CN117363721A - Composite detection primer set for measuring dynamic mutation of cerebellar ataxia related gene repetitive sequence and application - Google Patents

Abstract

The invention provides a composite detection primer set for measuring dynamic mutation of cerebellar ataxia related gene repetitive sequence and application thereof, belonging to the technical field of clinical molecular detection. The composite detection primer group for determining dynamic mutation of cerebellar ataxia related gene repetitive sequence provided by the invention comprises primer sequences shown as SEQ ID NO. 1-12. The detection system containing the primer sequence can detect the (CAG) n amplified mutation of the SCA1, SCA2, SCA3, SCA6, SCA12 and DRPLA related genes simultaneously. The subtypes can be accurately analyzed by single amplification, and the analysis flow is clear and simple.

Description

Composite detection primer set for measuring dynamic mutation of cerebellar ataxia related gene repetitive sequence and application

Technical Field

The invention relates to the technical field of clinical molecular detection, in particular to a composite detection primer group for measuring dynamic mutation of cerebellar ataxia related gene repetitive sequence and application thereof.

Background

Spinocerebellar ataxia (spinocerebellar ataxia, SCA) is a group of nervous system genetic diseases which are mainly clinically manifested by degeneration of cerebellum, brainstem and spinal cord caused by gene mutation, and progressive motor coordination function decline and imbalance. Both the definition of spinocerebellar ataxia initially and classical SCA are autosomal dominant inheritance, due to repeated amplification of abnormal copy numbers of the trinucleotide of the corresponding gene exon (CAG) to produce polyglutamine. Types of autosomal recessive inheritance, X-linked inheritance and mitochondrial inheritance (NARP, MERRF and CoQ10 deficiency) were later discovered. Tens of SCA causative genes have been found, but some types have not found definitive causative genes.

The method for detecting SCA dynamic mutation is PCR electrophoresis detection, capillary electrophoresis technology replaces traditional plate electrophoresis, multicolor fluorescent dye labeling is applied, and after PCR reaction, a DNA sequencer can analyze the generated PCR product and can determine the DNA fragment lengths of various different fluorescent dyes. The PCR product can be electrophoresed in one capillary, so that the influence of mobility difference between lanes is avoided, and the accuracy of electrophoresis detection is greatly improved.

Claudia Cagnoli et al (Cagnoli C. Spinocerebella rAlataxia Tethering PCR: ARapid Genetic Test for the Diagnosis of Spinocerebellar Ataxia Types 1,2,3,6,and 7by PCR and Capillary Electrophoresis.J Mol Diagn.2018May;20 (3): 289-297.) proposed multiple capillary electrophoresis amplification systems for SCA1, 2,3,6, 7 in 2018, but it divided SCA1,3,6 into one-tube amplifications, SCA2 into one-tube amplifications, SCA7 into one-tube amplifications, and a total of 3-tube amplification analyses were required, and the procedure was cumbersome. The complex amplification system proposed by LianM et al (Lian M.Single-Tube Screen for Rapid Detection of Repeat Expansions in Seven Common spinocelebella rAbtaxias. Clin chem.2022Jun 1;68 (6): 794-802.) was based on the principle of triplet-primed-PCR, and the peak pattern detected by the system had more peak type interferences between different detection subtypes, and had very high analysis requirements for general detection analysts, and could not simply, rapidly and effectively distinguish positive results. Aiming at the problems existing in the prior art, how to accurately, simply and efficiently complete the detection of the dynamic mutation of the cerebellar ataxia related gene repetitive sequence is the technical problem to be solved by the invention.

Disclosure of Invention

The invention aims to provide a composite detection primer group for measuring dynamic mutation of cerebellar ataxia related gene repetitive sequence and application thereof, which can simply, accurately and efficiently complete detection of cerebellar ataxia related gene.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a composite detection primer group for determining dynamic mutation of cerebellar ataxia related gene repetitive sequence, wherein the primer group comprises primer sequences shown as SEQ ID NO. 1-12.

Preferably, the primer set is used to detect (CAG) n amplified mutations of the SCA1, SCA2, SCA3, SCA6, SCA12 and DRPLA genes.

Preferably, when the primer set is used in an amplification reaction, the primer set is added to an amplification reaction system in the form of a mixed primer set.

Preferably, the concentration of SEQ ID NO. 1-2 in the mixed primer set is 0.5-0.7 nmol/μl, the concentration of SEQ ID NO. 3-4 is 0.9-1.1 nmol/μl, the concentration of SEQ ID NO. 5-6 is 1.3-1.5 nmol/μl, the concentration of SEQ ID NO. 7-8 is 0.9-1.1 nmol/μl, the concentration of SEQ ID NO. 9-10 is 2.1-2.3 nmol/μl, and the concentration of SEQ ID NO. 11-12 is 2.8-3.2 nmol/μl.

Preferably, the amplification reaction system is a PCR amplification reaction system; the PCR amplification reaction system comprises: KOD-FX 0.3-0.5. Mu.L, 2mM dNTP 3-5. Mu.L, 2 XPCR buffer 8-12. Mu.L, ddH2O 2.5-2.8. Mu.L, template DNA 0.8-1.2. Mu.L, and mixed primer set 1.5-2.5. Mu.L, with total volume of 17-23. Mu.L.

The invention also provides application of the primer group in preparing a detection reagent for measuring dynamic mutation of cerebellar ataxia related gene repetitive sequence.

Preferably, when the result of dynamic mutation of the cerebellar ataxia related gene repetitive sequence is interpreted, 100-300bp is a SCA1 detection interval, 300-600bp is a SCA2 detection interval, 100-400bp is a SCA3 detection interval, 400-600bp is a SCA6 detection interval, TRMRA fluorescent channel detects SCA12, ROX fluorescent channel detects DRPLA, and the highest peak in single cluster peaks appearing in each interval is recorded as a full-length product peak determined by a full-length amplification system.

Preferably, the CAG repeat number is calculated from the full-length product peak.

The detection system for measuring cerebellar ataxia provided by the invention can be used for simultaneously detecting (CAG) n amplified mutation of SCA1, SCA2, SCA3, SCA6, SCA12 and DRPLA related genes. The subtypes can be accurately analyzed by single amplification, and the analysis flow is clear and simple.

Drawings

FIG. 1 is a graph showing the fluorescence and data distribution of the detection of different SCA subtypes in example 1;

FIG. 2 shows the result of the negative sample test in example 2;

FIG. 3 shows the detection results of FAM fluorescence channel of SCA1 positive samples in example 2;

FIG. 4 shows the detection results of FAM fluorescence channel of SCA2 positive samples in example 2;

FIG. 5 shows the detection results of the HEX fluorescence channel of the SCA3 positive sample in example 2;

FIG. 6 shows the detection results of the HEX fluorescence channel of the SCA6 positive sample in example 2;

FIG. 7 shows the detection result of the TRMRA fluorescence channel of the SCA12 positive sample in example 2;

FIG. 8 shows the detection result of ROX fluorescence channel of DRPLA-positive sample in example 2.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1.1 primer design

The sequences of 6 pairs of specific primers obtained in this example are shown as SEQ ID NO. 1-12.

1.2PCR amplification reaction System and reaction procedure

The primers SEQ ID NO. 1-12 were proportioned according to the concentrations described in Table 1 to obtain a Primer mixture Primer-Mix for PCR amplification.

Table 1 6 pair specific primer sequences and concentrations

The PCR reaction system is shown in Table 2.

TABLE 2PCR reaction System

The components	Volume (mu L)
		Enzyme KOD FX (Toyobo)	0.4
2mM dNTP(Toyobo)	4
		2×PCR buffer(Toyobo)	10
ddH20	2.6
		Template DNA	1
Primer-Mix	2
		Total volume of	20

Each reaction tube was placed in a reaction tank of a PCR amplification apparatus, and the reaction system was set to 20. Mu.L. The PCR amplification procedure is shown in Table 3.

TABLE 3 PCR amplification procedure

Note that: in Table 3 "-1℃cycle" means a1℃decrease per Cycle.

1.3 capillary electrophoresis detection

A total of 9. Mu.L of a sample mixture (0.5. Mu.L of a molecular weight internal standard +8.5. Mu.L of formamide) containing SIZE 600 and formamide was prepared, 1. Mu.L of the amplified product was added, and after mixing, the mixture was denatured at 95℃for 5 minutes and ice-cooled for 3 minutes. The detection is performed according to the genetic analyzer user using manual steps. And detecting and setting the sample injection time to 10 seconds, the sample injection voltage to 3kV and the running time to 2000 seconds to obtain a detection result of capillary electrophoresis.

1.4 data analysis

Relevant files are imported into GeneMarker software, SIZE 600 internal Standard (Siez 600Size Standard of Rebaume Biotech company, hangzhou), capillary electrophoresis detection data (. Fsa file), and the files imported before are selected in relevant parameter selection columns, and the data are analyzed.

1.5 interpretation of results

Regional division was performed according to CAG repeat numbers of several subtypes of the chinese population currently reported to and studied earlier in the laboratory (fig. 1):

100-300bp of FAM fluorescent channel is SCA1 detection zone, 300-600bp is SCA2 detection zone; 100-400bp of HEX fluorescent channel is SCA3 detection zone, 400-600bp is SCA6 detection zone; detecting SCA12 by TRMRA fluorescent channel; the ROX fluorescence channel detects DRPLA. The highest peak of the single cluster peaks appearing in each interval is marked as the full-length product peak determined by the full-length amplification system and is marked as FL.

The calculation formula of the CAG repetition number of each SCA subtype is shown in Table 3.

TABLE 3 calculation formula for CAG repetition number for each SCA subtype

Example 2

Negative samples known in the laboratory and positive samples of SCA1, SCA2, SCA3, SCA6, SCA12, DRPLA were selected for testing to verify the correctness of the experimental system of example 1.

DNA extraction

The DNA of the peripheral blood sample was extracted using the Kangji blood genomic DNA extraction kit as an amplification template. And (5) after the DNA quality is qualified, the DNA is used for downstream experiments.

The PCR amplification reaction system, the reaction program, and the capillary electrophoresis detection method described in example 1 were used to carry out the detection, and the data analysis was carried out in the same manner as in example 1, and finally, the detection results were shown in tables 4 to 10.

TABLE 4 detection results for each subtype in negative samples

As can be seen from Table 1, all subtypes can be amplified accurately using the phylogenetic system to detect negative samples, without interaction between the subtypes (FIG. 2).

The SCA1 positive sample test results (table 5) show that one of the CAG alleles has 52 CAG repeats (the number of CAG repeats for one allele is excessive due to autosomal dominant inheritance, and thus the positive data detected is typically in allele 2) with greater than 45 repeats, within the pathogenic range (fig. 3).

TABLE 5SCA1 Positive sample detection results

The SCA2 positive sample detection results (table 6) show that one of the CAG alleles was 45 CAG repeats, greater than 33 repeats, within the pathogenic range (fig. 4).

TABLE 6SCA2 positive sample detection results

The SCA3 positive sample detection results (table 7) show that the allele of one CAG is 74 CAG repeats, greater than 60 repeats, within the pathogenic range (fig. 5).

TABLE 7SCA3 positive sample detection results

The SCA6 positive sample detection results (table 8) show that one of the CAG alleles was 22 CAG repeats, greater than 20 repeats, within the pathogenic range (fig. 6).

TABLE 8SCA6 Positive sample detection results

The results of the SCA12 positive sample detection (table 9) show that the allele of one CAG was 59 CAG repeats, greater than 51 repeats, within the pathogenic range (fig. 7).

TABLE 9SCA12 positive sample detection results

The DRPLA positive sample test results (table 10) show that the allele of one of the CAGs was 58 CAG repeats, greater than 48 repeats, within the pathogenic range (fig. 8).

TABLE 10DRPLA Positive sample detection results

In conclusion, the detection method can effectively detect the subtypes of SCA1, SCA2, SCA3, SCA6, SCA12 and DRPLA accurately, has no mutual interference and is simple and convenient in calculation method.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (8)

1. A composite detection primer group for determining dynamic mutation of cerebellar ataxia related gene repetitive sequence is characterized in that the primer group comprises primer sequences shown as SEQ ID NO. 1-12.

2. The primer set of claim 1, wherein the primer set is used to detect (CAG) n amplified mutations of SCA1, SCA2, SCA3, SCA6, SCA12 and DRPLA genes.

3. The primer set according to claim 2, wherein the primer set is added to an amplification reaction system as a mixed primer set when used in an amplification reaction.

4. The primer set according to claim 3, wherein the concentration of SEQ ID NO. 1-2 is 0.5-0.7 nmol/μl, the concentration of SEQ ID NO. 3-4 is 0.9-1.1 nmol/μl, the concentration of SEQ ID NO. 5-6 is 1.3-1.5 nmol/μl, the concentration of SEQ ID NO. 7-8 is 0.9-1.1 nmol/μl, the concentration of SEQ ID NO. 9-10 is 2.1-2.3 nmol/μl, and the concentration of SEQ ID NO. 11-12 is 2.8-3.2 nmol/μl.

5. The primer set of claim 4, wherein the amplification reaction system is a PCR amplification reaction system; the PCR amplification reaction system comprises: KOD-FX 0.3-0.5. Mu.L, 2mM dNTP 3-5. Mu.L, 2 XPCR buffer 8-12. Mu.L, ddH ₂ O2.5-2.8 mu L, template DNA 0.8-1.2 mu L, mixed primer 1.5-2.5 mu L and total volume 17-23 mu L.

6. Use of a primer set according to any one of claims 1 to 5 for the preparation of a detection reagent for determining dynamic mutations in repeated sequences of cerebellar ataxia-related genes.

7. The use according to claim 6, wherein in the interpretation of the results of dynamic mutations in cerebellar ataxia-related gene repeats, the highest peak in single cluster peaks occurring in each interval is recorded as the full-length product peak determined by the full-length amplification system according to 100-300bp for the FAM fluorescence channel as the SCA1 detection interval, 300-600bp for the SCA2 detection interval, 100-400bp for the HEX fluorescence channel as the SCA3 detection interval, 400-600bp for the SCA6 detection interval, TRMRA fluorescence channel as the SCA12, and ROX fluorescence channel as the full-length product peak determined by the full-length amplification system.

8. The use according to claim 7, wherein the CAG repeat number is calculated from the full-length product peak.