CN110872622A

CN110872622A - Kit for detecting MMACHC gene mutation sites

Info

Publication number: CN110872622A
Application number: CN201911200022.1A
Authority: CN
Inventors: 张岩; 胥慧; 郭涛; 常乐; 田茜茜
Original assignee: CapitalBio Technology Co Ltd
Current assignee: CapitalBio Technology Co Ltd
Priority date: 2019-11-29
Filing date: 2019-11-29
Publication date: 2020-03-10

Abstract

The invention discloses a kit for detecting MMACHC gene mutation sites. The invention provides a set of primers for detecting mutation sites of MMACHC genes, which consists of 13 primer groups from the following primer group 1 to 13, and establishes a mutation site high-throughput detection method of the MMACHC genes based on gene molecular typing according to the primers, so that the mutation typing of 13 mutation sites such as human MMACHC genes c.609G > A, c.658_660delAAG, c.567dupT, c.80A > G, c.482G > A, c.394C > T, c.1A > G, c.315C > G, c.446 _ del445, c.481C > T, c.217C > T, c.331C > T, c.365A > T and the like can be completed at the same time in the same reaction, the detection efficiency is improved, the cost is reduced, the detection throughput is high, the operation is simple and reliable, and the method is suitable for carrying out the detection of MMACHC genes related to the homomalonemia-associated cysteine in hospitals and medical detection laboratories.

Description

Kit for detecting MMACHC gene mutation sites

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a kit for detecting MMACHC gene mutation sites.

Background

Methyl Malonemia (MMA) is a common inherited metabolic disease caused by organic acid metabolic disorder, and belongs to autosomal recessive genetic disease, the hereditary MMA can be divided into 12 subtypes and 13 related genes at present, the most common subtypes are mutase defect and cblC defect, and the coding genes are MUT gene and MMACHC gene respectively. Mutase defects often occur in the neonatal period, are seriously ill and can threaten life. The ClbC defect is late in onset age compared with the mutase defect, but the ClbC defect usually occurs within the age of the year, and the children are usually accompanied with growth and development retardation.

CblC deficient methylmalonic acidemia combined with homocysteinemia is a metabolic disease of vitamin B12 caused by MMACHC deficiency. The MMACHC gene is located at 1p34.1, contains 5 exons and transcribes a 5.2kb mRNA, expressing a 282 amino acid protein. Molecular research experiments prove that the carboxyl terminal region of MMACHC gene expression protein is similar to the structure of the TonB protein of Escherichia coli, the structure of the TonB protein is related to energy transfer in the process of cobalamin uptake, MMACHC is mainly involved in converting vitamin B12 (cobalamin) obtained from food into a form which can be utilized by a human body, and the MMACHC gene mutation is presumed to cause cobalamin metabolic disorder through the mechanism. At present, nearly 80 MMACHC gene mutations are reported at home and abroad, and about 30 MMACHC gene mutations are reported at home.

Since MMA infants have different gene phenotypes, the specific treatment method is different. The gene diagnosis and the found pathogenic gene mutation are the gold standard for determining the disease. Therefore, the development of the detection of MMACHC gene mutation has clinical practical value and superior biological significance.

The types of mutations in the human MMACHC gene are as follows: c.609G > A, c.658_660delAAG, c.567dupT, c.80A > G, c.482G > A, c.394C > T, c.1A > G, c.315C > G, c.445_446delTG, c.481C > T, c.217C > T, c.331C > T, c.365A > T.

Disclosure of Invention

An object of the invention is to provide a set of primers for detecting mutation sites of MMACHC genes.

The primer set provided by the invention comprises 13 primer groups from the following primer group 1 to the primer group 13:

the primer group 1 consists of a specific primer 1-1, a specific primer 1-2 and a universal primer 1;

the primer group 2 consists of a specific primer 2-1, a specific primer 2-2 and a universal primer 2;

the primer group 3 consists of a specific primer 3-1, a specific primer 3-2 and a universal primer 3;

the primer group 4 consists of a specific primer 4-1, a specific primer 4-2 and a universal primer 4;

the primer group 5 consists of a specific primer 5-1, a specific primer 5-2 and a universal primer 5;

the primer group 6 consists of a specific primer 6-1, a specific primer 6-2 and a universal primer 6;

the primer group 7 consists of a specific primer 7-1, a specific primer 7-2 and a universal primer 7;

the primer group 8 consists of a specific primer 8-1, a specific primer 8-2 and a universal primer 8;

the primer group 9 consists of a specific primer 9-1, a specific primer 9-2 and a universal primer 9;

the primer group 10 consists of a specific primer 10-1, a specific primer 10-2 and a universal primer 10;

the primer group 11 consists of a specific primer 11-1, a specific primer 11-2 and a universal primer 11;

the primer group 12 consists of a specific primer 12-1, a specific primer 12-2 and a universal primer 12;

the primer group 13 consists of a specific primer 13-1, a specific primer 13-2 and a universal primer 13;

the specific primer 1-1 is 1-1-a) or 1-1-b) or 1-1-c) as follows:

1-1-a) a single-stranded DNA molecule shown as a sequence 1 in a sequence table;

1-1-b) single-stranded DNA molecules shown in 22 th to 43 th sites of a sequence 1 in a sequence table;

1-1-c) carrying out single-stranded DNA molecule obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequence of 1-1-b) or connecting the tail end of the sequence with a fluorescent sequence;

the specific primer 1-2 is 1-2-a) or 1-2-b) or 1-2-c):

1-2-a) a single-stranded DNA molecule shown as a sequence 2 in a sequence table;

1-2-b) single-stranded DNA molecules shown in 22 th to 43 th sites of a sequence 2 in a sequence table;

1-2-c) carrying out single-stranded DNA molecule obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequence of 1-2-b) or connecting the tail end of the sequence with a fluorescent sequence;

the universal primer 1 is 1-A) or 1-B) as follows:

1-A) a single-stranded DNA molecule shown as a sequence 3 in a sequence table;

1-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequence of 1-A);

the specific primer 2-1 is the following 2-1-a) or 2-1-b) or 2-1-c):

2-1-a) a single-stranded DNA molecule shown as a sequence 4 in a sequence table;

2-1-b) single-stranded DNA molecules shown in 22 th to 46 th sites of a sequence 4 in a sequence table;

2-1-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 2-1-b) or connecting the tail ends of the sequences with fluorescent sequences;

the specific primer 2-2 is the following 2-2-a) or 2-2-b) or 2-2-c):

2-2-a) a single-stranded DNA molecule shown as sequence 5 in the sequence table;

2-2-b) single-stranded DNA molecules shown in 22 th to 45 th sites of a sequence 5 in a sequence table;

2-2-c) carrying out 1 or several nucleotide residue substitution and/or deletion and/or addition on the sequence of 2-2-b) to obtain a single-stranded DNA molecule or connecting the tail end of the sequence with a fluorescent sequence;

the universal primer 2 is 2-A) or 2-B) as follows:

2-A) a single-stranded DNA molecule shown as a sequence 6 in a sequence table;

2-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequence of 2-A);

the specific primer 3-1 is 3-1-a) or 3-1-b) or 3-1-c):

3-1-a) a single-stranded DNA molecule shown as sequence 7 in the sequence table;

3-1-b) single-stranded DNA molecules shown in 22 th to 49 th sites of a sequence 7 in a sequence table;

3-1-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 3-1-b) or connecting the tail ends of the sequences with fluorescent sequences;

the specific primer 3-2 is 3-2-a) or 3-2-b) or 3-2-c) as follows:

3-2-a) a single-stranded DNA molecule shown as a sequence 8 in a sequence table;

3-2-b) single-stranded DNA molecules shown in 22 th to 49 th sites of a sequence 8 in a sequence table;

3-2-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of the 3-2-b) or connecting the tail ends of the sequences with fluorescent sequences;

the universal primer 3 is 3-A) or 3-B) as follows:

3-A) a single-stranded DNA molecule shown as a sequence 9 in a sequence table;

3-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequence of 3-A);

the specific primer 4-1 is 4-1-a) or 4-1-b) or 4-1-c) as follows:

4-1-a) a single-stranded DNA molecule shown as a sequence 10 in a sequence table;

4-1-b) single-stranded DNA molecules shown in 22 th-43 th sequence 10 in the sequence table;

4-1-c) carrying out substitution and/or deletion and/or addition of 1 or more nucleotide residues on the 4-1-b) nucleotide sequence to obtain a single-stranded DNA molecule or connecting the tail end of the sequence with a fluorescent sequence;

the specific primer 4-2 is 4-2-a) or 4-2-b) or 4-2-c) as follows:

4-2-a) a single-stranded DNA molecule shown as a sequence 11 in the sequence table;

4-2-b) single-stranded DNA molecules shown in 22 th-43 th sequence 11 in the sequence table;

4-2-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 4-2-b) or connecting the tail ends of the sequences with fluorescent sequences;

the universal primer 4 is 4-A) or 4-B) as follows:

4-A) a single-stranded DNA molecule shown as a sequence 12 in a sequence table;

4-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequence of 4-A);

the specific primer 5-1 is 5-1-a) or 5-1-b) or 5-1-c):

5-1-a) a single-stranded DNA molecule shown as sequence 13 in the sequence table;

5-1-b) single-stranded DNA molecules shown in 22 th to 39 th of a sequence 13 in a sequence table;

5-1-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 5-1-b) or connecting the tail ends of the sequences with fluorescent sequences;

the specific primer 5-2 is 5-2-a) or 5-2-b) or 5-2-c):

5-2-a) a single-stranded DNA molecule shown as a sequence 14 in a sequence table;

5-2-b) single-stranded DNA molecules shown in 22 th-39 th sequence 14 in the sequence table;

5-2-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 5-2-b) or connecting the tail ends of the sequences with fluorescent sequences;

the universal primer 5 is 5-A) or 5-B) as follows:

5-A) a single-stranded DNA molecule shown as a sequence 15 in a sequence table;

5-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues to the sequence of 5-A);

the specific primer 6-1 is 6-1-a) or 6-1-b) or 6-1-c):

6-1-a) a single-stranded DNA molecule shown as a sequence 16 in a sequence table;

6-1-b) single-stranded DNA molecules shown in 22 th to 43 th sites of a sequence 16 in a sequence table;

6-1-c) carrying out 1 or several nucleotide residue substitution and/or deletion and/or addition on the sequence of 6-1-b) to obtain a single-stranded DNA molecule or connecting the tail end of the sequence with a fluorescent sequence;

the specific primer 6-2 is 6-2-a) or 6-2-b) or 6-2-c):

6-2-a) a single-stranded DNA molecule shown as a sequence 17 in a sequence table;

6-2-b) single-stranded DNA molecules shown in 22 th to 43 th sites of a sequence 17 in a sequence table;

6-2-c) carrying out 1 or more nucleotide residue substitution and/or deletion and/or addition on the sequence of 6-2-b) to obtain a single-stranded DNA molecule or connecting the tail end of the sequence with a fluorescent sequence;

the universal primer 6 is 6-A) or 6-B) as follows:

6-A) a single-stranded DNA molecule shown as a sequence 18 in a sequence table;

6-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues to the sequence of 6-A);

the specific primer 7-1 is 7-1-a) or 7-1-b) or 7-1-c):

7-1-a) a single-stranded DNA molecule shown as a sequence 19 in the sequence table;

7-1-b) a single-stranded DNA molecule shown in 22 th to 43 th sites of a sequence 19 in a sequence table;

7-1-c) carrying out 1 or several nucleotide residue substitution and/or deletion and/or addition on the sequence of 7-1-b) to obtain a single-stranded DNA molecule or connecting the tail end of the sequence with a fluorescent sequence;

the specific primer 7-2 is 7-2-a) or 7-2-b) or 7-2-c):

7-2-a) a single-stranded DNA molecule shown as a sequence 20 in the sequence table;

7-2-b) a single-stranded DNA molecule shown in 22 th to 42 th sites of a sequence 20 in a sequence table;

7-2-c) carrying out 1 or several nucleotide residue substitution and/or deletion and/or addition on the sequence of 7-2-b) to obtain a single-stranded DNA molecule or connecting the tail end of the sequence with a fluorescent sequence;

the universal primer 7 is 7-A) or 7-B) as follows:

7-A) a single-stranded DNA molecule shown as a sequence 21 in a sequence table;

7-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequence of 7-A);

the specific primer 8-1 is the following 8-1-a) or 8-1-b) or 8-1-c):

8-1-a) a single-stranded DNA molecule shown as a sequence 22 in a sequence table;

8-1-b) single-stranded DNA molecules shown in 22 nd to 51 th sites of a sequence 22 in a sequence table;

8-1-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 8-1-b) or connecting the tail ends of the sequences with fluorescent sequences;

the specific primer 8-2 is 8-2-a) or 8-2-b) or 8-2-c) as follows:

8-2-a) a single-stranded DNA molecule shown as sequence 23 in the sequence table;

8-2-b) single-stranded DNA molecules shown in 22 th to 51 th sites of a sequence 23 in a sequence table;

8-2-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 8-2-b) or connecting the tail ends of the sequences with fluorescent sequences;

the universal primer 8 is the following 8-A) or 8-B):

8-A) a single-stranded DNA molecule shown as a sequence 24 in a sequence table;

8-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequence of 8-A);

the specific primer 9-1 is the following 9-1-a) or 9-1-b) or 9-1-c):

9-1-a) a single-stranded DNA molecule shown as sequence 25 in the sequence table;

9-1-b) single-stranded DNA molecules shown in 22 th to 42 th of a sequence 25 in a sequence table;

9-1-c) carrying out 1 or several nucleotide residue substitution and/or deletion and/or addition on the sequence of 9-1-b) to obtain a single-stranded DNA molecule or connecting the tail end of the sequence with a fluorescent sequence;

the specific primer 9-2 is the following 9-2-a) or 9-2-b) or 9-2-c):

9-2-a) a single-stranded DNA molecule shown as a sequence 26 in a sequence table;

9-2-b) single-stranded DNA molecules shown in 22 th to 41 th of a sequence 26 in a sequence table;

9-2-c) carrying out 1 or several nucleotide residue substitution and/or deletion and/or addition on the sequence of 9-2-b) to obtain a single-stranded DNA molecule or connecting the tail end of the sequence with a fluorescent sequence;

the universal primer 9 is the following 9-A) or 9-B):

9-A) a single-stranded DNA molecule shown as a sequence 27 in a sequence table;

9-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequence of 9-A);

the specific primer 10-1 is 10-1-a) or 10-1-b) or 10-1-c) as follows:

10-1-a) a single-stranded DNA molecule shown as sequence 28 in the sequence table;

10-1-b) single-stranded DNA molecules shown in 22 th to 39 th of a sequence 28 in a sequence table;

10-1-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 10-1-b) or connecting the tail ends of the sequences with fluorescent sequences;

the specific primer 10-2 is 10-2-a) or 10-2-b) or 10-2-c) as follows:

10-2-a) a single-stranded DNA molecule shown as a sequence 29 in a sequence table;

10-2-b) single-stranded DNA molecules shown in 22 th to 39 th of a sequence 29 in a sequence table;

10-2-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequence of 10-2-b) or connecting the tail ends of the sequence with fluorescent sequences;

the universal primer 10 is 10-A) or 10-B) as follows:

10-A) a single-stranded DNA molecule shown as a sequence 30 in a sequence table;

10-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequence of 10-A);

the specific primer 11-1 is the following 11-1-a) or 11-1-b) or 11-1-c):

11-1-a) a single-stranded DNA molecule shown as a sequence 31 in a sequence table;

11-1-b) single-stranded DNA molecules shown in 22 th to 42 th of a sequence 31 in a sequence table;

11-1-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 11-1-b) or connecting the tail ends of the sequences with fluorescent sequences;

11-1-c) connecting the tail end of the nucleotide sequence of 11-1-a) or 11-1-b) with a fluorescent sequence;

the specific primer 11-2 is the following 11-2-a) or 11-2-b) or 11-2-c):

11-2-a) a single-stranded DNA molecule shown as a sequence 32 in a sequence table;

11-2-b) single-stranded DNA molecules shown in 22 th-42 th sequence 32 in the sequence table;

11-2-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 11-2-b) or connecting the tail ends of the sequences with fluorescent sequences;

the universal primer 11 is the following 11-A) or 11-B):

11-A) a single-stranded DNA molecule shown as a sequence 33 in a sequence table;

11-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues to the sequence of 11-A);

the specific primer 12-1 is 12-1-a) or 12-1-b) or 12-1-c):

12-1-a) a single-stranded DNA molecule shown as a sequence 34 in a sequence table;

12-1-b) single-stranded DNA molecules shown in 22 th to 43 th of a sequence 34 in a sequence table;

12-1-c) carrying out substitution and/or deletion and/or addition of 1 or more nucleotide residues on the sequence of 12-1-b) to obtain a single-stranded DNA molecule or connecting the tail end of the sequence with a fluorescent sequence;

the specific primer 12-2 is 12-2-a) or 12-2-b) or 12-2-c):

12-2-a) a single-stranded DNA molecule shown as sequence 35 in the sequence table;

12-2-b) single-stranded DNA molecules shown in 22 th to 43 th of a sequence 35 in a sequence table;

12-2-b) carrying out 1 or several nucleotide residue substitution and/or deletion and/or addition on the sequence of 12-2-a) to obtain a single-stranded DNA molecule or connecting the tail end of the sequence with a fluorescent sequence;

the universal primer 12 is 12-A) or 12-B) as follows:

12-A) a single-stranded DNA molecule shown as a sequence 36 in a sequence table;

12-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues to the sequence of 12-A);

the specific primer 13-1 is 13-1-a) or 13-1-b) or 13-1-c):

13-1-a) a single-stranded DNA molecule shown as a sequence 37 in the sequence table;

13-1-b) single-stranded DNA molecules shown in 22 th to 39 th of a sequence 37 in a sequence table;

13-1-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 13-1-b) or connecting the tail ends of the sequences with fluorescent sequences;

the specific primer 13-2 is 13-2-a) or 13-2-b) or 13-2-c):

13-2-a) a single-stranded DNA molecule shown as sequence 38 in the sequence table;

13-2-b) single-stranded DNA molecules shown in 22 th-39 th sequence 38 in the sequence table;

13-2-c) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues on the sequences of 13-2-b) or connecting the tail ends of the sequences with fluorescent sequences;

the universal primer 13 is 13-A) or 13-B) as follows:

13-A) a single-stranded DNA molecule shown as a sequence 39 in a sequence table;

13-B) single-stranded DNA molecules obtained by substituting and/or deleting and/or adding 1 or more nucleotide residues to the sequence of 13-A).

The above-mentioned 1 or several nucleotide residue substitution and/or deletion and/or addition are not more than 10 nucleotide residue substitution and/or deletion and/or addition.

In the above primer set, the fluorescence sequences connected to the 2 specific primers in each primer set are different; in the embodiment of the invention, the specific primer for amplifying the wild-type locus is connected with the FAM fluorescent sequence, and the specific primer for amplifying the mutant locus is connected with the HEX fluorescent sequence.

In the above primer sets, each primer set is packaged separately.

Another objective of the invention is to provide a PCR reagent for detecting the mutation site of the MMACHC gene.

The PCR reagent provided by the invention consists of 13 PCR reagents;

each PCR reagent comprises a group of primers in the primer set.

The third purpose of the invention is to provide a PCR kit for detecting the mutation site of the MMACHC gene.

The kit provided by the invention comprises the primer set or the PCR reagent.

The application of the primer set or the PCR reagent or the kit in detecting the mutation site of the MMACHC gene is also within the protection scope of the invention;

or, the application of the above-mentioned primer set or the above-mentioned PCR reagent in the preparation of product for detecting mutation site of MMACHC gene is also the protection scope of the present invention.

The 4 th purpose of the invention is to provide a method for detecting the mutation site of the MMACHC gene in a sample to be detected.

The method provided by the invention comprises the following steps: and (3) carrying out KASP amplification on the genome DNA of the sample to be detected by using each primer group in the primer set, carrying out genotyping on the amplified product, and judging the MMACHC gene mutation site in the sample to be detected according to the KASP genotyping result.

The KASP amplification was divided into 2 types as follows:

A. carrying out amplification on an ABI7500 PCR instrument, wherein the amplification system is as follows:

the 10 μ L ABI7500 PCR amplification reaction system was as follows:

5 μ L KASP 2 × Master MIX (LGC Inc., cat # KBS-1016-)

1 μ L template

mu.L primer (final concentration of specific primer is 0.12-0.25. mu.M; universal primer is 0.5-1.0. mu.M)

3μL ddH2O

Judging MMACHC gene mutation sites in the sample to be detected according to the KASP genotyping result as follows: if the sample to be detected amplified by the primer group of a certain mutation site shows the color (Allole 1, red) corresponding to the FAM sequence, the base of the mutation site in the MMACHC gene of the sample to be detected is wild type; if the color (Allele2, blue) corresponding to the HEX sequence is displayed, the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type; and if the mixed color (green) of the FAM sequence and the HEX sequence is displayed, the base of the mutation site in the MMACHC gene of the sample to be detected is the heterozygous mutant.

B. Chip pool for KASP amplification

The primers (the final concentration of the specific primers is 0.12-0.25 mu M, the final concentration of the universal primers is 0.5-1.0 mu M) are spotted in a chip reaction pool (each sample to be detected is amplified by 13 groups of primers shown in the table 2 respectively), and PCR amplification reaction systems of different samples to be detected are added after film covering, wherein the PCR amplification reaction systems of the different samples to be detected are as follows:

20 μ L KASP 2 × Master MIX (LGC Inc., cat # KBS-1016-)

10 μ L of genomic DNA from different test samples (40 ng-300ng in total)

20μL ddH2O

The amplified chip was detected by using a confocal laser chip scanner (LuxScan 10K/D) manufactured by Doctorbo Crystal Limited technologies. If the color (red) of the FAM sequence is displayed in the sample to be detected amplified by the primer group of a certain mutation site, the base of the mutation site in the MMACHC gene of the sample to be detected is wild type; if the color (green) of the HEX sequence is displayed, the basic group of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type; if the mixed color (yellow) of the FAM sequence and the HEX sequence is displayed, the base of the mutation site in the MMACHC gene of the sample to be detected is the heterozygous mutant type.

In the above method, the sample to be tested is derived from a human.

In the above, the MMACHC gene mutation site is at least one of: c.609G > A, c.658_660delAAG, c.567dupT, c.80A > G, c.482G > A, c.394C > T, c.1A > G, c.315C > G, c.445_446delTG, c.481C > T, c.217C > T, c.331C > T, c.365A > T.

The application of the substance for detecting the MMACHC gene mutation site in the preparation of a product for assisting in judging the CblC defect type methyl malonic acidemia combined with homocysteinemia is also within the protection range of the invention;

the MMACHC gene mutation site is at least one of the following 13: c.609G > A, c.658_660delAAG, c.567dupT, c.80A > G, c.482G > A, c.394C > T, c.1A > G, c.315C > G, c.445_446delTG, c.481C > T, c.217C > T, c.331C > T, c.365A > T.

The invention provides a set of primers for detecting MMACHC gene mutation sites, and a gene molecule typing based high-throughput detection method for the MMACHC gene mutation sites is established according to the primers, so that mutation typing of 13 mutation sites such as human MMACHC genes c.609G > A, c.658_660delAAG, c.567dupT, c.80A > G, c.482G > A, c.394C > T, c.1A > G, c.315C > G, c.445_446delTG, c.481C > T, c.217C > T, c.331C > T, c.365A > T and the like can be completed at the same time in the same reaction, the detection efficiency is improved, and the cost is reduced. Compared with other detection methods, the method has the advantages of high detection flux, simple and reliable operation, clear and efficient result interpretation, is suitable for hospitals and medical detection laboratories to carry out the detection of MMACHC genes related to the combination of the methylmalonic acidemia and the homocysteinemia, and can be used for screening or auxiliary diagnosis. The method of the invention improves the coverage rate of the mutant site of the CblC defect type methyl malonic acidemia combined with the homocysteinemia on the MMACHC gene to 93.28 percent, covers the mutant site of Chinese population, and realizes the gene screening and diagnosis of the CblC defect type methyl malonic acidemia combined with the homocysteinemia. The primer system for detecting the MMACHC gene mutation can be widely applied to the detection and analysis process of the gene mutation condition of the MMACHC gene, is simple and convenient to operate, saves time, is beneficial to early discovering the genotype of an infant patient, provides scientific basis for later diagnosis and treatment, is beneficial to timely taking intervention measures, and effectively improves the life quality of the infant patient.

Drawings

FIG. 1 shows the result of detection of ABI7500 of c.609G > A.

FIG. 2 shows the detection results of c.609G > A chip.

FIG. 3 shows the result of ABI7500 detection of c.658_660 delAAG.

FIG. 4 shows the chip detection results of c.658_660 delaAG.

FIG. 5 shows the result of ABI7500 assay of c.567dupT.

FIG. 6 shows the results of chip detection of c.567dupT.

FIG. 7 shows the result of ABI7500 assay of c.80A > G.

FIG. 8 shows the chip detection results of c.80A > G.

FIG. 9 shows the result of ABI7500 assay of c.482G > A.

FIG. 10 shows the results of the chip detection of c.482G > A.

FIG. 11 shows the result of ABI7500 detection of c.394C > T.

FIG. 12 shows the chip detection results of c.394C > T.

FIG. 13 shows the result of ABI7500 detection of c.1A > G.

FIG. 14 shows the results of chip detection of c.1A > G.

Fig. 15 shows the ABI7500 detection result of c.315c > G.

FIG. 16 shows the results of chip detection of c.315C > G.

FIG. 17 shows the result of ABI7500 detection of c.445_446 delTG.

FIG. 18 shows the chip detection results of c.445_446 delTG.

FIG. 19 shows the result of ABI7500 detection of c.481C > T.

FIG. 20 shows the results of chip detection of c.481C > T.

Fig. 21 is the ABI7500 detection result of c.217c > T.

FIG. 22 shows the results of chip detection of c.217C > T.

FIG. 23 shows the result of ABI7500 detection of c.331C > T.

FIG. 24 shows the chip detection results of c.331C > T.

FIG. 25 shows the result of ABI7500 assay for c.365A > T.

FIG. 26 shows the results of chip detection of c.365A > T.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The chip platform instrument models used in the following examples are shown in table 1 below:

TABLE 1

Oil-free air compressor	QTS-750
		Centrifugal machine	MPC2000
Flat PCR amplification instrument	Mastercycler nexus flat
		Scanner	LuxScan-10K/D
Film laminating machine	TY360
		Pneumatic punching machine	XTY-300

Example 1 establishment of a set of KASP primers and detection method for detecting the genotype of mutant site of MMACHC gene

The genome sequence genbank number of the wild-type MMACHC gene is NG _ 013378;

table 2 below shows 13 mutation sites in the MMACHC gene (primer design according to genomic sequence).

Designing a group of KASP primers corresponding to each mutation site according to flanking sequences before and after 13 mutation sites in the table 2,

table 2 shows 13 sets of KASP primers for detecting the genotype of mutant site of MMACHC gene

In the above table, c.609G > A indicates that the 609 th G in the MMACHC gene on the autosome is mutated into A, and the similar analogy is repeated; wherein, the former is the base of the site which is the wild base, and the latter is the base of the site which is the mutant base;

c.658-660 delAAG denotes the deletion of AAG 658 to 660 of the MMACHC gene on the autosome,

c.567dupT denotes a repeat of the T base at position 567 in the MMACHC gene on the autosome.

In the above table, in the KASP primer set of each mutation site, the first 2 are two allele-specific primers, the last one is a universal primer, and the wild-type specific primer whose 5 'end is connected to the FAM sequence (bold), the mutant-type specific primer whose 5' end is connected to the HEX sequence (bold), and the universal primer are sequentially included.

Each of the above primers was synthesized.

Second, establishment of KASP detection method for detecting MMACHC gene mutation site genotype

1. Extraction of DNA

Genomic DNA is extracted from a sample to be tested such as blood spots or whole blood.

2. KASP amplification by ABI7500 PCR instrument

Adding the genomic DNA extracted in the step 1 and primers shown in the table 2 into an amplification system respectively, and adding a group of KASP primers of mutation sites into each system; performing KASP amplification on each sample to be detected by using 13 primer groups shown in the table 2 respectively; the method comprises the following specific steps:

the 10 μ L ABI7500 PCR amplification reaction system was as follows:

5 μ L KASP 2 × Master MIX (LGC Inc., cat # KBS-1016-)

1 μ L template

3μL ddH2O

MMACHC site blood slides.

The final concentrations of each primer in the above system were as follows:

c.609G > A specific primer: 0.28 uM; universal primer 0.7 uM.

c.658_660dclAAG specific primers: 0.28 uM; universal primer 0.7 uM.

567dupt specific primers: 0.28 uM; universal primer 0.7 uM.

80a > G specific primers: 0.28 uM; universal primer 0.7 uM.

c.482G > A specific primers: 0.28 uM; universal primer 0.7 uM.

c.394C > T specific primers: 0.28 uM; universal primer 0.7 uM.

c.1A > G specific primers: 0.15 uM; universal primer 0.4 uM.

c.315C > G specific primers: 0.28 uM; universal primer 0.7 uM.

c.445 — 446deTTG specific primers: 0.28 uM; universal primer 0.7 uM.

c.217c > T specific primer: 0.2 uM; universal primer 0.5 uM.

c.331C > T specific primers: 0.28 uM; universal primer 0.7 uM.

c.365A > T specific primer: 0.28 uM; universal primer 0.7 uM.

c.481C > T specific primers: 0.28 uM; universal primer 0.7 uM.

After the reaction system is prepared, subpackaging the mixture into eight rows, sealing the eight rows by using a flat cover to prevent the sample from evaporating, shaking, uniformly mixing and centrifuging; the sealed eight-row rows were placed on an ABI7500 PCR instrument for reaction, and the amplification program selected "Genotyping" as follows in Table 3 below:

TABLE 3

And (3) analyzing results after the amplification is finished:

if the sample to be detected amplified by the primer group of a certain mutation site shows the color (Allole 1, red) corresponding to the FAM sequence, the base of the mutation site in the MMACHC gene of the sample to be detected is wild type; if the color (Allele2, blue) corresponding to the HEX sequence is displayed, the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type; and if the mixed color (green) of the FAM sequence and the HEX sequence is displayed, the base of the mutation site in the MMACHC gene of the sample to be detected is the heterozygous mutant.

3. Chip pool for KASP amplification

20 μ L KASP 2 × Master MIX (LGC Inc., cat # KBS-1016-)

10 μ L of genomic DNA from different test samples (40 ng-300ng in total)

20μL ddH2O

The final concentrations of each primer in the above system were as follows:

c.609G > A specific primer: 0.28 uM; universal primer 0.7 uM.

c.658_660dclAAG specific primers: 0.28 uM; universal primer 0.7 uM.

567dupt specific primers: 0.28 uM; universal primer 0.7 uM.

80a > G specific primers: 0.28 uM; universal primer 0.7 uM.

c.482G > A specific primers: 0.28 uM; universal primer 0.7 uM.

c.394C > T specific primers: 0.28 uM; universal primer 0.7 uM.

c.1A > G specific primers: 0.15 uM; universal primer 0.4 uM.

c.315C > G specific primers: 0.28 uM; universal primer 0.7 uM.

c.445 — 446deTTG specific primers: 0.28 uM; universal primer 0.7 uM.

c.217c > T specific primer: 0.2 uM; universal primer 0.5 uM.

c.331C > T specific primers: 0.28 uM; universal primer 0.7 uM.

c.365A > T specific primer: 0.28 uM; universal primer 0.7 uM.

c.481C > T specific primers: 0.28 uM; universal primer 0.7 uM.

After the chip is sealed, the plate PCR amplification instrument performs amplification, and the amplification procedure is as follows:

TABLE 4

Hybrid mutant refers to 2 homologous chromosomes, in 1 homologous chromosome, MMACHC gene is mutation site (such as c.609A), and the other MMACHC gene is wild type site (such as c.609G); homozygous mutation means that 2 homologous chromosome MMACHC genes are all mutation sites (such as c.609A);

example 2 set of KASP primers for detecting MMACHC gene mutation site genotype and application of the detection method

1. Extraction of DNA

Genomic DNA is extracted from 39 samples of blood spots to be detected (sites are obtained by sequencing; each mutation site corresponds to 3 samples to be detected and is respectively known as a site wild type, a site heterozygous mutant and a site homozygous mutant) with known MMACHC gene mutation types as templates.

2. KASP amplification

1) The ABI7500 PCR instrument performs KASP amplification: amplification was performed according to the method of example 1.

2) Chip pool for KASP amplification: amplification was performed according to the method of example 1.

3. KASP amplification results

The ABI7500 detection result of the sample to be detected amplified by the primer group of the c.609G/A locus is shown in figure 1: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

c.609G/A site primer group amplified to the chip detection results of the sample to be detected are shown in figure 2, four rows (each row is 4 repeats) are sequentially blank control from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is wild type, the base of the mutation site in the MMACHC gene of the sample to be detected is heterozygous mutant type, and the base of the mutation site in the MMACHC gene of the sample to be detected is homozygous mutant type;

the ABI7500 detection result of the test sample amplified by the primer group of the c.658_660delaAG site is shown in FIG. 3: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

the chip detection results of the samples to be detected amplified by the primer group of the c.658_660delaAG site are shown in FIG. 4: blank control is sequentially arranged in four rows from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type;

the ABI7500 detection result of the test sample amplified by the primer group of the c.567dupT site is shown in FIG. 5: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is heterozygous mutant type, and X represents blank control

The chip detection result of the sample to be detected amplified by the primer group of the 567dupT site is shown in FIG. 6: blank control is sequentially arranged in four rows from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutation;

the detection result of ABI7500 of the sample to be detected amplified by the primer group of the 80A > G site is shown in FIG. 7: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is heterozygous mutant type, and X represents blank control

The chip detection result of the sample to be detected amplified by the primer group of the sites 80A and G is shown in FIG. 8: blank control is sequentially arranged in four rows from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutation;

the detection result of ABI7500 of the sample to be detected amplified by the primer group of the c.482G > A site is shown in FIG. 9: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

the chip detection results of the samples to be detected amplified by the primer sets at the c.482g > a sites are shown in fig. 10: the four rows are sequentially from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is heterozygous mutation, the base of the mutation site in the MMACHC gene of the sample to be detected is homozygous mutation, and the base of the mutation site in the MMACHC gene of the sample to be detected is wild type and blank control;

the ABI7500 detection result of the sample to be detected amplified by the primer group of the c.394C > T locus is shown in FIG. 11: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

the chip detection results of the samples to be detected amplified by the primer sets of the c.394C > T sites are shown in FIG. 12: blank control is sequentially arranged in four rows from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutation;

the detection result of ABI7500 of the sample to be detected amplified by the primer group of the c.1A > G sites is shown in FIG. 13: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

the chip detection result of the sample to be detected amplified by the primer group of the c.1A > G sites is shown in FIG. 14: the four rows are sequentially from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is heterozygous mutation, the base of the mutation site in the MMACHC gene of the sample to be detected is homozygous mutation, and the base of the mutation site in the MMACHC gene of the sample to be detected is wild type and blank control;

the detection result of ABI7500 of the sample to be detected amplified by the primer group of the c.315C > G site is shown in figure 15: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

the chip detection result of the sample to be detected amplified by the primer group of the c.315C > G site is shown in figure 16: blank control is sequentially arranged in four rows from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutation;

the detection result of ABI7500 of the test sample amplified by the primer group at the c.445_446delTG site is shown in FIG. 17: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

the chip detection results of the samples to be detected amplified by the primer group at the c.445_446delTG site are shown in FIG. 18: blank control is sequentially arranged in four rows from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutation;

the ABI7500 detection result of the sample to be detected amplified by the primer group of the c.481C > T site is shown in figure 19: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

the chip detection result of the sample to be detected amplified by the primer group of the c.481c > T site is shown in fig. 20: the four rows are sequentially from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is heterozygous mutation, the base of the mutation site in the MMACHC gene of the sample to be detected is homozygous mutation, and the base of the mutation site in the MMACHC gene of the sample to be detected is wild type and blank control;

the ABI7500 detection result of the test sample amplified by the primer group of c 217C > T site is shown in FIG. 21: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

c.217C > T site primer set amplified chip detection results of the samples to be detected are shown in FIG. 22: the four rows are sequentially from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is heterozygous mutation, the base of the mutation site in the MMACHC gene of the sample to be detected is homozygous mutation, and the base of the mutation site in the MMACHC gene of the sample to be detected is wild type and blank control;

the ABI7500 detection result of the sample to be detected amplified by the primer group of the c.331C > T site is shown in FIG. 23: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

the chip detection result of the sample to be detected amplified by the primer group of the c.331C > T site is shown in FIG. 24: the four rows are sequentially from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is heterozygous mutation, the base of the mutation site in the MMACHC gene of the sample to be detected is homozygous mutation, and the base of the mutation site in the MMACHC gene of the sample to be detected is wild type and blank control;

the detection result of ABI7500 of the sample to be detected amplified by the primer group of the c.365A > T site is shown in figure 25: red represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

the chip detection result of the sample to be detected amplified by the primer group of the c.365A > T site is shown in FIG. 26: blank control is sequentially arranged in four rows from left to right, the base of the mutation site in the MMACHC gene of the sample to be detected is a wild type, the base of the mutation site in the MMACHC gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MMACHC gene of the sample to be detected is a homozygous mutation.

Comparing the ABI7500 detection result and the chip detection result with the actual known gene mutation type of the sample to be detected, and finding that the ABI7500 detection result and the chip detection result of the mutation sites completely accord with the gene mutation type of the sample to be detected.

Example 3 application of sets of KASP primers for detecting the genotype of mutant site of MMACHC Gene

1. Extraction of DNA

Genomic DNA of a blood spot sample to be tested (the locus is obtained by sequencing) with known MMACHC gene mutation types in the table 5 is respectively extracted as a template.

2. KASP amplification

Chip pool for KASP amplification: amplification was performed according to the method of example 1.

3. KASP amplification results

The results are shown in table 5, and it can be seen that the mutation site chip detection results of the samples completely accord with the gene mutation types of the samples.

TABLE 5

SEQUENCE LISTING

<110> Beijing Boo Athens Biotech Ltd

<120> a kit for detecting MMACHC gene mutation site

<160>39

<170>PatentIn version 3.5

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<400>8

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<213>Artificial sequence

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<213>Artificial sequence

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<213>Artificial sequence

<400>27

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<212>DNA

<213>Artificial sequence

<400>28

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<210>29

<211>39

<212>DNA

<213>Artificial sequence

<400>29

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<212>DNA

<213>Artificial sequence

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<213>Artificial sequence

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<213>Artificial sequence

<400>32

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<213>Artificial sequence

<400>33

cccagatggt aggccacaca ctg 23

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<400>34

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<400>35

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<212>DNA

<213>Artificial sequence

<400>36

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<213>Artificial sequence

<400>37

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<210>38

<211>39

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<400>38

gaaggtcgga gtcaacggat tggcccagac agcagccct 39

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Claims

1. The complete set of primers for detecting the mutation site of the MMACHC gene consists of 13 primer sets from the following primer set 1 to the primer set 13:

the specific primer 1-1 is 1-1-a) or 1-1-b) or 1-1-c) as follows:

the specific primer 1-2 is 1-2-a) or 1-2-b) or 1-2-c):

the universal primer 1 is 1-A) or 1-B) as follows:

1-A) a single-stranded DNA molecule shown as a sequence 3 in a sequence table;

the specific primer 2-1 is the following 2-1-a) or 2-1-b) or 2-1-c):

the specific primer 2-2 is the following 2-2-a) or 2-2-b) or 2-2-c):

the universal primer 2 is 2-A) or 2-B) as follows:

2-A) a single-stranded DNA molecule shown as a sequence 6 in a sequence table;

the specific primer 3-1 is 3-1-a) or 3-1-b) or 3-1-c):

the specific primer 3-2 is 3-2-a) or 3-2-b) or 3-2-c) as follows:

the universal primer 3 is 3-A) or 3-B) as follows:

3-A) a single-stranded DNA molecule shown as a sequence 9 in a sequence table;

the specific primer 4-1 is 4-1-a) or 4-1-b) or 4-1-c) as follows:

the specific primer 4-2 is 4-2-a) or 4-2-b) or 4-2-c) as follows:

the universal primer 4 is 4-A) or 4-B) as follows:

4-A) a single-stranded DNA molecule shown as a sequence 12 in a sequence table;

the specific primer 5-1 is 5-1-a) or 5-1-b) or 5-1-c):

the specific primer 5-2 is 5-2-a) or 5-2-b) or 5-2-c):

the universal primer 5 is 5-A) or 5-B) as follows:

5-A) a single-stranded DNA molecule shown as a sequence 15 in a sequence table;

the specific primer 6-1 is 6-1-a) or 6-1-b) or 6-1-c):

the specific primer 6-2 is 6-2-a) or 6-2-b) or 6-2-c):

the universal primer 6 is 6-A) or 6-B) as follows:

6-A) a single-stranded DNA molecule shown as a sequence 18 in a sequence table;

the specific primer 7-1 is 7-1-a) or 7-1-b) or 7-1-c):

the specific primer 7-2 is 7-2-a) or 7-2-b) or 7-2-c):

the universal primer 7 is 7-A) or 7-B) as follows:

7-A) a single-stranded DNA molecule shown as a sequence 21 in a sequence table;

the specific primer 8-1 is the following 8-1-a) or 8-1-b) or 8-1-c):

the specific primer 8-2 is 8-2-a) or 8-2-b) or 8-2-c) as follows:

the universal primer 8 is the following 8-A) or 8-B):

8-A) a single-stranded DNA molecule shown as a sequence 24 in a sequence table;

the specific primer 9-1 is the following 9-1-a) or 9-1-b) or 9-1-c):

the specific primer 9-2 is the following 9-2-a) or 9-2-b) or 9-2-c):

the universal primer 9 is the following 9-A) or 9-B):

9-A) a single-stranded DNA molecule shown as a sequence 27 in a sequence table;

the specific primer 10-1 is 10-1-a) or 10-1-b) or 10-1-c) as follows:

the specific primer 10-2 is 10-2-a) or 10-2-b) or 10-2-c) as follows:

the universal primer 10 is 10-A) or 10-B) as follows:

the specific primer 11-1 is the following 11-1-a) or 11-1-b) or 11-1-c):

the specific primer 11-2 is the following 11-2-a) or 11-2-b) or 11-2-c):

the universal primer 11 is the following 11-A) or 11-B):

the specific primer 12-1 is 12-1-a) or 12-1-b) or 12-1-c):

the specific primer 12-2 is 12-2-a) or 12-2-b) or 12-2-c):

the universal primer 12 is 12-A) or 12-B) as follows:

the specific primer 13-1 is 13-1-a) or 13-1-b) or 13-1-c):

the specific primer 13-2 is 13-2-a) or 13-2-b) or 13-2-c):

the universal primer 13 is 13-A) or 13-B) as follows:

2. The set of primers according to claim 1, wherein: the fluorescent sequences connected with the 2 specific primers in each group of primers are different.

3. The set of primers according to claim 1 or 2, characterized in that: and each group of primers is packaged independently.

4. The PCR reagent for detecting the mutation site of the MMACHC gene consists of 13 PCR reagents;

a set of primers of the primer set of any one of claims 1-3 is included in each of the PCR reagents.

5. A PCR kit for detecting mutation sites of MMACHC gene, which comprises the primer set of any one of claims 1 to 3 or the PCR reagent of claim 4.

6. Use of the set of primers of any one of claims 1 to 3 or the PCR reagent of claim 4 or the kit of claim 5 for detecting a mutation site of the MMACHC gene;

or, the use of the primer set of any one of claims 1 to 3 or the PCR reagent of claim 4 for preparing a product for detecting mutation sites of MMACHC gene.

7. A method for detecting MMACHC gene mutation sites in a sample to be detected comprises the following steps: the set of primers of any one of claims 1-3, wherein the genomic DNA of the test sample is subjected to KASP amplification, and the mutation site of the MMACHC gene in the test sample is determined based on the KASP genotyping result.

8. The method of claim 7, wherein: the sample to be detected is from a human.

9. The set of primers according to any one of claims 1 to 3 or the PCR reagents of claim 4 or the kit of claim 5 or the use of claim 6 or the method of claim 7 or 8, characterized in that:

the MMACHC gene mutation site is at least one of the following: c.609G > A, c.658_660delAAG, c.567dupT, c.80A > G, c.482G > A, c.394C > T, c.1A > G, c.315C > G, c.445_446delTG, c.481C > T, c.217C > T, c.331C > T, c.365A > T.

10. The application of the substance for detecting the MMACHC gene mutation site in the preparation of a product for assisting in judging the CblC defective methyl malonic acidemia combined with homocysteinemia;