CN114854849A

CN114854849A - Method for detecting high-frequency mutation of MMACHC gene based on digital PCR platform

Info

Publication number: CN114854849A
Application number: CN202210719393.6A
Authority: CN
Inventors: 赵燕; 崔亚洲; 韩金祥; 杨海宁; 邹卉
Original assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Current assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Priority date: 2022-06-23
Filing date: 2022-06-23
Publication date: 2022-08-05

Abstract

The invention discloses a primer probe composition for detecting high-frequency mutation of MMACHC gene and application thereof, wherein different fluorescent reporter groups are used for respectively marking a wild type probe and a mutant type probe, and the mutation conditions of high-frequency mutation sites of the MMACHC gene, namely c.609G > A, c.482G > A, c.80A > G, c.567dupT and c.217C > T, can be detected by combining the detection result of a quality control product, so that the detection cost is low, the period is short, the operation is simple, the accuracy is high, the specificity is better, and the clinical application can be met.

Description

Method for detecting high-frequency mutation of MMACHC gene based on digital PCR platform

Technical Field

The invention relates to the technical field of biology, in particular to a kit for detecting high-frequency mutation sites of MMACHC genes.

Background

Methylmalonemia (MMA), also called methylmalonuria, is a common organic acid metabolic disease, and as methylmalonyl-coa in the catabolism process of valine, isoleucine, methionine, threonine and odd-carbon fatty acids in vivo cannot be converted into succinyl-coa, bypass metabolism is enhanced, toxic substances such as methylmalonate and methylcitrate are abnormally accumulated, so that multiple organ injury, especially nervous system injury, is caused, and life is seriously threatened. The disease belongs to autosomal recessive inheritance, the global incidence rate is about 1:48,000-1: 250000, the Chinese incidence rate is 1/28004, and the Shandong province incidence rate is 1/3920. MMA is classified into two types, namely, simple type methylmalonic acidemia and methylmalonic acidemia combined with homocysteinemia. The clinical manifestation of the disease has large individual difference, can show that multiple systems are affected, the disease can be developed from the fetal period to the adult period, the missed diagnosis and the misdiagnosis are easy, the gene detection is the reliable basis of clinical typing, and the early molecular detection and the prevention are particularly critical. At present, the clinical MMA detection method mainly adopts biochemical inspection tandem mass spectrometry detection, the genetic mutation detection method adopts expensive high-throughput sequencing, and the high-throughput sequencing detection of genetic mutation has certain difficulty in clinical popularization because the environmental requirements of a sequencing instrument are strict and are all outward delivery detection.

The combined MMA is most common in Chinese MMA patients, and comprises three subtypes of cb1C, cb1D and cb1F, wherein the cb1C is taken as the main type, and the coding gene is MMACHC, wherein c.609G > A, c.80A > G, c.567dupT, c.482G > A and c.217C > T5 mutation sites have high clinical detection rate, and the MMACHC gene covers more than 60 percent of mutations. Although the number of mutation sites causing MMA disease is large, the mutation types in the global range have regional difference, the most common type in foreign countries is MUT gene mutation, and the most common type in China is cblC type. However, no detection kit specially used for gene mutation in the hot spot mutation region of the gene exists in China at present. Therefore, how to establish a simple, rapid, economic and accurate combined type high-frequency mutation screening method for the methylmalonic acidemia gene and provide technical support for prevention, auxiliary clinical diagnosis and prognosis evaluation of MMA is a problem to be solved urgently.

Disclosure of Invention

Aiming at the practical requirements of clinical MMA on a gene detection method, the invention provides a primer probe composition for detecting high-frequency mutation of an MMACHC gene and application thereof, wherein different fluorescence reporter groups are used for respectively marking a wild type probe and a mutant type probe, and the mutation conditions of high-frequency mutation sites of the MMACHC gene, namely c.609G > A, c.482G > A, c.80A > G, c.567dupT and c.217C > T, can be detected by combining the detection result of a quality control product, and the primer probe composition has the advantages of low detection cost, short period, simple operation, high accuracy and better specificity, and can meet the clinical application.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides five primer probe compositions for detecting MMACHC gene high-frequency mutation based on a digital PCR platform, wherein the five primer probe compositions comprise a first primer probe composition, a second primer probe composition, a third primer probe composition, a fourth primer probe composition and a fifth primer probe composition;

in certain embodiments, the first primer probe composition is used to detect the c.609G > A site of the MMACHC gene; the first primer probe composition consists of a first forward primer, a first reverse primer, a first wild type probe and a first mutant type probe, wherein the nucleotide sequences of the first forward primer, the first reverse primer, the first wild type probe and the first mutant type probe are sequentially shown as SEQ ID NO. 1-SEQ ID NO. 4;

in certain embodiments, the second primer probe composition is used to detect the c.482g > a site of the MMACHC gene; the second primer probe composition consists of a second forward primer, a second reverse primer, a second wild type probe and a second mutant type probe, wherein the nucleotide sequences of the second forward primer, the second reverse primer, the second wild type probe and the second mutant type probe are sequentially shown as SEQ ID NO. 5-SEQ ID NO. 8;

in certain embodiments, the third primer probe composition is used to detect the c.80a > G site of the MMACHC gene; the third primer probe composition consists of a third forward primer, a third reverse primer, a third wild-type probe and a third mutant-type probe, wherein the nucleotide sequences of the third forward primer, the third reverse primer, the third wild-type probe and the third mutant-type probe are sequentially shown as SEQ ID NO. 9-SEQ ID NO. 12;

in certain embodiments, the fourth primer probe composition is used to detect the c.567dupt site of the MMACHC gene; the fourth primer probe composition consists of a fourth forward primer, a fourth reverse primer, a fourth wild type probe and a fourth mutant type probe, and the nucleotide sequences of the fourth forward primer, the fourth reverse primer, the fourth wild type probe and the fourth mutant type probe are sequentially shown as SEQ ID NO. 13-SEQ ID NO. 16;

in certain embodiments, the primer probe composition is used to detect the c.217c > T site of the MMACHC gene; the fifth primer probe composition consists of a fifth forward primer, a fifth reverse primer, a fifth wild-type probe and a fifth mutant probe, wherein the nucleotide sequences of the fifth forward primer, the fifth reverse primer, the fifth wild-type probe and the fifth mutant probe are sequentially shown as SEQ ID NO. 17-SEQ ID NO. 20.

In certain embodiments, the Tm value for each primer in the first primer probe composition, the second primer probe composition, the third primer probe composition, the fourth primer probe composition, and the fifth primer probe composition is independently 58-60 ℃. Preferably, the difference in Tm values between the primers is within 2 ℃ to facilitate amplification in subsequent experiments under the same PCR reaction conditions.

In certain embodiments, the probe is a Taqman probe, wherein the 5 'end of the Taqman probe is labeled with a fluorescent reporter group and the 3' end of the Taqman probe is labeled with a quencher group. Preferably, the fluorescent reporter group comprises FAM and/or VIC. Preferably, the quencher group comprises MGB. Preferably, the fluorescent reporter of the wild-type probe is different from the fluorescent reporter of the mutant-type probe.

In certain embodiments, the fluorescent reporter group of the wild-type c.609G > A, c.80A > G, c.567dupT, c.482G > A and c.394C > T probes comprises VIC.

In certain embodiments, the fluorescent reporter group of the mutant c.609G > A, c.80A > G, c.567dupT, c.482G > A and c.394C > T probes comprises FAM.

In certain embodiments, the wild-type probe is a wild-type specific probe and the mutant probe is a mutant specific probe.

In some embodiments, the digital PCR primer specifically amplifies a fragment with the length of 100-150 bp, so that the specificity is good and the result is accurate.

In some embodiments, the primer is used at 900nM and the fluorescent probe is used at 250 nM.

In a second aspect, the invention provides a detection kit of a primer probe composition for detecting high-frequency mutation of an MMACHC gene, and the detection kit comprises the primer probe composition of the first aspect.

In certain embodiments, the kit further comprises a PCR reaction solution. Preferably, the PCR reaction solution comprises DNA polymerase and Mg ²⁺ Buffer, dNTPs and water.

In certain embodiments, the test kit further comprises a quality control.

In certain embodiments, the quality control comprises enzyme-free water.

In a third aspect, the invention provides a method for using the primer probe composition for detecting high-frequency mutation of MMACHC gene of the second aspect of the invention, the method comprising: 1) extracting DNA of a sample to be detected; 2) carrying out digital PCR amplification and simultaneously detecting negative quality control products; 3) and (4) putting the amplified chip into a biochip reader, detecting a fluorescence signal and analyzing.

In certain embodiments, the DNA sample is from a plurality of blood-type nucleic acid samples, such as peripheral blood, dried blood spots, and the like.

In certain embodiments, the digital PCR reaction system comprises: DNA of a sample to be tested, a primer probe composition according to the first aspect of the present invention, and a PCR reaction solution according to the second aspect of the present invention.

In certain embodiments, the reaction conditions of the digital PCR are: first-stage pre-denaturation: 10min at 96 ℃ for 1 cycle; and (3) second-stage cyclic amplification: at 55 ℃ for 2min, at 98 ℃ for 30s, for 39 cycles; and a third stage extension: 2min at 55 ℃ for 1 cycle.

In some embodiments, the quality control product amplification detection is performed first in the detection, and whether the reaction system and the detection process are correct or not can be judged according to the fluorescent signal in the quality control product amplification. And then, according to comparison between the fluorescence signal of the sample to be detected and the scatter diagram in different samples, judging whether the sample is mutated or not and judging the type of the mutation.

In certain embodiments, the analyzing comprises: and comparing the fluorescence signal and the scatter diagram of the sample to be detected to determine the genotype of the sample to be detected, wherein the genotype refers to the sequence of the FAM marked mutant type and the sequence of the VIC marked wild type, and the mutation condition of the sample to be detected is determined through the fluorescence signal and the scatter diagram of the FAM and the VIC.

In some embodiments, the genotype of the test sample is determined from the fluorescence signals of FAM and VIC of the test sample: the fluorescence signal in the VIC range is strong, the fluorescence signal of FAM is weak or absent, and the sample to be detected is judged to be a wild type; the fluorescence signal in the FAM range is strong, the fluorescence signal of VIC is weak or none, and the sample to be detected is judged to be a homozygous mutant type; and (5) judging the sample to be detected to be the heterozygous mutant type when the fluorescence signal intensities of the FAM and the VIC are similar.

Compared with the prior art, the invention has the beneficial effects that:

1) the primer probe composition for detecting the high-frequency mutation of the MMACHC gene has good specificity and high amplification efficiency, can detect the mutation conditions of hot spot mutations of c.609G > A, c.80A > G, c.567dupT, c.482G > A and c.217C > T of the MMACHC gene of a sample, and has accurate result, shorter time and lower cost;

2) by adopting a specific primer and a novel probe technology and a double-fluorescence channel detection mode, the internal control gene is set to detect FAM and VIC signals, so that the quality of sample nucleic acid can be monitored while mutation is detected, and the judgment accuracy is improved.

3) Based on digital PCR technology, sensitive and accurate absolute target quantification is achieved by not using a reference or standard curve. After the chip of the PCR reaction system is processed, a sample is distributed into up to 20,000 independent reaction holes by utilizing the high-density nano-fluidic chip technology to detect the high-frequency mutation amplification state of the MMACHC gene, and the method has the advantages of good repeatability, absolute quantification, simple and convenient operation, high accuracy, good specificity, low cost and the like.

4) The detection kit and the detection method for the hot spot mutation region of the MMACHC gene can quickly, accurately and sensitively detect the mutation condition of the hot spot mutation. The experimental result has good repeatability and high precision. In addition, the invention has short detection time period, can finish detection within 4 hours to the utmost extent and obtain results, greatly saves the detection time, accelerates the clinical diagnosis efficiency, and is an efficient auxiliary detection means.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a scattergram of a quality control product in example 2 of the present invention.

FIG. 2 is a scattergram of sample 1 in example 2 of the present invention.

FIG. 3 is a scattergram of sample 2 in example 2 of the present invention.

FIG. 4 is a scattergram of sample 3 in example 2 of the present invention.

FIG. 5 is a scattergram of sample 4 in example 2 of the present invention.

FIG. 6 is a scattergram of sample 5 in example 2 of the present invention.

FIG. 7 is a scattergram of sample 6 in example 2 of the present invention.

FIG. 8 is a scattergram of samples 8, 9 and 10 in example 3 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It should be apparent that the described embodiments are only some of the embodiments of the present invention, and not all of them. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.

Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.

The method for detecting high-frequency mutation of MMACHC gene and the effect thereof are described in detail with reference to specific examples.

Example 1 design of primer Probe compositions

A primer probe composition for detecting high-frequency mutation of MMACHC Gene (NCBI Gene ID:25974) is prepared, which comprises the following steps:

1) obtaining a sequence: the gene sequence used was derived from NCBI (national center for Biotechnology information)

TABLE 1 hotspot mutation site information of MMACHC genes

Base change	Amino acid changes	rs number
			c.609G>A	p.Trp203Ter	rs587776889
c.80A>G	p.Gln27Arg	rs546099787
			c.567dupT	p.Ile190fs	rs1463495909
c.482G>A	p.Arg161Gln	rs121918243
			c.217C>T	p.Arg73Ter	rs796051995

2) The design method comprises the following steps: primer probes were designed using software such as Primer Express 3.0 based on the hot-spot mutations c.609G > A, c.80A > G, c.567dupT, c.482G > A and c.217C > T5 SNP mutation site sequences of MMACHC genes published by NCBI database (Table 1). Selecting a 100-150 bp amplification target fragment, designing a specific PCR primer, wherein the Tm value of the specific PCR primer is between 58 and 60 ℃; and designing a pair of probes according to each SNP locus, wherein the locus is positioned in the middle of the probes as much as possible, the normal sequence is a wild type, and the mutant sequence is a mutant type. The probe is a Taqman probe, a fluorescence reporter group is marked at the 5 'end of the Taqman probe, and a quenching group is marked at the 3' end of the Taqman probe. The sequences of the primer probe compositions for the 5 SNP mutation sites are shown in Table 2 below.

TABLE 2 sequence Listing of primer Probe compositions for 5 SNP sites

3) Sequence synthesis: the sequence obtained was synthesized by the company Lihuada, Heihe, Beijing by the company Huada, Heihe Huada, Japan.

Example 2 accuracy verification

The hot spot mutations c.609G > A, c.80A > G, c.567dupT, c.482G > A and c.217C > T of the MMACHC gene in a newborn dry blood spot sample are detected by using a kit of primer probe compositions for detecting the high-frequency mutation of the MMACHC gene, and specific primer probe compositions for detecting the high-frequency mutation of the MMACHC gene in the kit are prepared in the embodiment 1.

2.1 samples

Taking 1 sample of known normal type newborn dry blood spots; 5 samples of dried blood spots of neonates from patients with methylmalonemia of known mutation type (Fuzijinan Hospital, named sample 1, sample 2, sample 3, sample 4, sample 5 and sample 6, respectively, where sample 2 is known as the c.609G > A mutation, sample 3 is c.80A > G, sample 4 is the c.567dupT mutation, sample 5 is the c.482G > A mutation and sample 6 is c.217C > T).

2.2 extraction of DNA from samples to be tested

Using Magnetic Blood Spots DNA Kit (TIANGEN, Inc. of China, Cat No. DP344) to extract nucleic acid from the newborn dry Blood Spots, obtaining 30ul of DNA solution by the extraction step according to the product instruction, diluting the extracted nucleic acid to 14.5 ng/muL, and directly detecting or temporarily storing at 4 ℃; and simultaneously carrying out negative quality control product detection.

2.3 digital PCR amplification and quality control detection

Digital PCR reaction systems were prepared according to Table 3 using Applied Biosystems ^TM QuantStudio ^TM And (3) carrying out PCR amplification reaction liquid preparation by a 3D digital PCR system (Saimeishi), wherein 14.5 mu L of each reaction is carried out, uniformly mixing by blowing a pipette, and carrying out instantaneous centrifugation.

TABLE 3 digital PCR reaction System

Material	Volume(uL)	Stock(uM)	Final(nM)
				Master Mix	7.25	2×	1×
Wild-type probe	0.18	20	250
				Mutant probe	0.18	20	250
Forward primer	0.65	20	900
				Reverse primer	0.65	20	900
DNA	1.00
				Water	4.59
Total volume	14.50

The forward primer, reverse primer, wild-type probe, and mutant probe in the table correspond to the primer probe set in example 1. Sample 1 employed any one of the five primer probe compositions; sample 2 is probed with a first primer probe composition; sample 3 using a second primer probe composition; sample 4 was probed with a third primer probe composition; sample 5 with a fourth primer probe composition; sample 6 was probed with a fifth primer probe composition.

According to Quantstrudio ^TM And (3) completing the sample adding of the chip by using a 3D Digital PCR System standard process, wherein the sample adding comprises droplet generation oil, Digital PCR premix liquid and confining liquid. The loaded chips were transferred horizontally to a thermal cycler of a digital PCR amplification machine and the amplification program was run as per Table 4. (the Tm value of each primer in the kit is 58-60 ℃ independently, and the Tm value difference between each group of primers is within 2 ℃, so that the same PCR amplification condition is shared)

TABLE 4 digital PCR amplification conditions

The amplified chip is put into a biochip reader, and a fluorescence signal is detected and analyzed. Putting the amplified chip into a biochip reader, exporting the EDS experiment file to a USB driver, and using Analysissuite ^TM And uploading the cloud software to the cloud for analysis. Detect each coreAnd (4) carrying out fluorescence signal clustering on the amplification products by using software to obtain a scatter diagram.

Judging the genotype of the sample to be detected according to the fluorescence signals of FAM and VIC of the sample to be detected; the fluorescence signal in the VIC range is strong, the fluorescence signal of FAM is weak or absent, and the sample to be detected is judged to be wild type; the fluorescence signal in the FAM range is strong, the fluorescence signal of VIC is weak or none, and the sample to be detected is judged to be a homozygous mutant type; and (5) judging the sample to be detected to be the heterozygous mutant type when the fluorescence signal intensities of the FAM and the VIC are similar. The quality control samples are shown in the scattergram 1 and the detection results of 6 dry blood spot samples are shown in the scattergrams 2 to 7 in Table 5.

TABLE 5.6 test results of dried blood spot samples

Sample(s)	Clinical information	Fluorescent signal	Digital PCR interpretation
				1	Healthy person	VIC	No mutation
2	c.609G>A	FAM+VIC	Heterozygous mutations
				3	c.80A>G	FAM	Homozygous mutations
4	c.567dupT	FAM+VIC	Heterozygous mutations
				5	c.482G>A	FAM+VIC	Heterozygous mutations
6	c.217C>T	FAM	Homozygous mutations

The final result shows that the consistency of the detection result of 6 neonatal dry blood spot samples in the experiment and the detection result of clinical high-throughput sequencing is 100%. The detection method and the scattergram result of the embodiment both show that the primer probe composition for detecting the high-frequency mutation of the MMACHC gene and the application thereof are feasible when the high-frequency mutation of the MMACHC gene is detected.

Experimental example 3 sensitivity detection

3 blood samples of known normal type were collected (source laboratory members, named sample 8, sample 9, sample 10). Collecting venous blood with anticoagulant tube (blood collecting tube containing EDTA) of purple cranial tube, and storing at 4 deg.C.

Extracting DNA of a sample to be detected: taking 200 mu L of each sample, extracting nucleic acid according to a Vazyme Blood DNA Kit (Cat No. DC112 of Vazyme company in China) to obtain 30ul of DNA solution, diluting the extracted nucleic acid to the concentration of 14.5 ng/mu L, and directly detecting or temporarily storing at 4 ℃; and simultaneously carrying out negative quality control product detection.

Samples 8, 9 and 10 were used as templates, respectively, and the templates were diluted 2-fold and 10-fold. The total amount of DNA in each 1ul reaction system is shown in Table 6, and the genome of 9 samples to be tested was subjected to digital PCR amplification using a primer probe composition kit for detecting high-frequency mutation of MMACHC gene, wherein any one of five primer probe compositions was used, and the amplification reaction system and procedure were the same as those in step 3 of example 2.

TABLE 6 DNA amount per microliter in digital PCR amplification reaction system

Sample(s)	Stock solution (ng)	Dilute 2 times (ng)	Diluting 10 times (ng)
				8	1	0.5	0.1
9	1	0.5	0.1
				10	1	0.5	0.1

The amplified chip was placed in a biochip reader, and the fluorescence signal was detected and analyzed in the same manner as in step 4 of example 2.

Fig. 8 is a result picture of sample 8, sample 9, and sample 10.

The first row of FIG. 8 shows that the fluorescence signal of VIC is strong; FIG. 8 is a second row of the result picture after 2-fold dilution, and it can be seen that the fluorescence signal of FAM is strong; figure 8 third row is a 10-fold dilution of the results picture, and it can be seen that FAM is weak, but no mutation results can be judged. Further illustrates that when detecting a mutation at one site, multiple samples can be tested simultaneously. By combining the results, the kit for detecting the primer probe composition for detecting the high-frequency mutation of the MMACHC gene has the advantages of good sensitivity, accurate result, short time, simple and convenient operation and wider application range.

Experimental example 4 clinical sample assay

Blood samples of the subjects in 50 physical examination items of the women and children health care institute in Shannan are collected. The procedure of example 3 was followed to extract DNA from the sample.

And (3) performing corresponding 5 sets of primer probe composition amplification on the genome of each sample to be detected by using a primer probe composition kit for detecting the high-frequency mutation of the MMACHC gene, wherein the system and the procedure of the amplification reaction are the same as those of the corresponding steps in the example 2.

The amplified chips were placed in a biochip reader, the fluorescence signal of each chip was detected, and the amplified products were subjected to signal clustering using software to obtain a scattergram, which was the same as the corresponding procedure in example 2.

And analyzing the scatter diagram according to the statistical result, searching mutation sites, and recording the mutation types.

TABLE 7.50 clinical specimen test results

The final results showed (table 7) that 13 positive (5 patients, 8 carriers) were detected at the c.609G > A mutation site using digital PCR technique; 80A > G > 1 positive patient was detected at the mutant site; 567dupT mutant site 2 carriers were detected; c.482G > 10 positive mutations were detected in the A mutation site (2 patients, 8 carriers); 217C > T mutation site 2 carriers were detected.

From the results of examples 1-4, it can be seen that the primer probe composition for high-frequency mutation of MMACHC gene provided by the invention has good specificity, and can detect the mutation conditions of high-frequency mutation sites c.609G > A, c.80A > G, c.567dupT, c.482G > A and c.217C > T of MMACHC gene in a sample; the primer probe composition kit for detecting the high-frequency mutation of the MMACHC gene has good sensitivity, repeatability and specificity, can detect a plurality of samples at one time, is simple and convenient to operate, is low in cost, is short in time consumption and accurate in result, and has extremely high application value.

While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and the description is given here only for clarity, and those skilled in the art should integrate the description, and the embodiments may be combined appropriately to form other embodiments understood by those skilled in the art.

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Claims

1. Five primer probe compositions for detecting high-frequency mutation of MMACHC genes comprise a first primer probe composition, a second primer probe composition, a third primer probe composition, a fourth primer probe composition and a fifth primer probe composition;

the first primer probe composition is used for detecting c.609G > A sites of the MMACHC gene; the kit comprises a first forward primer, a first reverse primer, a first wild type probe and a first mutant type probe, wherein the nucleotide sequences of the first forward primer, the first reverse primer, the first wild type probe and the first mutant type probe are shown as SEQ ID NO. 1-SEQ ID NO.4 in sequence;

the second primer probe composition is used for detecting c.482G > A sites of the MMACHC gene and consists of a second forward primer, a second reverse primer, a second wild type probe and a second mutant type probe, wherein the nucleotide sequences of the second forward primer, the second reverse primer, the second wild type probe and the second mutant type probe are sequentially shown as SEQ ID No. 5-SEQ ID No. 8;

the third primer probe composition is used for detecting c.80A > G sites of the MMACHC gene and consists of a third forward primer, a third reverse primer, a third wild-type probe and a third mutant-type probe, wherein the nucleotide sequences of the third forward primer, the third reverse primer, the third wild-type probe and the third mutant-type probe are sequentially shown as SEQ ID No. 9-SEQ ID No. 12;

the fourth primer probe composition is used for detecting c.567dupT locus of MMACHC gene and consists of a fourth forward primer, a fourth reverse primer, a fourth wild type probe and a fourth mutant type probe, wherein the nucleotide sequences of the fourth forward primer, the fourth reverse primer, the fourth wild type probe and the fourth mutant type probe are shown as SEQ ID NO. 13-SEQ ID NO.16 in sequence;

the fifth primer probe composition is used for detecting the c.217C > T site of the MMACHC gene; consists of a fifth forward primer, a fifth reverse primer, a fifth wild-type probe and a fifth mutant-type probe, the nucleotide sequences of which are sequentially shown as SEQ ID NO. 17-SEQ ID NO. 20.

2. The primer probe combination of claim 1, wherein each primer in the first, second, third, fourth and fifth primer probe compositions independently has a Tm of 58 to 60 ℃; preferably, the difference in Tm values between the primers is within 2 ℃.

3. The primer probe combination of claim 1, wherein the probe is a Taqman probe, the 5 'end of the Taqman probe is labeled with a fluorescent reporter group, and the 3' end of the Taqman probe is labeled with a quencher group; preferably, the fluorescent reporter group comprises FAM and/or VIC; the quencher group includes MGB.

4. The primer probe combination of claim 3, wherein the fluorescent reporter of the wild-type probe is different from the fluorescent reporter of the mutant probe.

5. A kit for detecting high-frequency mutation of MMACHC gene, wherein the kit comprises the primer probe composition according to any one of claims 1 to 4.

6. The detection kit according to claim 5, wherein the kit further comprises a PCR reaction solution; preferably, the PCR reaction solution comprises DNA polymerase and Mg ²⁺ Buffer, dNTPs and water.

7. The test kit of claim 6, wherein the test kit further comprises a quality control substance.

8. The kit of claim 7, wherein the quality control substance comprises enzyme-free water.

9. The method of using the primer probe composition of claims 1-4 or the test kit of claims 5-8, wherein the method comprises the steps of: 1) extracting DNA of a sample to be detected; 2) performing digital PCR amplification and detecting negative quality control products at the same time; 3) and (4) putting the amplified chip into a biochip reader, detecting a fluorescence signal and analyzing.

10. The method of claim 9, wherein the analysis is performed by comparing the fluorescence signal of the test sample with a scattergram to determine the genotype of the test sample, and determining the mutation in the test sample from the fluorescence signal of FAM and VIC and the scattergram.