Detection of small amounts of target DNA in the presence of large amounts of interfering DNA and applications thereof

Abstract

The present application relates to the detection of a small amount of target DNA in the presence of a large amount of interfering DNA and its application. Specifically, the present application provides a method for detecting whether a gene has a mutation, comprising: providing an amplification product containing an amplification product of a target nucleic acid sequence and an amplification product of a background nucleic acid sequence; ionizing the amplification product and enriching the amplification product of the target nucleic acid sequence by using gas phase ion online separation technology; fragmenting the enriched amplification product and performing mass spectrometry analysis on the obtained fragment ions to record the fragment ion spectrum; comparing the fragment ion spectrum obtained in step (3) with the ion spectrum of the background nucleic acid sequence obtained by the same step; obtaining the part with m / z value ≥600 in the comparison spectrum, and calculating the P value of the three peaks ranked first in the difference peak intensity in this part; wherein, P value ≤0.05 indicates that the target nucleic acid sequence has a mutation, and P value >0.05 indicates that the target nucleic acid sequence does not have a mutation.

Description

Technical Field

[0001] This invention relates to the detection of small amounts of target DNA in the presence of a large amount of interfering DNA and its application. Background Technology

[0002] Currently, the main methods for detecting gene mutations include sequencing, digital droplet PCR, and DNA probe hybridization.

[0003] Direct sequencing is the most classic and widely applicable method for gene mutation detection. Sequencing directly provides the nucleotide sequence of the target DNA molecule, thus it is considered the gold standard for gene mutation detection. While Sanger sequencing enables low-cost and rapid detection of single samples, its sensitivity is limited, with a detection limit of 5-10% for mutated gene abundance, making it unsuitable for direct detection of ctDNA. Next-generation sequencing (NGS) can be performed in large-scale parallel processing, enabling sequencing while synthesizing; at conventional sequencing depths, NGS can provide effective data on mutation abundance ranging from 0.1% to above 0.5%. Newman et al. developed a tumor-specific deep sequencing analysis method. The core idea of ​​this method is to narrow the detection range of NGS. Through analysis of large population samples, a gene library representing approximately 0.04% of the target range is selected. High-depth NGS sequencing is then performed only on this gene library, achieving a detection limit of 0.02% and simultaneously detecting hundreds of gene mutations. However, this method still cannot meet the requirements for detecting early-stage cancer samples, and to achieve a detection limit of 0.02%, the sequencing depth needs to be more than 10,000 times.

[0004] Digital droplet PCR, building upon traditional PCR techniques, utilizes chips or droplets to achieve independent sample dispensing and PCR on individual droplets. The limit of detection (LOD) can reach 0.005-0.05%, making it the most sensitive method for detecting ctDNA to date. However, achieving this LOD requires extremely high precision in ultradispersing the original PCR system to microdroplets. To ensure accuracy, the LOD of this method is stably controlled between 0.01-0.1%, and in actual clinical use, it is controlled between 0.05-0.1%.

[0005] Fluorescent probe methods utilize artificially designed and synthesized single-stranded DNA probes labeled with fluorescent and quenching groups, respectively. These probes specifically recognize and bind to the target mutant strand in the system, emitting a fluorescent signal to detect the abundance of the mutant gene in the sample. Under typical assay conditions, the difference between the wild-type and mutant strands in the sample is only one base. The DNA probe's ability to distinguish this single base is limited, with a detection limit of approximately 10% in practical applications.

[0006] Because wild-type DNA lacks mutations, does not carry information related to key pathogenic genes, and cannot provide crucial information for early tumor screening or postoperative recurrence monitoring, it constitutes the vast majority of the detection system, causing significant background interference in the detection of mutant DNA in samples. Therefore, how to accurately detect small amounts of mutant DNA in the presence of a large amount of interfering DNA has always been a pressing problem in this field. Summary of the Invention

[0007] This invention aims to provide a method, composition, kit, and related apparatus and equipment for accurately detecting small amounts of target DNA even when a large amount of interfering DNA is present in the sample. This invention solves the problems of low throughput, long cycle time, high cost, and inaccuracy of existing detection methods, simplifies the detection method, reduces detection costs, and improves detection efficiency and accuracy. In particular, the method, composition, kit, and related apparatus and equipment of this invention can be used for screening for birth defects in early pregnancy (5-7 weeks of gestation).

[0008] Specifically, the present invention provides a method for detecting whether a gene has a mutation, the method comprising:

[0009] (1) Provide an amplification product containing an amplification product of a target nucleic acid sequence and an amplification product of a background nucleic acid sequence;

[0010] (2) Ionize the amplification product and enrich the amplification product of the target nucleic acid sequence using gas phase ionization online separation technology;

[0011] (3) Fragment the enriched amplified product and perform mass spectrometry analysis on the obtained fragment ions to record the fragment ion spectrum.

[0012] (4) Compare the fragment ion spectrum obtained in step (3) with the ion spectrum of the background nucleic acid sequence obtained in the same step;

[0013] (5) Obtain the portion of the comparison spectrum with an m / z value ≥600, preferably ≥700, calculate the P value of the three peaks with the highest difference in intensity in this portion, and determine whether the target nucleic acid sequence has a mutation relative to the background nucleic acid sequence based on the P value;

[0014] Wherein, a P value ≤ 0.05 indicates that the target nucleic acid sequence has a mutation, and a P value > 0.05 indicates that the target nucleic acid sequence does not have a mutation.

[0015] In one or more embodiments, the length of the amplified product does not exceed 80 bp.

[0016] In one or more embodiments, the amplification product is an amplification product of cfDNA.

[0017] In one or more embodiments, the cfDNA is derived from blood.

[0018] In one or more embodiments, the cfDNA is derived from the blood of a pregnant woman.

[0019] In one or more embodiments, the target nucleic acid sequence is the nucleic acid sequence of the offspring, and the background nucleic acid sequence is the nucleic acid sequence of the parent.

[0020] In one or more embodiments, the target nucleic acid sequence is the cfDNA sequence of the offspring, and the background nucleic acid sequence is the cfDNA sequence of the parent.

[0021] In one or more embodiments, the amplified product is obtained by nested polymerase chain reaction amplification.

[0022] In one or more embodiments, the amplification product is purified and desalted.

[0023] In one or more implementation schemes, primers are designed for polynucleotide polymorphism sites in the target nucleic acid sequence to perform PCR amplification.

[0024] A second aspect of the present invention provides the application of nucleic acid amplification reagents or detection kits containing such nucleic acid amplification reagents in detecting whether a gene has a mutation, or the application of nucleic acid amplification reagents in preparing detection kits for detecting whether a gene has a mutation; the detection is performed using the method for detecting whether a gene has a mutation as described in any embodiment of the present invention.

[0025] In one or more embodiments, the nucleic acid amplification reagent includes reagents required for performing PCR.

[0026] In one or more embodiments, the PCR is a nested polymerase chain reaction amplification reaction.

[0027] In one or more embodiments, the detection kit further includes one or more of the following: a sample acquisition device, reagents for isolating nucleic acids from a sample, reagents for purifying PCR amplification products, and reagents required for performing mass spectrometry.

[0028] A third aspect of the present invention provides an apparatus for detecting and analyzing gene mutations, the apparatus comprising a memory, a processor, and a computer program stored in the memory and executable by the processor, wherein the processor, when executing the computer program, performs the following: (1) acquiring a background nucleic acid sequence mass spectrometry spectrum, a sample nucleic acid sequence mass spectrometry spectrum, and an optional positive reference mass spectrometry spectrum; (2) comparing the background nucleic acid sequence mass spectrometry spectrum and the sample nucleic acid sequence mass spectrometry spectrum to obtain a portion with an m / z value ≥600, preferably ≥700, and calculating the P-values ​​of the three most intense difference peaks in that portion; (3) providing a conclusion on whether a gene mutation exists based on the P-values.

[0029] In one or more embodiments, the apparatus is used to implement the method for detecting the presence of mutations in a gene as described in any embodiment of this document. Attached Figure Description

[0030] Figure 1 : String mass spectra of 50NT oligonucleotide sequences.

[0031] Figure 2 Mass spectra of the amplified products tested with relative contents of 10%, 2%, 0.5%, 0.2%, 0.1%, 0.01%, and 0% against a background.

[0032] Figure 3 Significant differences in mass spectrometry detection results of amplified products with relative contents of 10%, 2%, 0.5%, 0.2%, 0.1%, 0.01%, and 0% against a background.

[0033] Figure 4 Mass spectrometry results of a single mutant cell in the background.

[0034] Figure 5 : A graph showing the significant differences in mass spectrometry results of a single mutant cell against the background.

[0035] Figure 6 Mass spectrometry results of non-pathogenic mutations detected during clinical gestation at 5-7 weeks.

[0036] Figure 7 : A graph showing the significant differences in mass spectrometry detection results of non-pathogenic mutations during clinical gestation at 5-7 weeks of gestation.

[0037] Figure 8 : PKU non-invasive screening results at 12-16 weeks of pregnancy.

[0038] Figure 9 : A graph showing significant differences in PKU non-invasive screening between 12 and 16 weeks of gestation. Detailed Implementation

[0039] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form preferred technical solutions.

[0040] This invention employs gas-phase enrichment integration tandem mass spectrometry, which can eliminate interference from a large amount of background DNA and accurately detect extremely small amounts of DNA mutations. In particular, the detection technology of this invention can perform highly reliable and accurate single-gene mutation detection in fetuses at 5-7 weeks of gestation. The method of this invention avoids the use of expensive fluorescent reagents and does not require extremely high sequencing depth coverage, thus reducing costs by tens of times. Furthermore, unlike conventional sequencing methods that require the accumulation of many samples to reduce costs, the method of this invention can detect one or more samples, thereby significantly reducing sample detection response time. In addition, the method of this invention is compatible with widely used automated pipetting systems for large-scale sample processing, making it suitable for large-scale automated high-throughput sample processing modes.

[0041] sample

[0042] Typically, the nucleic acids used in the methods, compositions, kits, and related apparatus and equipment described herein are isolated from samples obtained from the subject. In this document, the subject includes test subjects and reference subjects. A "test subject" refers to the subject to be evaluated for the presence of genetic variation. A "reference subject" refers to the subject used as the basis for comparison with the test subject. Reference subjects may or may not have the genetic variation of the test subject being evaluated. In this document, the subject generally refers to various organisms, including but not limited to humans, non-human animals, plants, bacteria, fungi, or protozoa. Non-human animals include, but are not limited to, mammals, reptiles, birds, amphibians, fish, ungulates, etc. Non-human animals can also be poultry and livestock, such as chickens, ducks, geese, pigs, cattle, sheep, and horses. Non-human animals can also be pets, such as cats and dogs. Plants can be any plant of interest, including but not limited to crops, landscape plants, and horticultural plants, such as rice, wheat, corn, potatoes, tomatoes, sorghum, soybeans, etc. Landscape or horticultural plants include various flowers. Plants of interest also include plants used for extracting edible oils, such as sesame, peanuts, rapeseed, rice, soybeans, etc.

[0043] Nucleic acids can be isolated from any type of suitable biological specimen or sample. Non-limiting examples of specimens derived from animals include fluids or tissues of the subject, including but not limited to peripheral blood, umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, cerebrospinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneum, ducts, ear, joints), biopsy samples (e.g., from pre-implantation embryos), intermembranous fluid samples, fetal nucleated cells or fetal cell remnants, female reproductive tract cleansing fluid, urine, feces, sputum, saliva, nasal mucosa, prostatic fluid, lavage fluid, semen, lymph, bile, tears, sweat, breast milk, mammary gland fluid, embryonic cells, and fetal cells (e.g., placental cells). Non-limiting examples of specimens derived from plants include seeds, leaves, petals, etc.

[0044] In some embodiments, the biological sample may be blood, including plasma or serum. Plasma refers to the fraction of whole blood obtained by centrifugation of blood treated with an anticoagulant. Serum refers to the watery fraction remaining after the blood sample has clotted. Liquid or tissue samples are typically collected according to standard methods followed in hospitals or clinical practice. In the case of blood, an appropriate amount of peripheral blood (e.g., 3-40 ml) is typically collected and preserved according to standard procedures before further preparation. Liquid or tissue samples used for nucleic acid extraction may be cell-free. In some embodiments, the liquid or tissue sample may contain cellular elements or cellular remnants. In some embodiments, the sample may contain fetal cells or cancer cells.

[0045] The sample can be heterogeneous, meaning that the sample contains more than one type of nucleic acid. For example, heterogeneous nucleic acids include, but are not limited to, (i) fetal and maternal nucleic acids, (ii) cancer and non-cancer nucleic acids, (iii) pathogen and host nucleic acids, and more commonly (iv) mutant and wild-type nucleic acids.

[0046] In a particularly preferred embodiment of the invention, the sample is derived from a pregnant woman, including her tissues or cells and her peripheral blood. In some embodiments, the sample is collected from pregnant women with a fetus at approximately 1 to approximately 45 weeks of gestation (e.g., fetuses at 1-4, 4-8 (e.g., 5-7 weeks), 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, or 40-44 weeks of gestation), more specifically, from pregnant women with a fetus at 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 29 weeks of gestation.

[0047] In some implementations, the sample includes a sample for generating a background signal and a sample for generating a sample signal (i.e., containing the sequence to be tested). Optionally, the sample also includes a sample for generating a positive reference signal. For example, when detecting the presence of mutations in a fetal gene sequence, the sample for generating the background signal could be a sample from the mother containing maternal genetic information, which typically does not contain fetal genetic information; the sample for generating the sample signal could be a sample from the mother containing the fetal sequence to be tested, which typically contains both fetal and current genetic information, such as both fetal cfDNA and maternal cfDNA. The sample for generating the positive reference signal could be a sample from the father. Typically, the sample for generating the sample signal could be maternal blood. The sample for generating the background signal could be from any tissue of the mother, and the sample for generating the positive reference signal could be from any tissue of the father.

[0048] The term "background nucleic acid" as used herein typically refers to nucleic acids in a sample other than the sequence to be tested, which generate "background signals" during the detection process that can interfere with the detection of the sequence to be tested. Preferably, the "background nucleic acid" sequence contains a wild-type sequence of the sequence to be tested, meaning that the wild-type sequence does not contain the mutations found in the sequence to be tested. In some embodiments, the background nucleic acid is maternal nucleic acid and / or paternal nucleic acid.

[0049] Nucleic acid isolation and processing

[0050] Nucleic acids can be obtained from one or more sample sources (such as cells) using methods known in the art. Cell lysis methods and reagents are known in the art and can generally be performed by chemical, physical, or electrolytic lysis methods. For example, chemical methods typically use a lysis agent to disrupt cells and extract nucleic acids from them, followed by treatment with a dissociative salt. Physical methods can also be used, such as grinding after freeze-thaw cycles, or using a cell crusher. High-salt lysis is also commonly used. For example, alkaline lysis can be used. The latter method conventionally involves the use of a phenol-chloroform solution, or alternatively, a phenol-chloroform-free method comprising three solutions. In the latter method, one solution may contain 15 mM Tris, pH 8.0; 10 mM EDTA and 100 μg / mL RNase A; the second solution may contain 0.2 N NaOH and 1% SDS; and the third solution may contain 3 M KOAc, pH 5.5. These methods can be found in sections 6.3.1–6.3.6 (1989) of *Current Protocols in Molecular Biology*, published by John Wiley & Sons, Inc., New York, which is included in full here. Commercially available reagents such as DNAzol can be used. TM To lyse the cells.

[0051] In this document, "nucleic acid" and "nucleic acid molecule" are used interchangeably to refer to nucleic acids in any composite form, such as: deoxyribonucleic acid (DNA, e.g., cDNA and genomic DNA), ribonucleic acid (RNA, e.g., mRNA, siRNA, rRNA), tRNA, microRNA, RNA highly expressed in the fetus or placenta, etc.) and / or DNA or RNA analogs (e.g., base analogs, sugar analogs, and / or non-natural backbones), RNA / DNA hybrids, and polyamide nucleic acids (PNA), all of which can be in single-stranded or double-stranded form. Typically, nucleic acids can encompass known analogs of natural nucleotides, some of which function in a manner similar to naturally occurring nucleotides. Nucleic acids can be in any form suitable for performing the methods described herein (e.g., linear, circular, supercoiled, single-stranded, double-stranded, etc.). In some embodiments, nucleic acids can be or may be derived from plasmids, bacteriophages, autonomously replicating sequences (ARS), centromeres, artificial chromosomes, chromosomes, or other nucleic acids capable of replicating or being replicated in vitro or in host cells, cells, the nucleus of cells, or the cytoplasm of cells. In some implementations, nucleic acids may be derived from a single chromosome (e.g., a nucleic acid sample may be derived from a chromosome of a sample obtained from a diploid organism). Nucleic acids may also include RNA or DNA derivatives, variants, and analogs synthesized, replicated, or amplified from single-stranded and double-stranded polynucleotides. Nucleic acids may be prepared using nucleic acids obtained from the subject as templates.

[0052] In some embodiments, nucleic acids include extracellular nucleic acids. "Extracellular nucleic acids" refers to nucleic acids isolated from essentially cell-free sources. Extracellular nucleic acids often contain undetectable cells and may contain cellular elements or cellular remnants. Non-limiting examples of cell-free sources of extracellular nucleic acids are plasma, serum, and urine. Without being theoretically limited, extracellular nucleic acids can be products of apoptosis and cell lysis, which often results in a wide range of serial lengths.

[0053] Extracellular nucleic acids can contain different types of nucleic acids. For example, the blood serum or plasma of a person with cancer may contain nucleic acids from cancer cells and nucleic acids from non-cancer cells, and the blood serum or plasma of a pregnant woman may contain maternal nucleic acids and fetal nucleic acids. Typically, fetal nucleic acids account for less than 50% of all nucleic acids, for example, 5-10%. Typically, the majority of fetal nucleic acids are less than 500 base pairs in length, more typically less than 250 base pairs, and even more typically less than 200 base pairs. In some embodiments, the majority of fetal nucleic acids are less than about 150 base pairs in length. In some embodiments, the majority of fetal nucleic acids are less than about 100 base pairs in length. The term "majority" typically means more than 80%, more than 85%, more than 90%, more than 95%, or 100%.

[0054] Nucleic acids can typically be extracted, isolated, purified, or amplified from a sample. As used herein, “isolation” refers to removing nucleic acids from their original environment (e.g., the natural environment in which nucleic acids occur or the host cell from which exogenous nucleic acids are expressed). Isolated nucleic acids contain fewer (e.g., less than 10%, less than 5%) non-nucleic acid components (e.g., proteins, lipids) compared to the component content present in the source sample. As used herein, “purification” refers to providing nucleic acids containing fewer types of nucleic acids compared to the sample from which they are derived. As used herein, “amplification” refers to processing nucleic acids in a sample to produce amplicon nucleic acids in a linear or exponential manner, the nucleotide sequence of which is identical or substantially identical to the nucleotide sequence of the nucleic acids in the sample or a portion thereof.

[0055] In some embodiments, the nucleic acid may be processed to produce nucleic acid fragments prior to being provided for use in the methods described herein. In some embodiments, the fragmented or cleaved nucleic acid may have 5-10,000 base pairs, 100-1,000 base pairs, 100-500 base pairs, or 50-180 base pairs. Fragments can be produced by any suitable method known in the art, and the average, geometric mean, or nominal length of the nucleic acid fragments can be controlled by selecting an appropriate fragmentation method.

[0056] Nucleic acids in a sample can be fragmented using various methods known in the art, including but not limited to physical, chemical, and enzymatic methods. Non-limiting examples of such methods are described in US 2005 / 0112590, the entire contents of which are incorporated herein by reference.

[0057] Amplification methods

[0058] This article describes the amplification of one or more nucleic acid molecules using appropriate amplification methods. Any suitable amplification technique can be used.

[0059] The term "amplification" refers to any in vitro method used to double copies of a nucleic acid target sequence. In some implementations, a limited amplification reaction, also known as pre-amplification, may be performed. Pre-amplification is a method where a limited amount of amplification occurs due to a small number of cycles (e.g., 10 cycles). Pre-amplification allows for some amplification but stops amplification before the exponential phase and typically produces about 500 copies of the desired one or more nucleotide sequences. Using pre-amplification also limits inaccuracies associated with reactant depletion in standard PCR reactions and reduces amplification bias caused by the nucleotide sequence or species abundance of the target.

[0060] Examples of polynucleotide amplification include, but are not limited to: polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR), amplification methods based on the application of Q-β replicase or template-dependent polymerase, helicase-dependent isothermal amplification, strand displacement amplification (SDA), amplification based on thermophilic SDA nucleic acid sequences (3SR or NASBA), and transcription-associated amplification (TAA). Non-limiting examples of PCR amplification methods include standard PCR, AFLP-PCR, allele-specific PCR, Alu-PCR, asymmetric PCR, colony PCR, heat-initiated PCR, inverse PCR (IPCR), in situ PCR (ISH), sequence-specific PCR (ISSR-PCR), long PCR, multiplex PCR, nested PCR, quantitative PCR, reverse transcriptase PCR (RT-PCR), real-time PCR, single-cell PCR, solid-phase PCR, and combinations thereof. Reagents and hardware for performing PCR are commercially available.

[0061] Typically, primers are contacted with the target nucleic acid, and then complementary sequences are annealed to each other. Primers may anneal to the target nucleic acid at or near the sequence of interest (e.g., adjacent, neighboring, etc.). A reaction mixture containing components necessary for enzyme function is added to the primer-target nucleic acid mixture, and amplification can then occur under appropriate conditions. Components of the amplification reaction may include, but are not limited to, primers (e.g., single primers, primer pairs, primer sets, etc.), polynucleotide templates (e.g., target nucleic acids), polymerases, dNTPs, buffers, etc. For example, in some embodiments, non-naturally occurring nucleotides or nucleotide analogs may be used, such as analogs containing detectable markers (e.g., fluorescent or colorimetric markers). Suitable polymerases may be selected, including polymerases for thermal cycling amplification, such as Taq DNA polymerase, Q-Bio™ Taq DNA polymerase (a recombinant truncated form of Taq DNA polymerase lacking 5'-3' exonuclease activity). For example, other enzyme components such as reverse transcriptase for transcription-mediated amplification (TMA) reactions may be added.

[0062] Typically, primers hybridize with about 10-30 nucleotides of the nucleic acid sequence of interest to generate an amplification product.

[0063] In some embodiments, the amplified nucleic acids each independently have a length of about 10 to about 500 base pairs. In some embodiments, the length of the amplified nucleic acids is about 20 to about 250 base pairs, such as 50 to 200 base pairs, 100 to 180 base pairs, etc.

[0064] The amplification products often have nucleotide sequences that are identical or substantially identical to the nucleotide sequence of the sample nucleic acid or its complement. The "substantially identical" nucleotide sequence in the amplification products usually has a high sequence identity with the amplified nucleic acid or its complement (e.g., sequence identity greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99%). Variations are sometimes caused by polymerase distortion during extension and / or amplification, or by the addition of additional nucleotide sequences to the primers used for amplification.

[0065] PCR conditions can vary depending on primer sequences, target abundance, and the desired amplification amount, thus those skilled in the art can choose from a variety of available PCR protocols (see, for example, US 4,683,195 and US 4,683,202). PCR is typically performed automatically using thermostable enzymes. In this process, the temperature of the reaction mixture is automatically cyclicated between denaturation, primer annealing, and extension reaction zones. Machines specifically suited for this purpose are commercially available. A non-limiting example of a PCR protocol suitable for the embodiments described herein is that the sample is treated at 95°C for 3 minutes, repeated for no more than 30 cycles: 95°C for 15 seconds, 60°C for 1 minute, and 72°C for 30 seconds; then the sample is treated at 72°C for 2 minutes. Multiple cycles are typically performed using a commercially available thermal cycler. In some embodiments, a known and selected suitable isothermal amplification method may also be applied.

[0066] In some embodiments, multiplex amplification methods can be used to amplify target nucleic acids, thereby simultaneously amplifying multiple amplicones in a single homogeneous reaction. As used herein, "multiplex amplification" refers to a variant of PCR that allows for the simultaneous amplification of multiple targets of interest within a single reaction vessel using more than one pair of primers (e.g., more than one set of primers). In some embodiments, multiplex amplification can be used to analyze deletions, mutations, and polymorphisms, or for quantitative assays. In some embodiments, multiplex amplification can be used for paralogous sequence imbalance detection, genotyping applications requiring simultaneous analysis of multiple biomarkers, detection of pathogens or genetically modified organisms, or for microsatellite analysis. In some embodiments, multiplex amplification can be coupled with another amplification method (e.g., PCR) (e.g., nested PCR or hot-start PCR) to improve amplification specificity and reproducibility. In other embodiments, multiplex amplification can be performed during replication to reduce the variation introduced by the amplification.

[0067] Amplification can be performed on a solid support. In some embodiments, primers can bind to the solid support. In some embodiments, target nucleic acids (such as nucleic acids) can bind to the solid support. Nucleic acids (primers or targets) bound to a solid support are generally referred to as solid-phase nucleic acids.

[0068] In some embodiments, nucleic acid molecules for amplification are provided in a “microreactor.” As used herein, a “microreactor” refers to a spaced space that enables nucleic acid molecules to hybridize with a solid support. Examples of microreactors include, but are not limited to, emulsion spheres and cavities in a matrix. In some embodiments, cavities in the matrix can be pits, holes, or pores (e.g., micropores, nanopores, picometer pores, microholes, or nanoholes) in a matrix constructed from a solid material suitable for containing liquids (e.g., plastics such as polypropylene, polyethylene, polystyrene, or silicon). Emulsion spheres are separated by an immiscible phase. The sphere shape can be spheres, near-spheres, or hemispheres.

[0069] Primers

[0070] This invention provides primers for amplifying nucleic acids. As used herein, the term "primer" refers to a nucleic acid whose nucleotide sequence hybridizes or anneals to a target nucleic acid at or near a specific region of interest (e.g., adjacent). "Specific" refers to the recognition, contact, and formation of a stable complex between two molecules, compared to a significantly lower likelihood of recognition, contact, or complex formation between either molecule and other molecules. The term "annealing" as used herein refers to the formation of a stable complex between two molecules. When referring to primers, the terms "primer," "oligonucleotide," or "oligonucleotide" may be used interchangeably throughout the text.

[0071] In embodiments of the present invention, primer sets comprising at least one primer pair may be used. In some embodiments, the primer set may comprise a third or fourth nucleic acid (e.g., two primer pairs, or, for example, a nested primer set). In some embodiments, the primer set may contain multiple primer pairs. In some embodiments, multiple primer sets may be used, each containing multiple primer pairs. In a particularly preferred embodiment of the present invention, the primer set used for nucleic acid amplification of the present invention includes primer pairs for amplifying nucleic acid sequences from the mother and primer pairs for amplifying nucleic acid sequences from the fetus; preferably. The primer pairs for amplifying nucleic acid sequences from the mother are the same as the primer pairs for amplifying nucleic acid sequences from the fetus. In some embodiments, the primers of the present invention further include primer pairs for amplifying nucleic acid sequences from the father. Preferably, the primer pairs for amplifying nucleic acid sequences from the mother, the primer pairs for amplifying nucleic acid sequences from the fetus, and the primer pairs for amplifying nucleic acid sequences from the father are the same.

[0072] Primers can be designed and synthesized using suitable methods and can have any length suitable for hybridizing with the nucleotide sequence of interest (e.g., nucleic acid in liquid phase or bound to a solid support) and performing the analytical methods described herein. Primers can be designed based on the target nucleotide sequence. In some embodiments, primers can be 10-100 nucleotides, about 10-70 nucleotides, 10-50 nucleotides, or 15-30 nucleotides long. Primers can consist of naturally occurring and / or non-naturally occurring nucleotides (e.g., labeled nucleotides) or mixtures thereof. Primers suitable for the embodiments described herein can be synthesized and labeled using known techniques. Oligonucleotides (e.g., primers) can be chemically synthesized by solid-phase phosphoramidite trimerization. Purification of oligonucleotides can be achieved by natural acrylamide gel electrophoresis or by anion-exchange high-performance liquid chromatography (HPLC).

[0073] In some implementations, all or part of the primer's nucleic acid sequence is substantially complementary to the target nucleic acid. "Substantially complementary" as used herein refers to nucleotide sequences capable of hybridization. The stringency of hybridization conditions can be varied to allow for different amounts of sequence mismatches.

[0074] Primers that are substantially complementary to the target nucleic acid sequence are also substantially identical to the complement of the target nucleic acid sequence. That is, the primers are substantially identical to the antisense strand of the nucleic acid. "Substantially identical" as used herein refers to a similarity of nucleotide sequences to each other of 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. One test for determining whether two nucleotide sequences are substantially identical is to determine the percentage of identical nucleotide sequences they share.

[0075] Primer sequences and lengths can affect hybridization with target nucleic acid sequences. Depending on the degree of mismatch between the primers and target nucleic acids, low, medium, and high stringency conditions can be used to achieve primer / target annealing. As used herein, the term "stringency conditions" refers to the conditions for hybridization and washing. Methods for optimizing hybridization reaction temperature conditions are known to those skilled in the art and can be found in sections 6.3.1–6.3.6 of John Wiley & Sons' *New Laboratory Manual for Molecular Biology* (New York), 1989. A non-limiting example of stringent hybridization conditions is hybridization at approximately 45°C in 6X sodium chloride / sodium citrate (SSC), followed by one or more washes at 50°C with 0.2X SSC and 0.1% SDS. Another example of stringent hybridization conditions is hybridization at approximately 45°C in 6X sodium chloride / sodium citrate (SSC), followed by one or more washes at 55°C with 0.2X SSC and 0.1% SDS. Another example of stringent hybridization conditions is hybridization in 6X sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by washing once or multiple times at 60°C with 0.2X SSC and 0.1% SDS.

[0076] In this paper, primers can be designed according to different purposes. For example, to detect whether a mutation exists at a known site, primers can be designed targeting that site to amplify nucleotide fragments containing that site, and then the mass spectrometry analysis described in this paper can be performed. Alternatively, for unknown mutations, multiple primer pairs covering the entire gene can be designed to perform PCR, amplify different fragments of the gene, and then the mass spectrometry analysis described in this paper can be performed.

[0077] In some embodiments, the primers of this invention are designed such that the length of the amplification product is less than 80 bp.

[0078] Purification and desalting

[0079] Before mass spectrometry, the amplified products need to be purified and desalted to eliminate the influence of contained ions on the mass spectrometry results. Purification and desalting can be performed using methods well-known in the art. For example, purification can be performed using an HLB packing material and a solid-phase extraction column, including sequential treatment with reagents such as NH4OAc, MeOH, and ammonia. Exemplary purification and desalting processes can be found in the "Purification and Desalting" section of the experimental part of this application.

[0080] After purification and desalting, the resulting product is usually dried for later use.

[0081] Mass spectrometry and data processing

[0082] The PCR amplification products of this invention can be detected using tandem mass spectrometry. Suitable tandem mass spectrometry methods include electrospray quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS), electrospray ion-hydrazine mass spectrometry (ESI-IT-MS), and Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS).

[0083] Typically, samples need to be redissolved. For example, samples can be redissolved using an aqueous solution containing ACN (acetonitrile), TEA (triethanolamine), and imidazole. The concentration of the ACN aqueous solution can be 40-60%, the concentration of TEA can be 0.5-2%, and the concentration of imidazole can be 0.5-2%. In an exemplary embodiment, a dried sample is dissolved using a 50% ACN aqueous solution + 1% TEA + 1% imidazole, and vortexed to ensure complete redissolved dissolution. After redissolved dissolution, the sample can be centrifuged at room temperature, and the resulting supernatant is used for mass spectrometry analysis.

[0084] Before mass spectrometry analysis, the syringe can be cleaned using standard methods. For example, clean the syringe needle and internal tubing with MeOH solution. A blank reference of approximately 50% ACN aqueous solution can be used for mass spectrometry analysis.

[0085] Typically, primary mass spectrometry can be performed in MCA mode with the following parameters set: Temperature = 230-270℃, e.g., approximately 250℃; Scan type: TOF MS; Accumulation time = 0.9-1.3s, e.g., approximately 1.1s; Duration = 0.3-0.8min, e.g., 0.5min; TOF Masses (Da): 410-1300; Flow rate = 5-10μL / min, e.g., approximately 7μL / min.

[0086] First, inject the buffer solution and click "Acquire". If almost no impurity peaks appear above m / z = 600, it indicates that the cleaning is complete. At this point, inject an appropriate amount of sample and wait for a signal to appear and stabilize before clicking "Acquire".

[0087] The primary mass spectrometry (PMS) spectrum determines whether the sample contains the target sequence. After PMS, secondary mass spectrometry is performed. Secondary mass spectrometry involves enriching the target nucleic acid sequence, fragmenting and enriching the nucleic acid sequence, and analyzing the resulting fragment ions using mass spectrometry, recording the fragment ion spectra.

[0088] The parameter settings for secondary mass spectrometry include: Scan type: Product Ion; Accumulation time = 0.8-1.2s, e.g., approximately 1s; Duration = 0.8-1.2min, e.g., approximately 1min; TOF mass (Da): 510-1400; Masses set to 550 (Da); Time bins to sum: 14-18, e.g., 16; Collision energy (CE) = -20 to -28, e.g., -24.00. In secondary mass spectrometry, the required m / z values ​​for the fragments need to be set in the MS interface, then the sample is injected, and the Acquire parameter is clicked to obtain the corresponding spectrum.

[0089] Typically, each fragment should be repeated at least three times, and each sample at least twice. Before proceeding with the next cleaning and subsequent sample injection, the mass spectrometry parameters can be adjusted back to the primary mass spectrometry parameters, and then primary mass spectrometry acquisition can be performed.

[0090] In an exemplary embodiment, the present invention uses a Trpletof 5600 mass spectrometer to acquire nucleic acid mass spectrometry data, including background signal (twice), sample signal and positive reference signal.

[0091] The acquired spectra can be converted into a text file (mgf) for processing using a Python script.

[0092] This paper establishes three criteria to remove spectra unsuitable for further processing. These three criteria are: 1) molecular weight > 600 Da and total ionic intensity < 4000; 2) < 600 T.h and total ionic intensity > 4000; and 3) mass shift > 0.1 Da. Spectra meeting any one of these three criteria will be discarded.

[0093] The spectrum can be corrected using the three-point method (taking the three strongest mass spectral peaks, comparing them with their standard molecular weights, and calculating their accurate molecular weights through linear regression), thus achieving high-precision mass spectrometry detection of MS1 and MS2 ions.

[0094] Next, the spectrum was subjected to high-density block generation (BIN), which merges all signals within the 0.5 Da range into a single signal for easier comparison. Then, normalization was performed by summing the peak values ​​of the 100 strongest peaks in the spectrum.

[0095] Further, data for the positive reference were collected, and the difference peak between the positive reference and the background was obtained. A T-test was used to test the significance of the difference peak between the positive reference and the background. If the T-test value between two background tests was <0.3, or the T-test value between the positive control and the background was >0.01, the data set was discarded, and data was collected again. Conversely, a T-test was performed on the tandem spectra of the tested samples. Finally, if the T-test value was <0.05, it indicated that the tested sample contained trace impurities; if the T-test value was >0.05, it indicated that the tested sample did not contain trace impurities.

[0096] Results Analysis

[0097] The presence of mutations in a sample can be analyzed based on the spectra obtained from secondary mass spectrometry. Typically, the sample spectrum is compared with the background spectrum, and the portion of the comparison spectrum with m / z ≥ 600 (preferably ≥ 700) is selected. The p-values ​​of the top three intensity peaks in this portion are calculated. If the p-value is ≤ 0.05, it indicates that a mutation exists in the tested nucleic acid sequence compared to the background nucleic acid sequence. If the p-value is > 0.05, it indicates that no mutation exists in the tested nucleic acid sequence compared to the background nucleic acid sequence.

[0098] method

[0099] Therefore, in some embodiments, the present invention provides a method for detecting whether a gene has a mutation, the method comprising: (1) providing an amplification product containing an amplification product of a target nucleic acid sequence and an amplification product of a background nucleic acid sequence; (2) ionizing the amplification product and enriching the amplification product of the target nucleic acid sequence using gas phase ion online separation technology; (3) fragmenting the enriched amplification product and performing mass spectrometry analysis on the obtained fragment ions to record the fragment ion spectrum; (4) comparing the fragment ion spectrum obtained in step (3) with the ion spectrum of the background nucleic acid sequence obtained using the same step; (5) obtaining the portion of the comparison spectrum with an m / z value ≥600, preferably ≥700, calculating the P value of the top three peaks with the highest difference in intensity in this portion, and judging whether the target nucleic acid sequence has a mutation relative to the background nucleic acid sequence based on the P value; wherein, a P value ≤0.05 indicates that the target nucleic acid sequence has a mutation, and a P value >0.05 indicates that the target nucleic acid sequence does not have a mutation.

[0100] The amplification product can be obtained by using the method described in the "Amplification Methods" section of this document; in a preferred embodiment, the amplification product is obtained by nested polymerase chain reaction (PCR). Typically, the length of the amplification product does not exceed 80 bp. In some embodiments, the amplification product is an amplification product of cfDNA. In some embodiments, the target nucleic acid sequence is the nucleic acid sequence of the offspring (e.g., children), and the background nucleic acid sequence is the nucleic acid sequence of the mother. Maternal peripheral blood can be obtained, the gene sequence isolated from it, and primers designed for fetal cfDNA can be used for PCR amplification. This PCR amplification can amplify both fetal cfDNA and maternal cfDNA, and the amplification product is used in step (2).

[0101] The amplification products of the target nucleic acid sequence can be enriched based on their molecular weight. It should be understood that the enriched products usually also contain amplification products of background nucleic acid sequences, but their proportion of all amplification products is much lower than their proportion in the initial sample.

[0102] Step (3) above can be implemented using methods well known in the art. The ion spectrum of the background nucleic acid sequence mentioned in step (4) usually refers to the ion spectrum obtained by using a sample from a parent (such as the mother) that does not contain the target nucleic acid sequence or the sequence of the mutation to be tested, according to the methods in steps (1)-(3). For example, the sample can be a cell or tissue sample from the mother, from which the mother's gene sequence (i.e., the background nucleic acid sequence) is isolated, and amplified using primers used to amplify the target nucleic acid sequence to obtain the amplification product. After enriching the amplification product, mass spectrometry is performed to obtain its ion spectrum.

[0103] In some implementations, step (5) involves obtaining the portion of the comparison spectrum with an m / z value ≥ 700.

[0104] The p-values ​​mentioned in this article were obtained using the t-test method.

[0105] In some implementations, two background signals (i.e., ion spectra of background nucleic acid sequences, typically derived from maternal tissue or cells) are acquired, and the difference peak between the two background signals is obtained. A T-test is used to assess the significance of the difference peak. If the T-test value between the two background detections is <0.3, the data set is discarded, and data is reacquired. Otherwise, steps (4) and (5) of this implementation are performed.

[0106] In some embodiments, the method of the present invention further includes the step of providing an ion spectrum of the corresponding paternal sequence as a positive reference. Specifically, a paternal sample can be obtained, its nucleic acid sequence can be isolated using the method of the present invention, and amplified using primers used to amplify the target nucleic acid sequence to obtain an amplification product. After enriching the amplification product, mass spectrometry is performed to obtain its ion spectrum. The difference peak between the positive control and the background can be obtained, and the significance of the difference peak can be tested using the T-test method. If the T-test value between the positive control and the background is >0.01, the data set is invalidated. Data is reacquired. Otherwise, steps (4) and (5) of the present invention are performed.

[0107] The method of this invention does not include a diagnostic step.

[0108] Device and its application

[0109] This article also includes a device for detecting and analyzing gene mutations, the device comprising a memory, a processor, and a computer program stored in the memory and executable by the processor, wherein the processor, when executing the computer program, performs the following: (1) acquiring a background nucleic acid sequence mass spectrometry spectrum, a sample nucleic acid sequence mass spectrometry spectrum, and an optional positive reference mass spectrometry spectrum; (2) comparing the background nucleic acid sequence mass spectrometry spectrum and the sample nucleic acid sequence mass spectrometry spectrum, acquiring a portion with an m / z value ≥600, preferably ≥700, and calculating the P-values ​​of the three peaks with the highest intensity in this portion; (3) giving a conclusion on whether a gene mutation exists based on the P-values.

[0110] The background nucleic acid, sample nucleic acid sequence (i.e., target nucleic acid sequence), and positive reference (such as a nucleic acid sequence from the father) are as described in the various embodiments herein. Preferably, when the processor executes the computer program, it can implement steps (4) and (5) of the detection method described in any embodiment herein.

[0111] In some embodiments, the apparatus further includes a data input device and a data output device. The data input device may include, for example, various suitable interfaces for connection to other data storage and / or transmission devices. The data output device includes, for example, a display.

[0112] In some embodiments, the present invention also relates to the use of the device of the present invention in a method for detecting the presence of mutations in genes, and to the use of the device of the present invention in the preparation of an apparatus for detecting the presence of mutations in genes. Preferably, the apparatus further includes, for example, a mass spectrometer, especially a tandem mass spectrometer.

[0113] Reagent test kit

[0114] This document provides a kit that can be used to implement the method for detecting the presence of gene mutations as described in any embodiment of this document.

[0115] The kit of the present invention may comprise one or more containers containing one or more of the components described herein. The kit may contain one or more components in any number of individual containers, pouches, tubes, vials, multiwell plates, etc., or the components may be combined in different combinations within such containers. The kits described herein typically include at least one or more of the following reagents: (1) primers for amplifying the target nucleic acid sequence and the background nucleic acid sequence; and (2) reagents required for amplification, such as polymerase, buffer, dNTPs, etc. Amplification may be as described herein; preferably, amplification is a multiplex amplification method.

[0116] More preferably, the kit described herein may also include reagents required for DNA extraction, including but not limited to reagents for cell lysis, DNA precipitation, DNA washing, and DNA dissolution. More preferably or optionally, the kit described herein may also include reagents for purifying and desalting PCR products, including, for example, HLB packing material, NH4OAc solution, NH4HCO3 solution, and MeOH solution. More preferably or optionally, the kit described herein may also include reagents required for mass spectrometry analysis, including, for example, ACN aqueous solution, TEA solution, and imidazole solution.

[0117] The kit can be used to perform the methods described herein. The kit may also include instructions. The instructions may be in tangible form (e.g., paper) or electronic form (e.g., a computer-readable document on tangible media such as a compressed disk), and may be included in the kit insert.

[0118] In a preferred embodiment, the kit described herein is used for prenatal testing and contains at least primers for amplifying fetal cfDNA and primers for amplifying maternal nucleic acid sequences, and optionally includes any one or more of the reagents described above for PCR reactions, reagents for DNA extraction, reagents for PCR product purification and desalting, and reagents required for mass spectrometry analysis. In some embodiments, the kit further includes primers for amplifying nucleic acid sequences from the father. Preferably, the amplified product obtained using the primers is ≤80 bp in length. Preferably, the primers for amplifying fetal cfDNA, the primers for amplifying maternal nucleic acid sequences, and the primers for amplifying paternal nucleic acid sequences are the same.

[0119] use

[0120] The methods, compositions, kits, and apparatus of this invention can accurately detect small amounts of target DNA carrying mutations even when a large amount of interfering DNA is present in the sample. In a particularly preferred embodiment, this invention utilizes blood from pregnant women in early pregnancy to amplify the fetal gene sequence contained in the blood via nested polymerase chain reaction. Therefore, this invention overcomes the time limitation of examination and can determine whether the fetus in the pregnant woman's body has gene mutations at an early stage of pregnancy.

[0121] In some embodiments, the present invention provides the use of reagents in the preparation of kits for detecting small amounts of target DNA in the presence of large amounts of interfering DNA in a sample. It should be understood that "large amounts" as used herein generally refers to the presence of interfering DNA that significantly affects the accuracy of target DNA detection. Generally, "large amounts" as used herein means that the interfering DNA constitutes more than 70% of the total DNA in the sample, preferably more than 80%, more preferably more than 90%, and even more preferably more than 95%. For example, in early pregnancy, fetal cfDNA may account for less than 5% (in absolute terms) of the total cfDNA in a mother's blood. The reagents include one or more of the reagents described above, such as primers, reagents for DNA extraction and purification, reagents for PCR reactions, and reagents for mass spectrometry analysis. The methods described herein can be used to detect small amounts of target DNA in the presence of large amounts of interfering DNA in a sample.

[0122] In some embodiments, the present invention provides reagents and kits for carrying out the detection methods described herein. The reagents include one or more of the reagents described above, such as primers, reagents for DNA extraction and purification, reagents for PCR reactions, and reagents for mass spectrometry analysis. The kits may be kits containing one or more of the reagents described herein, or may be one or more known kits or combinations thereof.

[0123] In some embodiments, the present invention provides the use of nucleic acid amplification reagents or detection kits containing such nucleic acid amplification reagents in detecting the presence of gene mutations, or the use of nucleic acid amplification reagents in preparing detection kits for detecting the presence of gene mutations; the detection is performed using the method for detecting the presence of gene mutations described in any embodiment herein. Preferably, the nucleic acid amplification reagents include reagents required for performing PCR; more preferably, the PCR is a nested polymerase chain reaction amplification reaction. In some embodiments, the detection kit further includes one or more of the following: a sample acquisition device, reagents for isolating nucleic acids from a sample, reagents for purifying PCR amplification products, and reagents required for performing mass spectrometry.

[0124] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. The reagents and materials used in the embodiments are all conventional reagents and materials in the art and are available from commercial sources.

[0125] I. DNA Extraction

[0126] 1. Cell lysis

[0127] For whole blood samples smaller than 100 μL, add 1 mL of DNAzol and gently pipette to lyse cells. For whole blood samples larger than 100 μL, precipitate cells and wash with 0.9% NaCl. Reprecipitate cells again and resuspend in one volume of cold (4°C) hypotonic solution [20 mM Tris HCl (pH 8.0), 10 mM EDTA]. Centrifuge at 4000 rpm for 10 min at 4°C. Discard the supernatant and repeat every 1–3 × 10⁻⁶ cells. 7 Add 1 mL of DNAzol to each cell and gently pipette to fully lyse the cells.

[0128] 2. Centrifugation (optional)

[0129] Centrifuge at 10000g at room temperature or 4°C for 10 min, and transfer the viscous supernatant to a new test tube to remove insoluble tissue fragments, RNA and excess polysaccharides from the lysate.

[0130] 3. DNA precipitation

[0131] Add half the volume of 100% ethanol (the same volume as the DNAzol reagent used previously) to the lysate to precipitate the DNA. Invert the tube to mix the sample thoroughly and incubate at room temperature for 1-3 minutes. You will see the DNA rapidly turn into a cloudy precipitate. Use a pipette tip to transfer the DNA precipitate into a new EP tube.

[0132] 4. DNA washing

[0133] Add 0.8-1.0 mL of 75% ethanol, invert the EP tube 3-6 times to suspend the DNA in the ethanol, then let the EP tube stand for 0.5-1 min to allow the DNA to precipitate at the bottom of the EP tube. Aspirate and discard the supernatant. Repeat this washing process twice.

[0134] 5.DNA dissolution

[0135] After washing the DNA precipitate, open the EP tube cap and let it stand for 5-15 seconds to allow the ethanol to evaporate completely. Add 8mM NaOH and gently pipette to dissolve the DNA precipitate completely.

[0136] 6. DNA quantification

[0137] A spectrophotometer was used to measure the A260 and A280 of the resulting solution to determine the amount and purity of the extracted DNA.

[0138] II. PCR Amplification

[0139] 1. PCR system:

[0140] The PCR reaction system is shown in Table 1 below:

[0141] Table 1

[0142] 10× buffer solution 5 DNA polymerase 0.8 dNTP 6 <![CDATA[H2O]]> 31.2 Primers 1.5+1.5 template 4 total 50

[0143] 2. PCR reaction conditions

[0144] The PCR reaction conditions were: 95℃ for 3 minutes; 95℃ for 15 seconds, 60℃ for 1 minute and 72℃ for 30 seconds, for a total of 30 rounds; 72℃ for 2 minutes.

[0145] A large number of target nucleic acid fragments were obtained by performing two consecutive PCR operations for subsequent mass spectrometry analysis.

[0146] III. Purification and Desalting

[0147] PCR products (approximately 400 μL of PCR amplification products from two rounds) were used with 20 μL of HLB packing material.

[0148] 1. Take the required volume of HLB packing material, briefly centrifuge to remove the supernatant, and wash with 300 μL of 20 mM NH4OAc by blowing and mixing. Briefly centrifuge to remove the supernatant, and repeat twice.

[0149] 2. Resuspend the HLB precipitate in 20 mM NH4OAc and add it evenly to the enzyme digestion system;

[0150] 3. Add 20mM NH4OAc to bring the total volume of the solution in the EP tube to about 1mL;

[0151] 4.37℃, 1300rpm, shake for 30 minutes;

[0152] 5. Transfer all the suspension in the EP tube into the holes of the filter plate, and cover the remaining empty holes with a paraffin film to increase the vacuum pressure, so that the washing liquid can be drawn more cleanly and the subsequent elution and filtration can be more complete.

[0153] 6. Turn on the vacuum pump to completely drain the liquid from the well containing the sample suspension;

[0154] 7. Add 500 μL of 100 mM NH4OAc to each sample well, turn on the vacuum pump until the washing solution is completely drained, and repeat three times.

[0155] 8. Add 500 μL of 40 mM NH4HCO3 to each sample well, turn on the vacuum pump until the washing solution is completely drained, and repeat three times.

[0156] 9. Add 500 μL of 20% MeOH to each sample well, turn on the vacuum filtration pump until the washing solution is completely drained, repeat five times, and the last filtration time should be slightly longer to ensure that the washing solution is completely filtered out without affecting the subsequent elution.

[0157] 10. Add 150 μL of 1% NH3·H2O (prepared by diluting Fluka 25% NH3·H2O) to each sample well and let stand for 3 min to allow the nucleotide fragments adsorbed on the filtration membrane to dissolve as much as possible in the 1% NH3·H2O.

[0158] 11. Turn on the vacuum filtration pump and continue for 2 minutes to elute the nucleotide fragments completely.

[0159] IV. Drying

[0160] 1. Dry at 60℃ for about 2 hours to completely dry the solution inside the EP tube;

[0161] 2. Immediately reconstitute the dried tube for mass spectrometry analysis, or temporarily store it in a -20°C freezer until subsequent reconstitution and mass spectrometry analysis.

[0162] V. Mass Spectrometry Analysis

[0163] 1. Redissolution: Dissolve the dried sample in each EP tube with 380 μL of 50% ACN aqueous solution + 1% TEA + 1% imidazole, vortex to completely redissolve, centrifuge at 14000g at room temperature for 10 min, and transfer the supernatant to a new EP tube for mass spectrometry analysis.

[0164] 2. Mass spectrometry analysis

[0165] Mass spectrometry experimental procedures:

[0166] (1) Clean the syringe and injection needle with MeOH three times, 200 μL each time.

[0167] (2) Clean the internal tubing of the mass spectrometer three times with MeOH.

[0168] (3) Use a 50% ACN aqueous solution (about 100 μL) as a BLANK reference.

[0169] (4) First-order mass spectrometry parameter settings: Temperature = 250℃, Scan type: TOF MS, Accumulation time = 1.1s, Duration = 0.5min, TOF Masses (Da): 410~1300, Select MCA mode, Syringediameter (mm) = 3.25, Flow rate = 7μL / min.

[0170] (5) After the part above m / z = 600 has almost no extraneous peaks, click Acquire.

[0171] (6) Take about 220 μL of sample, remove air bubbles from the syringe, and place the syringe on the holder for injection.

[0172] (7) Perform the first-level mass spectrometry operation as described above. After the signal appears and stabilizes, click Acquire.

[0173] (8) Cascade mass spectrometry parameter settings: Scan type: Product Ion, Accumulation time = 1s, Duration = 1min, TOF Masses: 510~1400, Masses set to 550 (Da), Time bins to sum: 16, Collision Energy (CE) = -24.00.

[0174] (9) Return to the MS interface and set the m / z values ​​of the fragments to be fragmented (559.2, 699.3, 843.4).

[0175] (10) Inject the sample and click Acquire.

[0176] (11) Each fragment is repeated three times, and each sample is repeated twice.

[0177] (12) Before performing the next cleaning and subsequent injection operations, switch back to Scan type: TOP MS and modify the following parameters: Accumulation time = 1.1s, Duration = 0.5min, TOF Masses (Da): 410~1300.

[0178] (13) Return to the Advanced MS interface and click suggest to restore the default parameters for subsequent first-stage mass spectrometry acquisition.

[0179] (14) Remove the syringe and return the remaining sample to the EP tube.

[0180] (15) Repeat all the above steps to perform mass spectrometry on the next sample.

[0181] VI. Data Processing

[0182] 1. As mentioned above, nucleic acid mass spectrometry data were acquired using a Trpletof 5600 mass spectrometer, including two background signals (pure DNA extracted from the mother's tissue or cells), one sample signal (containing maternal blood cfDNA + fetal cfDNA), and one positive control signal (pure DNA from the father).

[0183] 2. Convert the acquired spectra into a text file format (mgf) for processing using a Python script.

[0184] 3. Three criteria were established to remove spectra that were not suitable for further processing: 1) (>600 Da) total ion intensity less than 4000; 2) (<600 T.h) total ion intensity greater than 4000; 3) mass shift greater than 0.1 Da.

[0185] 4. The 3-point method is used to correct the spectrum, achieving high-precision mass spectrometry detection of MS1 and MS2 ions.

[0186] 5. High-density block partitioning (BIN) of the spectrum: In order to facilitate comparison, all signals within the 0.5 Da interval are merged into one signal.

[0187] 6. Normalization: The acquired spectrum is normalized by summing the peak values ​​of the 100 strongest peaks in the spectrum.

[0188] 7. Collect data from the positive control and obtain the difference peak between the positive control and the background.

[0189] 8. Use the T-test to test the significance of the differences obtained in step 7. If the T-test value between two background tests is <0.3, or the T-test value between the positive control and the background is >0.01, then the data set is invalid. Data should be collected again. Conversely, perform a T-test on the tandem spectra of the tested samples. Finally, if the T-test value is <0.05, it indicates that the tested sample contains trace impurities; if the T-test value is >0.05, it indicates that the tested sample does not contain trace impurities.

[0190] 9. Compare the sample spectrum with the background spectrum, and obtain the P-values ​​of the three peaks with the highest m / z values ​​(≥600, preferably ≥700) and the largest differences in intensity in the comparison spectrum. Based on these P-values, determine the relative intensity of the sample spectrum compared to the background nucleic acid sequence.

[0191] VII. Experimental Results

[0192] 1. Cascade analysis of 50NT oligonucleotide sequences

[0193] The above method was used to perform skewing analysis on the 50NT oligonucleotide sequences (SEQ ID NO: 1-4, with a hydroxyl group at the 5' end) shown in the table below:

[0194]

[0195] The 24th base of each sequence is T, A, C and G respectively, and the bases at the other positions are the same.

[0196] The results are as follows Figure 1 As shown in the figure. The results show that tandem mass spectrometry can resolve single-base mutations very well, especially in the case of longer nucleotide sequences (50-80 NT).

[0197] 2. Results of single-base mutation detection of oligonucleotides under strong background interference.

[0198] The above method was used to test the ability of this invention to distinguish oligonucleotide single-base mutations under strong background interference. The primary and secondary primers shown in the table below were used, and the corresponding product sequences were obtained:

[0199]

[0200]

[0201] The relative concentrations of the tested amplified products against the background were 10%, 2%, 0.5%, 0.2%, 0.1%, 0.01%, and 0%, respectively. The results are as follows: Figure 2 and 3 As shown in the figure. The results indicate that gas phase ion enrichment combined with tandem mass spectrometry can better resolve single-base mutation sequences under strong background interference, and even when the amount of background oligonucleotides is 200 times that of the detected oligonucleotides, statistically reproducible results can still be obtained.

[0202] 3. Detection results of a single mutant cell in the background

[0203] The above method was used to test the ability of this invention to distinguish oligonucleotide single-base mutations under strong background interference. The primary and secondary primers shown in the table below were used, and the corresponding product sequences were obtained:

[0204]

[0205]

[0206]

[0207] The results are as follows Figure 4 and 5 As shown in the figure. The results show that gas phase ion enrichment combined with tandem mass spectrometry can achieve extremely high sensitivity. Even under strong background interference, single-base mutation sequences with only two copies can still be detected, and the results are statistically significant.

[0208] 4. Clinical experimental validation results of non-pathogenic mutations at 5-7 weeks of gestation

[0209] Maternal blood was obtained from women at 5-7 weeks of gestation. Using the method of this invention, the blood underwent two PCR amplifications, followed by enzymatic digestion and purification, and then gas phase ion enrichment combined with tandem mass spectrometry as described in this invention. The obtained data were statistically analyzed. The results were compared with Sanger sequencing results from aborted fetuses. The primers used for the two PCRs and the amplification products are shown in the table below:

[0210]

[0211]

[0212] The results are as follows Figure 6 and 7 As shown in the figure. The results show that this invention can distinguish whether the fetal DNA contained in the mother's cfDNA contains paternal single-base mutations. In a total of 148 maternal blood samples, 106 samples contained paternal single-base mutations, and 42 samples did not. All results were validated by Sanger sequencing of abortion tissue, with an accuracy of 100%.

[0213] 5. Results of PKU non-invasive screening at 12-16 weeks of pregnancy

[0214] Peripheral blood samples from pregnant women carrying PKU were analyzed using gas chromatography-ion enrichment combined with tandem mass spectrometry. Primers were designed and used to detect the PAH gene on chromosome 12, and the results were compared with Sanger sequencing results from chorionic villus tissue. The primary and secondary primers shown in the table below were used, and the corresponding product sequences were obtained.

[0215]

[0216]

[0217]

[0218] The results are as follows Figure 8 and 9 As shown in the figure. The results show that this invention can distinguish whether the fetal DNA contained in the mother's cfDNA contains a paternally pathogenic PAH gene mutation. In a total of 14 maternal blood samples, 8 samples contained paternally pathogenic mutations, and 6 samples did not. All results were validated by Sanger sequencing of chorionic villus tissue, with an accuracy of 100%.

[0219] Mass spectrometry is a highly sensitive and precise instrument that can not only detect molecular weight but also fragment molecules using its tandem mass spectrometry function to obtain fragment spectra. In the detection of nucleic acid molecules, we can use electrospray ionization to ionize neutral nucleic acid molecules, giving them a negative charge, and then send them into the mass spectrometer for detection. Due to the characteristics of electrospray ionization, we often obtain multi-charged molecular ion peaks for nucleic acid molecules. These multi-charged molecular ion peaks reduce the mass-to-charge ratio difference between two single-base mutation nucleic acid molecules. Therefore, to detect single-base mutations in genetic variations, we need high-resolution mass spectrometry to meet our requirements. However, high-resolution mass spectrometry alone cannot yield the desired results because the detection of fetal cfDNA is subject to massive interference from maternal cfDNA. Data shows that in early pregnancy, fetal cfDNA accounts for less than 5% of the total cfDNA in the mother's blood. Under such circumstances, mass spectrometry cannot directly detect fetal cfDNA because the dynamic range of detection exceeds the limits achievable by mass spectrometry. The signal from a large amount of maternal cfDNA overwhelms the signal from fetal cfDNA, resulting in unsatisfactory detection results. Therefore, this invention employs a gas chromatography-enrichment combined with tandem mass spectrometry fragmentation method to detect the cascade signal of fetal cfDNA to examine whether it contains mutations of interest.

[0220] The method of the present invention will have the following advantages:

[0221] 1. Mass spectrometry gas phase enrichment can effectively enrich fetal cfDNA ions from the huge maternal cfDNA background ions, so that these signals can be effectively detected by mass spectrometry.

[0222] 2. The gas phase enrichment system is very fast, enriching the fetal cfDNA signal while simultaneously transporting ions.

[0223] 3. The enriched fetal cfDNA is fragmented. This involves applying energy to the fetal cfDNA and then performing mass spectrometry analysis on the fragment ions, recording the fragment ion spectra. These spectra are called tandem spectra. Since the mutations being detected are often single-base mutations, the molecular weight differences are not significant. Furthermore, electrospray ionization generates multi-charged ions, further reducing the mass-charge ratio differences of the molecular ion peaks. Tandem spectra, due to differences in individual bases, produce completely different fragments, thus helping to further eliminate interference from maternal cfDNA and obtain highly reliable detection of fetal cfDNA base mutations.

[0224] 4. Digital PCR or high-depth sequencing often suffers from errors generated by PCR, affecting the interpretation of final results. High-resolution tandem mass spectrometry effectively removes the influence of PCR erroneous products, resulting in highly reliable and accurate measurements. Research data from this invention's high-reliability sample studies show that the accuracy rate was 100% in the detection of over 100 samples.

[0225] 5. The entire experiment only requires a standard PCR kit. It does not require expensive fluorescent materials, digital PCR equipment, or equally expensive sequencing chips and materials.

[0226] 6. Data analysis is simple, reliable, and fast.

[0227] 7. The entire testing process can be fully automated with the help of a pipetting workstation, thereby greatly improving throughput.

[0228] 8. It is fast; the entire test can be completed within 12 hours.

[0229] 9. Testing can be performed with only one sample, greatly reducing sample detection reaction time. sequence list <110> Shanghai Junyi Biotechnology Co., Ltd. <120> Detection and application of small amounts of target DNA in the presence of abundant interfering DNA. <130> 215132 <160> 44 <170> SIPOSequenceListing 1.0 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <400> 1 aactccgcag tttcttttct ccctctccca acctacgtag ggtccttcat 50 <210> 2 <211> 50 <212> DNA <213> Artificial Sequence <400> 2 aactccgcag tttcttttct cccactccca acctacgtag ggtccttcat 50 <210> 3 <211> 50 <212> DNA <213> Artificial Sequence <400> 3 aactccgcag tttcttttct ccccctccca acctacgtag ggtccttcat 50 <210> 4 <211> 50 <212> DNA <213> Artificial Sequence <400> 4 aactccgcag tttcttttct cccgctccca acctacgtag ggtccttcat 50 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 tgcttacgta cgggcaatga 20 <210> 6 <211> twenty one <212> DNA <213> Artificial Sequence <400> 6 gcacaggtgc caaattctgt t 21 <210> 7 <211> 60 <212> DNA <213> Artificial Sequence <400> 7 tgcttacgta cgggcaatga ctgagaaaag gatcgtcata acagaatttg gcacctgtgc 60 <210> 8 <211> twenty four <212> DNA <213> Artificial Sequence <400> 8 tgcttacgta cgggggatga atga 24 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <400> 9 gcacaggtgc caaaggatgt ctgtt 25 <210> 10 <211> 69 <212> DNA <213> Artificial Sequence <400> 10 tgcttacgta cgggggatga atgactgaga aaaggatcgt cataacagac atcctttggc 60 acctgtgca 69 <210> 11 <211> 69 <212> DNA <213> Artificial Sequence <400> 11 aacgaatgca tgccccctac ttactgactc ttttcctagc agtattgtct gtaggaaacc 60 gtggacacg 69 <210> 12 <211> twenty four <212> DNA <213> Artificial Sequence <400> 12 tgcaaatgtt ctttcctttc caca 24 <210> 13 <211> twenty one <212> DNA <213> Artificial Sequence <400> 13 tcagccaaag actcagaaca c 21 <210> 14 <211> 104 <212> DNA <213> Artificial Sequence <400> 14 tgcaaatgtt ctttcctttc cacaacttct acttttgttc cgatttcttc agattctaaa 60 aaggtatcct catacttctc gttgtgttct gagtctttgg ctga 104 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <400> 15 ttttgttccg atttcggatg tcagattct 29 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <400> 16 gccaaagact ggatgagaac acaac 25 <210> 17 <211> 79 <212> DNA <213> Artificial Sequence <400> 17 ttttgttccg atttcggatg tcagattcta aaaaggtatc ctcatacttc tcgttgtgtt 60 ctcatccagt ctttggcta 79 <210> 18 <211> 79 <212> DNA <213> Artificial Sequence <400> 18 aaaaacaagg ctaaagccta cagtctaaga tttttccata ggagtatgaa gagcaacaca 60 agagtaggtc agaaaccga 79 <210> 19 <211> twenty one <212> DNA <213> Artificial Sequence <400> 19 ggatttgaat tcgggcgtct g 21 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <400> 20 tcctcagagt ggcattctgc 20 <210> twenty one <211> 101 <212> DNA <213> Artificial Sequence <400> twenty one ggatttgaat tcgggcgtct gctggagtgt gcccaatgct atatgtcagt tgaggttcta 60 agacttggaa gccacagaaa tgcagaatgc cactctgagg a 101 <210> twenty two <211> 27 <212> DNA <213> Artificial Sequence <400> twenty two tgtgcccaat gctatggatg tgtcagt 27 <210> twenty three <211> 25 <212> DNA <213> Artificial Sequence <400> twenty three tctgcatttc tggatgtggc ttcca 25 <210> twenty four <211> 68 <212> DNA <213> Artificial Sequence <400> twenty four tgtgcccaat gctatggatg tgtcagttga ggttctaaga cttggaagcc acatccagaa 60 atgcagaa 68 <210> 25 <211> 68 <212> DNA <213> Artificial Sequence <400> 25 aacacgggtt acgataccta cacagtcaac tccaagattc tgaaccttcg gtgtaggtct 60 ttacgtct 68 <210> 26 <211> twenty two <212> DNA <213> Artificial Sequence <400> 26 tcagtggtga tgagctttga gt 22 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <400> 27 gaaagcaggc cagccaca 18 <210> 28 <211> 88 <212> DNA <213> Artificial Sequence <400> 28 tcagtggtga tgagctttga gttttctttc ttcttttcat cccagcttgc actggtttcc 60 gcctccgacc tgtggctggc ctgctttc 88 <210> 29 <211> twenty four <212> DNA <213> Artificial Sequence <400> 29 gtttccgcct cggatggacc tgtg 24 <210> 30 <211> 64 <212> DNA <213> Artificial Sequence <400> 30 gtttccgcct cggatggacc tgtggctggc ctgctttcct ctcgggattc atcccttggg 60 tgga 64 <210> 31 <211> twenty four <212> DNA <213> Artificial Sequence <400> 31 ccacccaagg gatgaatccc gaga 24 <210> 32 <211> 64 <212> DNA <213> Artificial Sequence <400> 32 acaaaggcgg agcctacctg gacaccgacc ggacgaaagg agagccctaa gtagggaacc 60 CACC 64 <210> 33 <211> twenty three <212> DNA <213> Artificial Sequence <400> 33 tttgtgcctg tattctagtg ggc 23 <210> 34 <211> 65 <212> DNA <213> Artificial Sequence <400> 34 tttgtgcctg tattctagtg ggcagcccat ccctcgagtg gaatacatgc atccaggaag 60 aaaaa 65 <210> 35 <211> 29 <212> DNA <213> Artificial Sequence <400> 35 ttttcttcct ggatgcatgt attccactc 29 <210> 36 <211> 65 <212> DNA <213> Artificial Sequence <400> 36 aaaacacgga cataagatca cccgtcgggt agggagctca ccttatgtac gtaggtcctt ctttt 65 <210> 37 <211> 29 <212> DNA <213> Artificial Sequence <400> 37 agagttttaa tgatgccgga tgaggaga <210> 38 <211> 72 <212> DNA <213> Artificial Sequence <400> 38 aggttttaa tgatgccgga tgaggagaaa gtaaggtgag gtggtgacaa agcatcctga gccactagct about <210> 39 <211> 26 <212> DNA <213> Artificial Sequence <400> 39 gagctagtgg ctcaggatgc tttgtc <210> 40 <211> 72 <212> DNA <213> Artificial Sequence <400> 40 atctcaaaat tactacggcc tactcctctt tcattccact ccaccactgt ttcgtaggac tcggtgatcg 72 <210> 41 <211> twenty four <212> DNA <213> Artificial Sequence <400> 41 tttcatccca gcggatgtgc actg 24 <210> 42 <211> 63 <212> DNA <213> Artificial Sequence <400> 42 tttcatccca gcggatgtgc actggtttcc gcctccgacc tgtggctgca tcccctgctt 60 tca 63 <210> 43 <211> twenty three <212> DNA <213> Artificial Sequence <400> 43 gaaagcaggg gatgcagcca cag 23 <210> 44 <211> 63 <212> DNA <213> Artificial Sequence <400> 44 aaaagtaggg tcgcctacac gtgaccaaag gcggaggctg gacaccgacg taggggacga 60 aag 63

Claims

1. A method for detecting the presence of mutations in a target nucleic acid sequence relative to a background nucleic acid sequence for non-diagnostic purposes, the method comprising: (1) Provide an amplification product containing an amplification product of a target nucleic acid sequence and an amplification product of a background nucleic acid sequence, wherein the amplification product is an amplification product of cfDNA from the blood of a pregnant woman, the target nucleic acid sequence is the cfDNA sequence of the offspring, and the background nucleic acid sequence is the cfDNA sequence of the mother. (2) Ionize the amplification product and enrich the amplification product of the target nucleic acid sequence using gas phase ionization online separation technology; (3) Fragment the enriched amplified product and perform tandem mass spectrometry analysis on the obtained fragment ions, and record the fragment ion spectrum. The tandem mass spectrometry is electrospray quadrupole time-of-flight mass spectrometry, electrospray ion hydrazine mass spectrometry or Fourier transform ion cyclotron resonance mass spectrometry. (4) Compare the fragment ion spectrum obtained in step (3) with the ion spectrum of the background nucleic acid sequence obtained in the same step; (5) Obtain an m / z value ≥ 600 in the comparison spectrum, calculate the P value of the three peaks with the highest difference in intensity in this part, and determine whether the target nucleic acid sequence has a mutation relative to the background nucleic acid sequence based on the P value; Wherein, a P value ≤ 0.05 indicates that the target nucleic acid sequence has a mutation, and a P value > 0.05 indicates that the target nucleic acid sequence does not have a mutation.

2. The method as described in claim 1, characterized in that, The length of the amplified product does not exceed 80 bp.

3. The method as described in claim 1, characterized in that, (5) is: to obtain the part of the comparison spectrum with an m / z value ≥ 700, calculate the P value of the three peaks with the highest difference peak intensity in this part, and judge whether the target nucleic acid sequence has a mutation relative to the background nucleic acid sequence based on the P value; wherein, a P value ≤ 0.05 indicates that the target nucleic acid sequence has a mutation, and a P value > 0.05 indicates that the target nucleic acid sequence does not have a mutation.

4. The method according to any one of claims 1-3, characterized in that, The amplified product was obtained by nested polymerase chain reaction.

5. The method as described in claim 4, characterized in that, The amplification product was purified and desalted.

6. The method according to any one of claims 1-3, characterized in that, Primers were designed targeting polynucleotide polymorphism sites in the target nucleic acid sequence for PCR amplification.

7. The application of a nucleic acid amplification reagent or a detection kit containing said nucleic acid amplification reagent in non-diagnostic purposes for detecting the presence of mutations in a target nucleic acid sequence relative to a background nucleic acid sequence; characterized in that, The detection of whether the target nucleic acid sequence has a mutation relative to the background nucleic acid sequence is carried out by any one of claims 1-6.

8. The application as described in claim 7, characterized in that, The nucleic acid amplification reagents include those required for PCR.

9. The application as described in claim 8, characterized in that, The PCR was a nested polymerase chain reaction amplification reaction.

10. The application as described in any one of claims 7-9, characterized in that, The test kit also includes one or more of the following: a sample acquisition device, reagents for isolating nucleic acids from a sample, reagents for purifying PCR amplification products, and reagents required for performing mass spectrometry.

11. An apparatus for detecting and analyzing gene mutations, the apparatus comprising a memory, a processor, and a computer program stored in the memory and executable by the processor, wherein the processor, when executing the computer program, performs: (1) acquiring a background nucleic acid sequence mass spectrometry spectrum and a sample nucleic acid sequence mass spectrometry spectrum; (2) comparing the background nucleic acid sequence mass spectrometry spectrum and the sample nucleic acid sequence mass spectrometry spectrum to obtain a portion with an m / z value ≥ 600, and calculating the P-values ​​of the three most significant peaks in that portion; (3) providing a conclusion on the presence or absence of a gene mutation based on the P-values, wherein the apparatus is used to implement the method of any one of claims 1-6.

12. An apparatus for detecting and analyzing gene mutations, the apparatus comprising a memory, a processor, and a computer program stored in the memory and executable by the processor, wherein the processor, when executing the computer program, performs: (1) acquiring a background nucleic acid sequence mass spectrometry spectrum, a sample nucleic acid sequence mass spectrometry spectrum, and a positive reference mass spectrometry spectrum; (2) comparing the background nucleic acid sequence mass spectrometry spectrum and the sample nucleic acid sequence mass spectrometry spectrum to obtain a portion with an m / z value ≥ 600, and calculating the P-values ​​of the three most significant peaks in that portion; (3) providing a conclusion on the presence or absence of a gene mutation based on the P-values, wherein the apparatus is used to implement the method of any one of claims 1-6.

Images