阅读说明：本技术 一种基于核酸质谱平台体细胞突变超敏检测方法 (Somatic mutation hypersensitivity detection method based on nucleic acid mass spectrometry platform ) 是由 叶绍云 蒋峻峰 杨小娟 刘雨 于 2021-09-01 设计创作，主要内容包括：本发明公开了一种基于核酸质谱平台体细胞突变超敏检测方法,所述方法包括以下步骤：获取待检测基因；对待检测基因执行分析操作,得到待测突变位点；根据待测突变位点设计PCR扩增引物、SAP反应试剂、单碱基延伸阻遏引物和野生型阻遏探针；对待检测基因执行扩增操作,得到PCR产物；对PCR产物执行SAP反应操作,得到消化产物；对消化产物执行延伸反应操作,得到延伸产物；对延伸产物执行点样操作,获得点样数据；对点样数据进行分析,获取待检测基因突变类型；通过上述方式,本发明成本较低,检测时间耗时较短,可以灵活设计突变位点,超高的灵敏度,较高的突变检测通量,可同时检测近百种突变类型,适用面广,可以适用于不同的样本类型。(The invention discloses a nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method, which comprises the following steps: obtaining a gene to be detected; performing analysis operation on a gene to be detected to obtain a mutation site to be detected; designing a PCR amplification primer, an SAP reaction reagent, a single base extension repression primer and a wild type repression probe according to the mutation site to be detected; performing amplification operation on a gene to be detected to obtain a PCR product; performing SAP reaction operation on the PCR product to obtain a digestion product; performing extension reaction operation on the digestion product to obtain an extension product; performing sample application operation on the extension product to obtain sample application data; analyzing the sample application data to obtain the gene mutation type to be detected; through the mode, the method is low in cost, short in detection time consumption, capable of flexibly designing mutation sites, ultrahigh in sensitivity, high in mutation detection flux, capable of detecting nearly hundreds of mutation types simultaneously, wide in application range and suitable for different sample types.)

1. A somatic mutation hypersensitivity detection method based on a nucleic acid mass spectrometry platform is characterized by comprising the following steps:

obtaining a gene to be detected;

performing sequence analysis operation on the gene to be detected to obtain a mutation site to be detected;

designing a PCR amplification primer, an SAP reaction reagent, a single base extension repression primer and a wild type repression probe according to the mutation site to be detected;

performing amplification operation on the gene to be detected through the PCR amplification primer to obtain a PCR product;

performing SAP reaction operation on the PCR product through the SAP reaction reagent to obtain a digestion product;

performing an extension reaction operation on the digestion product by the single-base extension repressor primer to obtain an extension product;

performing sample application operation on the extension product to obtain sample application data;

and analyzing the sample application data to obtain the gene mutation type to be detected.

2. The nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method according to claim 1, characterized in that:

the step of performing a sequence analysis operation further comprises: and analyzing the regional DNA sequence and the amino acid sequence of the gene to be detected to obtain the sequence information of the mutation site to be detected, so as to obtain the mutation site to be detected.

3. The nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method according to claim 1, characterized in that:

the PCR amplification primers comprise a first PCR amplification primer 1st-PCRP and a second PCR amplification primer 2 nd-PCRP.

4. The nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method according to claim 3, characterized in that:

the 5' end of the forward primer or the reverse primer in the first PCR amplification primer and the second PCR amplification primer is added with a universal sequence ACGTTGGATG.

5. The nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method according to claim 4, characterized in that:

the step of performing an amplification operation further comprises: and mixing the first PCR amplification primer and the second PCR amplification primer of the mutation site to be detected to obtain a mixed working solution, and amplifying the DNA of the gene to be detected by using a single primer with the concentration of 0.5-1 mu M to obtain the PCR product.

6. The nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method according to claim 1, characterized in that:

the step of performing the SAP reaction operation further comprises: and mixing the reaction system after the amplification operation is carried out with the SAP reaction reagent, and eliminating the residual primers and dNTPs in the reaction system to obtain the digestion product.

7. The nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method according to claim 1, characterized in that:

the step of performing an extension reaction operation further comprises: mixing the digestion product with the single base extension repressor primer to obtain the extension product;

8. the nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method according to claim 1, characterized in that:

the 5' end of the wild type repression probe is modified by 5' C3 Spacer or 5' P, the 3' end is modified by 3' MGB, and the melting point temperature value ranges from 65 ℃ to 72 ℃.

Technical Field

The invention relates to the technical field of gene detection, in particular to a nucleic acid mass spectrum platform-based somatic mutation hypersensitivity detection method.

Background

With the continuous deepening and development of human genomics, pharmacogenomics and tumor molecular biology research, the mode of tumor treatment gradually moves from empirical guided therapy to gene-oriented individualized therapy.

The existing gene detection technology mainly comprises the following steps:

the direct sequencing method has the advantages of low cost and visual detection result; the disadvantages are that the procedure is cumbersome, time consuming and limited sensitivity.

The pyrosequencing method has the advantages that a short target fragment can be rapidly and accurately determined without carrying out electrophoresis and carrying out fluorescence labeling on a DNA fragment; the disadvantages are short sequencing length and easy pollution.

The probe amplification blocking mutation system has the advantages of higher sensitivity and better consistency of the detection result and the clinical curative effect; the disadvantage is that special probes are required, only one mutation at a time being detectable.

The denatured high performance liquid chromatography has the advantages of simplicity, rapidness and high sensitivity, and can be used for not only detecting known mutation but also scanning unknown mutation; the method has the defects that only the existence of mutation can be detected, homologous mutation and mutation type can not be detected, the judgment of the detection result is easy to make mistakes, and when a plurality of fragments need to be detected, a plurality of steps of detection are needed due to a plurality of melting temperatures, so that the workload is increased.

The high-resolution melting curve analysis technology has the advantages of high sensitivity, no need of a sequence specific probe, no limitation of mutant base sites and types, simple operation, no need of PCR subsequent operation and good selectivity, and can be used for detecting somatic mutation and screening mutation before sequencing; the method has the disadvantages that only small-fragment DNA with single purity can be analyzed, enough templates are required, and stable and reliable results can be obtained when the content of the templates is not less than 1 ng.

Polymerase chain reaction-single strand conformation polymorphism analysis has the advantages of higher sensitivity, no need of special instruments and capability of detecting unknown mutation; the method has the defects of long electrophoresis time, complicated operation steps, capability of only performing qualitative analysis, parallel standard control, great influence by experimental conditions and easy occurrence of false negative.

Mutant enrichment PCR has the advantages of higher mutation sensitivity, stronger specificity and capability of detecting one in a thousand of mutations; the disadvantages are two PCR, complicated operation, long time consumption and easy pollution.

The micro-digital PCR has the advantages that compared with the result of histology, the sensitivity and the specificity of the method for detecting the plasma specimen are respectively as high as 92 percent and 100 percent; the disadvantage is the high cost.

Disclosure of Invention

The invention mainly solves the problems that the existing gene detection technology has complex detection process, long time consumption, easy pollution, inaccurate detection result, high cost and the like.

In order to solve the above problems, the present invention adopts a technical solution that: provides a somatic mutation hypersensitivity detection method based on a nucleic acid mass spectrometry platform, which comprises the following steps:

obtaining a gene to be detected;

performing sequence analysis operation on the gene to be detected to obtain a mutation site to be detected;

designing a PCR amplification primer, an SAP reaction reagent, a single base extension repression primer and a wild type repression probe according to the mutation site to be detected;

performing amplification operation on the gene to be detected through the PCR amplification primer to obtain a PCR product;

performing SAP reaction operation on the PCR product through the SAP reaction reagent to obtain a digestion product;

performing an extension reaction operation on the digestion product by the single-base extension repressor primer to obtain an extension product;

performing sample application operation on the extension product to obtain sample application data;

and analyzing the sample application data to obtain the gene mutation type to be detected.

Further, the step of performing a sequence analysis operation further comprises: and analyzing the regional DNA sequence and the amino acid sequence of the gene to be detected to obtain the sequence information of the mutation site to be detected, so as to obtain the mutation site to be detected.

Further, the PCR amplification primers comprise a first PCR amplification primer 1st-PCRP and a second PCR amplification primer 2 nd-PCRP.

Further, a universal sequence ACGTTGGATG is added to the 5' end of the forward primer or the reverse primer in the first PCR amplification primer and the second PCR amplification primer.

Further, the step of performing an amplification operation further comprises: and mixing the first PCR amplification primer and the second PCR amplification primer of the mutation site to be detected to obtain a mixed working solution, and amplifying the DNA of the gene to be detected by using a single primer with the concentration of 0.5-1 mu M to obtain the PCR product.

Further, the step of performing the SAP reaction operation further comprises: and mixing the reaction system after the amplification operation is carried out with the SAP reaction reagent, and eliminating the residual primers and dNTPs in the reaction system to obtain the digestion product.

Further, the step of performing an extension reaction operation further comprises: mixing the digestion product with the single base extension repressor primer to obtain the extension product;

further, the 5' end of the wild-type suppression probe is modified by 5' C3 Spacer or 5' P, and the 3' end is modified by 3' MGB, wherein the melting point temperature value ranges from 65 ℃ to 72 ℃.

The invention has the beneficial effects that:

the nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method disclosed by the invention is low in cost, short in detection time consumption, capable of flexibly designing mutation sites according to different genes to be detected, ultrahigh in sensitivity, capable of detecting mutations with 1-2 copies, high in mutation detection flux, capable of detecting nearly a hundred mutation types simultaneously, wide in application range and suitable for different sample types.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a flow chart of a nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method according to an embodiment of the present invention;

FIG. 2 is a configuration table of PCR primers for the nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method according to the embodiment of the present invention;

FIG. 3 is a reagent table of SAP reaction based on the nucleic acid mass spectrometry platform somatic mutation hypersensitivity detection method according to the embodiment of the present invention;

FIG. 4 is a reagent table of extension reaction based on the nucleic acid mass spectrometry platform somatic mutation hypersensitivity detection method according to the embodiment of the present invention;

FIG. 5 is a schematic diagram of the design and operation of a repression probe and a single-base extension repression primer for a somatic mutation hypersensitivity detection method based on a nucleic acid mass spectrometry platform according to an embodiment of the present invention;

FIG. 6 is a design sequence table for detecting EGFR _ L858R and EGFR _ T790M sites by using the nucleic acid mass spectrometry platform-based somatic mutation hypersensitivity detection method described in example 1 of the present invention;

FIG. 7 is a graph showing the detection result of the UltraCap technique when the mutation abundance of the T790M site of the gene EGFR detected by using the detection method is 0.005 in example 1 of the present invention;

FIG. 8 is a graph showing the detection result of the control group when the mutation abundance of the T790M site of the gene EGFR detected by using the detection method is 0.005 in example 1 of the present invention;

FIG. 9 is a graph showing the detection result of the UltraCap technique when the mutation abundance of the T790M site of the gene EGFR detected by using the detection method is 0.001, according to example 1 of the present invention;

FIG. 10 is a graph showing the results of detection in the control group when the abundance of mutation at the T790M site of the gene EGFR detected by the detection method is 0.001, according to example 1 of the present invention;

FIG. 11 is a graph showing the detection results of a wild-type sample of the UltraCap technique when the mutation abundance of the T790M site of the gene EGFR detected by the detection method is 0, according to example 1 of the present invention;

FIG. 12 is a graph showing the results of detection of a wild-type sample of a control group at the T790M site of the gene EGFR with a mutation abundance of 0 using the detection method of example 1 of the present invention;

FIG. 13 is a graph showing the detection results of blank control of the UltraCap technique when the mutation abundance of the T790M site of the gene EGFR detected by the detection method is 0, according to example 1 of the present invention;

FIG. 14 is a graph showing the results of blank control in the control group when the mutation abundance at the T790M site of the gene EGFR detected by the detection method is 0 according to example 1 of the present invention;

FIG. 15 is a graph showing the detection result of the UltraCap technique when the mutation abundance of the L858R locus of the gene EGFR detected by the detection method is 0.005 in example 1 of the present invention;

FIG. 16 is a graph showing the detection result of the control group when the mutation abundance of the L858R site of the gene EGFR detected by the detection method is 0.005 in example 1 of the present invention;

FIG. 17 is a graph showing the detection result of the UltraCap technique when the mutation abundance of the L858R locus of the gene EGFR detected by the detection method is 0.001 according to example 1 of the present invention;

FIG. 18 is a graph showing the results of detection in the control group when the mutation abundance at the L858R site of the gene EGFR detected by the detection method is 0.001, according to example 1 of the present invention;

FIG. 19 is a graph showing the detection results of a wild-type sample of the UltraCap technique when the mutation abundance of the L858R site of the gene EGFR detected by the detection method is 0, according to example 1 of the present invention;

FIG. 20 is a graph showing the results of detection of a wild-type sample of a control group at the L858R site of the gene EGFR at a mutation abundance of 0 according to example 1 of the present invention;

FIG. 21 is a graph showing the detection results of blank control of the UltraCap technique when the mutation abundance of the L858R site of the gene EGFR detected by the detection method is 0 according to example 1 of the present invention;

FIG. 22 is a graph showing the results of blank control in the control group when the mutation abundance at L858R locus of gene EGFR detected by the present detection method is 0 according to example 1 of the present invention;

FIG. 23 is a sequence listing of the design for detecting KRAS _ G12D and KRAS _ G13D sites using the detection method described in example 2 of the present invention;

FIG. 24 is a graph showing the detection result of the UltraCap technique when the mutation abundance of the G12D locus of the gene KRAS detected by the detection method is 0.001 according to example 2 of the present invention;

FIG. 25 is a graph showing the detection results of the control group when the mutation abundance of the G12D locus of KRAS gene detected by the detection method is 0.001 in example 2 of the present invention;

FIG. 26 is a diagram showing the detection result of a wild-type sample of the UltraCap technique when the mutation abundance of the G12D locus of the gene KRAS detected by the detection method is 0 according to example 2 of the present invention;

FIG. 27 is a graph showing the results of detection of a wild-type sample of a control group using the detection method of the present invention, which is described in example 2, when the mutation abundance of the G12D locus of gene KRAS is 0;

FIG. 28 is a graph showing the detection results of blank controls of the UltraCap technique when the mutation abundance of the G12D locus of the gene KRAS detected by the detection method is 0 according to example 2 of the present invention;

FIG. 29 is a graph showing the results of blank control in the control group when the mutation abundance of the G12D site of the gene KRAS detected by the detection method is 0 as described in example 2 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the description of the present invention, it is to be noted that, unless otherwise specifically defined and limited, terms such as "gene", "sequence", "primer", "extension primer", "mixture", "amplification", "mutation site", "reaction system", "elimination", "product", "spotting", "analysis", and the like are to be construed broadly. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature.

It should be noted that, in the description of the present invention,

MTB (Mycobacterium tuberculosis) is Mycobacterium tuberculosis,

MTBC (Mycobacterium tuberculosis complex) is a complex of Mycobacterium tuberculosis,

NTM (Non-tuberculosis mycobacteriosis) is a Non-tuberculous mycobacterium,

SNP (Single Nucleotide polymorphism) is a single Nucleotide polymorphism,

PCR (polymerase Chain reaction) is a polymerase Chain reaction,

DNA (deoxyribonucleic acid) is deoxyribonucleic acid,

dNTP (deoxy-riboside triphosphate) is deoxyribonucleoside triphosphate.

Example 1

The embodiment of the present invention provides a method for detecting somatic mutation hypersensitivity based on a nucleic acid mass spectrometry platform, please refer to fig. 1 to 22, comprising the following steps:

it should be noted that, in this example, the selected gene to be tested is EGFR, the selected test sites are L858R and T790M, and the designed sequence information is shown in fig. 6.

And (3) acquiring a gene EGFR to be detected, and performing sequence analysis operation on the gene EGFR to be detected, namely analyzing a regional DNA sequence and an amino acid sequence of the EGFR gene to obtain mutation sites L858R and T790M to be detected.

PCR amplification primers, SAP reaction reagents, single base extension repression primers and wild type repression probes are designed according to mutation sites L858R and T790M to be detected of EGFR gene, the reaction system of PCR amplification primers is shown in figure 2, the reaction system of SAP reaction reagents is shown in figure 3, the reaction system of single base extension repression primers is shown in figure 4, and the design of wild type repression probes is shown in figure 5.

The PCR amplification primers comprise a first PCR amplification primer 1st-PCRP and a second PCR amplification primer 2nd-PCRP, and the 5' ends of forward primers or reverse primers in the first PCR amplification primer and the second PCR amplification primer are added with a universal sequence ACGTTGGATG, so that the specificity of the primers and the amplification efficiency are enhanced.

The single base extension primer and its product are designed according to the principle that the difference of molecular weight is greater than 16 dalton.

Performing PCR amplification operation on a gene to be detected, namely mixing a first PCR amplification primer and a second PCR amplification primer to obtain a mixed working solution, putting the gene to be detected and the mixed working solution into a PCR instrument, and amplifying DNA of the gene to be detected through a single primer with the concentration of 0.5-1 mu M, wherein the PCR amplification process comprises the following steps:

pre-denaturation at 95 ℃ for 2 min; performing a cyclic reaction operation; extension at 72 ℃ for 5 min; keeping the temperature at 4 ℃;

the process of the cyclic reaction operation is as follows: denaturation at 95 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, and extension at 72 ℃ for 60 seconds; the cyclic reaction operation was performed 45 times in total.

Obtaining the PCR product after PCR amplification.

Mixing the PCR product with an SAP reaction reagent, putting the mixture into a PCR instrument for SAP reaction, and eliminating the residual primers and dNTP in a PCR amplification reaction system, wherein the SAP reaction process comprises the following steps:

the time is 40 minutes at 37 ℃ and 5 minutes at 85 ℃.

And performing SAP reaction on the PCR product to obtain a digestion product.

Mixing the digestion product with the single-base extension repression primer, putting the mixture into a PCR instrument for extension reaction, wherein the extension reaction process comprises the following steps:

the time duration is 30 seconds at 95 ℃; executing a large-cycle reaction operation; the time is 5 minutes at 72 ℃; keeping the temperature at 4 ℃;

the process of the large-cycle reaction operation is as follows: the time is 5 seconds at 95 ℃; performing an internal circulation reaction operation; the large-cycle reaction operation is carried out for 40 times;

the process of the internal circulation reaction operation is as follows: the time duration is 5 seconds at 52 ℃ and 5 seconds at 80 ℃; the internal circulation reaction operation was performed 5 times in total.

And performing extension operation on the digestion product to obtain an extension product.

Starting a sample applicator, applying the sample of the purified extension product to a chip, and scanning the chip by a MassARRAY nucleic acid detection mass spectrometer to obtain a detection result;

and performing typing operation on the detection result through TYPER4.0 software and outputting to obtain a typing result, and identifying the gene type through a mass spectrum peak of the typing result.

It should be noted that the MassARRAY nucleic acid detection mass spectrometer and TYPER4.0 software are only limited herein for the purpose of clearly explaining the present invention, and this does not represent that the scope of the present invention is limited to the MassARRAY nucleic acid detection mass spectrometer and TYPER4.0 software.

The test results of the T790M site of the gene EGFR are shown in fig. 7 to 14, and the test results of the L858R site of the gene EGFR are shown in fig. 15 to 22;

note that, in fig. 7 to 10 and 15 to 18, the left vertical broken line of each picture represents the undetected mutant template or UEP peak map position, and the right vertical broken line of each picture represents the detected mutant template; in fig. 11 to 14 and 19 to 22, the dotted line of each picture represents the undetected mutation template or UEP peak map position.

Example 2

The embodiment of the present invention provides a method for detecting somatic mutation hypersensitivity based on a nucleic acid mass spectrometry platform, please refer to fig. 1 to 5 and fig. 23 to 29, comprising the following steps:

it should be noted that, in this example, the selected gene to be tested is KRAS, the selected test sites are G12D and G13D, and the designed sequence information is shown in fig. 23.

And (3) obtaining a gene KRAS to be detected, and performing sequence analysis operation on the gene KRAS, namely analyzing a regional DNA sequence and an amino acid sequence of the KRAS gene to obtain mutation sites G12D and G13D to be detected.

PCR amplification primers, SAP reaction reagents, single base extension repression primers and wild-type repression probes are designed according to the mutation sites G12D and G13D to be detected of the KRAS gene, the reaction system of the PCR amplification primers is shown in figure 2, the reaction system of the SAP reaction reagents is shown in figure 3, the reaction system of the single base extension repression primers is shown in figure 4, and the design of the wild-type repression probes is shown in figure 5.

The PCR amplification primers comprise a first PCR amplification primer 1st-PCRP and a second PCR amplification primer 2nd-PCRP, and the 5' ends of forward primers or reverse primers in the first PCR amplification primer and the second PCR amplification primer are added with a universal sequence ACGTTGGATG, so that the specificity of the primers and the amplification efficiency are enhanced.

The single base extension primer and its product are designed according to the principle that the difference of molecular weight is greater than 16 dalton.

Performing PCR amplification operation on a gene to be detected, namely mixing a first PCR amplification primer and a second PCR amplification primer to obtain a mixed working solution, putting the gene to be detected and the mixed working solution into a PCR instrument, and amplifying DNA of the gene to be detected through a single primer with the concentration of 0.5-1 mu M, wherein the PCR amplification process comprises the following steps:

pre-denaturation at 95 ℃ for 2 min; performing a cyclic reaction operation; extension at 72 ℃ for 5 min; keeping the temperature at 4 ℃;

the process of the cyclic reaction operation is as follows: denaturation at 95 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, and extension at 72 ℃ for 60 seconds; the cyclic reaction operation was performed 45 times in total.

Obtaining the PCR product after PCR amplification.

Mixing the PCR product with an SAP reaction reagent, putting the mixture into a PCR instrument for SAP reaction, and eliminating the residual primers and dNTP in a PCR amplification reaction system, wherein the SAP reaction process comprises the following steps:

the time is 40 minutes at 37 ℃ and 5 minutes at 85 ℃.

And performing SAP reaction on the PCR product to obtain a digestion product.

Mixing the digestion product with the single-base extension repression primer, putting the mixture into a PCR instrument for extension reaction, wherein the extension reaction process comprises the following steps:

the time duration is 30 seconds at 95 ℃; executing a large-cycle reaction operation; the time is 5 minutes at 72 ℃; keeping the temperature at 4 ℃;

the process of the large-cycle reaction operation is as follows: the time is 5 seconds at 95 ℃; performing an internal circulation reaction operation; the large-cycle reaction operation is carried out for 40 times;

the process of the internal circulation reaction operation is as follows: the time duration is 5 seconds at 52 ℃ and 5 seconds at 80 ℃; the internal circulation reaction operation was performed 5 times in total.

And performing extension operation on the digestion product to obtain an extension product.

Starting a sample applicator, applying the sample of the purified extension product to a chip, and scanning the chip by a MassARRAY nucleic acid detection mass spectrometer to obtain a detection result;

and performing typing operation on the detection result through TYPER4.0 software and outputting to obtain a typing result, and identifying the gene type through a mass spectrum peak of the typing result.

It should be noted that the MassARRAY nucleic acid detection mass spectrometer and TYPER4.0 software are only limited herein for the purpose of clearly explaining the present invention, and this does not represent that the scope of the present invention is limited to the MassARRAY nucleic acid detection mass spectrometer and TYPER4.0 software.

The test results of the G12D site of the gene KRAS are shown in fig. 24 to fig. 29, it should be noted that the left vertical dotted line in fig. 24 and fig. 25 represents the undetected mutant template or UEP peak map position, and the right vertical dotted line represents the detected mutant template; the dashed lines in fig. 26-29 represent undetected mutant template or UEP peak map positions.

The numbers of the embodiments disclosed in the embodiments of the present invention are merely for description, and do not represent the merits of the embodiments.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.