CN113046434A - Primer pair, kit and detection method for SCA subtype gene detection - Google Patents

Abstract

The application belongs to the technical field of molecular biology, and particularly discloses a primer pair, a kit and a detection method for detecting SCA subtype genes. The primer pair includes one or more of primer pair 1 for ATXN1, primer pair 2 for ATXN2, primer pair 3 for ATXN3, primer pair 4 for CACNA1A, and primer pair 5 for ATXN 7. The application has at least one of the following beneficial effects: the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4 and the primer pair 5 can accurately detect the repeated conditions of the CAG in the genes ATXN1, ATXN2, ATXN3, CACNA1A and ATXN7 related to the SCA, and the primer pair has high specificity and high accuracy, and the error of the number of the repeated CAG is about plus or minus 1 time.

Description

Primer pair, kit and detection method for SCA subtype gene detection

Technical Field

The application belongs to the technical field of molecular biology, and particularly relates to a primer pair, a kit and a detection method for detecting an SCA subtype gene.

Background

Spinocerebellar ataxia (SCA) is a group of autosomal dominant hereditary neurological diseases that exhibit high clinical and genetic heterogeneity, mainly characterized by progressively aggravated ataxia of limbs, dysarthria, dysoculomotor, and the like. The pathogenesis molecular basis of SCA is mainly dynamic mutation, namely unstable continuous amplification of CAG end tandem repeat sequence copy number in disease genes, so that the coded abnormal protein containing polyglutamine finally causes cell apoptosis and neuron degeneration. The age difference of the SCA is not large, and the clinical symptoms overlap each other, so that the clinical typing is difficult to confirm, and the diagnosis of the Huntington's chorea, Multiple System Atrophy (MSA) and other neuromuscular diseases is difficult to distinguish.

At present, the disease diagnosis is carried out according to the medical history, physical signs, family history and imaging characteristics in clinic, and the genetic detection is needed for specific typing. Aiming at spinocerebellar ataxia, no specific treatment method exists, and the treatment method is mainly used for symptomatic treatment, so that the gene detection is not only beneficial to confirming the SCA subtype of a patient, but also can be used for presymptomatic diagnosis and early clinical intervention on family members of the patient, and provides a basis for prenatal diagnosis.

Several subtypes such as SCA 1-26 have been found so far according to the genotype classification. Epidemiological data display, SCA 3: (ATXN3) Is the most common subtype, accounting for about 54.6-72.5%, and is followed by SCA 1: (ATXN1) About 5.9%, SCA 2: (ATXN2) About 5.7-6.7%, SCA6 (C:)CACNA1A) And SCA7 (ATXN7) Respectively account for 1.6-3.3 percent and 0.8-4.8 percent, and the subtypes such as SCA8, SCA10, SCA17 and the like are not detected in China at present, so that the subtypes SCA3, SCA1, SCA2, SCA6 and SCA7 account for about 70-80 percent, and therefore, the five subtypes account forThe detection of the seed typing has important guiding significance for clinical medication.

Disclosure of Invention

The inventor finds that the existing SCA detection methods are all used for detecting single subtype of SCA due to the fact that CAG repetition number detection experiments are difficult and the gene characteristics related to different subtypes are different. When the subtype of a patient is confirmed, different subtype-related genes need to be detected one by one, and the experimental operation process is complicated and consumes a long time. Most of the existing CAG detection methods adopt a detection method combining PCR and agarose gel electrophoresis, and CAG is repeatedly estimated through an electrophoresis result, so that estimation has certain errors. Therefore, an object of the present invention is to provide primers for detecting the subtypes SCA3, SCA1, SCA2, SCA6, and SCA7, thereby reducing errors and improving accuracy.

The application is realized by the following scheme:

the first aspect of the application provides a primer pair for SCA subtype gene detection, which is characterized by comprising a primer pair for SCA subtype gene detectionATXN1Primer set 1, primer pairATXN2Primer set 2, primer pairATXN3Primer set 3, directed againstCACNA1APrimer pair 4 andATXN7wherein the nucleotide sequence of the upstream primer 1 of the primer pair 1 is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer 1 of the primer pair is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the upstream primer 2 of the primer pair 2 is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer 2 of the primer pair is shown as SEQ ID NO: 4 is shown in the specification; the nucleotide sequence of the upstream primer 3 of the primer pair 3 is shown as SEQ ID NO: 5, the nucleotide sequence of the downstream primer 3 of the primer pair is shown as SEQ ID NO: 6 is shown in the specification; the nucleotide sequence of the upstream primer 4 of the primer pair 4 is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer 4 of the primer pair is shown as SEQ ID NO: 8 is shown in the specification; the nucleotide sequence of the upstream primer 5 of the primer pair 5 is shown as SEQ ID NO: 9, the nucleotide sequence of the downstream primer 5 of the primer pair is shown as SEQ ID NO: shown at 10.

The primer pairs can be used in combination with each other for simultaneously detecting different SCA subtypes targeted by a plurality of primer pairs, and can also be used individually for detecting the SCA subtypes targeted by the primer pairs.

In a specific embodiment of the present application, the primer pair comprises a pairATXN1Primer pair 1 andATXN2primer set 2 of (4).

In one embodiment of the present application, the primer pair 1 and the primer pair 2 are linked to different fluorophores for simultaneous detectionATXN1AndATXN2。

in a specific embodiment of the present application, the primer pair comprises a pairATXN3Primer set 3, directed againstCACNA1APrimer pair 4 andATXN7the primer set 5.

In one embodiment of the present application, the primer pair 3, the primer pair 4 and the primer pair 5 are respectively connected with different fluorophores for simultaneous detectionATXN3、CACNA1AAndATXN7。

in a specific embodiment of the present application, the primer pair comprises a pairATXN1Primer set 1, primer pairATXN2Primer set 2, primer pairATXN3Primer set 3, directed againstCACNA1APrimer pair 4 andATXN7the primer set 5.

In one embodiment of the present application, the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4 and the primer pair 5 are respectively connected with different fluorophores for respectively detectingATXN1、ATXN2、ATXN3、CACNA1AAndATXN7。

another aspect of the invention provides for simultaneous detectionATXN1、ATXN2、ATXN3、CACNA1AAndATXN7the reagent comprises a primer pair group A and a primer pair group B, wherein the primer pair group A comprises the primer pair 1 and the primer pair 2, and the primer pair 1 and the primer pair 2 are respectively connected with different fluorescent groups; the primer pair group B comprises the primer pair 3, the primer pair 4 and the primer pair 5, and the primer pair 3, the primer pair 4 and the primer pair 5 are respectively connected with different fluorescent groups.

In another aspect, the present application provides a kit for detecting an SCA gene, which includes one or more of the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, or the primer pair 5.

In one embodiment of the present application, the primer pair is linked to a fluorophore.

In one embodiment of the present application, the kit comprises primer pair 1 and primer pair 2 for simultaneous detectionATXN1AndATXN2。

in one embodiment of the present application, the primer pair 1 and the primer pair 2 are respectively connected with different fluorophores.

In one embodiment of the present application, the kit comprises primer pair 3, primer pair 4 and primer pair 5 for simultaneous detectionATXN3、CACNA1AAndATXN7。

in one embodiment of the present application, the primer pair 3, the primer pair 4 and the primer pair 5 are respectively connected with different fluorophores.

In a specific embodiment of the present application, the kit includes a primer pair group a and a primer pair group B, the primer pair group a includes the primer pair 1 and the primer pair 2, and the primer pair 1 and the primer pair 2 are respectively connected to different fluorophores; the primer pair group B comprises the primer pair 3, the primer pair 4 and the primer pair 5, and the primer pair 3, the primer pair 4 and the primer pair 5 are respectively connected with different fluorescent groups.

In one embodiment of the present application, the kit further comprises a DNA polymerase.

In a specific embodiment of the present application, the DNA polymerase is a T6 enzyme.

In one embodiment of the present application, the kit further comprises dntps, PCR reaction buffer and Mg²⁺One or more of (a).

In a specific embodiment of the present application, the kit further comprises a wild-type control and/or a negative control.

In a specific embodiment of the present application, the wild-type control is a plasmid comprising five wild-type amplified target genes.

In one embodiment of the present application, the negative control is Tirs-EDTA buffer.

In a specific embodiment of the present application, the kit further comprises an internal standard.

In a specific embodiment of the present application, the internal standard is a GS500-LIZ internal standard.

In another aspect, the present application also provides a method for detecting SCA genotyping for non-disease diagnostic purposes, comprising the steps of:

s1: adding a sample to be detected into the primer pair, and carrying out PCR amplification on a corresponding gene sequence to obtain an amplification product;

s2: subjecting the amplification product of step S1 to capillary electrophoresis;

s3: the data and results in step S2 are analyzed.

In one embodiment of the present application, in step S1, the PCR amplification reaction conditions 1 of primer pair 1 and primer pair 2 are as follows: 5 minutes at 98 ℃; 30 seconds at 98 ℃ and 30 seconds at 63 ℃, and 20 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 12 cycles; finally 5 minutes at 72 ℃.

In one embodiment of the present application, in step S1, the PCR amplification reaction conditions 2 of primer pair 3, primer pair 4 and primer pair 5 are as follows: 5 minutes at 98 ℃; 10 cycles: 30 seconds at 98 ℃ and 30 seconds at 60 ℃, and 10 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 25 cycles: 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 25 cycles; finally 5 minutes at 72 ℃.

In one embodiment of the present application, the capillary electrophoresis of step S2 specifically includes the following steps:

s2-1: and (3) diluting an amplification product: diluting the amplification product;

s2-2: preparing a mixed solution: mixing a GS500-LIZ internal standard and highly deionized formamide (HIDI) according to a volume ratio of 1:90, and mixing the mixed solution and the diluted amplification product in the step S2-1 according to a ratio of V: V =9:1 to prepare an upper machine mixed solution;

s2-3: pre-denaturation: running the prepared on-machine mixed solution on a PCR instrument at 95 ℃ for 4 minutes, and rapidly cooling the mixed solution in an ice-water mixture for 5 minutes for denaturation treatment;

s2-4: capillary electrophoresis: the upper computer mixed liquor is detected in an ABI 3500xl Dx sequencer, grouping is carried out by adopting G5 color, and the typing parameters in the ABI 3500xl Dx sequencer are set as follows: detecting constant Temperature OVEN _ Temperature of 60 ℃ and detecting hole Temperature Detection Cell Temperature of 50 ℃; the pre-electrophoresis Voltage PreRun _ Voltage 19.5kV and the pre-electrophoresis Time PreRun _ Time 180 s; the Injection Voltage Injection _ Voltage is 1.2 kV; injection Time Injection _ Time is 10 s; the First reading First _ ReadOut _ Time 200 ms; second reading Second _ ReadOut _ Time 200 ms; the electrophoresis Voltage Run _ Voltage 19.5 kV; the Number of leap steps, Voltage _ Number _ of _ steps, is set to 30 steps; the kick Voltage _ steps _ Interval is set to 15 s; the Voltage Tolerance Voltage _ Tolerance is set to 0.7 kV; date Delay Data _ Delay 1 s; run _ Time 1330s at electrophoresis.

In one embodiment of the present application, the data analysis in step S3 uses genemarker2.2 software to interpret the typing results, and the analysis method uses the software default settings (GS 500).

In one embodiment of the present application, the method further includes step S0: sample genomic DNA was prepared.

In one embodiment of the present application, the sample is derived from peripheral blood or various tissues, such as skin, liver, kidney, etc., as long as the sample contains genomic DNA.

In one embodiment of the present application, the sample has a concentration of 50-100 ng/. mu.L.

In one embodiment of the present application, a simultaneous detection is providedATXN1AndATXN2the method specifically comprises the following steps:

s0: preparing sample genome DNA, and taking Tirs-EDTA buffer solution as a genome DNA solvent;

s1: adding primer pair 1, primer pair 2 (TMR labeled primer pair 1; HEX labeled primer pair 2), T6 enzyme, dNTP, PCR reaction buffer and MgCl into sample genome DNA₂And (3) PCR amplification:

the PCR amplification reaction conditions 1 were as follows: 5 minutes at 98 ℃; 30 seconds at 98 ℃ and 30 seconds at 63 ℃, and 20 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 12 cycles; finally 5 minutes at 72 ℃;

s2-1: adding deionized water into the amplification product for dilution;

s2-2: mixing an LIZ internal standard and highly deionized formamide (HIDI) according to a volume ratio of 1:90, and mixing the mixed solution and the diluted amplification product in the step S2-1 according to a ratio of V: V =9:1 to prepare an upper machine mixed solution;

s2-3: pre-denaturation: running the prepared on-machine mixed solution on a PCR instrument at 95 ℃ for 4 minutes, and rapidly cooling the mixed solution in an ice-water mixture for 5 minutes for denaturation treatment;

s2-4: capillary electrophoresis: the upper computer mixed liquor is detected in an ABI 3500xl Dx sequencer, grouping is carried out by adopting G5 color, and the typing parameters in the ABI 3500xl Dx sequencer are set as follows: detecting constant Temperature OVEN _ Temperature of 60 ℃ and detecting hole Temperature Detection Cell Temperature of 50 ℃; the pre-electrophoresis Voltage PreRun _ Voltage 19.5kV and the pre-electrophoresis Time PreRun _ Time 180 s; the Injection Voltage Injection _ Voltage is 1.2 kV; injection Time Injection _ Time is 10 s; the First reading First _ ReadOut _ Time 200 ms; second reading Second _ ReadOut _ Time 200 ms; the electrophoresis Voltage Run _ Voltage 19.5 kV; the Number of leap steps, Voltage _ Number _ of _ steps, is set to 30 steps; the kick Voltage _ steps _ Interval is set to 15 s; the Voltage Tolerance Voltage _ Tolerance is set to 0.7 kV; date Delay Data _ Delay 1 s; run _ Time 1330s during electrophoresis;

s3: and (3) data analysis: judging the typing result in GeneMarker2.2 software, adopting the default setting (GS 500) of the software for the analysis method, and defining after checking and correcting the internal standard peak:

ATXN1gene: TMR mark, loading the electrophoresis data 'show color' of the sample PCR amplification system A to be analyzed to select 'yellow'; marking the position of 146bp as a CAG repeat number 1, judging that the CAG repeat number corresponding to the first highest peak after the CAG repeat number 1 is the first allele according to the number of fluorescence cluster peaks appearing at about every 3bp, and selecting the CAG repeat number corresponding to the highest peak in five-finger peaks if the fluorescence cluster peaks existPlural as the second allele according toATXN1Calculating the number of CAG repeats of the numerical values corresponding to the two peaks of the gene;

ATXN2gene: HEX mark, loading electrophoresis data 'show color' of the sample PCR amplification system A to be analyzed to select 'green'; the position of 186bp is marked with CAG repetition number 1, and the CAG repetition number corresponding to the first highest peak after the CAG repetition number 1 is judged as the first allele according to the number of fluorescence cluster peaks appearing at about every 3 bp. Then if there is a fluorescence peak, selecting the CAG repeat number corresponding to the highest peak in the five-finger peak as the second allele according toATXN2Calculating the number of CAG repeats of the numerical values corresponding to the two peaks of the gene.

In one embodiment of the present application, a simultaneous detection is providedATXN3、CACNA1AAndATXN7the detection method specifically comprises the following steps:

s0: preparing sample genome DNA, and taking Tirs-EDTA buffer solution as a genome DNA solvent;

s1: the above primer set 3, primer set 4 and primer set 5 (FAM-labeled primer set 3; ROX-labeled primer set 4; TMR-labeled primer set 5), T6 enzyme, dNTP, PCR reaction buffer and MgCl were added to the genomic DNA of the sample₂And (3) PCR amplification:

PCR amplification reaction conditions 2 were as follows: 5 minutes at 98 ℃; 10 cycles: 30 seconds at 98 ℃ and 30 seconds at 60 ℃, and 10 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 25 cycles: 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 25 cycles; finally 5 minutes at 72 ℃;

s2-1: adding deionized water into the amplification product for dilution;

s2-2: mixing an LIZ internal standard and highly deionized formamide (HIDI) according to a volume ratio of 1:90, and mixing the mixed solution and the diluted amplification product in the step S2-1 according to a ratio of V: V =9:1 to prepare an upper machine mixed solution;

s2-3: pre-denaturation: running the prepared on-machine mixed solution on a PCR instrument at 95 ℃ for 4 minutes, and rapidly cooling the mixed solution in an ice-water mixture for 5 minutes for denaturation treatment;

s2-4: capillary electrophoresis: the upper computer mixed liquor is detected in an ABI 3500xl Dx sequencer, grouping is carried out by adopting G5 color, and the typing parameters in the ABI 3500xl Dx sequencer are set as follows: detecting constant Temperature OVEN _ Temperature of 60 ℃ and detecting hole Temperature Detection Cell Temperature of 50 ℃; the pre-electrophoresis Voltage PreRun _ Voltage 19.5kV and the pre-electrophoresis Time PreRun _ Time 180 s; the Injection Voltage Injection _ Voltage is 1.2 kV; injection Time Injection _ Time is 10 s; the First reading First _ ReadOut _ Time 200 ms; second reading Second _ ReadOut _ Time 200 ms; the electrophoresis Voltage Run _ Voltage 19.5 kV; the Number of leap steps, Voltage _ Number _ of _ steps, is set to 30 steps; the kick Voltage _ steps _ Interval is set to 15 s; the Voltage Tolerance Voltage _ Tolerance is set to 0.7 kV; date Delay Data _ Delay 1 s; run _ Time 1330s during electrophoresis;

s3: and (3) data analysis:

ATXN3gene: FAM mark, loading electrophoresis data 'show color' of the sample PCR amplification system B to be analyzed to select 'blue'; marking the 181bp part as a CAG repeat number 1 part, judging that the CAG repeat number corresponding to the first highest peak after the CAG repeat number 1 is the first allele according to the number of fluorescence cluster peaks appearing at intervals of about 3bp, and then selecting the CAG repeat number corresponding to the highest peak in the five-finger peaks as the second allele if the fluorescence cluster peaks exist; according toATXN3Calculating the number of CAG repeats of the numerical values corresponding to the two peaks of the gene;

CACNA1Agene: ROX mark, loading the electrophoresis data 'show color' of the sample PCR amplification system B to be analyzed to select 'red'; marking 298bp as CAG repeat number 1, judging CAG repeat number corresponding to the first highest peak after CAG repeat number 1 as the first allele according to the number of fluorescence cluster peaks appearing at intervals of about 3bp, and selecting CAG repeat number corresponding to the highest peak in five-finger peaks as the second allele if fluorescence cluster peaks exist; according toCACNA1ACalculating the number of CAG repeats of the numerical values corresponding to the two peaks of the gene.

ATXN7Gene: TMR mark, loading the electrophoresis data 'show color' of the sample PCR amplification system B to be analyzed to select 'yellow'; the position of 306bp is marked as the position of 1 CAG repetition number, and the number of fluorescence peaks appearing at about every 3bp is countedAfter judging the number of the CAG repeats 1, the number of the CAG repeats corresponding to the first highest peak is the first allele, and then if the fluorescence cluster exists, the number of the CAG repeats corresponding to the highest peak in the five-finger peak is selected as the second allele; according toATXN7Calculating the number of CAG repeats of the numerical values corresponding to the two peaks of the gene.

In one embodiment of the present application, a simultaneous detection is providedATXN1、ATXN2、ATXN3、CACNA1AAndATXN7the method specifically comprises the following steps:

s0: preparing sample genomic DNA, and preparing two copies of each sample genomic DNA;

s1-1: mixing a primer pair 1 and a primer pair 2 to form a primer pair group A, mixing a primer pair 3, a primer pair 4 and a primer pair 5 to form a primer pair group B, wherein the primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4 and the primer pair 5 are respectively connected with different fluorescent groups;

s1-2: adding a primer pair A, T6 enzyme, dNTP, PCR reaction buffer and MgCl into genomic DNA of one sample₂And (3) PCR amplification: the PCR amplification reaction conditions 1 were as follows: 5 minutes at 98 ℃; 30 seconds at 98 ℃ and 30 seconds at 63 ℃, and 20 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 12 cycles; finally, amplification product 1 is obtained after 5 minutes at 72 ℃;

to another sample genomic DNA was added the primer set B, T6 enzyme, dNTP, PCR reaction buffer and MgCl₂And (3) PCR amplification: PCR amplification reaction conditions 2 were as follows: 5 minutes at 98 ℃; 10 cycles: 30 seconds at 98 ℃ and 30 seconds at 60 ℃, and 10 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 25 cycles: 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 25 cycles; finally, amplification product 2 is obtained after 5 minutes at 72 ℃;

and then, respectively carrying out capillary electrophoresis on the amplification product 1 and the amplification product 2 for detection, or mixing the amplification product 1 and the amplification product 2 and then carrying out capillary electrophoresis detection.

The primer pair provided by the application has at least one of the following beneficial effects:

the application providesATXN1Primer set 1, primer pairATXN2Primer set 2, primer pairATXN3Primer set 3, directed againstCACNA1APrimer pair 4 andATXN7the primer set 5 of (a), which can accurately detect SCA-relatedATXN1、ATXN2、ATXN3、CACNA1A、ATXN7The repeated condition of CAG in the gene, high specificity of the primer pair and high accuracy, and the error of the number of CAG repeats is about plus or minus 1 time.

Drawings

FIG. 1 is a drawing provided in example 1 of the present applicationATXN1Gene agarose gel electrophoresis results.

FIG. 2 is a drawing provided in example 1 of the present applicationATXN1Partial result chart of gene Sanger sequencing.

FIG. 3 is a drawing provided in example 1 of the present applicationATXN1And (5) gene capillary electrophoresis result chart.

FIG. 4 is a drawing provided in example 1 of the present applicationATXN2And (5) gene capillary electrophoresis result chart.

FIG. 5 is a drawing provided in example 1 of the present applicationATXN3And (5) gene capillary electrophoresis result chart.

FIG. 6 is a drawing provided in example 1 of the present applicationCACNA1AAnd (5) gene capillary electrophoresis result chart.

FIG. 7 is a drawing provided in example 1 of the present applicationATXN7And (5) gene capillary electrophoresis result chart.

FIG. 8 is a diagram showing the results of capillary electrophoresis in different combinations of primer pairs, as provided in example 2 of the present application.

FIG. 9 is a diagram showing the results of capillary electrophoresis in the detection of primer set group A provided in example 2 of the present application.

FIG. 10 is a diagram showing the results of capillary electrophoresis in the detection of primer set group B provided in example 2 of the present application.

FIG. 11 is a diagram showing the results of capillary electrophoresis in the case of detection of the primer set group A in an inappropriate PCR reaction system, which is provided in example 3 of the present application.

FIG. 12 is a drawing provided in example 3 of the present applicationATXN1And (5) gene capillary electrophoresis result chart.

FIG. 13 is a drawing provided in example 3 of the present applicationATXN2Gene capillary electricityAnd (5) an electrophoresis result graph.

FIG. 14 is a drawing provided in example 3 of the present applicationATXN3And (5) gene capillary electrophoresis result chart.

FIG. 15 is a drawing provided in example 3 of the present applicationCACNA1AAnd (5) gene capillary electrophoresis result chart.

FIG. 16 is a drawing provided in example 3 of the present applicationATXN7And (5) gene capillary electrophoresis result chart.

Detailed Description

Some, but not all embodiments of the invention are described. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In the present application, it is preferred that,

nucleotide sequence of the upstream primer 1 (F1-2) of primer pair 1: tggaggcctattccactct, respectively;

nucleotide sequence of downstream primer 1 (R1-2) of primer pair 1: aatgtggacgtactggttctg, respectively;

nucleotide sequence of the upstream primer 2 of primer pair 2: gttccggcgtctccttg, respectively;

nucleotide sequence of downstream primer 2 of primer pair 2: aggagaccgaggacgag, respectively;

nucleotide sequence of the upstream primer 3 of primer pair 3: agactaactgctcttgcattct, respectively;

nucleotide sequence of the downstream primer 3 of primer pair 3: atggatgtgaactctgtcctg, respectively;

nucleotide sequence of the upstream primer 4 of primer pair 4: ccacacgtgtcctattccc, respectively;

nucleotide sequence of the downstream primer 4 of primer pair 4: atcggcctcgtcgtagt, respectively;

nucleotide sequence of upstream primer 5 of primer pair 5: gagcggaaagaatgtcgga, respectively;

nucleotide sequence of the downstream primer 5 of primer pair 5: ccttcccaggaagtttggaa are provided.

Example 1 screening of Single primer pairs for detection of SCA subtype genes

To one and twoATXN1Screening of primer pairs for Gene detection

ATXN1The CAG repetitive region of the gene is positioned in exon 7 of the gene, and the specific sequence is shown as SEQ ID NO: shown at 11. According toATXN1The primers designed by using PrimerQuest Tool software are as follows:

F1-1：tggaggcctattccactct

R1-1：gaactggaaatgtggacgtact

in order to adapt to the later-stage primer pair combination and simultaneously detect different SCA subtypes, primers obtained by software design are adjusted, and the adjusted primer sequences are various, wherein two pairs of primers shown as the following are taken as an example for description.

F1-2：tggaggcctattccactct

R1-2：aatgtggacgtactggttctg

F1-3：cgggacacaaggctgag

R1-3：gttctgctgggctggtg

1. Sample preparation

Randomly selecting 3 peripheral blood samples, extracting human genome DNA, using Tirs-EDTA buffer solution as genome DNA solvent, and the sample concentration is 50 ng/. mu.L.

2. PCR amplification

(1) Preparation of reagents: the reagent is stored at-20 ℃ and repeated freeze thawing is reduced as much as possible. The reagent needs to be kept in the dark and balanced to the room temperature before use;

(2) preparing a reagent: uniformly mixing primers 2 mu L F1-2 and 2 mu L R1-2, and PCR amplification reagent 20 mu L (containing dNTP, DNA polymerase, magnesium ions and the like) (referred to as primer 1-2); uniformly mixing primers 2 mu L F1-3 and 2 mu L R1-3, and PCR amplification reagent 20 mu L (containing dNTP, DNA polymerase, magnesium ions and the like) (referred to as primer 1-3 for short) for later use;

(3) adding a sample: adding 1 mu L of genome DNA into the primers 1-2 and 1-3 respectively;

(4) PCR amplification conditions:

5 minutes at 98 ℃; 30 seconds at 98 ℃ and 30 seconds at 60 ℃, and 10 cycles of cooling at 0.3 ℃ and 45 seconds at 72 ℃ in each cycle; 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 25 cycles; 5 minutes at 72 ℃.

DNA polymerase: taq enzyme or T6 enzyme.

The amplified products were detected by agarose gel electrophoresis, and the results are shown in FIG. 1. In FIG. 1, 2 and 3 are the primer pair 1-2, and the results of Taq enzyme detection on three samples are shown; 4. 5 and 6 are a detection result diagram of the primer pair 1-3 and the Taq enzyme pair three samples; m1 and M2 are markers; 7. 8 and 9 are detection result graphs of the primer pair 1-2 and the T6 enzyme on three samples; 10. 11 and 12 are the detection results of the primer pair 1-3 and the T6 enzyme on three samples.

As can be seen from FIG. 1, under the same experimental conditions, a plurality of non-specific amplified bands are likely to appear in the primer pairs 1-3, and since the number of repeats is determined by the size of the amplified bands in the laboratory, the non-specific bands affect the size recognition of the target fragment. Thus, primer pair 1-2 is more specific.

The primer pair 1-2 can effectively amplify both Taq enzyme and T6 enzyme, and compared with the prior art, the Taq enzyme has clean amplification band, the T6 enzyme has clearer and brighter amplification band, but a vague fuzzy band is arranged below a target band, the amplification band is non-specific amplification and can be shown as a miscellaneous peak during capillary electrophoresis, the amount of template DNA needs to be reduced or the annealing temperature needs to be properly increased, and the amplification effect of the Taq enzyme is better.

The primer pair 1-2 and Taq enzyme were used to perform Sanger sequencing on the PCR product amplified from sample 2, and the experimental results are shown in FIG. 2. Sanger sequencing charts can show the base sequence of the amplified bands. As can be seen from FIG. 2, when compared with the reference genome, it was confirmed that the amplified band was the target region, and the amplification specificity of the primer was high. Determining the length of the flanking region by combining the sequencing result of Sanger and the design of the primerATXN1The calculation formula of the gene repetition number n1 is: n1= (L1-146)/3, wherein L1 isATXN1The length of the gene amplification band is the value corresponding to the capillary electrophoresis peak.

Synthesizing TMR labeled fluorescent primers according to the sequences of the primer pairs 1-2, and performing capillary electrophoresis on PCR products amplified from the sample 2 by using the TMR labeled fluorescent primers and Taq enzyme, wherein the specific process is as follows:

(1) diluting: 4-fold dilution is carried out on the amplification product;

(2) preparing a mixed solution: mixing LIZ internal standard and highly deionized formamide (HIDI) according to a volume ratio of 1:90, and mixing 9 μ L of mixed solution with 1 μ L of diluted sample in (1) to obtain upper machine mixed solution;

(3) pre-denaturation: after the prepared on-machine mixed solution runs for 4 minutes at 95 ℃ on a PCR instrument, rapidly cooling the mixed solution in an ice-water mixture for 5 minutes for denaturation treatment to be analyzed;

(4) capillary electrophoresis: the upper computer mixed liquor is detected in an ABI 3500xl Dx sequencer, grouping is carried out by adopting G5 color, and the typing parameters in the ABI 3500xl Dx sequencer are set as follows: detecting constant Temperature OVEN _ Temperature of 60 ℃ and detecting hole Temperature Detection Cell Temperature of 50 ℃; the pre-electrophoresis Voltage PreRun _ Voltage 19.5kV and the pre-electrophoresis Time PreRun _ Time 180 s; the Injection Voltage Injection _ Voltage is 1.2 kV; injection Time Injection _ Time is 10 s; the First reading First _ ReadOut _ Time 200 ms; second reading Second _ ReadOut _ Time 200 ms; the electrophoresis Voltage Run _ Voltage 19.5 kV; the Number of leap steps, Voltage _ Number _ of _ steps, is set to 30 steps; the kick Voltage _ steps _ Interval is set to 15 s; the Voltage Tolerance Voltage _ Tolerance is set to 0.7 kV; date Delay Data _ Delay 1 s; run _ Time 1330s at electrophoresis.

(5) And (3) data analysis, namely judging the typing result in GeneMarker2.2 software, adopting the default setting (GS 500) of the software in the analysis method, and defining after checking and correcting the internal standard peak:

ATXN1gene: TMR mark, loading the electrophoretic data "show color" of the sample to be analyzed to select "yellow"; marking the position of 146bp as (CAG) repeat number 1, judging that the CAG repeat number corresponding to the first highest peak after the CAG repeat number (CAG)1 is the first allele according to the number of fluorescence cluster peaks appearing at every 3bp or so, and then selecting the CAG repeat number corresponding to the highest peak in five-finger peaks as the second allele if the fluorescence cluster peaks exist. The specific primer selected by sequencing can calculate the number of CAG repeats in the region according to the size of the fragment by a calculation formula set during primer design.

The detection result is shown in FIG. 3, the number of CAG repeats is 1 at 146bp, and the determination is made according to the number of fluorescence peaks appearing at intervals of about 3bpThe number of the CAG repeats corresponding to the first highest peak after the number of the CAG repeats 1 is the first allele, and then if there is a fluorescence cluster peak, the number of the CAG repeats corresponding to the highest peak in the five-finger peaks is selected as the second allele according to the result thatATXN1Calculating the number of CAG repeats of the numerical values corresponding to two peaks of the gene:

（218-146）/3=24；（294-146）/3=49。

by calculation, the number of repeats of this sample was 24 and 49, respectively, which is the same as the Sanger sequencing result.

From the above, detection in the present embodimentATXN1When in gene, the primer pair 1-2 and Taq enzyme are finally selected,ATXN1the calculation formula of the gene repetition number n1 is: n1= (L1-146)/3, wherein L1 is the length of the amplification band.

II, aiming atATXN2Screening of primer pairs for Gene detection

ATXN2The dynamic mutation of the CAG repetitive sequence of the gene is positioned in exon 1 of the gene. According to the human reference genome (GRCh 38/hg 38)ATXN2Gene sequence (NM — 002973) see SEQ ID NO: 16, screening againstATXN2Primer pairs for genes. Except the following steps and aims atATXN1The genes were screened differently and the others were identicalATXN1And (4) a gene screening process.

Sample preparation:

randomly selecting 3 peripheral blood samples, extracting human genome DNA, using Tirs-EDTA buffer solution as genome DNA solvent, and the sample concentration is 50 ng/. mu.L. HEX labels the primer pair.

ATXN2In the gene detection, the PCR amplification conditions are as follows:

5 minutes at 98 ℃; 30 seconds at 98 ℃ and 30 seconds at 64 ℃, and 20 cycles of cooling at 0.3 ℃ and 45 seconds at 72 ℃ in each cycle; 30 seconds at 98 ℃, 30 seconds at 58 ℃ and 45 seconds at 72 ℃ for 15 cycles; 5 minutes at 72 ℃; DNA polymerase: taq enzyme or T6 enzyme.

Capillary electrophoresis:ATXN2gene: HEX mark, loading electrophoresis data 'show color' of the sample PCR amplification system A to be analyzed to select 'green'; the position of 186bp is marked as the position of 1 CAG repetition number, and the first CAG repetition number after 1 is judged according to the number of fluorescence cluster peaks appearing at about every 3bpThe number of CAG repeats corresponding to the highest peak is the first allele. Then if there is a fluorescence peak, selecting the CAG repeat number corresponding to the highest peak in the five-finger peak as the second allele according toATXN2Calculating the number of CAG repeats of the numerical values corresponding to the two peaks of the gene. The experimental results are shown in FIG. 4.ATXN2Calculation formula of gene repetition number n 2: n2= (L2-186)/3, wherein L2 isATXN2The length of the gene amplification band is the value corresponding to the capillary electrophoresis peak.

To is directed atATXN2Primer pair 2 final selection of genes: nucleotide sequence of the upstream primer 2: gttccggcgtctccttg, respectively; nucleotide sequence of the downstream primer 2: aggagaccgaggacgag, DNA polymerase is selected from Taq enzyme and T6 enzyme, and the calculation formula of the gene repetition number n2 of ATXN2 is as follows: n2= (L2-186)/3, wherein L2 is the length of the ATXN2 gene amplification band.

III, toATXN3Screening of primer pairs for Gene detection

ATXN3The dynamic mutation of the CAG repetitive sequence of the gene is positioned in exon 9 of the gene. According to the human reference genome (GRCh 38/hg 38)ATXN3Gene sequence (NM _ 030660) see SEQ ID NO: 17 screening againstATXN3Primer pairs for genes. Except the following steps and aims atATXN1The genes were screened differently and the others were identicalATXN1And (4) a gene screening process. FAM labeled primer pairs.

Sample preparation:

randomly selecting 3 peripheral blood samples, extracting human genome DNA, using Tirs-EDTA buffer solution as genome DNA solvent, and the sample concentration is 50 ng/. mu.L.

ATXN3In the gene detection, the PCR amplification conditions are as follows: 5 minutes at 98 ℃; 35 cycles: 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃; finally 5 minutes at 72 ℃; DNA polymerase: taq enzyme, T6 enzyme, GoldStar enzyme or Pfu enzyme.

Capillary electrophoresis:ATXN3gene: FAM mark, loading electrophoresis data 'show color' of the sample PCR amplification system B to be analyzed to select 'blue'; the marker 181bp is the CAG repeat number 1, and the first maximum after the CAG repeat number 1 is judged according to the number of fluorescence series peaks appearing at about every 3bpThe number of the CAG repeats corresponding to the peak is the first allele, and then if the fluorescence peak still exists, the number of the CAG repeats corresponding to the highest peak in the five-finger peak is selected as the second allele; according toATXN3Calculating the number of CAG repeats of the numerical values corresponding to the two peaks of the gene. The experimental results are shown in fig. 5.ATXN3The calculation formula of the gene repetition number n3 is: n3= (L3-181)/3, wherein L3 isATXN3The length of the gene amplification band is the value corresponding to the capillary electrophoresis peak.

To is directed atATXN3Primer pair 3 for the gene was finally determined as: nucleotide sequence of the forward primer 3: agactaactgctcttgcattct, respectively; nucleotide sequence of the downstream primer 3: atggatgtgaactctgtcctg, which can be specifically amplified by the combination with Taq enzyme, T6 enzyme, GoldStar enzyme or Pfu enzymeATXN3The gene(s) is (are),ATXN3the calculation formula of the gene repetition number n3 is: n3= (L3-181)/3, wherein L3 isATXN3Length of gene amplification band.

Fourthly, aim atCACNA1AScreening of primer pairs for Gene detection

CACNA1ASee SEQ ID NO: 18 screening againstCACNA1APrimer pairs for genes. Except the following steps and aims atATXN1The genes were screened differently and the others were identicalATXN1And (4) a gene screening process.

Sample preparation:

randomly selecting 3 peripheral blood samples, extracting human genome DNA, using Tirs-EDTA buffer solution as genome DNA solvent, and the sample concentration is 50 ng/. mu.L. The primer pair is labeled by ROX.

CACNA1AIn the gene detection, the PCR amplification conditions are as follows: 5 minutes at 98 ℃; 10 cycles: 30 seconds at 98 ℃ and 30 seconds at 61 ℃, and 10 cycles of cooling at 0.3 ℃ and 45 seconds at 72 ℃ in each cycle; 25 cycles: 30 seconds at 98 ℃, 30 seconds at 58 ℃ and 45 seconds at 72 ℃ for 25 cycles; finally 5 minutes at 72 ℃; DNA polymerase: taq enzyme or T6 enzyme.

Capillary electrophoresis:CACNA1Agene: ROX mark, loading the electrophoresis data 'show color' of the sample PCR amplification system B to be analyzed to select 'red'; the position of 298bp is CAG repeat number 1, and fluorescence appears at about every 3bpJudging the number of the cross peaks, wherein the number of the CAG repeats corresponding to the first highest peak after the number of the CAG repeats 1 is a first allele, and then if the fluorescence cross peaks exist, selecting the number of the CAG repeats corresponding to the highest peak in the five-finger peaks as a second allele; according toCACNA1ACalculating the number of CAG repeats of the numerical values corresponding to the two peaks of the gene. The experimental results are shown in fig. 6.CACNA1AThe calculation formula of the gene repetition number n4 is: n4= (L4-298)/3, wherein L4 isCACNA1AThe length of the gene amplification band is the value corresponding to the capillary electrophoresis peak.

To is directed atCACNA1APrimer pair 4 for the gene was finally determined as: nucleotide sequence of the forward primer 4: ccacacgtgtcctattccc, respectively; nucleotide sequence of the downstream primer 4: atcggcctcgtcgtagt, which can be specifically amplified by complexing with T6 enzymeCACNA1AThe gene(s) is (are),CACNA1Athe calculation formula of the gene repetition number n4 is: n4= (L4-298)/3, wherein L4 isCACNA1ALength of gene amplification band.

Fifthly, aim atATXN7Screening of primer pairs for Gene detection

ATXN7See SEQ ID NO: 19 screening againstATXN7Primer pairs for genes. Except the following steps and aims atATXN1The genes were screened differently and the others were identicalATXN1And (4) a gene screening process.

Sample preparation:

randomly selecting 3 peripheral blood samples, extracting human genome DNA, using Tirs-EDTA buffer solution as genome DNA solvent, and the sample concentration is 50 ng/. mu.L. TMR labeled primer pair.

ATXN7In the gene detection, the PCR amplification conditions are as follows: 5 minutes at 98 ℃; 10 cycles: 30 seconds at 98 ℃ and 30 seconds at 60 ℃, and 10 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 25 cycles: 30 seconds at 98 ℃, 30 seconds at 55 ℃ and 45 seconds at 72 ℃ for 25 cycles; finally 5 minutes at 72 ℃; DNA polymerase: taq enzyme or T6 enzyme.

Capillary electrophoresis:ATXN7gene: TMR mark, loading the electrophoresis data 'show color' of the sample PCR amplification system B to be analyzed to select 'yellow'; the position of 306bp is marked as 1 position of CAG repetition number according to the interval of about 3bpJudging the number of the appeared fluorescence cluster peaks, wherein the number of the CAG repeats corresponding to the first highest peak after the number of the CAG repeats 1 is the first allele, and then if the fluorescence cluster peaks exist, selecting the number of the CAG repeats corresponding to the highest peak in the five-finger peaks as the second allele; according toATXN7Calculating the number of CAG repeats of the numerical values corresponding to the two peaks of the gene. The experimental results are shown in FIG. 7.ATXN7The calculation formula of the gene repetition number is as follows: n5= (L5-306)/3, wherein L5 is the length of the amplification band, i.e. the value corresponding to the capillary electrophoresis peak.

To is directed atATXN7Primer pair 5 for genes final selection: nucleotide sequence of the forward primer 5: gagcggaaagaatgtcgga, respectively; nucleotide sequence of the downstream primer 5: ccttcccaggaagtttggaa which can be specifically amplified by cooperation with Taq enzyme or T6 enzymeATXN7The gene(s) is (are),ATXN7the calculation formula of the gene repetition number is as follows: n5= (L5-306)/3, wherein L5 is the length of the amplification band.

Example 2 screening of primer pair combinations

The primers aiming at the single subtype are used for carrying out PCR amplification on the five subtypes of the SCA respectively and then carrying out capillary electrophoresis detection analysis in sequence, and the experimental process is relatively complicated. However, since primers for detecting different subtypes differ in the most suitable enzyme, the most suitable amplification condition, and the specificity of each primer pair, it is not possible to simultaneously detect different subtypes by simply mixing primer pairs for a single subtype. Therefore, the inventors of the present application have attempted to provide a primer pair combination and a detection method that can simultaneously detect multiple subtypes, so as to simplify the experimental steps and reduce the detection cost. Because the fluorescence channel of the current capillary electrophoresis detection equipment is mostly a 5-color fluorescence channel, the molecular internal standard LIZ used in the experiment needs to occupy one fluorescence channel, and the primer pairs of five subtypes cannot be mixed and detected simultaneously, the inventor of the application combines the primer pairs of five subtypes two by two or combines the primer pairs of five subtypes.

Using 5 pairs of primer pairs selected in example 1 for different subtypes of SCA, the 5 pairs of primer pairs were combined in pairs or three pairs under the following amplification conditions using T6 as DNA polymerase.

The PCR amplification conditions were: 5 minutes at 98 ℃; 10 cycles: 30 seconds at 98 ℃ and 30 seconds at 60 ℃, and 10 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 25 cycles: 30 seconds at 98 ℃, 30 seconds at 55 ℃ and 45 seconds at 72 ℃ for 25 cycles; finally 5 minutes at 72 ℃; DNA polymerase: t6 enzyme.

The inventor of the application finds out through experiments whether the combination of different primer pairs can detect different subtypes of SCA at the same time, and the results are different. For example, toATXN1Primer pair 1 (TMR-labeled, black fluorescent) of (1), forATXN3(FAM-labeled, blue-fluorescent) primer pair 3 andCACNA1Awhen the primer pair 4 (ROX-labeled, red fluorescence) is mixed and the number of CAG repeats is detected,ATXN3(blue fluorescence) andATXN1the (black fluorescence) gene has an interpretable peak, butCACNA1A(Red fluorescence) non-specific amplification signals appear around 200bp, and the signal intensity is equal toATXN3(blue fluorescence) andATXN1the (black fluorescence) gene signal intensities were comparable, affecting the interpretation of the results, as shown by the arrows in FIG. 8.

Through continuous trial and error, will be aimed at finallyATXN1Primer pair 1 andATXN2the primer pair 2 of (a) is combined to form a primer pair group A,ATXN1andATXN2all had clear specific fluorescent signals (as shown in FIG. 9). Will be directed toATXN3Primer set 3, directed againstCACNA1APrimer pair 4 andATXN7the primer pair 5 of (a) forms a primer pair group B,ATXN3、CACNA1A、ATXN7all had clear specific fluorescent signals (as shown in FIG. 10).

Although the primer pairs are combined in this embodiment, the optimal experimental conditions of the 5 primer pairs are different, and the reaction failure or the occurrence of false negative/false positive results can be caused under inappropriate conditions, so that the inventors of the present application respectively determine the PCR reaction conditions for the primer pair group a and the primer pair group B, thereby ensuring that the SCA subtype can be detected and avoiding the occurrence of false negative/false positive.

Example 3 determination of PCR reaction conditions

The inventors of the present application searched for PCR reactions that are relatively suitable for the primer pairs in the primer pair group A and the primer pair group B, respectivelyAnd (3) conditions for avoiding reaction failure or false negative/false positive results caused by PCR amplification reaction. For example, as shown in FIG. 11, when the PCR reaction conditions are: 5 minutes at 98 ℃; 30 seconds at 98 ℃ and 30 seconds at 62 ℃, and the temperature is reduced by 0.3 ℃ and 45 seconds at 72 ℃ in each cycle for 17 cycles; 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 15 cycles; at the temperature of 72 ℃ for 5 minutes,ATXN2except for the specific amplification signal, the gene has non-specific amplification (indicated by an arrow in FIG. 11) about 380bp, so that the gene is not easy to be distinguished from a mutation peak, and the result interpretation is influenced. Therefore, the conditions for PCR reaction of the primer set group A are adjusted so that the primer set group A can be specifically amplifiedATXN1AndATXN2the gene can not be amplified non-specifically.

After different adjustment of PCR reaction conditions, the finally determined PCR reaction conditions of the primer pair group A are as follows: 5 minutes at 98 ℃; 30 seconds at 98 ℃ and 30 seconds at 63 ℃, and 20 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 12 cycles; finally 5 minutes at 72 ℃.

Under the conditions of the reaction, the reaction solution is,ATXN1the capillary electrophoresis pattern of the gene is shown in FIG. 12,ATXN2the capillary electrophoresis pattern of the gene is shown in FIG. 13. As is clear from FIGS. 12 and 13, when simultaneous detection is carried out using primer set 1 and primer set 2 in primer set A, specific detection is possibleATXN1AndATXN2the number of CAG repeats of the gene was consistent with that obtained using Sanger sequencing assays.

After different adjustment of PCR reaction conditions, the finally determined PCR reaction conditions of the primer pair group B are as follows: 5 minutes at 98 ℃; 10 cycles: 30 seconds at 98 ℃ and 30 seconds at 60 ℃, and 10 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 25 cycles: 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 25 cycles; finally 5 minutes at 72 ℃.

Under the conditions of the reaction, the reaction solution is,ATXN3the capillary electrophoresis pattern of the gene is shown in FIG. 14,CACNA1Athe capillary electrophoresis pattern of the gene is shown in FIG. 15,ATXN7the capillary electrophoresis pattern of the gene is shown in FIG. 16. As is clear from FIGS. 14, 15 and 16, when the primer set B was used for detection, the detection could be simultaneously and specifically detectedATXN3、CACNA1AAndATXN7the number of CAG repeats of the gene was consistent with that obtained using Sanger sequencing assays.

As described above, the embodiments of the present application provideATXN1Primer set 1, primer pairATXN2Primer set 2, primer pairATXN3Primer set 3, directed againstCACNA1APrimer pair 4 andATXN7the primer pair 5 can accurately detect SCA related by combining with a capillary electrophoresis analysis methodATXN1、ATXN2、ATXN3、CACNA1A、ATXN7The repeated condition of CAG in the gene, high specificity of the primer pair and high accuracy, and the error of the number of CAG repeats is about plus or minus 1 time.

And the primer pair 1 and the primer pair 2 are mixed to simultaneously detectATXN1AndATXN2the primer pair 3, the primer pair 4 and the primer pair 5 are mixed to simultaneously detectATXN3、CACNA1AAndATXN7therefore, the experiment operation process that 5 times is needed for detecting five different subtypes in the prior art can be completed by utilizing the primer pair and the two PCR amplification reaction systems, the operation process is greatly reduced, the detection time is shortened, and the detection of five subtypes, namely SCA3, SCA1, SCA2, SCA6 and SCA7, can be completed in 5-6 hours by only using the two PCR amplification reaction systems.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

<110> third Hospital of Beijing university (third clinical medical college of Beijing university)

<120> primer pair, kit and detection method for SCA subtype gene detection

<160> 19

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<211> 19

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer 1

<400> 1

tggaggccta ttccactct 19

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer 1

<400> 2

aatgtggacg tactggttct g 21

<210> 3

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer 2

<400> 3

gttccggcgt ctccttg 17

<210> 4

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer 2

<400> 4

aggagaccga ggacgag 17

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer 3

<400> 5

agactaactg ctcttgcatt ct 22

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer 3

<400> 6

atggatgtga actctgtcct g 21

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer 4

<400> 7

ccacacgtgt cctattccc 19

<210> 8

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer 4

<400> 8

atcggcctcg tcgtagt 17

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer 5

<400> 9

gagcggaaag aatgtcgga 19

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer 5

<400> 10

ccttcccagg aagtttggaa 20

<210> 11

<211> 850

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ATXN1 Gene

<400> 11

gggcaaccct ggtggccggg gccacggggg cgggaggcat gggccggcag ggacctcggt 60

ggagcttggt ttacaacagg gaataggttt acacaaagca ttgtccacag ggctggacta 120

ctccccgccc agcgctccca ggtctgtccc cgtggccacc acgctgcctg ccgcgtacgc 180

caccccgcag ccagggaccc cggtgtcccc cgtgcagtac gctcacctgc cgcacacctt 240

ccagttcatt gggtcctccc aatacagtgg aacctatgcc agcttcatcc catcacagct 300

gatcccccca accgccaacc ccgtcaccag tgcagtggcc tcggccgcag gggccaccac 360

tccatcccag cgctcccagc tggaggccta ttccactctg ctggccaaca tgggcagtct 420

gagccagacg ccgggacaca aggctgagca gcagcagcag cagcagcagc agcagcagca 480

gcagcatcag catcagcagc agcagcagca gcagcagcag cagcagcagc agcagcacct 540

cagcagggct ccggggctca tcaccccggg gtccccccca ccagcccagc agaaccagta 600

cgtccacatt tccagttctc cgcagaacac cggccgcacc gcctctcctc cggccatccc 660

cgtccacctc cacccccacc agacgatgat cccacacacg ctcaccctgg ggcccccctc 720

ccaggtcgtc atgcaatacg ccgactccgg cagccacttt gtccctcggg aggccaccaa 780

gaaagctgag agcagccggc tgcagcaggc catccaggcc aaggaggtcc tgaacggtga 840

gatggagaag 850

<210> 12

<211> 19

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<213> Artificial Sequence (Artificial Sequence)

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<223> F1-1

<400> 12

tggaggccta ttccactct 19

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> R1-1

<400> 13

gaactggaaa tgtggacgta ct 22

<210> 14

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> F1-3

<400> 14

cgggacacaa ggctgag 17

<210> 15

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> R1-3

<400> 15

gttctgctgg gctggtg 17

<210> 16

<211> 1050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ATXN2 Gene

<400> 16

ggccaccgag tctcgccgct tcgccgcagc caggtggccc gggtggcgct cgctccagcg 60

gccggcgcgg cggagcgggc ggggcggcgg tggcgcggcc ccgggaccgt atccctccgc 120

cgcccctccc ccgcccggcc ccggcccccc tccctcccgg cagagctcgc ctccctccgc 180

ctcagactgt tttggtagca acggcaacgg cggcggcgcg tttcggcccg gctcccggcg 240

gctccttggt ctcggcgggc ctccccgccc cttcgtcgtc ctccttctcc ccctcgccag 300

cccgggcgcc cctccggccg cgccaacccg cgcctccccg ctcggcgccc gcgcgtcccc 360

gccgcgttcc ggcgtctcct tggcgcgccc ggctcccggc tgtccccgcc cggcgtgcga 420

gccggtgtat gggcccctca ccatgtcgct gaagccccag cagcagcagc agcagcagca 480

gcagcagcag cagcagcaac agcagcagca gcagcagcag cagcagccgc cgcccgcggc 540

tgccaatgtc cgcaagcccg gcggcagcgg ccttctagcg tcgcccgccg ccgcgccttc 600

gccgtcctcg tcctcggtct cctcgtcctc ggccacggct ccctcctcgg tggtcgcggc 660

gacctccggc ggcgggaggc ccggcctggg caggtgggtg tcggcacccc agccccctcc 720

gctccgggcc cggcgtcccc tcccccgcgg cccgcgccgc cgtccccgcc ccgtgacccg 780

ccgggctacc cggggtgggc tgggggccgg cagcgcgggg gagactcgct cgggcctgag 840

ccccgaggct cggccggtgg gcgcagccgg ggtcctctgg gattgtcagg cctgtccagc 900

ctcccgcagc atccccgccc cctcccccgg cggtcaagat ggagggagcg ggcggcctcc 960

cctccccacg cgtgttggga ggggttctcg ggtagcggcg atggtcagcc ccggctcccc 1020

cttccgcacg atcctccgcc cgcagcgtgg 1050

<210> 17

<211> 900

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ATXN3 Gene

<400> 17

ggctaatttt tgtattttta gtagagatgg ggtttcaccg tgttgtccag gctcgtgtca 60

aacttctgac ctcaagccat ccacccgcct cggcctccca aagtgctggg attacaggtg 120

tgagccacca ctcctggcca tgataggtta ttttgtgatg aaaataccta cctcttaatt 180

tgtctgataa atttaaattt tatgtctaga tttcctaaga tcagcacttc catattttaa 240

agtaatctgt atcagactaa ctgctcttgc attcttttaa taccagtgac tactttgatt 300

cgtgaaacaa tgtattttcc ttatgaatag tttttctcat ggtgtattta ttcttttaag 360

ttttgttttt taaatatact tcacttttga atgtttcaga cagcagcaaa agcagcaaca 420

gcagcagcag cagcagcagc agggggacct atcaggacag agttcacatc catgtgaaag 480

gccagccacc agttcaggag cacttgggag tgatctaggt aaggcctgct caccattcat 540

catgttcgct accttcacac tttatctgac atacgagctc catgtgattt ttgctttaca 600

ttattcttca ttccctcttt aatcatatta agaatcttaa gtaaatttgt aatctactaa 660

atttccctgg attaaggagc agttaccaaa agaaaaaaaa aaaaaaaagc tagatgtggt 720

ggctcacatc tgtaatccca gcactttggg aaaccaaggc aggagaggat tgctagaaca 780

tttaatgaat actttaacat aataatttaa acttcacagt aatttgtaca gtctccaaaa 840

attccttaga catcatggat atttttcttt ttttgagatg gagtcttgct ctgtcaccca 900

<210> 18

<211> 900

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CACNA1A Gene

<400> 18

tgttgtgttg gttttcgact cctcccctcc ctgtctcact ccccctcctc ccctccctcc 60

tccctgtggc tgttgctttt ttccattcaa tgtcctgtgt cccccctctc ctcctcctcc 120

tcctcctccc cctccccctc ctccctctcc tcccggcccc tctcccttcg ctcccctctc 180

ttcctcccaa tcccgtgtct cctttgattt tgttgtatct ttttttttga tttcctttgt 240

ttcaattttc gtgtagggca gtagttccgt aagtggaagc ccagccccct caacatctgg 300

taccagcact ccgcggcggg gccgccgcca gctcccccag accccctcca ccccccggcc 360

acacgtgtcc tattcccctg tgatccgtaa ggccggcggc tcggggcccc cgcagcagca 420

gcagcagcag cagcagcagc agcagcagca ggcggtggcc aggccgggcc gggcggccac 480

cagcggccct cggaggtacc caggccccac ggccgagcct ctggccggag atcggccgcc 540

cacggggggc cacagcagcg gccgctcgcc caggatggag aggcgggtcc caggcccggc 600

ccggagcgag tcccccaggg cctgtcgaca cggcggggcc cggtggccgg catctggccc 660

gcacgtgtcc gaggggcccc cgggtccccg gcaccatggc tactaccggg gctccgacta 720

cgacgaggcc gatggcccgg gcagcggggg cggcgaggag gccatggccg gggcctacga 780

cgcgccaccc cccgtacgac acgcgtcctc gggcgccacc gggcgctcgc ccaggactcc 840

ccgggcctcg ggcccggcct gcgcctcgcc ttctcggcac ggccggcgac tccccaacgg 900

<210> 19

<211> 1336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ATXN7 Gene

<400> 19

ggagccggcc gggtgccgcc gctcggacgg acgcgcggac ggaaggaagg agcggggcag 60

ccgggccggg cccggggatg caagcggccg aagggtgggc agctggaggt cctggggtgc 120

ggctcgggct tccccgcgcg ggctgccatg gtggggcgcg gggttggagc cgggccgctc 180

cggcgctggc ctccgcgcca ggtcctctga gcagaagcag gcaggggacc cagcgccgcg 240

gtggcgggcc gcctgctgcc cgtcccctcc ctcgggcggc cgcgggagtc gaaagcgaaa 300

gctagcccgc gccgcggact ttgagcccgg ggcggggggt ggccttgagg aggcgggctc 360

ggggggctgg gcggccatgg gggcgctgtc agcgtgcccc acccggtccg cgggccgcgc 420

acgccgccgg aactccctgg cgcctcctta aaaaacggcc cccgcgcgac tctttccccc 480

ttttttttgt tacattgtag gagcggaaag aatgtcggag cgggccgcgg atgacgtcag 540

gggggagccg cgccgcgcgg cggcggcggc gggcggagca gcggccgcgg ccgcccggca 600

gcagcagcag cagcagcagc agcagcagcc gccgcctccg cagccccagc ggcagcagca 660

cccgccaccg ccgccacggc gcacacggcc ggaggacggc gggcccggcg ccgcctccac 720

ctcggccgcc gcaatggcga cggtcgggga gcgcaggcct ctgcccagtc ctgaagtgat 780

gctgggacag tcgtggaatc tgtgggttga ggcttccaaa cttcctggga aggacggtga 840

gtgtccacgc cctcctcccc ccttcacccc ctcgcgaccc cctcctctct cctcccctcc 900

cccctgcccc cctcctgtga cccgccccct cgaggggcag agatgctatc gtttgctggg 960

ttgcggaacg cggaggtgcc cacacctacc ccgtgcgtgc gtgagtgtgc gtcacactcc 1020

tggccactga cctgcctctc ccctcctcct gtgtgtgtat atctcctagg gacagaattg 1080

gacgaaagtt tcaaggagtt tgggaaaaac cgcgaagtca tggggctctg tcgggaaggt 1140

gagtccagcc cccctgatgg agtttgtaca aacccctggg aagtttcatt gacagttcac 1200

tgggaccggg aacatcagcc caccataccg actccccgac tccccgtgcc tgcgaagatg 1260

ctgcctgagg agggagggag ggggcagagc gcttggaaag tttggtttgg gggcctcctg 1320

taatgagagc gtccgg 1336

Sequence listing

<110> third Hospital of Beijing university (third clinical medical college of Beijing university)

<120> primer pair, kit and detection method for SCA subtype gene detection

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer 1

<400> 1

tggaggccta ttccactct 19

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer 1

<400> 2

aatgtggacg tactggttct g 21

<210> 3

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer 2

<400> 3

gttccggcgt ctccttg 17

<210> 4

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer 2

<400> 4

aggagaccga ggacgag 17

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer 3

<400> 5

agactaactg ctcttgcatt ct 22

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer 3

<400> 6

atggatgtga actctgtcct g 21

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer 4

<400> 7

ccacacgtgt cctattccc 19

<210> 8

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer 4

<400> 8

atcggcctcg tcgtagt 17

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer 5

<400> 9

gagcggaaag aatgtcgga 19

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer 5

<400> 10

ccttcccagg aagtttggaa 20

<210> 11

<211> 850

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ATXN1 Gene

<400> 11

gggcaaccct ggtggccggg gccacggggg cgggaggcat gggccggcag ggacctcggt 60

ggagcttggt ttacaacagg gaataggttt acacaaagca ttgtccacag ggctggacta 120

ctccccgccc agcgctccca ggtctgtccc cgtggccacc acgctgcctg ccgcgtacgc 180

caccccgcag ccagggaccc cggtgtcccc cgtgcagtac gctcacctgc cgcacacctt 240

ccagttcatt gggtcctccc aatacagtgg aacctatgcc agcttcatcc catcacagct 300

gatcccccca accgccaacc ccgtcaccag tgcagtggcc tcggccgcag gggccaccac 360

tccatcccag cgctcccagc tggaggccta ttccactctg ctggccaaca tgggcagtct 420

gagccagacg ccgggacaca aggctgagca gcagcagcag cagcagcagc agcagcagca 480

gcagcatcag catcagcagc agcagcagca gcagcagcag cagcagcagc agcagcacct 540

cagcagggct ccggggctca tcaccccggg gtccccccca ccagcccagc agaaccagta 600

cgtccacatt tccagttctc cgcagaacac cggccgcacc gcctctcctc cggccatccc 660

cgtccacctc cacccccacc agacgatgat cccacacacg ctcaccctgg ggcccccctc 720

ccaggtcgtc atgcaatacg ccgactccgg cagccacttt gtccctcggg aggccaccaa 780

gaaagctgag agcagccggc tgcagcaggc catccaggcc aaggaggtcc tgaacggtga 840

gatggagaag 850

<210> 12

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> F1-1

<400> 12

tggaggccta ttccactct 19

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> R1-1

<400> 13

gaactggaaa tgtggacgta ct 22

<210> 14

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> F1-3

<400> 14

cgggacacaa ggctgag 17

<210> 15

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> R1-3

<400> 15

gttctgctgg gctggtg 17

<210> 16

<211> 1050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ATXN2 Gene

<400> 16

ggccaccgag tctcgccgct tcgccgcagc caggtggccc gggtggcgct cgctccagcg 60

gccggcgcgg cggagcgggc ggggcggcgg tggcgcggcc ccgggaccgt atccctccgc 120

cgcccctccc ccgcccggcc ccggcccccc tccctcccgg cagagctcgc ctccctccgc 180

ctcagactgt tttggtagca acggcaacgg cggcggcgcg tttcggcccg gctcccggcg 240

gctccttggt ctcggcgggc ctccccgccc cttcgtcgtc ctccttctcc ccctcgccag 300

cccgggcgcc cctccggccg cgccaacccg cgcctccccg ctcggcgccc gcgcgtcccc 360

gccgcgttcc ggcgtctcct tggcgcgccc ggctcccggc tgtccccgcc cggcgtgcga 420

gccggtgtat gggcccctca ccatgtcgct gaagccccag cagcagcagc agcagcagca 480

gcagcagcag cagcagcaac agcagcagca gcagcagcag cagcagccgc cgcccgcggc 540

tgccaatgtc cgcaagcccg gcggcagcgg ccttctagcg tcgcccgccg ccgcgccttc 600

gccgtcctcg tcctcggtct cctcgtcctc ggccacggct ccctcctcgg tggtcgcggc 660

gacctccggc ggcgggaggc ccggcctggg caggtgggtg tcggcacccc agccccctcc 720

gctccgggcc cggcgtcccc tcccccgcgg cccgcgccgc cgtccccgcc ccgtgacccg 780

ccgggctacc cggggtgggc tgggggccgg cagcgcgggg gagactcgct cgggcctgag 840

ccccgaggct cggccggtgg gcgcagccgg ggtcctctgg gattgtcagg cctgtccagc 900

ctcccgcagc atccccgccc cctcccccgg cggtcaagat ggagggagcg ggcggcctcc 960

cctccccacg cgtgttggga ggggttctcg ggtagcggcg atggtcagcc ccggctcccc 1020

cttccgcacg atcctccgcc cgcagcgtgg 1050

<210> 17

<211> 900

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ATXN3 Gene

<400> 17

ggctaatttt tgtattttta gtagagatgg ggtttcaccg tgttgtccag gctcgtgtca 60

aacttctgac ctcaagccat ccacccgcct cggcctccca aagtgctggg attacaggtg 120

tgagccacca ctcctggcca tgataggtta ttttgtgatg aaaataccta cctcttaatt 180

tgtctgataa atttaaattt tatgtctaga tttcctaaga tcagcacttc catattttaa 240

agtaatctgt atcagactaa ctgctcttgc attcttttaa taccagtgac tactttgatt 300

cgtgaaacaa tgtattttcc ttatgaatag tttttctcat ggtgtattta ttcttttaag 360

ttttgttttt taaatatact tcacttttga atgtttcaga cagcagcaaa agcagcaaca 420

gcagcagcag cagcagcagc agggggacct atcaggacag agttcacatc catgtgaaag 480

gccagccacc agttcaggag cacttgggag tgatctaggt aaggcctgct caccattcat 540

catgttcgct accttcacac tttatctgac atacgagctc catgtgattt ttgctttaca 600

ttattcttca ttccctcttt aatcatatta agaatcttaa gtaaatttgt aatctactaa 660

atttccctgg attaaggagc agttaccaaa agaaaaaaaa aaaaaaaagc tagatgtggt 720

ggctcacatc tgtaatccca gcactttggg aaaccaaggc aggagaggat tgctagaaca 780

tttaatgaat actttaacat aataatttaa acttcacagt aatttgtaca gtctccaaaa 840

attccttaga catcatggat atttttcttt ttttgagatg gagtcttgct ctgtcaccca 900

<210> 18

<211> 900

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CACNA1A Gene

<400> 18

tgttgtgttg gttttcgact cctcccctcc ctgtctcact ccccctcctc ccctccctcc 60

tccctgtggc tgttgctttt ttccattcaa tgtcctgtgt cccccctctc ctcctcctcc 120

tcctcctccc cctccccctc ctccctctcc tcccggcccc tctcccttcg ctcccctctc 180

ttcctcccaa tcccgtgtct cctttgattt tgttgtatct ttttttttga tttcctttgt 240

ttcaattttc gtgtagggca gtagttccgt aagtggaagc ccagccccct caacatctgg 300

taccagcact ccgcggcggg gccgccgcca gctcccccag accccctcca ccccccggcc 360

acacgtgtcc tattcccctg tgatccgtaa ggccggcggc tcggggcccc cgcagcagca 420

gcagcagcag cagcagcagc agcagcagca ggcggtggcc aggccgggcc gggcggccac 480

cagcggccct cggaggtacc caggccccac ggccgagcct ctggccggag atcggccgcc 540

cacggggggc cacagcagcg gccgctcgcc caggatggag aggcgggtcc caggcccggc 600

ccggagcgag tcccccaggg cctgtcgaca cggcggggcc cggtggccgg catctggccc 660

gcacgtgtcc gaggggcccc cgggtccccg gcaccatggc tactaccggg gctccgacta 720

cgacgaggcc gatggcccgg gcagcggggg cggcgaggag gccatggccg gggcctacga 780

cgcgccaccc cccgtacgac acgcgtcctc gggcgccacc gggcgctcgc ccaggactcc 840

ccgggcctcg ggcccggcct gcgcctcgcc ttctcggcac ggccggcgac tccccaacgg 900

<210> 19

<211> 1336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ATXN7 Gene

<400> 19

ggagccggcc gggtgccgcc gctcggacgg acgcgcggac ggaaggaagg agcggggcag 60

ccgggccggg cccggggatg caagcggccg aagggtgggc agctggaggt cctggggtgc 120

ggctcgggct tccccgcgcg ggctgccatg gtggggcgcg gggttggagc cgggccgctc 180

cggcgctggc ctccgcgcca ggtcctctga gcagaagcag gcaggggacc cagcgccgcg 240

gtggcgggcc gcctgctgcc cgtcccctcc ctcgggcggc cgcgggagtc gaaagcgaaa 300

gctagcccgc gccgcggact ttgagcccgg ggcggggggt ggccttgagg aggcgggctc 360

ggggggctgg gcggccatgg gggcgctgtc agcgtgcccc acccggtccg cgggccgcgc 420

acgccgccgg aactccctgg cgcctcctta aaaaacggcc cccgcgcgac tctttccccc 480

ttttttttgt tacattgtag gagcggaaag aatgtcggag cgggccgcgg atgacgtcag 540

gggggagccg cgccgcgcgg cggcggcggc gggcggagca gcggccgcgg ccgcccggca 600

gcagcagcag cagcagcagc agcagcagcc gccgcctccg cagccccagc ggcagcagca 660

cccgccaccg ccgccacggc gcacacggcc ggaggacggc gggcccggcg ccgcctccac 720

ctcggccgcc gcaatggcga cggtcgggga gcgcaggcct ctgcccagtc ctgaagtgat 780

gctgggacag tcgtggaatc tgtgggttga ggcttccaaa cttcctggga aggacggtga 840

gtgtccacgc cctcctcccc ccttcacccc ctcgcgaccc cctcctctct cctcccctcc 900

cccctgcccc cctcctgtga cccgccccct cgaggggcag agatgctatc gtttgctggg 960

ttgcggaacg cggaggtgcc cacacctacc ccgtgcgtgc gtgagtgtgc gtcacactcc 1020

tggccactga cctgcctctc ccctcctcct gtgtgtgtat atctcctagg gacagaattg 1080

gacgaaagtt tcaaggagtt tgggaaaaac cgcgaagtca tggggctctg tcgggaaggt 1140

gagtccagcc cccctgatgg agtttgtaca aacccctggg aagtttcatt gacagttcac 1200

tgggaccggg aacatcagcc caccataccg actccccgac tccccgtgcc tgcgaagatg 1260

ctgcctgagg agggagggag ggggcagagc gcttggaaag tttggtttgg gggcctcctg 1320

taatgagagc gtccgg 1336

Claims (10)

1. A primer pair for SCA subtype gene detection, which is characterized by comprising one or more of a primer pair 1 aiming at ATXN1, a primer pair 2 aiming at ATXN2, a primer pair 3 aiming at ATXN3, a primer pair 4 aiming at CACNA1A and a primer pair 5 aiming at ATXN7,

wherein the content of the first and second substances,

the nucleotide sequence of the upstream primer 1 of the primer pair 1 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer 1 of the primer pair is shown as SEQ ID NO: 2 is shown in the specification;

the nucleotide sequence of the upstream primer 2 of the primer pair 2 is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer 2 of the primer pair is shown as SEQ ID NO: 4 is shown in the specification;

the nucleotide sequence of the upstream primer 3 of the primer pair 3 is shown as SEQ ID NO: 5, the nucleotide sequence of the downstream primer 3 of the primer pair is shown as SEQ ID NO: 6 is shown in the specification;

the nucleotide sequence of the upstream primer 4 of the primer pair 4 is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer 4 of the primer pair is shown as SEQ ID NO: 8 is shown in the specification;

the nucleotide sequence of the upstream primer 5 of the primer pair 5 is shown as SEQ ID NO: 9, the nucleotide sequence of the downstream primer 5 of the primer pair is shown as SEQ ID NO: shown at 10.

2. The primer pair of claim 1, wherein the primer pair comprises primer pair 1 for ATXN1 and primer pair 2 for ATXN2 for simultaneous detection of ATXN1 and ATXN 2.

3. The primer pair of claim 1, wherein the primer pair comprises primer pair 3 for ATXN3, primer pair 4 for CACNA1A and primer pair 5 for ATXN7, and is used for simultaneously detecting ATXN3, CACNA1A and ATXN 7.

4. The primer pair of claim 1, wherein the primer pair comprises primer pair 1 for ATXN1, primer pair 2 for ATXN2, primer pair 3 for ATXN3, primer pair 4 for CACNA1A, and primer pair 5 for ATXN 7.

5. The primer pair of claims 1-4, wherein a fluorophore is attached to the primer pair.

6. A kit for detecting a SCA subtype gene, comprising the primer set according to any one of claims 1 to 5.

7. The kit of claim 6, wherein the kit further comprises a DNA polymerase.

8. The kit of claim 7, wherein the DNA polymerase is T6 enzyme.

9. A method for detecting SCA genotyping for non-disease diagnostic purposes, comprising the steps of:

s1: adding the primer pair of claim 1, and performing PCR amplification on the corresponding gene sequence to obtain an amplification product;

s2: subjecting the amplification product of step S1 to capillary electrophoresis;

s3: the data and results in step S2 are analyzed.

10. The detection method according to claim 9,

the PCR amplification reaction conditions 1 of the primer pair 1 and the primer pair 2 are as follows: 5 minutes at 98 ℃; 30 seconds at 98 ℃ and 30 seconds at 63 ℃, and 20 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 12 cycles; finally 5 minutes at 72 ℃;

the PCR amplification reaction conditions 2 of the primer pair 3, the primer pair 4 and the primer pair 5 are as follows:

5 minutes at 98 ℃; 10 cycles: 30 seconds at 98 ℃ and 30 seconds at 60 ℃, and 10 cycles of cooling at 0.5 ℃ and 45 seconds at 72 ℃ in each cycle; 25 cycles: 30 seconds at 98 ℃, 30 seconds at 57 ℃ and 45 seconds at 72 ℃ for 25 cycles; finally 5 minutes at 72 ℃.

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