CN110904211A

CN110904211A - Kit for detecting MUT gene mutation site related to methyl malonic acidemia

Info

Publication number: CN110904211A
Application number: CN201911201201.7A
Authority: CN
Inventors: 张岩; 常乐; 胥慧; 郭涛; 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: 2020-03-24

Abstract

The invention discloses a kit for detecting MUT gene mutation sites related to methylmalonic acidemia. The invention provides a set of primers for detecting MUT gene mutation sites, which consists of 13 primer groups from the following primer group 1 to 13, and establishes a mutation site high-throughput detection method of MUT genes based on gene molecule typing according to the primers, so that mutation typing of 13 mutation sites such as human MUT genes c.729_730instT, c.914T > C, c.1106G > A, c.2080C > T, c.2179C > T, c.494A > G, c.441T > A, c.1280G > A, c.323G > A, c.398_399 GA, c.755dupA, c.970G > A, c.1630_1631 > TA 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, and the operation is simple and reliable.

Description

Kit for detecting MUT gene mutation site related to methyl malonic acidemia

Technical Field

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

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 MUT 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.

The MMA infant patients are treated by special diet, supplemented with lifetime treatment and periodically detected in urine content of methylmalonic acid. The methylmalonic acidemia causes the increase of toxic organic acids such as methylmalonic acid, propionic acid and the like in vivo, metabolic acidosis, hyperammonemia, hypoglycemia, hyperglycoxyacidosis and the like can be caused, the accumulation of the toxic substances often causes the damage of the central nervous system, a series of nervous system damage symptoms are caused, and serious patients can die. Because the abnormal metabolites of MMA in children can cause mitochondrial dysfunction, energy generation and insufficient supply are caused, and the function of each tissue and organ is damaged, the clinical manifestations of patients are various, and the patients are easy to misdiagnose clinically, so that the treatment time is delayed, and the prognosis of the children is seriously influenced. Because of the harmfulness, the heritability and the difficult treatment of the MMA, the research on the MMA pathogenic gene is developed, and a theoretical basis is provided for the early diagnosis, the early intervention and the genetic consultation of MMA patients.

The literature reports that MUT gene mutation types are more than 250, and the simple MMA is proved to be caused by MUT gene mutation. MUT gene mutation can cause the reduction of mutase activity, and a series of clinical symptoms appear in children patients. MMA caused by MUT gene mutation is ineffective to treat vitamin B12, 80% of children have symptoms within 1 week of birth, 90% of children have symptoms within 1 month of birth, and the fatality rate and the complication incidence rate are extremely high.

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 MUT gene SNP has clinical practical value and superior biological significance.

The types of mutations in the human MUT gene are as follows: c.729_730instT, c.914T > C, c.1106G > A, c.2080C > T, c.2179C > T, c.494A > G, c.441T > A, c.1280G > A, c.323G > A, c.398_399delGA, c.755dupA, c.970G > A, c.1630_1631GG > TA.

Disclosure of Invention

An object of the present invention is to provide a set of primers for detecting MUT gene mutation sites.

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 48 th sites of a sequence 1 in a sequence table;

1-1-c)1-1-b) through 1 or several nucleotide residue substitution and/or deletion and/or addition to obtain single-stranded DNA molecule or connecting the sequence end thereof with 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-49 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 56 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 56 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) a single-stranded DNA molecule shown in 22 th to 48 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 48 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-45 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-46 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 46 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-46 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 44 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 43 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 42 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 42 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-42 th sequence 26 in the 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 51 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 55 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 30in 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 57 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-61 th sequence 32 in a 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;

11-2-c) connecting the tail end of the nucleotide sequence of 11-2-a) or 11-2-b) with a fluorescent sequence;

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 56 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 56 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-b) 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 43 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-46 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 set of primers, the fluorescent sequences connected to the 2 specific primers in each set of primers are different in the embodiment of the present invention, the specific primers for amplifying the wild-type locus are connected to the FAM fluorescent sequence, and the specific primers for amplifying the mutant locus are connected to 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 MUT gene mutation sites.

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

a set of primers of the primer set of

claim

1 or 2 is included in each of the PCR reagents.

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

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 MUT gene mutation sites is also within the protection scope of the invention;

or, the application of the primer set or the PCR reagent set in the preparation of the product for detecting MUT gene mutation sites is also within the protection scope of the invention.

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

The method provided by the invention comprises the following steps: and (3) performing KASP amplification on the genome DNA of the sample to be detected by using each primer group in the complete set of primers, genotyping the amplified product, and judging MUT gene mutation sites 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 MUT 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 of the FAM sequence (Allole 1, red), the base of the mutation site in the MUT gene of the sample to be detected is wild type; if the color of the HEX sequence is displayed (allee 2, blue), the base of the mutation site in the MUT 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 MUT 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)

10μL 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 of the sample to be detected amplified by the primer group of a certain mutation site is displayed on a scanning result picture, the basic group of the mutation site in the MUT gene of the sample to be detected is a wild type; if the color (green) of the HEX sequence is displayed, the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant; and if the mixed color (yellow) of the FAM sequence and the HEX sequence is displayed, the base of the mutation site in the MUT gene of the sample to be detected is the heterozygous mutant.

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

In the above, the MUT gene mutation site is at least one of: c.729_730instT, c.914T > C, c.1106G > A, c.2080C > T, c.2179C > T, c.494A > G, c.441T > A, c.1280G > A, c.323G > A, c.398_399delGA, c.755dupA, c.970G > A, c.1630_1631GG > TA.

The application of the substance for detecting MUT gene mutation sites in the preparation of the product for assisting in judging the simplex type methyl malonic acidemia is also within the protection range of the invention;

the MUT gene mutation site is at least one of the following 13: c.729_730instT, c.914T > C, c.1106G > A, c.2080C > T, c.2179C > T, c.494A > G, c.441T > A, c.1280G > A, c.323G > A, c.398_399delGA, c.755dupA, c.970G > A, c.1630_1631GG > TA.

The invention provides a set of primers for detecting MUT gene mutation sites, and establishes a mutation site high-throughput detection method of MUT genes based on gene molecule typing according to the primers, so that mutation typing of 13 mutation sites such as human MUT genes c.729_730instT, c.914T > C, c.1106G > A, c.2080C > T, c.2179C > T, c.494A > G, c.441T > A, c.1280G > A, c.323G > A, c.398_399 GA, c.755dupA, c.970G > A, c.1630_1631GG > TA and the like can be completed at the same time in the same reaction, and 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 the MUT gene related to the simplex type methyl malonic acidemia, and can be used for screening or auxiliary diagnosis. The primer system for MUT gene mutation detection can be widely applied to the detection and analysis process of the gene mutation condition of the MUT gene, is simple and convenient to operate, saves time, is beneficial to early finding the genotype of the 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 ABI7500 detection of c.729_730 instT.

FIG. 2 shows the chip test result of c.729_730 instT.

FIG. 3 shows the result of ABI7500 detection of c.914T > C.

FIG. 4 shows the chip detection results of c.914T > C.

FIG. 5 shows the result of ABI7500 assay of c.1106G > A.

FIG. 6 shows the results of the chip detection of c.1106G > A.

FIG. 7 shows the result of detection of ABI7500 at c.2080C > T.

FIG. 8 shows the results of chip detection at c.2080C > T.

FIG. 9 shows the result of ABI7500 detection of c.2179C > T.

FIG. 10 shows the results of chip detection of c.2179C > T.

FIG. 11 shows the result of ABI7500 detection of c.494A > G.

FIG. 12 shows the results of chip detection for c.494A > G.

FIG. 13 shows the result of ABI7500 detection of c.441T > A.

FIG. 14 shows the results of chip detection of c.441T > A.

FIG. 15 shows the result of detection of ABI7500 in c.1280G > A.

FIG. 16 shows the results of the chip detection of c.1280G > A.

FIG. 17 shows the result of ABI7500 assay for c.323G > A.

FIG. 18 shows the chip detection results of c.323G > A.

FIG. 19 shows the result of ABI7500 detection of c.398_399 delGA.

FIG. 20 shows the chip detection results of c.398_399 delGA.

FIG. 21 shows the result of ABI7500 detection of c.755dupA.

FIG. 22 shows the results of chip detection of c.755dupA.

FIG. 23 shows the result of detection of ABI7500 in c.970G > A.

FIG. 24 shows the results of chip detection for c.970G > A.

FIG. 25 shows the result of ABI7500 detection of c.1630-1631 GG > TA.

FIG. 26 shows the results of chip detection of c.1630_1631GG > TA.

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, set of KASP primers and detection method for detecting MUT gene mutation site genotype to establish genome sequence genbank number NG _007100 of wild type MUT gene;

table 2 below shows the 13 mutation sites in the MUT 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 MUT gene mutation site genotype

In the above table, c.729_730insTT represents insertion of TT between 729 and 730in the MUT gene on autosome, and so on;

c.914T > C represents that the 914 th T mutation in the MUT gene on the autosome is C, wherein the base of the site in front is wild base, and the base of the site in back is mutant base; the like is analogized in turn;

c.398-399 delGA denotes the GA deletion at positions 398 to 999 in the MUT gene on the autosome,

c.755dupA represents the repeat of the A base at position 755 in the MUT 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 MUT 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

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

c.729_730insTT specific primer: 0.28 uM; universal primer 0.7 uM.

c.914T > C specific primers: 0.2 uM; universal primer 0.5 uM.

c.1106G > A specific primer: 0.28 uM; universal primer 0.7 uM.

c.2080C > T specific primer: 0.28 uM; universal primer 0.7 uM.

c.2179C > T specific primers: 0.28 uM; universal primer 0.7 uM.

c.494A > G specific primers: 0.15 uM; universal primer 0.4 uM.

c.441t > a specific primers: 0.28 uM; universal primer 0.7 uM.

c.1280G > A specific primers: 0.28 uM; universal primer 0.7 uM.

c.323G > A specific primers: 0.28 uM; universal primer 0.7 uM.

c.398-399 delGA specific primers: 0.2 uM; universal primer 0.5 uM.

c.755dupA specific primer: 0.28 uM; universal primer 0.7 uM.

c.970G > A specific primer: 0.28 uM; universal primer 0.7 uM.

c.1630-1631 TGG > TA 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 of the FAM sequence (Allole 1, red), the base of the mutation site in the MUT gene of the sample to be detected is wild type; if the color of the HEX sequence is displayed (allee 2, blue), the base of the mutation site in the MUT 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 MUT gene of the sample to be detected is the heterozygous mutant.

3. Chip pool for KASP amplification

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

10μL ddH2O

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

c.729_730insTT specific primer: 0.28 uM; universal primer 0.7 uM.

c.914T > C specific primers: 0.2 uM; universal primer 0.5 uM.

c.1106G > A specific primer: 0.28 uM; universal primer 0.7 uM.

c.2080C > T specific primer: 0.28 uM; universal primer 0.7 uM.

c.2179C > T specific primers: 0.28 uM; universal primer 0.7 uM.

c.494A > G specific primers: 0.15 uM; universal primer 0.4 uM.

c.441t > a specific primers: 0.28 uM; universal primer 0.7 uM.

c.1280G > A specific primers: 0.28 uM; universal primer 0.7 uM.

c.323G > A specific primers: 0.28 uM; universal primer 0.7 uM.

c.398-399 delGA specific primers: 0.2 uM; universal primer 0.5 uM.

c.755dupA specific primer: 0.28 uM; universal primer 0.7 uM.

c.970G > A specific primer: 0.28 uM; universal primer 0.7 uM.

c.1630-1631 TGG > TA 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

The heterozygous mutant refers to 2 homologous chromosomes, the MUT gene in 1 homologous chromosome is a mutant site (c.914C), and the other MUT gene is a wild-type site (c.914T); homozygous mutation means that 2 homologous chromosome MUT genes are all mutation sites (such as c.914C);

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

1. Extraction of DNA

Genomic DNA is extracted from 39 samples of the blood spots to be detected (the locus is obtained by sequencing; each mutation locus corresponds to 3 samples to be detected and is respectively known as the wild type of the locus, the heterozygous mutant of the locus and the homozygous mutant of the locus) with known MUT 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

c.729-730 insTT locus, the detection result of ABI7500 of the sample to be detected amplified by the primer group is shown in FIG. 1: red represents that the base of the mutation site in the MUT gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutant type, and X represents a blank control;

c, the chip detection result of the sample to be detected amplified by the primer group of the 729_730insTT locus is shown in fig. 2, four rows (4 repeats in each row) are sequentially blank control from left to right, the base of the mutation locus in the MUT gene of the sample to be detected is a wild type, the base of the mutation locus in the MUT gene of the sample to be detected is a heterozygous mutant, and the base of the mutation locus in the MUT gene of the sample to be detected is a homozygous mutant;

the ABI7500 detection result of the sample to be detected amplified by the primer group of the c.914T > C locus is shown in figure 3: red represents that the base of the mutation site in the MUT gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant type, green represents that the base of the mutation site in the MUT 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.914T > C locus is shown in figure 4: blank control is sequentially arranged from left to right in four rows, the base of the mutation site in the MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutant, and the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant;

the detection result of ABI7500 of the sample to be detected amplified by the primer group of the c.1106G > A site is shown in figure 5: red represents that the base of the mutant site in the MUT gene of the sample to be detected is wild type, blue represents that the base of the mutant site in the MUT gene of the sample to be detected is homozygous mutant, green represents that the base of the mutant site in the MUT gene of the sample to be detected is heterozygous mutant, and X represents blank control

The chip detection result of the sample to be detected amplified by the primer group of the c.1106G > A site is shown in figure 6: blank control is sequentially arranged from left to right in four rows, the base of the mutation site in the MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MUT 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 with c.2080C > T sites is shown in FIG. 7: red represents that the base of the mutant site in the MUT gene of the sample to be detected is wild type, blue represents that the base of the mutant site in the MUT gene of the sample to be detected is homozygous mutant, green represents that the base of the mutant site in the MUT gene of the sample to be detected is heterozygous mutant, and X represents blank control

The chip detection results of the sample to be detected amplified by the primer sets at the c.2080C > T sites are shown in FIG. 8: blank control is sequentially arranged from left to right in four rows, the base of the mutation site in the MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MUT 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 of c.2179C > T sites is shown in FIG. 9: red represents that the base of the mutation site in the MUT gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant, green represents that the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutant, and X represents a blank control;

the chip detection results of the samples to be detected amplified by the primer sets of the c.2179c > T sites are shown in fig. 10: blank control is sequentially arranged from left to right in four rows, the base of the mutation site in the MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MUT 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.494A > G sites is shown in FIG. 11: red represents that the base of the mutation site in the MUT gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant, green represents that the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutant, and X represents a blank control;

the chip detection results of the samples to be detected amplified by the primer groups of the c.494A > G sites are shown in FIG. 12: blank control is sequentially arranged from left to right in four rows, the base of the mutation site in the MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MUT 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.441T > A site is shown in FIG. 13: red represents that the base of the mutation site in the MUT gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant, green represents that the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutant, and X represents a blank control;

the chip detection result of the sample to be detected amplified by the primer group of the c.441T > A site is shown in FIG. 14: blank control is sequentially arranged from left to right in four rows, the base of the mutation site in the MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MUT 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.1280G > A locus is shown in FIG. 15: red represents that the base of the mutation site in the MUT gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant, green represents that the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutant, and X represents a blank control;

the chip detection results of the samples to be detected amplified by the primer sets at the c.1280G > A sites are shown in FIG. 16: blank control is sequentially arranged from left to right in four rows, the base of the mutation site in the MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MUT 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.323G > A site is shown in figure 17: red represents that the base of the mutation site in the MUT gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant, green represents that the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutant, and X represents a blank control;

the chip detection result of the sample to be detected amplified by the primer group of the c.323G > A site is shown in figure 18: blank control is sequentially arranged from left to right in four rows, the base of the mutation site in the MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutation;

the result of ABI7500 detection of the test sample amplified by the primer set at the position of 398_399delG is shown in FIG. 19: red represents that the base of the mutation site in the MUT gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant, green represents that the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutant, and X represents a blank control;

the chip detection results of the samples to be detected amplified by the primer set at the position of the delG site of 398_399 are shown in FIG. 20: blank control is sequentially arranged from left to right in four rows, the base of the mutation site in the MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MUT 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 755dupA locus is shown in figure 21: red represents that the base of the mutation site in the MUT gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant, green represents that the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutant, and X represents a blank control;

the chip detection result of the sample to be detected amplified by the primer group of the c.755dupA locus is shown in FIG. 22: blank control is sequentially arranged from left to right in four rows, the base of the mutation site in the MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MUT 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.970G > A locus is shown in FIG. 23: red represents that the base of the mutation site in the MUT gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant, green represents that the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutant, and X represents a blank control;

the chip detection results of the samples to be detected amplified by the primer sets at the c.970G > A sites are shown in FIG. 24: blank control is sequentially arranged from left to right in four rows, the base of the mutation site in the MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MUT 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.1630-1631 GG > TA site is shown in FIG. 25: red represents that the base of the mutation site in the MUT gene of the sample to be detected is a wild type, blue represents that the base of the mutation site in the MUT gene of the sample to be detected is a homozygous mutant, green represents that the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutant, and X represents a blank control;

the chip detection result of the sample to be detected amplified by the primer group with c.1630_1631GG > TA sites 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 MUT gene of the sample to be detected is a wild type, the base of the mutation site in the MUT gene of the sample to be detected is a heterozygous mutation, and the base of the mutation site in the MUT 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 MUT Gene mutation site

1. Extraction of DNA

Genomic DNA of a blood spot sample to be tested (the locus is obtained by sequencing) with known MUT 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> kit for detecting MUT gene mutation sites related to methylmalonic acidemia

<160>39

<170>PatentIn version 3.5

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Claims

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

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

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: each set of primers was packaged separately.

4. The PCR reagent for detecting MUT gene mutation sites 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 MUT gene mutation site, comprising the primer set of any one of claims 1 to 3 or the PCR reagent of claim 4.

6. Use of the primer set 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 MUT 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 MUT gene mutation sites.

7. A method for detecting MUT gene mutation sites in a sample to be detected comprises the following steps: KASP amplification of genomic DNA of a test sample is carried out using each of the primer sets of claims 1 to 3, and based on the KASP genotyping results, MUT gene mutation sites in the test sample are determined.

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 MUT gene mutation site is at least one of the following: c.729_730instT, c.914T > C, c.1106G > A, c.2080C > T, c.2179C > T, c.494A > G, c.441T > A, c.1280G > A, c.323G > A, c.398_399delGA, c.755dupA, c.970G > A, c.1630_1631GG > TA.

10. The application of the substance for detecting MUT gene mutation sites in the preparation of a product for assisting in judging simplex methylmalonic acidemia;