A method for detecting gene amplification based on nucleic acid mass spectrometry

Abstract

The application discloses a gene amplification detection method based on nucleic acid mass spectrometry technology, comprising the following steps: setting a target gene region, configuring a reference gene, and confirming experimental primers based on the target gene region and the reference gene; obtaining an experimental sample, performing a primer reaction processing operation based on the experimental primers and the experimental sample, and obtaining a sample to be analyzed; setting an interpretation interval, configuring a sample applicator and a nucleic acid mass spectrometer, performing a data analysis and interpretation operation based on the sample to be analyzed, the interpretation interval, the sample applicator and the nucleic acid mass spectrometer, and obtaining a gene amplification interpretation result; the application can achieve a detection accuracy higher than that of the fluorescence in situ hybridization technology and the immunohistochemical technology in the prior art, a detection throughput higher than that of the fluorescent quantitative PCR technology and the digital PCR technology, a detection cost lower than that of the fluorescent quantitative PCR technology and the digital PCR technology, and a detection time shorter than that of the second-generation sequencing NGS technology.

Description

Technical Field

[0001] This invention relates to the field of gene amplification detection technology, and in particular to a gene amplification detection method based on nucleic acid mass spectrometry technology. Background Technology

[0002] In existing technologies, commonly used methods for determining copy number variations in the HER2 and MET genes include: fluorescence in situ hybridization, quantitative PCR, immunohistochemistry, digital PCR, and next-generation sequencing (NGS).

[0003] Specifically, the principles and disadvantages of the above technologies are as follows:

[0004] Fluorescence in situ hybridization (FISH) is based on the principle of complementary base pairing, which allows probes carrying fluorescent substances to bind to target DNA. The gene copy number can then be determined based on the location of the DNA. However, its experimental operation is complex, requires high technical skills, and has low accuracy.

[0005] Quantitative real-time PCR (qPCR) is a technique based on conventional PCR combined with fluorescently labeled probes. It collects a fluorescence intensity signal after each PCR cycle and monitors the change in product quantity based on the change in fluorescence intensity, thereby enabling the monitoring of gene copy number. However, it has low throughput and high experimental cost.

[0006] Immunohistochemistry is based on the principle of antigen-antibody reaction and chemical color development. It observes the distribution and content of antigens on antigen-antibody reactants to determine gene copy number. However, it cannot directly quantify the results, and the interpretation of experimental results is highly subjective, which also reduces the accuracy.

[0007] Digital PCR technology divides DNA or cDNA samples into many individual, parallel PCR reactions, and then uses TaqMan chemical reagents and dye-labeled probes to detect specific target sequences in the PCR reaction. Finally, based on the target molecules and PCR analysis, the gene copy number is determined. This method has the same disadvantages as real-time PCR technology: low throughput and high experimental cost.

[0008] Next-generation sequencing (NGS) technology is a high-throughput sequencing method used to rapidly sequence the base pairs of DNA or RNA samples. Based on this method, it can also be used to determine gene copy number, but its experimental operation and data analysis process is extremely time-consuming and the experimental cost is high.

[0009] In summary, the present invention aims to provide a method for determining copy number variations of the HER2 and MET genes that is superior to existing technologies in all aspects. Summary of the Invention

[0010] The main objective of this invention is to develop a method for determining copy number variations in the HER2 and MET genes that has high accuracy and throughput while having low detection cost and time.

[0011] To achieve the above objectives, one technical solution adopted by the present invention is to provide a gene amplification detection method based on nucleic acid profiling technology, comprising the following steps:

[0012] Experimental primer preparation steps:

[0013] Set the target gene region, configure the reference gene, and confirm the experimental primers based on the target gene region and the reference gene;

[0014] Primer reaction processing steps:

[0015] Obtain experimental samples, and perform primer reaction processing based on the experimental primers and the experimental samples to obtain samples to be analyzed;

[0016] Data analysis and interpretation steps:

[0017] Set the interpretation interval, configure the spotting instrument and nucleic acid mass spectrometer, and perform data analysis and interpretation operations based on the sample to be analyzed, the interpretation interval, the spotting instrument and the nucleic acid mass spectrometer to obtain the gene amplification interpretation results.

[0018] As an improved approach, the reference genes include: a MET-specific reference gene, the MET gene, an ERBB2-specific reference gene, the ERBB2 gene, an internal reference gene, and a universal reference gene; the experimental primers include: PCR amplification primers and extension primers.

[0019] As an improved approach, the step of confirming the experimental primers based on the target gene region and the reference gene further includes:

[0020] Based on the target gene region, primer information for several regions matching the MET gene, the ERBB2 gene, and the internal reference gene was identified; primers for amplifying the target gene and primers for amplifying the internal reference gene were configured based on the primer information for the several regions; both primers for amplifying the target gene and primers for amplifying the internal reference gene were the PCR amplification primers.

[0021] Based on the target gene region, several first gene regions were identified that matched the MET-specific reference gene, the MET gene, the ERBB2-specific reference gene, the ERBB2 gene, and the universal reference gene, respectively. Based on the several first gene regions, several first extension gene primers were screened from the MET-specific reference gene, the MET gene, the ERBB2-specific reference gene, the ERBB2 gene, and the universal reference gene. All of the several first extension gene primers were extension primers.

[0022] As an improved approach, the step of obtaining experimental samples further includes:

[0023] Confirm the experimental technology platform information, confirm the sample characteristic information based on the experimental technology platform information, and obtain a tumor tissue sample based on the sample characteristic information; the tumor tissue sample is the experimental sample.

[0024] As an improved approach, the primer reaction processing operation includes:

[0025] Set the first primer quality information; perform HPLC purification on the PCR amplification primers and the extension primers, and perform mass spectrometry quality control on the PCR amplification primers and the extension primers after HPLC purification to obtain the second primer quality information; determine whether the second primer quality information matches the first primer quality information, and if they match, execute the amplification reaction step.

[0026] As an improved approach, the amplification reaction step includes:

[0027] The PCR reaction procedure is set up by mixing the target gene amplification primers and the internal reference gene amplification primers after HPLC purification to form the PCR working solution.

[0028] The DNA of the experimental sample was amplified based on the PCR reaction procedure and the PCR working solution to obtain PCR amplification products.

[0029] A digestion reaction procedure is set up, and the PCR amplification product is subjected to an SAP reaction according to the digestion reaction procedure. The remaining PCR amplification primers and dNTPs of the PCR amplification product in the SAP reaction are detected to see if they are all digested. If they are all digested, the amplification digestion product is obtained.

[0030] An extension reaction procedure is set up, and the amplification digestion product is extended based on the extension reaction procedure and the extension primers purified by HPLC to obtain the sample to be analyzed.

[0031] As an improved approach, the interpretation interval includes: the target gene copy number interval, the proprietary internal reference gene copy number interval, and the ploidy copy number interval;

[0032] The target gene copy number interval includes: a first target gene interpretation interval and a second target gene interpretation interval; the proprietary internal reference gene copy number interval includes: a first proprietary internal reference interpretation interval and a second proprietary internal reference interpretation interval; the ploidy copy number interval includes: a first ploidy interpretation interval and a second ploidy interpretation interval;

[0033] The gene amplification interpretation results include: a first interpretation result, a second interpretation result, and a third interpretation result; the first interpretation result includes: normal copy number, no gene amplification, and normal ploidy; the second interpretation result includes: gene amplification; the third interpretation result includes: ploidy duplication.

[0034] As an improved approach, the data analysis and interpretation operation includes:

[0035] The sample to be analyzed is spotted onto the spotting chip of the spotting instrument; the nucleic acid mass spectrometer is controlled to excite the sample to be analyzed on the spotting chip with high-energy laser to obtain the gene data to be analyzed;

[0036] Based on the interpretation interval, the gene data to be analyzed is interpreted to obtain the gene amplification interpretation result.

[0037] As an improved approach, the result interpretation step includes:

[0038] Identify the copy number of the first target gene, the copy number of the first proprietary internal reference gene, and the copy number of the first ploid gene in the gene data to be analyzed;

[0039] If the copy number of the first target gene is within the first target gene interpretation interval, the copy number of the first proprietary internal reference gene is within the first proprietary internal reference interpretation interval, and the copy number of the first ploidy is within the first ploidy interpretation interval, then the gene amplification interpretation result is set as the first interpretation result.

[0040] If the copy number of the first target gene is within the second target gene interpretation interval, and the copy number of the first ploidy is within the second ploidy interpretation interval, then the gene amplification interpretation result is set as the second interpretation result;

[0041] If the copy number of the first target gene is within the first target gene interpretation interval, and the copy number of the first ploidy is within the second ploidy interpretation interval, then the gene amplification interpretation result is set as the third interpretation result.

[0042] As an improved approach, the gene data to be analyzed includes: MET gene data and ERBB2 gene data.

[0043] The beneficial effects of this invention are:

[0044] The gene amplification detection method based on nucleic acid mass spectrometry described in this invention can achieve higher detection accuracy than existing technologies such as fluorescence in situ hybridization and immunohistochemistry, higher detection throughput than quantitative PCR and digital PCR, and lower detection cost than quantitative PCR and digital PCR. At the same time, it has a shorter detection time compared with next-generation sequencing (NGS) technology. The operation procedure of this invention is simple, the detection results are objective, and the comprehensive data of accuracy, detection throughput and detection cost are superior to existing technologies, which has extremely high experimental value. Attached Figure Description

[0045] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0046] Figure 1 This is a flowchart of the gene amplification and detection method based on nucleic acid mass spectrometry technology described in Embodiment 1 of the present invention;

[0047] Figure 2 This is a schematic diagram of the specific process of the gene amplification and detection method based on nucleic acid mass spectrometry technology described in Embodiment 1 of the present invention;

[0048] Figure 3 This is the primer synthesis code table described in Embodiment 1 of the present invention;

[0049] Figure 4 This is the extended primer information table described in Embodiment 1 of the present invention;

[0050] Figure 5 This is the experimental sample information table described in Embodiment 1 of the present invention;

[0051] Figure 6 This is the MET gene data table described in Embodiment 1 of the present invention;

[0052] Figure 7 This is the ERBB2 gene data table described in Embodiment 1 of the present invention;

[0053] Figure 8 This is the interpretation standard information table described in Embodiment 1 of the present invention;

[0054] Figure 9 This is the MET mass spectrometry peak diagram of Embodiment 1 of the present invention;

[0055] Figure 10 This is the ERBB2 mass spectrum peak diagram of Embodiment 1 of the present invention. Detailed Implementation

[0056] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0057] In the description of this invention, it should be noted that the embodiments described in this invention are only some embodiments of this invention, not all embodiments; based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0058] In the description of this invention, it should be noted that the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0059] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "target gene region," "reference gene," "experimental primer," "primer reaction processing operation," "sample to be analyzed," "interpretation interval," "data analysis and interpretation operation," "gene amplification interpretation result," "MET-specific reference gene," "ERBB2-specific reference gene," "internal reference gene," "universal reference gene," "PCR amplification primer," "extension primer," "technology platform information," "sample characteristic information," "HPLC purification processing," "primer quality control information," "mass spectrometry quality control operation," "PCR reaction process," "PCR working solution," "digestion reaction process," "SAP reaction," "remaining PCR amplification primers," "extension reaction process," and "high-energy laser excitation" should be interpreted broadly. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0060] In the description of this invention, it should be noted that:

[0061] NDUFA4 is an antibody; TRA2A is a protein antibody; ANKFY1 is a reference gene; RPP30 is a universal reference gene; PSMB6 is the proteasome subunit β6; ERBB2 is tyrosine kinase receptor 2; ACTB is a cytoskeletal actin.

[0062] DNA (Deoxyribonucleic acid) is deoxyribonucleic acid;

[0063] PCR (Polymerase Chain Reaction) is a polymerase chain reaction;

[0064] NGS (High-throughput sequencing) is a high-throughput sequencing technology;

[0065] FISH (Fluorescence in situ hybridization) is a fluorescent in situ hybridization technique.

[0066] dNTP (deoxy-ribonucleoside triphosphate) is a deoxyribonucleoside triphosphate;

[0067] HPLC (High Performance Liquid Chromatography) is a high-performance liquid chromatography method.

[0068] GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a glyceraldehyde-3-phosphate dehydrogenase.

[0069] MET (Metronidazole) is 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole and 2-methyl-5-nitro-1H-imidazole-1-ethanol.

[0070] Example 1

[0071] This embodiment provides a gene amplification detection method based on nucleic acid mass spectrometry technology, such as Figures 1-10 As shown, it includes:

[0072] It should be noted that this embodiment is based on nucleic acid mass spectrometry technology, and its main working principle is as follows: after the sample analyte covalently binds with the chip matrix, i.e., silicon compound, to form crystals, high-energy laser excitation is performed in the vacuum chamber of the nucleic acid mass spectrometer, causing the nucleic acid molecules to desorb into single-charged ions. The flight time of these single-charged ions in the electric field is inversely proportional to their mass; therefore, the precise molecular weight of the sample analyte can be calculated by detecting the flight time of the desorbed nucleic acid molecules in the vacuum chamber, thereby obtaining the genotype information of the analyte. Correspondingly, in this embodiment, the platform adopts a vertical flight mode. Specifically, the specific steps of a gene amplification and detection method based on nucleic acid mass spectrometry technology in this embodiment are as follows:

[0073] S100, Experimental primer preparation steps, specifically including:

[0074] S110. Set the target gene region, configure the reference gene, and confirm the experimental primers based on the target gene region and the reference gene.

[0075] Specifically, the reference genes include: a MET-specific reference gene, the MET gene, an ERBB2-specific reference gene, the ERBB2 gene, an internal reference gene, and a universal reference gene; the experimental primers include: PCR amplification primers and extension primers; in this embodiment, the MET-specific reference genes are NDUFA4, TRA2A, and ACTB, the ERBB2-specific reference genes are ANKFY1 and PSMB6, and the universal reference genes are GAPDH and RPP30;

[0076] Specifically, based on the target gene region, different regional primers matching the MET gene, the ERBB2 gene, and the internal reference gene are identified. Through optimization, information on several regional primers matching these different regional primers is confirmed. In this embodiment, the specific information on the regional primers is detailed below. Figure 3 The primer synthesis code table; the target gene amplification primers and the internal reference gene amplification primers are configured according to the primer information of several regions; the target gene amplification primers and the internal reference gene amplification primers are all the PCR amplification primers;

[0077] Based on the target gene region, several first gene regions were identified that matched the MET-specific reference gene, the MET gene, the ERBB2-specific reference gene, the ERBB2 gene, and the universal reference gene, respectively. Correspondingly, several first extension gene primers corresponding to several different UEP sequences were screened from the MET-specific reference gene, the MET gene, the ERBB2-specific reference gene, the ERBB2 gene, and the universal reference gene according to the several first gene regions. All of the several first extension gene primers are extension primers. In this embodiment, specific information about the extension primers can be found in [link to documentation]. Figure 4 Extended primer information table.

[0078] S200, primer reaction processing steps, specifically including:

[0079] S210. Obtain experimental samples, and perform primer reaction processing based on the experimental primers and the experimental samples to obtain samples to be analyzed.

[0080] Specifically, the experimental technology platform information is confirmed, sample characteristic information is confirmed based on the experimental technology platform information, and tumor tissue samples are obtained based on the sample characteristic information; the tumor tissue samples are the experimental samples; in this embodiment, if a FISH technology platform or an NGS technology platform is used, positive and negative samples are selected for testing. In this embodiment, the specific information and sample number of the tumor tissue samples are as follows: Figure 5 Experimental sample information table;

[0081] Specifically, the primer reaction processing is performed using nucleic acid mass spectrometry technology on a nucleic acid mass spectrometry platform, and the primer reaction processing includes:

[0082] The process involves setting first primer quality information; purifying the PCR amplification primers and extension primers using HPLC, and then performing mass spectrometry quality control on the purified primers to obtain second primer quality information; determining whether the second primer quality information matches the first primer quality information; if they match, then executing the amplification reaction step; in this embodiment, the first primer quality information is specifically set according to the configuration company and configuration type of the configured PCR amplification primers and extension primers, with the purpose of detecting the quality of the PCR amplification primers and extension primers. The mass spectrometry quality control operation involves detecting the quality of the PCR amplification primers and extension primers using a mass spectrometer; if the detected second primer quality information matches the first primer quality information, it indicates that the PCR amplification primers and extension primers required for the experiment meet the standards and can be used for the corresponding reaction, thus executing the amplification reaction step; if the detected second primer quality information does not match the first primer quality information, it indicates that the PCR amplification primers and extension primers required for the experiment do not meet the standards and need to be reconfigured;

[0083] Specifically, the amplification reaction steps include:

[0084] A PCR reaction procedure is set up, in which the target gene amplification primers and the internal reference gene amplification primers, purified by HPLC, are mixed to form a PCR working solution. In this embodiment, the PCR reaction procedure is the reaction program or strategy corresponding to the PCR instrument. Specifically, the PCR reaction procedure includes: pre-denaturation at a first temperature for 2 minutes; performing 45 cycles; extending at a second temperature for 5 minutes; and holding at a third temperature. Correspondingly, the first temperature ranges from 91℃ to 96℃, set according to specific conditions; the second temperature ranges from 70℃ to 73℃, set according to specific conditions; and the third temperature ranges from 2℃ to 5℃, set according to specific conditions. Specifically, the cycle operation includes: denaturation at a first temperature for 30 seconds, annealing at a fourth temperature for 30 seconds, and extending at a second temperature for 60 seconds. Correspondingly, the fourth temperature ranges from 51℃ to 57℃, set according to specific conditions.

[0085] Based on the PCR reaction procedure and the PCR working solution, the DNA of the experimental sample is amplified to obtain PCR amplification products. In this embodiment, primers with a single primer concentration of 0.5 μM-1 μM in the PCR working solution are selected to amplify the DNA of the tumor tissue sample to obtain the corresponding PCR amplification products. These PCR amplification products contain residual PCR amplification primers and dNTPs after the PCR amplification reaction. Therefore, a digestion reaction procedure is set up, and the PCR amplification products are subjected to SAP reaction according to the digestion reaction procedure. The residual PCR amplification primers and dNTPs of the PCR amplification products in the SAP reaction are detected to see if they are all digested. If they are all digested, the amplification digestion product is obtained. In this embodiment, the digestion reaction procedure is to control the temperature at the fifth temperature for 40 minutes of SAP reaction, and then control the temperature at the sixth temperature for 5 minutes of SAP reaction. Correspondingly, in this embodiment, the temperature range of the fifth temperature is between 35℃ and 38℃. The temperature range of the sixth temperature is between 83℃ and 86℃, and is set according to specific circumstances. An extension reaction process is established, and based on the extension reaction process and the extension primers purified by HPLC, the amplification digestion product is extended to obtain the sample to be analyzed. In this embodiment, the extension reaction process includes: controlling the temperature at the first temperature for 30 seconds; performing 40 first-cycle reactions; controlling the temperature at the second temperature for 5 minutes, and then maintaining the temperature at the third temperature; the first-cycle reaction is: controlling the temperature at the first temperature for 5 seconds, followed by 5 second-cycle reactions; the second-cycle reaction is: controlling the temperature at the seventh temperature for 5 seconds, and then controlling the temperature at the eighth temperature for 5 seconds; in this embodiment, the temperature range of the seventh temperature is between 50℃ and 53℃, and is set according to specific circumstances; the temperature range of the eighth temperature is between 78℃ and 81℃, and is set according to specific circumstances; the final sample to be analyzed needs to be subjected to mass spectrometry excitation analysis.

[0086] S300, Data Analysis and Interpretation Steps, specifically include:

[0087] S310. Set the interpretation interval, configure the spotting instrument and the nucleic acid mass spectrometer, and perform data analysis and interpretation operations based on the sample to be analyzed, the interpretation interval, the spotting instrument and the nucleic acid mass spectrometer to obtain the gene amplification interpretation results;

[0088] Specifically, the interpretation intervals include: the target gene copy number interval, the proprietary internal reference gene copy number interval, and the ploidy copy number interval; in this embodiment, the target gene copy number is Tpa-ICpa = target gene peak area / proprietary reference gene; the proprietary internal reference gene copy number is ICpa-NCpa = proprietary reference gene / universal reference gene; the ploidy copy number is Tpa-NCpa = target gene peak area / universal reference gene; in this embodiment, the interpretation intervals are set based on the wild-type sample analysis module, gene amplification standards, and ploidy repeat standards; in this embodiment, the target gene copy number intervals include: a first target gene interpretation interval and a second target gene interpretation interval; the first target gene interpretation interval is 1.8. The second target gene interpretation interval is ≥2.0; the proprietary internal reference gene copy number interval includes: a first proprietary internal reference interpretation interval and a second proprietary internal reference interpretation interval; the first proprietary internal reference interpretation interval is 0.8~1.5, and the second proprietary internal reference interpretation interval is ≥1.5; the ploidy copy number interval includes: a first ploidy interpretation interval and a second ploidy interpretation interval; the first ploidy interpretation interval is 1.1~1.8, and the second ploidy interpretation interval is ≥1.8; correspondingly, the gene amplification interpretation results include: a first interpretation result, a second interpretation result, and a third interpretation result; the first interpretation result includes: normal copy number, no gene amplification, and normal ploidy; the second interpretation result includes: gene amplification; the third interpretation result includes: ploidy duplication;

[0089] Specifically, the data analysis and interpretation operation includes: spotting the sample to be analyzed onto the spotting chip of the spotting instrument; controlling the nucleic acid mass spectrometer to excite the sample to be analyzed on the spotting chip with a high-energy laser to obtain the gene data to be analyzed; in this embodiment, the gene data to be analyzed includes: MET gene data and ERBB2 gene data, and the specific information of MET gene data and ERBB2 gene data are respectively provided in [the relevant documentation]. Figure 6 MET gene data table and Figure 7 The ERBB2 gene data table; based on the target gene copy number, proprietary internal reference gene copy number, and ploidy copy number at the end of the table, the gene amplification results can be interpreted; therefore, based on the interpretation interval, the gene data to be analyzed is interpreted to obtain the gene amplification interpretation results;

[0090] Specifically, the result interpretation step includes: identifying the copy number of the first target gene, the copy number of the first proprietary internal reference gene, and the copy number of the first ploidy gene in the gene data to be analyzed; if the copy number of the first target gene is within the first target gene interpretation interval, the copy number of the first proprietary internal reference gene is within the first proprietary internal reference interpretation interval, and the copy number of the first ploidy gene is within the first ploidy interpretation interval, then the gene amplification interpretation result is set as the first interpretation result; if the copy number of the first target gene is within the second target gene interpretation interval, and the copy number of the first ploidy gene is within the second ploidy interpretation interval, then the gene amplification interpretation result is set as the second interpretation result; if the copy number of the first target gene is within the first target gene interpretation interval, and the copy number of the first ploidy gene is within the second ploidy interpretation interval, then the gene amplification interpretation result is set as the third interpretation result; in this embodiment, interpretation criteria are set according to the interpretation interval and the gene amplification interpretation result, see details below. Figure 8 The interpretation criteria information table; finally, this embodiment also plotted mass spectrometry peak diagrams based on MET gene data and ERBB2 gene data, as detailed in the table below. Figure 9 MET mass spectrum peaks and Figure 10 The ERBB2 mass spectrometry peak diagram is shown. In this embodiment, the horizontal axis of the mass spectrometry peak diagram is Mass, and the vertical axis is Intensity. In this embodiment, comprehensive data analysis is performed by combining the interpretation standard information table, the MET gene data table, and the ERBB2 gene data table. The obtained interpretation results have a 100% consistency rate with the detection results of the NGS platform and the FISH platform, with extremely high accuracy, which makes up for the shortcomings of the existing technology. At the same time, the gene amplification detection method based on nucleic acid mass spectrometry technology described in this embodiment can also be used for subsequent reagent kit development and methodological expansion, etc., and has extremely high experimental value.

[0091] Unlike existing technologies, the gene amplification detection method based on nucleic acid mass spectrometry proposed in this application can achieve higher detection accuracy than existing technologies such as fluorescence in situ hybridization and immunohistochemistry, higher detection throughput than quantitative PCR and digital PCR, and lower detection cost than quantitative PCR and digital PCR. At the same time, it has a shorter detection time compared with next-generation sequencing (NGS) technology. The operation procedure of this application is simple, the detection results are objective, and the comprehensive data of accuracy, detection throughput and detection cost are superior to existing technologies, which has extremely high experimental value.

[0092] The embodiment numbers disclosed in the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0093] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A gene amplification detection method based on nucleic acid mass spectrometry technology, characterized in that, The method described is not intended for the diagnosis or treatment of disease, and includes the following steps: Experimental primer preparation steps: Set the target gene region, configure the reference gene, and confirm the experimental primers based on the target gene region and the reference gene; The reference genes include: the MET proprietary reference gene, the MET gene, the ERBB2 proprietary reference gene, the ERBB2 gene, the internal reference gene, and the universal reference gene; the experimental primers include: PCR amplification primers and extension primers; The MET-specific reference genes are NDUFA4, TRA2A, and ACTB; the ERBB2-specific reference genes are ANKFY1 and PSMB6; and the universal reference genes are GAPDH and RPP30. Primer reaction processing steps: Obtain experimental samples, and perform primer reaction processing based on the experimental primers and the experimental samples to obtain samples to be analyzed; Data analysis and interpretation steps: Set the interpretation interval, configure the spotting instrument and the nucleic acid mass spectrometer, and perform data analysis and interpretation operations based on the sample to be analyzed, the interpretation interval, the spotting instrument and the nucleic acid mass spectrometer to obtain the gene amplification interpretation results; The interpretation intervals include: the target gene copy number interval, the proprietary internal reference gene copy number interval, and the ploidy copy number interval; The target gene copy number interval includes: a first target gene interpretation interval and a second target gene interpretation interval; the proprietary internal reference gene copy number interval includes: a first proprietary internal reference interpretation interval and a second proprietary internal reference interpretation interval; the ploidy copy number interval includes: a first ploidy interpretation interval and a second ploidy interpretation interval; The first target gene interpretation interval is 1.8~2.0, and the second target gene interpretation interval is ≥2.0; the first proprietary internal reference interpretation interval is 0.8~1.5, and the second proprietary internal reference interpretation interval is ≥1.5; the first ploidy interpretation interval is 1.1~1.8, and the second ploidy interpretation interval is ≥1.8; The gene amplification interpretation results include: a first interpretation result, a second interpretation result, and a third interpretation result; the first interpretation result is: normal copy number, no gene amplification, and normal ploidy; the second interpretation result is: gene amplification; the third interpretation result is: ploidy duplication; The data analysis and interpretation operation includes: spotting the sample to be analyzed onto the spotting chip of the spotting instrument; controlling the nucleic acid mass spectrometer to excite the sample to be analyzed on the spotting chip with high-energy laser to obtain the gene data to be analyzed; and performing a result interpretation step on the gene data to be analyzed based on the interpretation interval to obtain the gene amplification interpretation result. The result interpretation step includes: identifying the copy number of the first target gene, the copy number of the first proprietary internal reference gene, and the copy number of the first ploidy gene in the gene data to be analyzed; if the copy number of the first target gene is within the first target gene interpretation interval, the copy number of the first proprietary internal reference gene is within the first proprietary internal reference interpretation interval, and the copy number of the first ploidy gene is within the first ploidy interpretation interval, then the gene amplification interpretation result is set as the first interpretation result; if the copy number of the first target gene is within the second target gene interpretation interval, and the copy number of the first ploidy gene is within the second ploidy interpretation interval, then the gene amplification interpretation result is set as the second interpretation result; if the copy number of the first target gene is within the first target gene interpretation interval, and the copy number of the first ploidy gene is within the second ploidy interpretation interval, then the gene amplification interpretation result is set as the third interpretation result.

2. The gene amplification detection method based on nucleic acid mass spectrometry according to claim 1, characterized in that, The step of confirming the experimental primers based on the target gene region and the reference gene further includes: Based on the target gene region, primer information for several regions matching the MET gene, the ERBB2 gene, and the internal reference gene was identified; primers for amplifying the target gene and primers for amplifying the internal reference gene were configured based on the primer information for the several regions; both primers for amplifying the target gene and primers for amplifying the internal reference gene were the PCR amplification primers. Based on the target gene region, several first gene regions were identified that matched the MET-specific reference gene, the MET gene, the ERBB2-specific reference gene, the ERBB2 gene, and the universal reference gene, respectively. Based on the several first gene regions, several first extension gene primers were screened from the MET-specific reference gene, the MET gene, the ERBB2-specific reference gene, the ERBB2 gene, and the universal reference gene. All of the several first extension gene primers were extension primers.

3. The gene amplification detection method based on nucleic acid mass spectrometry according to claim 2, characterized in that, The step of obtaining experimental samples further includes: Confirm the experimental technology platform information, confirm the sample characteristic information based on the experimental technology platform information, and obtain a tumor tissue sample based on the sample characteristic information; the tumor tissue sample is the experimental sample.

4. The gene amplification detection method based on nucleic acid mass spectrometry according to claim 3, characterized in that, The primer reaction processing operation includes: Set the first primer quality information; perform HPLC purification on the PCR amplification primers and the extension primers; perform mass spectrometry quality control on the HPLC-purified PCR amplification primers and the extension primers to obtain the second primer quality information; determine whether the second primer quality information matches the first primer quality information, and if they match, execute the amplification reaction step.

5. The gene amplification detection method based on nucleic acid mass spectrometry according to claim 4, characterized in that, The amplification reaction steps include: The PCR reaction procedure is set up by mixing the target gene amplification primers and the internal reference gene amplification primers after HPLC purification to form the PCR working solution. The DNA of the experimental sample was amplified based on the PCR reaction procedure and the PCR working solution to obtain PCR amplification products. A digestion reaction procedure is set up, and the PCR amplification product is subjected to an SAP reaction according to the digestion reaction procedure. The remaining PCR amplification primers and dNTPs of the PCR amplification product in the SAP reaction are detected to see if they are all digested. If they are all digested, the amplification digestion product is obtained. An extension reaction procedure is set up, and the amplification digestion product is extended based on the extension reaction procedure and the extension primers purified by HPLC to obtain the sample to be analyzed.

6. The gene amplification detection method based on nucleic acid mass spectrometry according to claim 1, characterized in that, The gene data to be analyzed includes: MET gene data and ERBB2 gene data.

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