CN117683876A - Primer group, method and application for detecting SCAs9 subtype based on multiplex fluorescence TP-PCR and capillary electrophoresis technology - Google Patents

Abstract

The invention aims to provide SCAs which are a group of inherited neurodegenerative diseases with obvious clinical and genetic heterogeneity, and the risk generation is higher than the risk generation, so that clinical diagnosis cannot be carried out according to clinical manifestations alone; therefore, according to the literature and database data, 9 common or relatively common SCA subtypes caused by repeated expansion of CAG three bases in more than 40 SCA subtypes are selected, namely SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA8, SCA12, SCA17 and DRPLA are selected, and the primer group, the screening method and the application thereof for detecting the SCAs9 subtype can be rapidly and simply used at one time, have low cost and are easy to comprehensively popularize and screen.

Description

Primer group, method and application for detecting SCAs9 subtype based on multiplex fluorescence TP-PCR and capillary electrophoresis technology

Technical Field

The invention relates to the field of gene detection, in particular to a primer group, a method and application thereof for detecting common SCAs9 subtype based on multiplex fluorescence TP-PCR and capillary electrophoresis technology.

Background

Spinocerebellar ataxia (Spinocerebellar ataxia, SCAs) is a common ataxia genetic disorder, lesions mainly involve spinal cord, brain stem and cerebellum, cerebellar degenerative disease and impaired afferent and efferent pathways thereof are key to causing characteristic progressive ataxia, and patients may be accompanied by dysarthria, intention tremor, eye muscle paralysis, etc. Genetic patterns of SCAs include autosomal dominant inheritance, which is the most common genetic pattern of SCAs, and companion X-linked recessive inheritance.

SCAs are largely divided into two classes: SCAs caused by repeat expansion and SCAs caused by non-repeat mutation. More than 40 subtypes have been discovered, with SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA17 and DRPLA being due to repeated amplification mutations in the CAG coding region of the pathogenic gene, which encode pure glutamine fragments in the respective disease protein; SCA8 and SCA12 are caused by CAG/CTG and CAG repeated amplification mutation of non-coding region of pathogenic gene; these diseases share a common feature, namely that the symptoms are severe from generation to generation. According to the data, the prevalence rate of SCAs is between 0 and 5.6/10000, and the average value reaches 2.7/10000; polyglutamine SCAs (SCA 1, SCA2, SCA3/MJD, SCA6, SCA7, SCA17 and DRPLA) are the most common mutant subtypes of SCA; the largest ratio of SCA3 subtype in Chinese population, and second SCA2, SCA1, SCA7 and SCA6, other subtypes are rare, but sporadic reports are also presented.

SCAs are mostly developed in middle-aged people, but the development period of SCAs runs through the whole life cycle, and the aspects of growth, development, life and the like of a carrier are seriously damaged. There is currently no therapeutic approach to slow down or stop SCAs (many SCAs lead to premature death), and clinical care of SCA patients is focused on controlling symptoms by physical therapy, occupational therapy, and linguistic therapy. Early discovery, early treatment, early intervention can significantly improve the prognosis of the infant and can avoid the recurrence of the same patient in the family by providing prenatal genetic counseling.

The detection methods reported in the literature at present are fluorescence PCR-capillary electrophoresis, southern blot and the recently emerging third generation sequencing technology. The fluorescent PCR-capillary electrophoresis method also comprises the steps of amplifying a long sequence and a repeated sequence, and calculating the CAG repeated number according to the length or the peak number of the capillary electrophoresis; the southern blot can be used for determining the number of large fragment repeated sequences, but can not determine the repeated sequences smaller than 10, and has the advantages of complex operation and low flux; three-generation sequencing techniques can detect all repetitive sequences, but are costly and have a long cycle.

The Shenzhen large gene (CN 102206701A A20111005) selects a PCR primer designed by the coding sequence of the transglutaminase 6 gene or the 10 th exon sequence thereof; the aprataxin gene was also selected (EP 1293570A2 a2 20030319); selecting a single detection site for SCA7 or SCA2 or SCA5 (JPH 11206393AA19990803; US2007224624A 1A 1 20070927); a method for diagnosing huntington's disease, kennedy's disease, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, dentate nucleus-pallidum atrophy, fragile X syndrome, fragile E site, myotonic dystrophy, friedreich's ataxia or spinocerebellar ataxia type 8 by microcapillary electrophoresis (WO 03014396A1 a1 20030220); the same literature for multiplex detection of several SCA subtypes is not retrieved.

How to establish a rapid, economical and efficient method for pre-mutation and total mutation screening and prenatal diagnosis is the basis for prevention, auxiliary diagnosis and genetic blocking. Thus, according to the literature (Jiang Hong, tang Beisha, xu Bo, etc.) mutation frequency analysis of each subtype of SCA of the Chinese continental Han population and clinical and molecular characteristics of SCA6 [ J ]. J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.J.3.3. Shu Anli, pu Quanzhou, yin Jiahui, etc.. J.J.J.J.J.J.J.J.J.J.J.J.J.J.62.62.J.J.J.62 (2013,21 (11) 18-20.DOI: 10.13404/j.cnki.cjbhh.2013.11.008) and database data, 9 types of SCA subtypes, SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA8, SCA12, SCA17 and DRPLA, are selected from among the 40 types of SCA subtypes of SCA commonly seen or relatively seen in more than 40 types of SCA population, a simple, fast, simple, high-speed, accurate detection, easy to implement, and stable screening.

SCAs are a group of genetic neurodegenerative diseases with obvious clinical and genetic heterogeneity, and the risk generation is higher than the generation, so that clinical diagnosis cannot be carried out according to clinical manifestations alone; the primer group, the screening method and the application thereof can be used for detecting the SCAs9 subtype in a one-time, quick and simple way, have low cost and are easy to comprehensively popularize and screen.

Disclosure of Invention

The invention provides a primer group capable of detecting SCAs9 subtype caused by CAG three-base repeated sequence mutation at one time;

the primer group sequences comprise multiplex amplification primer sequences TP-PCR general primer sequences shown as SEQ ID NO.1-18 and TP-PCR general primer sequences shown as SEQ ID NO.19-20;

the multiplex amplification primer sequences described above are divided into 3 groups:

the first set of multiplex amplification primer sequences includes seq_ID No.1-4, 11-12;

the second set of multiplex amplification primer sequences includes seq_ID No.5-8, 15-16;

the third set of multiplex amplification primer sequences includes seq_ID No.9-10, 13-14, 17-18;

the above-mentioned TP-PCR general primer sequence 3 groups are added:

the first set of TP-PCR universal primers includes seq_ID No.19-20;

the second set of TP-PCR universal primers includes seq_ID No.19-20;

the third set of TP-PCR universal primers includes seq_ID No.19-20;

the invention also provides a method for detecting SCAs9 subtype, which comprises the steps of detecting 9SCA variant subtypes in a genome of a sample to be detected by using the primer group;

the invention also provides application of the primer group in preparation of SCAs9 subtype screening products.

Further, multiplex fluorescence TP-PCR is carried out on the genome of the object to be detected by using the primer group for screening the SCAs9 subtype, and then PCR products are identified by capillary electrophoresis, so that whether the SCAs9 subtype of the genome of the object to be detected has CAG repeated expansion variation is determined.

The beneficial effects of the invention are as follows: the primer group for detecting the SCAs9 subtype is obtained through sample test screening, and can accurately detect whether the SCAs9 subtype has CAG repeated sequence expansion variation; the SCAs9 subtype primer group provided by the invention covers 9 SCAs: SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA8, SCA12, SCA17 and DRPLA;

the method for screening SCAs9 subtype diseases can realize specific detection on the mutation sites, and has the advantages of high accuracy, high sensitivity, good repeatability, low cost, short detection period, visual result, no need of message intervention and the like;

the invention overcomes the defect of few SCAs subtype detection at one time in the prior art, has low cost and short detection period, and is suitable for wide popularization; the sample required by the invention has strong adaptability, and can be well detected on peripheral blood, dry blood slices and the like; the detection efficiency is greatly improved, the method is particularly suitable for batch detection, and a scientific and effective clinical basis is provided for screening SCAs9 subtype variation;

the invention is suitable for infants and fertility-required people, can help the testees with suspected family history to screen pathogenic variation, evaluate pathogenic efficacy and assist reproduction; the high-risk carrier can take targeted measures early, so that the treatment effect is improved, the risk of neonatal diseases is reduced, and an effective scientific basis is provided for prevention and treatment of SCAs.

Drawings

Exemplary figure of the number of CAG repeats of ATXN1 gene:

FIG. 1SCA1 subtype negative sample example_26/29;

FIG. 2SCA1 subtype positive sample example_26/56;

FIG. 3 example of SCA1 subtype plasmid_SCA1 quality control-60;

SCA2: schematic of the number of CAG repeats of ATXN2 gene:

FIG. 4SCA2 subtype negative sample example_22/22;

FIG. 5SCA2 subtype positive sample example_22/43;

FIG. 6 example of SCA2 subtype plasmid_SCA2 quality control-32;

SCA3: schematic of the number of CAG repeats of ATXN3 gene:

FIG. 7SCA3 subtype negative sample example_14/28;

FIG. 8SCA3 subtype positive sample example_14/67;

FIG. 9 example of SCA3 subtype plasmid_SCA3 quality control-49;

SCA6: schematic of CACNA1A CAG repetition number:

FIG. 10SCA6 subtype negative sample example_11/13;

FIG. 11SCA6 subtype positive sample example_11/22;

FIG. 12 example of SCA6 subtype plasmid_SCA6 quality control-19;

SCA7: schematic of the number of CAG repeats of ATXN7 gene:

FIG. 13SCA7 subtype negative sample example_10/12;

FIG. 14 example of SCA7 subtype plasmid_SCA7 quality control of_33;

SCA8: schematic representation of the number of repeats of ATXN8OS/ATXN8 gene (CTA. TAG) n (CTG. CAG) n:

FIG. 15SCA8 subtype negative sample example_27/29;

FIG. 16 example of SCA8 subtype plasmid_SCA8 quality control-61;

schematic of CAG repeat number of SCA12 PPP2R2B gene:

FIG. 17SCA12 subtype negative sample example_13/17;

FIG. 18 example of SCA12 subtype plasmid_SCA12 quality control-33;

SCA17: schematic of the number of CAG repeats of TBP gene:

FIG. 19SCA17 subtype negative sample example_24/37;

FIG. 20SCA17 subtype positive sample example_38/41;

FIG. 21 example of quality control for the SCA17 subtype plasmid_SCA17_41;

DRPLA: schematic of the number of CAG repeats of the ATN1 gene:

FIG. 22DRPLA subtype negative sample example_19/22;

FIG. 23DRPLA subtype plasmid_DRPLA quality control example_36 g/. Mu.L; FIG. 24 is a diagram of an example SCAs limit of detection (except SCA 12);

FIG. 25SCA12 shows the detection limit result (peak at arrow).

Detailed Description

The main reagent information used in the examples of the present invention is as follows:

the main instrument information used in the embodiment of the invention is as follows:

example 1 selection of SCAs detection subtypes

According to literature reports and related database records, the SCA subtype caused by repeated expansion of CAG three bases is selected from more than 40 SCA subtypes, and the SCA variant subtype is highly developed or reported in Chinese people. The variant subtype selection to be tested of the invention requires:

1) Referring to databases of 1000genome, dbSNP, clinVar, HGMD and the like, the SCA mutation is confirmed to have definite pathogenic effect through literature demonstration;

2) And screening SCA variant subtypes with Chinese crowd screening significance by combining with the characteristics of Chinese crowd.

The invention then determines the mutation site including clinically accepted pathogenicity through clinical verification,

SCAs9 subtype genes and variation information are shown in the following table:

TABLE 1 SCAs9 subtype genes and their repetitive sequence variation information table

Example 2 design of primers and establishment of preliminary reaction System

Firstly, the invention designs the repetitive sequences and flanking sequences of ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, ATXN8OS/ATXN8, PPP2R2B, TBP and ATN1 genes covering SCAs9 subtype through an online primer design platform (https:// www.ncbi.nlm.nih.gov/tools/primer-blast /), and uses NCBI and MFE online platform (MFEprimer-3.1:PCR Primer Quality Control-Specificity, hairpin & Dimers (igetech.com)) to complete the design of specific primers and multiplex PCR primers.

Synthesizing primers, preparing amplification primers with the concentration of 10uM according to the requirement, and uniformly mixing for later use.

The primer group for detecting SCAs9 subtype variation is a pathogenic gene aiming at SCAs9 subtype: the CAG repeated sequences and flanking sequences thereof in the ATXN1, ATXN2, ATXN3, CACNA1A, ATXN, ATXN8OS/ATXN8, PPP2R2B, TBP and ATN1 genes are designed, so that specific detection of SCAs9 subtype can be realized, the accuracy is high, and the detection results cover 9SCA subtypes: SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA8, SCA12, SCA17 and DRPLA can greatly shorten the detection period and reduce the detection cost.

The primer group provided by the invention is obtained through modification, optimization and sample testing and screening, and all primer sequences can accurately detect samples, so that rapid and effective detection of SCAs9 subtype by using multiple fluorescence TP-PCR-capillary electrophoresis is realized.

TABLE 2 multiplex amplification primer sequences

The multiplex amplification primer components were divided into 3 groups as follows:

the first set comprises amplification primer sets seq_ID No.1-4, 11-12 for detecting SCA1, SCA2, SCA 8;

the second set comprises amplification primer sets seq_ID No.5-8, 15-16 for detecting SCA3, SCA6, SCA 17;

the third group comprises amplification primer groups seq_ID No.9-10, 13-14, 17-18 for detecting SCA7, SCA12 and DRPLA;

the grouping comprehensively considers the factors such as the specificity of the amplified primers, and the like, so that the mutual interference among the primers in each group is avoided or the interaction does not influence the final result;

the 3 groups also need to be added with TP-PCR general primer groups, and the sequences are shown as SEQ ID NO.19-20.

TABLE 3 TP-PCR general primer sequences

The specific operation of primer test and optimization is as follows:

1) The reaction system was prepared and labeled as follows, centrifuged, and PCR thermocycling was performed:

table 4PCR amplification reaction system sca_1_2_8 configuration table:

table 5PCR amplification reaction system sca_3_6_17 configuration table:

table 6 PCR amplification reaction system sca_7_12_drpla configuration table:

table 7PCR reaction procedure (sca_1_2_8):

table 8PCR reaction procedure (sca_3_6_17/sca_7_12_drpla):

2) Capillary electrophoresis detection: in Liz500: hi-di=2.5: 97.5, preparing capillary electrophoresis loading buffer solution in proportion; PCR amplification product and capillary electrophoresis loading buffer at 1:9, proportional loading detection; after electrophoresis, data are exported;

3) And (3) result analysis and interpretation: geneMark, see FIGS. 1-23.

Example 3 Performance verification

1) And (3) accuracy verification: randomly picking 20 peripheral blood samples which are detected by a sanger, and comparing and verifying the detection by a multiplex fluorescence TP-PCR and capillary electrophoresis combined technology with a sanger result to ensure that the accuracy reaches 100%;

table 8 accuracy verification results_sca1_2_8:

table 9 accuracy verification results_sca3_6_17:

table 10 accuracy validation results_sca7_12_drpla:

2) And (3) verifying the detection limit: 1 sample with the concentration higher than 200ng/uL is selected, and the detection limit is verified according to concentration gradients of 200ng/uL, 100ng/uL, 50ng/uL, 25ng/uL, 12.5ng/uL, 6.25ng/uL and 3.125ng/uL, and the result shows that: SCA12 shows that the concentration is lower than 12.5ng/uL due to the existence of the impurity peak, and the impurity peak signal is higher than the result peak signal as shown in FIG. 24; the remaining subtypes have low requirements on DNA concentration, as shown in FIG. 25, and can still judge the detection result to 3.125. Thus the method requires no less than or more than 12.5ng/uL for detecting DNA concentration.

3) And (5) repeatability verification: the above peripheral blood samples were randomly selected for 5 cases: d204631, D204636, D204638, D204648, D204650 for repeatability verification; within lot repeatability: the results of the three repeated experiments of the same batch are shown in Table 11; batch-to-batch repeatability: the results of the 3 samples measured in 3 times are shown in Table 12. The results showed that the method was stable, including both intra-batch and inter-batch repetition maintaining consistent results.

TABLE 11 within-batch repeatability verification results

TABLE 12 results of batch-to-batch repeatability verification

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (6)

1. A primer group for detecting SCAs9 subtype caused by CAG three-base repetitive sequence mutation at one time; the primer group sequences comprise multiplex amplification primer sequences TP-PCR primer sequences shown as SEQ ID NO.1-18 and TP-PCR universal primer sequences shown as SEQ ID NO.19-20;

the multiplex amplification primer sequences described above are divided into 3 groups:

the first set of multiplex amplification primer sequences includes seq_ID No.1-4, 11-12;

the second set of multiplex amplification primer sequences includes seq_ID No.5-8, 15-16;

the third set of multiplex amplification primer sequences includes seq_ID No.9-10, 13-14, 17-18;

the above-mentioned TP-PCR general primer sequence 3 groups are added:

the first set of TP-PCR universal primers includes seq_ID No.19-20;

the second set of TP-PCR universal primers includes seq_ID No.19-20;

the third set of TP-PCR universal primers included seq_ID No.19-20.

2. A composition for detecting SCAs9 subtype, characterized in that: the composition comprising the primer set of claim 1.

3. A kit for detecting SCAs9 subtype, characterized in that: the composition comprising the primer set of claim 1.

4. A method for detecting SCAs9 subtype, comprising the step of detecting 9SCA variant subtypes in a genome of a sample to be detected by using the primer set.

5. Use of the primer set of claim 1, the composition of claim 2, the kit of claim 3 for preparing a product for screening for SCAs9 subtype.

6. The method according to claim 5, wherein the primer set for screening SCAs9 subtype is used for multiplex fluorescence TP-PCR amplification of the genome of the object to be detected, and the PCR product is identified by capillary electrophoresis to determine whether the SCAs9 subtype of the genome of the object to be detected has CAG repeated expansion variation.