCh38 / hg38, reference transcript: NM_004993.6). Exon 10 of the ATXN3 gene contains a (CAG)n trinucleotide repeat sequence encoding polyglutamine. Abnormal amplification of the (CAG)n trinucleotide leads to an abnormal increase in the polyglutamine chain (PolyQ) near the carboxyl terminus of the encoded protein, resulting in disease. In normal individuals, the number of (CAG)n repeats in exon 10 of the ATXN3 gene is typically 12-44, while in patients it is typically 52-86, with intermediate repeat counts of 45-51 (which may be asymptomatic or have incomplete penetrance, but the repeat sequence is unstable; when passed on to the next generation, the increased repeat count can develop into the pathogenic repeat range). The ATXN3 gene has a high GC content in the (CAG)n trinucleotide repeat sequence in exon 10. The biallelic gene is mostly heterozygous. When it expands abnormally, first-generation sequencing analysis is difficult or impossible. At the same time, this region is a repetitive sequence region, and second-generation sequencing cannot accurately detect it or cannot detect it at all.

[0006] Due to the characteristics of the (CAG)n trinucleotide repeat sequence in exon 10 of the ATXN3 gene and its low incidence, no commercially available kits have been found. Currently, the recommended primary detection method in the literature is fluorescent PCR-capillary electrophoresis for fragment length analysis. The principle involves using specific primers designed flanking the (CAG)n repeat region of the ATXN3 gene for polymerase chain reaction (PCR), with one primer labeled with fluorescence. After PCR amplification, capillary electrophoresis is performed. This recommended method is simple and rapid, but when amplifying a single normal fragment, it cannot distinguish whether it is a homozygous allele or a normal allele with an excessively long repeat that fails to amplify. First-generation sequencing may be inaccurate for extremely long (CAG)n repeat sequences and can be used as a supplementary method. Because this region is a repeat sequence, second-generation sequencing has limitations or cannot detect it.

[0007] Patent 200910182735.X discloses primers and a PCR amplification method for detecting dynamic mutations in the CAG repeat sequence of the ATXN3 gene. This method uses a specific pair of primers for PCR amplification, followed by gel electrophoresis. The quantity and size of the products are observed to determine the dynamic mutation status of the CAG repeat sequence in the ATXN3 gene. This method estimates fragment size through gel electrophoresis, but is affected by primer sequences or electrophoresis conditions, resulting in low fragment resolution, inability to distinguish similar bands, and large calculation errors. Furthermore, the CAG repeat sequence of the ATXN3 gene has a high GC content and easily forms secondary structures; therefore, even with a specific pair of primers for amplification, the aforementioned inaccurate results still exist, and the precision and reliability need further improvement. Summary of the Invention

[0008] This invention aims to overcome the shortcomings of existing technologies and provide a primer set, kit, and method for accurately detecting the number of trinucleotide repeats in the ATXN3 gene (CAG)n. The first technical problem solved by this invention is to provide a primer set for detecting the number of trinucleotide repeats in the ATXN3 gene (CAG)n.

[0009] The primer set of the present invention for detecting the number of trinucleotide repeats of the ATXN3 gene (CAG)n includes 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. 3, and the reverse primer sequence is shown in SEQ ID NO. 4.

[0010] The first primer pair is used to amplify the target fragment containing the CAGn trinucleotide repeat region of exon 10 of the ATXN3 gene and its 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 genomic location is chr14:92070901_92071204.

[0011] The second primer pair is a triplet-like primer pair, used to amplify the core region of the (CAG)n trinucleotide repeat sequence and generate a ladder-like amplicon with a 3bp difference. Its forward primer sequence is shown in SEQ ID NO. 3, and its reverse primer sequence is shown in SEQ ID NO. 4. Its amplification reference genome position is chr14:92070999_92071170.

[0012] The second technical problem solved by this invention is to provide a kit for detecting the number of trinucleotide repeats in the ATXN3 gene (CAG)n.

[0013] The present invention provides a kit for detecting the number of trinucleotide repeats in the ATXN3 gene (CAG)n, comprising the primer set described above.

[0014] Preferably, the concentration of each primer in the primer set is 10 μM.

[0015] Preferably, the kit further includes components necessary for the PCR reaction, namely PCR reaction reagents. Preferably, the PCR reaction reagents include 2×F-PCR Master Mix, GC Enhancer, and DMSO, wherein the 2×F-PCR Master Mix includes DNA polymerase, buffer, and dNTPs.

[0016] The DNA polymerase is preferably a mixture that has rapid elongation capability and is suitable for templates with high GC content.

[0017] The present invention also provides a method for in vitro detection of the number of trinucleotide repeats of the ATXN3 gene (CAG)n for non-disease diagnostic purposes.

[0018] This invention discloses a method for in vitro detection of the trinucleotide repeat number of the ATXN3 gene (CAG)n for non-disease diagnostic purposes, comprising the following steps:

[0019] (1) Provide a genomic DNA sample from the subject;

[0020] (2) Using genomic DNA as a template, PCR amplification is performed using the first primer pair and the second primer pair as described in any one of claims 1 to 3 to obtain amplification products;

[0021] (3) Perform capillary electrophoresis analysis on the amplification products obtained in step (2);

[0022] (4) Based on the capillary electrophoresis results, analyze the length of the fragments and calculate the number of repeats of (CAG)n trinucleotides.

[0023] In one embodiment of the present invention, in step (2), the PCR amplification system is as follows: based on a total PCR reaction system of 25 μL, it includes 12.5 μL of 2×F-PCR Master Mix, 2 μL of primers, 1 μL of DNA template, 2 μL of GC Enhancer, 2 μL of DMSO, and ultrapure water to make up to 25 μL; the primers are either the first primer pair or the second primer pair, and the concentration of the primers is 10 μmol / L; the concentration of the DNA template is 10–20 ng / μL.

[0024] In one embodiment of the present invention, in step (2), the procedure for PCR amplification using the first primer pair includes: pre-denaturation at 95°C for 2 minutes; followed by 20 to 30 cycles, each cycle including denaturation at 95°C for 1.5 minutes, annealing at 55 to 62°C for 1.5 minutes, extension at 72°C for 3 minutes; and finally final extension at 72°C for 10 minutes.

[0025] The PCR amplification procedure using the second primer pair is a landing PCR, which includes: pre-denaturation at 95°C for 5 minutes; followed by 10–15 landing phase cycles, each cycle including denaturation at 95°C for 1 minute, annealing at 65–58°C for 2 minutes, and extension at 72°C for 3 minutes, wherein the annealing temperature is decreased in each cycle; followed by extension at 72°C for 3 minutes; followed by 20–25 cycles, each cycle including denaturation at 95°C for 1 minute, annealing at 58°C for 2 minutes, and extension at 72°C for 3 minutes; and finally, a final extension at 72°C for 7 minutes.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) Comprehensive and reliable detection results: This invention achieves complementary verification through the design of two primer pairs. The first primer pair (P1 / P2) amplifies the conserved sequences on both sides of the (CAG)n trinucleotide repeat region. The amplification product contains the entire sequence of the (CAG)n repeat region, which can be relatively quantified and the number of (CAG)n repeats can be calculated. The second primer pair (triple primer-like P3 / P4) has one side of the primer located on a conserved sequence and the other side contains part of the conserved sequence and part of the repeat sequence. The amplification product consists of multiple amplicons that differ by 3 nucleotides, which has a similar amplification effect to the triple primer and produces ladder-like amplicons. This effectively avoids amplification failure caused by excessive repeat times and ensures the detection of large fragment alleles.

[0028] (2) Significant technical advantages: The detection results of this invention make up for the technical deficiencies of first-generation sequencing and second-generation sequencing in the detection of long fragments and high GC content repetitive sequences. Moreover, the detection cost is low and the detection cycle is short, making it suitable for clinical testing services. Attached Figure Description

[0029] Figure 1 The capillary electrophoresis diagram of amplicon 1 in Example 3 of the present invention is as follows: A is a homozygous allele sample (normal), B is a family sample - husband (pathogenic (CAG)n duplicate, heterozygous), C is a family sample of wife (normal, heterozygous), D is a positive control sample (pathogenic (CAG)n duplicate, heterozygous), and E is a negative control sample (normal, heterozygous).

[0030] Figure 2 The capillary electrophoresis diagram of amplicon 2 in Example 3 of the present invention is as follows: A is a homozygous allele sample (normal), B is a family sample - husband (pathogenic (CAG)n duplicate, heterozygous), C is a family sample of wife (normal, heterozygous), D is a positive control sample (pathogenic (CAG)n duplicate, heterozygous), and E is a negative control sample (normal, heterozygous).

[0031] Figure 3 The images show the results of three different time-time tests on negative control sample E in Example 3 of this invention; the left side shows the capillary electrophoresis image of amplicon 1, and the right side shows the capillary electrophoresis image of amplicon 2.

[0032] Figure 4 The images show the results of three different detection times for positive control sample D in Example 3 of this invention; the left side shows the capillary electrophoresis image of amplicon 1, and the right side shows the capillary electrophoresis image of amplicon 2.

[0033] Figure 5This is a diagram showing the first-generation sequencing results of the amplification products of homozygous allele sample A and negative control sample E in Example 3 of the present invention. Detailed Implementation

[0034] 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.

[0035] Example 1 Primer Sequence

[0036] The primer sequences used in this invention are shown in Table 1 below. All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0037] Table 1

[0038]

[0039] Example 2 Composition of the detection kit

[0040] The detection kit of the present invention comprises the following components:

[0041] 1. Primer Mixture 1: Primers containing the sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2, with a concentration of 10 μM for each primer.

[0042] 2. Primer mixture 2: Primers containing the sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4, with a concentration of 10 μM for each primer.

[0043] 3. 2×F-PCR Master Mix: Contains fast, high-fidelity DNA polymerase, buffer, and dNTPs.

[0044] 4. GC Enhancer.

[0045] 5. DMSO.

[0046] 6. Ultrapure water.

[0047] Example 3: Method for detecting the number of repeats in the ATXN3 gene (CAG)n

[0048] This embodiment provides a detailed description using one homozygous allele sample, one family (couple) sample, one positive control sample, and one negative control sample.

[0049] I. Genomic DNA Extraction from Samples

[0050] 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 NanoDrop ultraviolet spectrophotometer, and the concentration was diluted to 10-20 ng / μL for later use. This DNA sample served as a template for the F-PCR reaction.

[0051] II. F-PCR amplification of the (CAG)n trinucleotide repeat region of exon 10 of the ATXN3 gene

[0052] The primers for amplifying the trinucleotide repeat region of exon 10 (CAG)n of the ATXN3 gene are designed and synthesized as shown in Table 2.

[0053] Table 2

[0054]

[0055] The PCR reaction reagents for amplifying the ATXN3 gene (CAG)n trinucleotide sequence include a 2×F-PCR Master Mix, which contains DNA polymerase, buffer, and dNTPs; GC Enhancer; DMSO (dimethyl sulfoxide); primers Mix 1 (P1 / P2) and Mix 2 (P3 / P4). The total PCR reaction volume is 25 μL, including 12.5 μL of 2×F-PCR Master Mix, 2 μL of primer Mix 1 or primer Mix 2 (concentration 10 μmol / L), 1 μL of DNA template (DNA amount 10-20 ng), 2 μL of GC Enhancer, 2 μL of DMSO, and ultrapure water to a final volume of 25 μL.

[0056] The first primer pair (P1 / P2) and the second primer pair (P3 / P4) need to be amplified separately in different reaction tubes to obtain amplicon 1 and amplicon 2.

[0057] Amplicon 1 PCR cycling parameter settings:

[0058] 95℃ for 2 min, 1 cycle;

[0059] Denaturation at 95℃ for 1.5 min, annealing at 58℃ for 1.5 min, extension at 72℃ for 3 min, 20-30 cycles;

[0060] Extend at 72℃ for 10 minutes.

[0061] Amplicon 2 PCR cycling parameter settings:

[0062] 95℃ for 5 minutes, 1 cycle;

[0063] Denaturation at 95℃ for 1 min, annealing at 65-58℃ (fall PCR) for 2 min each, extension at 72℃ for 3 min, touch-down cycle number 10-15;

[0064] Extend at 72℃ for 3 minutes;

[0065] Denaturation at 95℃ for 1 min, annealing at 58℃ for 2 min, extension at 72℃ for 3 min, cycle number 20-25;

[0066] Extend at 72℃ for 7 minutes.

[0067] III. Capillary electrophoresis detection using ABI 3500Dx.

[0068] Take 0.5 μL of F-PCR product and mix it with 9.5 μL of molecular weight standard mixture (containing 9.0 μL HIDI and 0.5 μL LIZ600 standard). Perform capillary electrophoresis using an ABI 3500Dx gene analyzer. Results are shown below. Figures 1 to 5 .in, Figure 1 This is a capillary electrophoresis image of five samples amplified using the first primer pair (P1 / P2) (i.e., amplicon 1). Figure 2 This is a capillary electrophoresis image of five samples amplified using the second primer pair (P3 / P4) (i.e., amplicon 2). Figure 3 The results of three tests at different times for negative control sample E demonstrate that the present invention has good repeatability. Figure 4 The results of three tests at different times for positive control sample D demonstrate that the present invention has good repeatability. Figure 5 First-generation sequencing was performed on the amplification products of homozygous allele sample A and negative control sample E. The difference between the first-generation sequencing results and the fragment analysis results was one replicate, proving that the accuracy of this test is within the acceptable range. In the figure, A is the homozygous allele sample, B is the husband of the family sample (pathogenic (CAG)n replicate), C is the wife of the family sample, D is the positive control sample, and E is the negative control sample.

[0069] IV. Results Analysis

[0070] Fragment analysis was performed on the electrophoresis results using GeneMapper analysis software.

[0071] Results analysis: The number of repeats of (CAG)n trinucleotides 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.

[0072] Calculation formula: (CAG)n repeat number = (amplifier fragment length - number of conserved amino acids) / 3.

[0073] Calculation reference: The number of conserved amino acids in amplicon 1 is 262 bp; the number of conserved amino acids in amplicon 2 is 130 bp.

[0074] Note: Amplicon 2 consists of multiple fragments differing by 3 bp. It is recommended to calculate (CAG)n trinucleotides as long fragments.

[0075] Figure 1 and Figure 2 The specific calculation process and results of the number of repeats in sample (CAG)n are shown in Table 3.

[0076] Table 3

[0077]

[0078] Figure 3 The difference in fragment length in negative control sample E at three different time points was less than 1 bp. Figure 4 The difference in fragment length between three detection times for positive control sample D was less than 2 bp (long amplicon fragments were not included in the comparison due to their large number), which demonstrates the good repeatability of this detection method.

[0079] Figure 5 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 The homozygous allele sample A had a CAG n repeat count of 14 in its first-generation sequencing results, while the negative control sample E had a CAG n repeat count of 19 / 27. Compared with the detection method of the present invention, the CAG n repeat count differs by 1, which is within the allowable range. Therefore, the detection method of the present invention has good accuracy.

[0080] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A primer set for detecting the number of (CAG)n trinucleotide repeats of the ATXN3 gene, characterized by: The primer set comprises a first primer pair and a second primer pair, The forward primer sequence of the first primer pair is shown as SEQ ID NO. 1, and the reverse primer sequence is shown as SEQ ID NO. 2; The forward primer sequence of the second primer pair is shown as SEQ ID NO. 3, and the reverse primer sequence is shown as SEQ ID NO.

4.

2. Kit for detecting the number of (CAG)n trinucleotide repeats of the ATXN3 gene, characterized by: The primer set comprises a first primer pair and a second primer pair, 3. The kit for detecting the number of (CAG)n trinucleotide repeats of the ATXN3 gene according to claim 2, characterized in that: The concentration of each primer in the primer set is 10 μM.

4. The kit for detecting the number of (CAG)n trinucleotide repeats of the ATXN3 gene according to claim 2, characterized in that: The PCR reaction reagent further comprises 2×F-PCR Master Mix, GC Enhancer and DMSO, wherein the 2×F-PCR Master Mix comprises DNA polymerase, buffer and dNTPs.

5. A method for detecting the number of (CAG)n trinucleotide repeats of the ATXN3 gene in vitro for non-disease diagnostic purposes, characterized by, The method comprises the following steps: (1) providing a genomic DNA sample from a subject; (2) using the first primer pair and the second primer pair of claim 1 to perform PCR amplification respectively with the genomic DNA as a template to obtain an amplification product; (3) performing capillary electrophoresis analysis on the amplification product obtained in step (2); (4) analyzing the length of the fragment and calculating the number of (CAG)n trinucleotide repeats according to the capillary electrophoresis result.

6. The method for in vitro detection of the number of (CAG)n trinucleotide repeats of the ATXN3 gene for non-disease diagnostic purposes according to claim 5, characterized by the fact that: In step (2), the PCR amplification system is as follows: taking the total PCR reaction system as 25 μL, comprising 2×F-PCR Master Mix 12.5 μL, primer 2 μL, DNA template 1 μL, GC Enhancer 2 μL, DMSO 2 μL, and ultrapure water to make up to 25 μL system; The primer is the first primer pair or the second primer pair, and the concentration of the primer is 10 μmol / L; the DNA concentration of the DNA template is 10-20 ng / uL.

7. The method for in vitro detection of the number of (CAG)n trinucleotide repeats of the ATXN3 gene for non-disease diagnostic purposes according to claim 6, characterized by the fact that: In step (2), The program for performing PCR amplification using the first primer pair comprises: 95℃ pre-denaturation for 2 minutes; then 20-30 cycles, each cycle comprising 95℃ denaturation for 1.5 minutes, 55-62℃ annealing for 1.5 minutes, and 72℃ extension for 3 minutes; finally, 72℃ final extension for 10 minutes; The program for performing PCR amplification using the second primer pair is drop PCR, comprising: 95℃ pre-denaturation for 5 minutes; then 10-15 cycles of drop stage, each cycle comprising 95℃ denaturation for 1 minute, 65-58℃ annealing for 2 minutes, and 72℃ extension for 3 minutes, wherein the annealing temperature decreases in each cycle; then 72℃ extension for 3 minutes; then 20-25 cycles, each cycle comprising 95℃ denaturation for 1 minute, 58℃ annealing for 2 minutes, and 72℃ extension for 3 minutes; finally, 72℃ final extension for 7 minutes.

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