Tumor circulating nucleic acid standard substance for liquid biopsy as well as synthesis method and application of tumor circulating nucleic acid standard substance

Abstract

The invention relates to a tumor circulating nucleic acid standard substance suitable for liquid biopsy as well as a synthesis method and application of the tumor circulating nucleic acid standard substance. By systematically optimizing the synthesis process of the tumor circulating nucleic acid standard substance, the application performance and stability of the tumor circulating nucleic acid standard substance in liquid biopsy are remarkably improved. The standard substance synthesized by the method provided by the invention is superior to a traditional preparation mode in the aspects of detection sensitivity, repeatability and storage stability, and has stronger experimental controllability and clinical availability.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to tumor circulating nucleic acid standards suitable for liquid biopsy, as well as synthesis methods and applications thereof. Background Art

[0002] In recent years, liquid biopsies have become increasingly widely used in the clinical diagnosis and treatment of cancer, enabling precision medicine for tumors. Liquid biopsy samples vary widely, including small fragments of nucleic acid (DNA) and RNA (RNA) that are free in peripheral blood. Effective and accurate standards are essential for these tests.

[0003] Tumor circulating nucleic acid standards are artificially prepared simulated samples containing known tumor gene mutations. They are used as reference materials to evaluate, validate, monitor, and standardize liquid biopsy technologies and detect the performance, sensitivity, and accuracy of circulating tumor DNA or RNA in the blood. In the existing technology, the preparation of tumor circulating nucleic acid standards mainly relies on artificial molecular cloning, in vitro transcription, or synthetic oligonucleotide fragments followed by splicing. However, these methods have certain defects that affect the stability, consistency, and feasibility of large-scale preparation of the standards. In addition, the preparation process of existing methods often requires manual operation and is complex, making it difficult to achieve automation and high-throughput production, resulting in large batch-to-batch variability and making it difficult to meet the consistency and reproducibility requirements of standards for clinical testing and large-scale scientific research applications.

[0004] Therefore, there is an urgent need for an optimized tumor nucleic acid standard and its preparation method. The obtained standard can be preserved for a long time and has high sensitivity and good reproducibility, which can meet the requirements of large-scale synthesis and application. Summary of the Invention

[0005] To solve the above technical problems, the present application provides a tumor circulating nucleic acid standard suitable for liquid biopsy, which is not easily degraded by external pollutants and can be preserved for a long time.

[0006] The present application also provides a method for synthesizing tumor circulating nucleic acid standards suitable for liquid biopsy. The method can ensure the integrity and uniformity of the final synthesized standard, and has high synthesis efficiency, and is suitable for large-scale synthesis and application.

[0007] The present application also provides a use of the above-mentioned tumor circulating nucleic acid standard in detecting tumors. When using the above-mentioned tumor circulating nucleic acid standard for cancer detection, it has high sensitivity, good reproducibility, good experimental controllability and clinical applicability.

[0008] The present application provides a tumor circulating nucleic acid standard suitable for liquid biopsy, the standard comprising standard 1 and standard 2;

[0009] The standard 1 includes RNA with the sequence shown below:

[0010] SEQ ID NO: 1: 5'-AUGCGUCUUCACCUGCUGCUGGGUGCGAUG-3'; and / or

[0011] SEQ ID NO: 2: 5'-AUGCGUCUUCACCUGCAGCUGGGUGCGAUG-3';

[0012] The standard 2 includes RNA with the sequence shown below:

[0013] SEQ ID NO: 3: 5′-GAGAGACAAUGAAUUAAGGGAAAGAAGAAA-3′; and / or

[0014] SEQ ID NO: 4: 5'-GAGAGACAAUGAAUUAAGGAAAAGAAGAAA-3'.

[0015] Preferably, the above-mentioned tumor circulating nucleic acid standard further comprises a lysis protective agent.

[0016] Preferably, the lysis protection agent comprises at least one of RNAlater stabilizing solution, glycerol and DMSO.

[0017] Preferably, the above-mentioned tumor includes at least one of lung cancer, colon cancer and breast cancer.

[0018] The present application also provides the use of the above-mentioned tumor circulating nucleic acid standard in the preparation of a kit for detecting tumors in a subject.

[0019] Preferably, the above-mentioned standard 1 is used to detect the EGFR L858R mutation, and the standard 2 is used to detect the PIK3CA E545K mutation.

[0020] The present application also provides a tumor circulating nucleic acid kit suitable for liquid biopsy, including the above-mentioned tumor circulating nucleic acid standard.

[0021] The present application also provides a method for synthesizing a tumor circulating nucleic acid standard, comprising the following steps:

[0022] Obtaining a DNA template: Selecting a sequence containing a cancer driver gene and a high-frequency mutation site as the original target sequence, optimizing it, and amplifying the optimized target sequence to obtain the DNA template;

[0023] In vitro transcription: preparing a transcription system solution and using the transcription system solution to transcribe the DNA template in vitro, and purifying the transcription product to obtain the RNA product;

[0024] RNA fragmentation: The RNA product is fragmented to obtain RNA standards, and the RNA standards are mixed with a lysis protective agent to obtain the tumor circulating nucleic acid standards.

[0025] Preferably, the step of optimizing the original target sequence includes: avoiding the homology region and selecting a 15-22 bp region containing the mutation site as the target sequence; adjusting the CG content of the target sequence to less than 60%; eliminating the secondary structure of the target sequence to obtain an optimized target sequence.

[0026] Preferably, the method further comprises the step of designing an automated synthesis system; the automated synthesis system comprises a microfluidic chip system or a robotic platform.

[0027] This application has the following beneficial effects:

[0028] This application significantly improves the application performance and batch stability of tumor circulating nucleic acid standards in liquid biopsies by systematically optimizing the synthesis process. Specifically, in this application, high-frequency mutation sites are selected and sequence optimization is performed in combination with the human genome database to ensure that the standards have high versatility and detection specificity; the DNA template is amplified and the T7 promoter is introduced to ensure RNA transcription efficiency, and the product quality is improved by purification; further modifications are added after RNA synthesis to enhance its structural stability and simulate the characteristics of real mRNA; the RNA fragmentation step accurately controls the length to 50-300nt, which is closer to the state of plasma free nucleic acid. The entire synthesis process can be automated through microfluidic chips to accurately control the reaction volume and time, significantly improving the synthesis efficiency and consistency. Verified by qPCR and NGS, the standards synthesized by this method are superior to traditional preparation methods in terms of detection sensitivity, repeatability and storage stability, and have stronger experimental controllability and clinical usability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0030] Figure 1 This is the structural integrity test result of the RNA sample in one embodiment of the present application;

[0031] Figure 2 This is an electrophoresis peak diagram obtained by length detection after RNA fragmentation in one embodiment of the present application;

[0032] Figure 3 This is a standard curve diagram for setting the threshold value of digital PCR (Droplet Digital PCR, ddPCR) in one embodiment of the present application;

[0033] Figure 4 This is a standard curve graph established in an embodiment of the present application using circulating free DNA (cfDNA) from healthy individuals as the background;

[0034] Figure 5 This is a graph showing the sensitivity of next-generation sequencing (NGS) in one embodiment of the present application;

[0035] Figure 6 Standard curve graph for commercially available standards used for digital PCR threshold setting;

[0036] Figure 7 The stability comparison results of the RNA standard and cfRNA extracted by TRIzol in this application are shown.

[0037] The above drawings illustrate specific embodiments of the present application, which will be described in more detail below. These drawings and the textual description are not intended to limit the scope of the present application in any way, but rather to illustrate the concepts of the present application to those skilled in the art by reference to specific embodiments. DETAILED DESCRIPTION

[0038] To enable those skilled in the art to better understand the solutions of the present application, the present application is further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present application. The examples cited are only used to explain the present application and do not limit the scope of the present application. Based on the embodiments of the present application, all other implementation methods obtained by those of ordinary skill in the art without making creative efforts are within the scope of protection of this application.

[0039] The present application provides a tumor circulating nucleic acid standard suitable for liquid biopsy, the standard comprising standard 1 and standard 2;

[0040] The standard 1 includes RNA with the sequence shown below:

[0041] SEQ ID NO: 1: 5'-AUGCGUCUUCACCUGCUGCUGGGUGCGAUG-3'; and / or

[0042] SEQ ID NO: 2: 5'-AUGCGUCUUCACCUGCAGCUGGGUGCGAUG-3';

[0043] The standard 2 includes RNA with the sequence shown below:

[0044] SEQ ID NO: 3: 5′-GAGAGACAAUGAAUUAAGGGAAAGAAGAAA-3′; and / or

[0045] SEQ ID NO: 4: 5'-GAGAGACAAUGAAUUAAGGAAAAGAAGAAA-3'.

[0046] In the above RNA sequences, SEQ ID NO: 1 and SEQ ID NO: 2 are standards for detecting EGFR L858R mutation; SEQ ID NO: 3 and SEQ ID NO: 4 are standards for detecting PIK3CA E545K mutation.

[0047] In an alternative embodiment, the standard further comprises a lysis-protective agent.

[0048] In an optional embodiment, the lysis protection agent includes at least one of RNAlater stabilizing solution, glycerol and DMSO.

[0049] In an alternative embodiment, the content of RNAlater Stabilizing Solution in the standard is the same as the volume of the RNA standard.

[0050] In an alternative embodiment, the final concentration of glycerol in the standard is 8-12% (v / v), preferably 9-11% (v / v).

[0051] In an alternative embodiment, the final concentration of glycerol in the standard is 10% (v / v).

[0052] In an alternative embodiment, the final concentration of DMSO in the standard is 3-7% (v / v), preferably 4-6% (v / v).

[0053] In an alternative embodiment, the final concentration of DMSO in the standard is 5% (v / v).

[0054] RNAlater Stabilizing Solution inhibits nucleases, preventing standards from cleavage and loss of efficacy. Glycerol reduces ice crystal damage to nucleic acid structures during freezing. DMSO enhances stability during freeze-thaw cycles, preventing standards from losing efficacy due to repeated freeze-thaw cycles.

[0055] In an optional embodiment, the standard is stored as follows:

[0056] Short-term storage (<1 week): Store at 2-8°C away from light;

[0057] Medium-term storage (<3 months): cryopreserved at -15°C to -25°C;

[0058] Long-term storage: below -80°C, avoid repeated freezing and thawing.

[0059] In an optional embodiment, the above-mentioned standard is suitable for detecting at least one of lung cancer, colon cancer and breast cancer.

[0060] The present application also provides the use of tumor circulating nucleic acid standards suitable for liquid biopsy in the preparation of a kit for detecting tumors in a subject.

[0061] The present application also provides a kit for detecting circulating tumor nucleic acids for liquid biopsy, comprising the standard described above.

[0062] The present application provides a method for synthesizing a tumor circulating nucleic acid standard suitable for liquid biopsy, comprising the following steps:

[0063] Obtaining a DNA template: Selecting a sequence containing cancer driver genes and high-frequency mutation sites from NCBI or other gene libraries as the original target sequence, optimizing it, and then amplifying the optimized target sequence to obtain the DNA template;

[0064] In vitro transcription: Prepare a transcription system solution and transcribe the DNA template in vitro using the transcription system solution, and purify the transcription product to obtain an RNA product; wherein the transcription system solution includes transcription buffer, NTP mixture, RNA polymerase, DNA template, RNase inhibitor, etc.

[0065] RNA quality testing: testing the concentration, structure and integrity of the obtained RNA product to see if it meets the requirements;

[0066] RNA fragmentation: The RNA product is fragmented to obtain RNA standards, and the RNA standards are mixed with a lysis protective agent to obtain tumor circulating nucleic acid standards.

[0067] In this application, "tumor circulating nucleic acid standards" refers to

[0068] In an optional embodiment, the selected cancer driver genes and high-frequency mutation sites can be common in clinical practice. For example, cross-comparisons can be performed using public databases such as COSMIC, TCGA, ClinVar, and dbSNP, and combined with meta-analysis to screen for common and highly specific mutations. Methods for selecting cancer driver genes and high-frequency mutation sites are conventional techniques in the art and are not limited here.

[0069] In an optional embodiment, the cancer driver gene includes at least one of TP53, EGFR, KRAS, PIK3CA, BRAF, and ALK.

[0070] In an optional embodiment, the high-frequency mutation sites include:

[0071] TP53: c.743G>A, R248Q;

[0072] EGFR: c.2573T>G, L858R;

[0073] KRAS: c.35G>T, G12V;

[0074] PIK3CA: c.1624G>A, E545K.

[0075] In an alternative embodiment, the method for optimizing the original target sequence is as follows:

[0076] Based on the human genome annotations in Gencode v38 and COSMIC v96, the sequences were analyzed for splice variants, and fragments with high expression abundance and strong conservation in the exon region were selected;

[0077] BLAST was used to perform specific alignment of the entire genome to ensure the uniqueness of the target region and avoid nonspecific amplification or hybridization.

[0078] In an optional embodiment, the optimization of the original target sequence comprises the following steps:

[0079] Truncation target region: avoid homology regions and select a 15-22 bp region containing the mutation site;

[0080] Adjusting GC content: replacing some bases in high GC regions, for example, replacing G with A. A GC content of less than 40% is considered low GC content; a GC content of 40%-60% is considered medium GC content; a GC content of more than 60% is considered high GC content; and a GC content of more than 70% is considered very high GC content.

[0081] Elimination of secondary structure: disruption of the hairpin structure by point mutation.

[0082] Among them, point mutations include the following methods:

[0083] Use oligonucleotide primers carrying the mutated base;

[0084] Mutations were inserted using PCR and gene synthesis.

[0085] The above point mutations can be precisely located at the specified base, and subsequent sequencing can be used to verify whether the mutation is successful.

[0086] In an alternative embodiment, the target sequence after amplification optimization can be amplified using chemical methods and / or PCR.

[0087] In an alternative embodiment, for target sequence DNA fragments ≤ 200 bp in length, chemical synthesis is employed. The chemical synthesis of DNA templates can be performed using the following four-step synthesis cycle mechanism:

[0088] 1. Deprotection: Remove the DMT (4,4'-dimethoxytrityl) protecting group at the 5'-end to expose the hydroxyl group for the next round of connection;

[0089] 2. Base coupling: Add the required phosphoramide monomer base (A, T, C, G) and react with the 5'-hydroxyl group of the previous base under the action of an alkaline catalyst to form a phosphotriester bond;

[0090] 3. Oxidation: Use a mixture of iodine / water / pyridine / tetrahydrofuran (THF) to oxidize the unstable phosphorus-hydrogen bond (P(III)) to a stable phosphate bond (P(V));

[0091] 4. End-capping: Block unreacted 5'-OH to prevent the generation of impurities with missing sequences;

[0092] Chemical synthesis reference: Caruthers MH. Gene synthesis machines: DNA chemistry and its uses. Science. 1985 May 3;230(4723):281-285.

[0093] In an alternative embodiment, target DNA fragments greater than 200 bp in length can be amplified by PCR; the amplified fragment is then inserted into the conventional vector pUC19 or pGEM-T Easy2. For detailed methods, see Sambrook, J., & Russell, DW Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001.

[0094] Other methods in the prior art may also be used to amplify the DNA template, and this application does not impose any specific limitation thereto.

[0095] In an alternative embodiment, the DNA template may introduce a T7 promoter sequence to support in vitro transcription. The T7 promoter sequence may be:

[0096] SEQ ID NO: 5: 5'-TAATACGACTCACTATAGGG-3'.

[0097] In an optional embodiment, the amplified target sequence is purified and detected: residual primers and polymerase in the PCR amplification product are removed by a column method or a magnetic bead method; the DNA concentration and purity are measured using a Nanodrop or Qubit quantifier to ensure that the OD260 / 280 is between 1.8 and 2.0 and the OD260 / 230 is >1.5.

[0098] For the column method, Qiagen PCR Purification Kit was used for purification. The specific steps are as follows:

[0099] Add 500 μL of binding buffer (Buffer PB in the kit) to every 100 μL of DNA to be purified. Mix thoroughly, then transfer to a QIAquick silica spin column. Centrifuge at 13,000 rpm for 1 minute and discard the filtrate. Then, add 750 μL of wash buffer (Buffer PE in the kit) and centrifuge again at 13,000 rpm for 1 minute to remove impurities. Spin for another 1 minute to remove residual ethanol. Finally, place the column in a clean 1.5 mL centrifuge tube and add 30-50 μL of elution buffer (Buffer EB in the kit). Incubate at room temperature for 1 minute, then centrifuge for 1 minute to collect the purified product. The PCR product purification steps described above can be performed according to the QIAGEN QIAquick PCR Purification Kit instructions.

[0100] Among them, for the magnetic bead method, AMPure XP beads are used for purification. The specific steps are as follows:

[0101] Add magnetic beads to the reaction solution to be purified, incubate at room temperature for binding, separate using a magnetic stand, wash twice with 80% ethanol, dry, and elute with an appropriate amount of eluent. The resulting supernatant is the purified product. For related methods, see the reference: Rohland N, Reich D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 2012;22(5):939-946.

[0102] In an optional embodiment, the transcription system includes the following components: template DNA, RNA polymerase, NTP mixture and transcription buffer.

[0103] In an optional embodiment, the components of the transcription buffer in the transcription system include the following reagents:

[0104] Tris-HCl, final concentration 35-45 mM;

[0105] MgCl2, final concentration 5.5-6.5 mM;

[0106] DTT, final concentration 5-10 mM;

[0107] Spermidine, final concentration 1.5-2.5 mM;

[0108] NaCl / KCl, final concentration 50-100 mM.

[0109] In an optional embodiment, the final concentration of the template DNA in the transcription system is 0.8-1.2 μg / μL.

[0110] In an optional embodiment, the final concentration of RNA polymerase in the transcription system is 1.8-2.2 U / μL.

[0111] In an optional embodiment, the final concentration of the NTP mixture in the transcription system is 0.8-1.2 mM each of ATP, CTP, GTP, and UTP.

[0112] In an optional embodiment, the transcription system further includes an RNase inhibitor with a final concentration of 0.8-1.2 U / μL.

[0113] In an optional embodiment, a transcription buffer is used to adjust the pH value of the transcription system to 7.5-8.

[0114] In an optional embodiment, the pH value of the transcription system is adjusted to 7.9 using a transcription buffer.

[0115] In an alternative embodiment, in vitro transcription is performed using the NEB HiScribe T7 kit.

[0116] In an optional embodiment, modified nucleotides are added to the transcription system, including m1Ψ-UTP, i.e., N1-methylpseudouridine; 5mC-CTP, i.e., 5-methylcytidine; or a combination of the two.

[0117] In an optional embodiment, the concentration of the modified nucleotide added to the transcription system is 0.8-1.2 mM.

[0118] In an optional embodiment, the concentration of the modified nucleotide added to the transcription system is 1 mM.

[0119] In an optional embodiment, a reaction buffer comprising DTT, MgCl2 or a combination thereof is added to the transcription system.

[0120] In an optional embodiment, the reaction conditions for in vitro transcription are 35-39° C. for 2-4 h.

[0121] In an alternative embodiment, the reaction temperature of in vitro transcription is 37°C.

[0122] In an optional embodiment, the in vitro transcription further includes a post-processing step: adding DNase I to remove the template DNA; and then performing LiCl precipitation or magnetic bead purification of the RNA.

[0123] In an alternative embodiment, the RNA is modified during in vitro transcription.

[0124] In an alternative embodiment, the modification of the RNA includes capping at the 5' end and adding Poly(A) at the 3' end.

[0125] In an alternative embodiment, the 5' end is capped using Vaccinia Capping enzyme, adding Cap 0, ie, m7GpppN; or Cap 1, ie, m7GpppNm.

[0126] In an alternative embodiment, Poly(A) is added to the 3' end using Poly(A) polymerase to add 150-200 A tails to mimic the structure of eukaryotic mRNA.

[0127] In an alternative embodiment, a 200 A tail is added to mimic the eukaryotic mRNA structure.

[0128] In an alternative embodiment, the quality of the RNA is tested before RNA fragmentation:

[0129] The concentration was measured using a Nanodrop micro-spectrophotometer;

[0130] The structure was evaluated using a Bioanalyzer and RNA 6000 Nano kit;

[0131] The integrity of the RNA was determined by the RNA Integrity Number (RIN) value; a RIN value > 8 indicated high-quality RNA.

[0132] In an alternative embodiment, RNA is fragmented using a metal ion induced method:

[0133] To achieve controlled fragmentation of RNA fragments, first prepare the reaction system: take the RNA template, add the buffer and a solution containing metal ions, and finally fill to the required volume with RNase-free ultrapure water. After thoroughly mixing the reaction mixture, place it in a thermal cycler and heat it at high temperature to induce RNA fragmentation. After the reaction is complete, immediately cool it to terminate the fragmentation reaction.

[0134] In an alternative embodiment, a buffer is used to adjust the pH of the RNA fragmentation reaction system to 7-8.

[0135] In an alternative embodiment, a buffer is used to adjust the pH of the RNA fragmentation reaction system to 7.5.

[0136] In an alternative embodiment, cooling in an ice bath is performed for more than 5 minutes.

[0137] RNA can also be fragmented using existing methods such as ultrasonic disruption and heat induction, which are not limited here.

[0138] In an alternative embodiment, the length of the fragmented RNA is 50-300 nt.

[0139] In an alternative embodiment, the length of the fragmented RNA is 150-200 nt.

[0140] In an optional embodiment, the above method further comprises the step of designing an automated synthesis system; the automated synthesis system comprises a microfluidic chip system or a robotic platform.

[0141] In an alternative embodiment, a microfluidic chip system made of PDMS, containing independent reaction chambers and an integrated temperature control module can achieve a single-chip daily output of >1,000 standard samples.

[0142] In an optional embodiment, the robotic platform includes the following modules:

[0143] Magnetic bead purification module for DNA / RNA purification.

[0144] Real-time fluorescence monitoring module for dynamic tracking of transcription efficiency.

[0145] The reagents used in the following examples are all conventional reagents in the art and, unless otherwise specified, were obtained from commercial sources. Specific preparation and testing methods, etc., are all conventional methods in the art or performed according to the instructions for use of the instrument / kit, unless otherwise specified.

[0146] Example 1

[0147] Preparation of RNA standards for detecting EGFR L858R mutation

[0148] 1. Obtain DNA template

[0149] (1) Obtaining target sequence

[0150] Download the EGFR gene from the NCBI database, which contains the L858R mutation site sequence:

[0151] SEQ ID NO: 6: 5'- TCC AGT GTC CCA AAC CAG GAC GTA CTC GTC-3' (T mutated to G resulting in L858R).

[0152] The wild-type sequence is:

[0153] SEQ ID NO: 7: 5'- TCC AGT GTC CCA AAC CAG AAC GTA CTC GTC-3'. Analysis revealed a CG content of 52% and a ΔG of -2.1 kcal / mol at the mutation site. BLAST analysis revealed an 8-bp match with EGFR exon 19, indicating partial homology. Therefore, this sequence was optimized.

[0154] Specifically, the homology region is avoided for truncation; some bases "GAC" in the high GC region are replaced with "GAT"; and the hairpin structure is destroyed by point mutation of the "C" site to "A" and "T".

[0155] The optimized target sequence is:

[0156] SEQ ID NO: 8: 5'-TCC AGT GTC CCA AAA TAG GAT GTA CTC GTC-3'.

[0157] Probes were designed based on the optimized target sequence for detection of target sequences in subsequent steps. The probe sequences are as follows:

[0158] Mutation probe:

[0159] SEQ ID NO: 9: 5'-CAG GAC GTA CTC GTC CCA AA-3';

[0160] GC content: 48%, Tm=65°C, ΔG = -0.3 kcal / mol.

[0161] Wild-type probe (control):

[0162] SEQ ID NO: 10: 5'-CAG AAC GTA CTC GTC CCA AA-3'.

[0163] The above-mentioned probes can be synthesized using techniques known in the art, which are not limited here.

[0164] (2) Construction of DNA template

[0165] The DNA template was the chr7: 55,240,500 - 55,239,000 (GRCh38) segment of the EGFR gene, totaling 1501bp.

[0166] The DNA template can be obtained by molecular cloning. After amplification by PCR, the DNA template is inserted into the pUC19 vector and a promoter sequence is introduced. For specific methods, refer to the book: Sambrook, J., & Russell, DW Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press, 2001.

[0167] After synthesis, the residual primers and polymerase in the DNA template solution were removed using magnetic beads (AMPure XP beads) to obtain the purified DNA template. For related methods, see the reference: Rohland N, Reich D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 2012;22(5):939-946. Magnetic beads were added to the PCR reaction solution, incubated at room temperature for binding, separated using a magnetic stand, and washed twice with 80% ethanol. After drying, the supernatant was eluted with an appropriate amount of elution buffer.

[0168] Determination of DNA concentration and purity: The template purity was determined using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Product Number: Q32851) according to the instructions. The result was 50 ng / μL, which is higher than the qualified standard of 20 ng / ml and meets the standard.

[0169] The OD260 / 280 measured by Nanodrop was 1.92, within the range of 1.8 to 2.0; the OD260 / 230 was 1.78, >1.5, indicating that the obtained DNA template was of good purity.

[0170] 2. In vitro transcription and modification

[0171] The primers synthesized for in vitro transcription were:

[0172] Upstream primer (containing T7 promoter):

[0173] SEQ ID NO: 11:

[0174] 5'-TAATACGACTCACTATAGGGGCAAGTGCCAGTGTGTGAAA-3';

[0175] Downstream primer:

[0176] SEQ ID NO: 12: 5'-CTGGCCATGGTGCCTGTA-3'.

[0177] In vitro transcription was performed using the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, NEB, Cat. No. E2050S / L) according to the manufacturer's instructions.

[0178] The composition of the transcription system is shown in Table 1:

[0179] Table 1 Composition of transcription system

[0180]

[0181] The composition of the T7 transcription buffer is shown in Table 2.

[0182] Table 2 Composition of T7 transcription buffer

[0183]

[0184] In vitro transcription was performed at 37°C for 3 hours. After transcription, the RNA concentration was measured using the Qubit RNA HS Assay and found to be 65 ng / µL, significantly exceeding the acceptable level of 50 ng / µL, indicating sufficient RNA for downstream modification.

[0185] After transcription is complete, DNase I (2 U, NEB, Cat. No. M0303S) is added to the reaction system and incubated at 37°C for 15 minutes to fully degrade any residual template DNA. RNA is then purified using magnetic beads. AMPure XP beads are used for RNA purification. The purification steps are as follows: add the beads to the transcription reaction, mix thoroughly according to the recommended ratio, and incubate at room temperature to allow full binding of the RNA to the beads. The beads are then separated on a magnetic stand and washed twice with 80% ethanol. After drying, the beads are eluted with an appropriate amount of RNase-free water or TE buffer. The supernatant is collected as the purified RNA sample.

[0186] Further modification of the purified RNA sample:

[0187] 5' end capping: Cap 0 (m7GpppN) using Vaccinia Capping Enzyme; incubate at 37°C for 30 min; the reaction system is shown in Table 3:

[0188] Table 3 5' end capping reaction system

[0189]

[0190] The 5' capping rate was determined to be 95% by LC-MS, exceeding the qualified standard of 90%. This truly simulates the mRNA structure and helps improve the stability of the final RNA standard.

[0191] Add Poly(A) to the 3' end: Use Poly(A) Polymerase to add 200 A tails to simulate the eukaryotic mRNA structure; incubate at 37°C for 40 min; the reaction system is shown in Table 4.

[0192] Table 4 3' end addition Poly (A) reaction system

[0193]

[0194] 3'Poly A addition efficiency was determined by RT-qPCR, using eukaryotic mRNA as a control. Reference: Livak KJ, et al. (1995). Oligonucleotides with fluorescent dyes for real-time PCR. PCR Methods and Applications, 4(6): 357-362.

[0195] The obtained results showed that ΔCt < 1, which was close to that of the control group, indicating that the tail length was sufficient and consistent.

[0196] 3. RNA quality testing

[0197] The in vitro transcribed and modified RNA was subjected to the following quality checks:

[0198] Purity detection: The OD260 / 280 was measured by Nanodrop micro-spectrophotometer and was 2.01, indicating that the obtained RNA was of high purity and had almost no protein contamination.

[0199] Structural evaluation: The Bioanalyzer bioanalyzer was used with the RNA 6000 Nano Kit to test the structural integrity of the transcribed and modified RNA samples. Figure 1 As shown. Figure 1 It can be seen that the main RNA peak is clear, there are no obvious degradation fragments, and the overall distribution is uniform, indicating that the RNA structure is well intact.

[0200] Integrity assessment: The RIN value (RNA Integrity Number) was 8.9, which is higher than the qualified standard of 8, indicating that the obtained RNA is of excellent quality and has not been significantly degraded.

[0201] 4. RNA fragmentation

[0202] (1) Fragmented RNA

[0203] Use the metal ion-induced method to fragment RNA as follows:

[0204] To achieve controlled fragmentation of RNA fragments, first prepare a 50 μL reaction system: Dispense the modified RNA to 50 ng / μL. Take 5 μL of this 50 ng / μL RNA, add 0.5 μL of 1 M Tris-HCl buffer (pH 7.5) and 2.5 μL of 1 M MgCl₂ solution, and top up to a total volume of 50 μL with RNase-free ultrapure water. Mix thoroughly and heat in a thermal cycler at 95°C for 8 minutes to induce RNA fragmentation. After the reaction is complete, immediately cool the reaction tube in an ice bath for at least 5 minutes to terminate the fragmentation reaction. After fragmentation, the RNA standard is obtained.

[0205] This standard contains the following core sequences:

[0206] Reference type: SEQ ID NO: 1:

[0207] 5'-AUGCGUCUUCACCUGCUGCUGGGUGCGAUG-3'.

[0208] Based on the aforementioned wild-type template, the optimized target sequence was introduced by site-directed mutagenesis primer PCR to prepare a mutant sequence, which includes the following core sequence:

[0209] Mutant: SEQ ID NO: 2:

[0210] 5'-AUGCGUCUUCACCUGCAGCUGGGUGCGAUG-3'.

[0211] (2) Verify whether the fragmented RNA standard contains mutant / reference core sequences

[0212] To verify whether the standards obtained by RNA fragmentation contain the designed reference and mutant core sequences, RT-qPCR was used for detection. The specific steps are as follows:

[0213] (i) Primer design

[0214] For SEQ ID NO: 1 and SEQ ID NO: 2, use Primer3Plus to design specific forward and reverse primers, respectively, and ensure that the Tm value is 58-60°C and the GC content is 40%-55%. The primer sequences are as follows:

[0215] Reference primers:

[0216] SEQ ID NO: 13: Upstream primer: 5′-ATGCGTCTTCACCTGCTGCT-3′;

[0217] SEQ ID NO: 14: Downstream primer: 5'-CATCAGCACCCAGCAGCAGG-3'.

[0218] Mutation primer pairs:

[0219] SEQ ID NO: 15: Upstream primer: 5′-ATGCGTCTTCACCTGCAGCT-3′;

[0220] SEQ ID NO: 16: Downstream primer: 5'-CATCAGCACCCAGCTGCTGG-3'.

[0221] (ii) RNA template preparation

[0222] Take the fragmented RNA standard and dilute it to 10 ng / μL with RNase-free water for use as a template for RT-qPCR.

[0223] (iii) RT-qPCR reaction

[0224] One-Step RT-qPCR Kit (Takara PrimeScript™ One Step RT-PCR Kit) was used for reverse transcription and real-time quantitative reaction. The reaction system is shown in Table 5:

[0225] Table 5 RT-qPCR reaction system

[0226]

[0227] The RT-qPCR reaction conditions were set as follows:

[0228] Reverse transcription: 42°C, 10 min;

[0229] Hot start: 95°C, 2 min;

[0230] PCR cycles (40 cycles):

[0231] 95℃, 5 sec;

[0232] 60°C, 30 sec (collect fluorescence signals).

[0233] The ΔCq method was used to determine whether the target sequence was detected. If the Cq value (the cycle number at which the fluorescence signal first exceeded the background threshold) in the experimental group was significantly lower than that in the negative control, and the reference / mutant primers only produced amplification signals for the corresponding templates, the fragmented RNA contained the corresponding sequence. The test results are shown in Table 6.

[0234] Table 6 RT-qPCR test results

[0235]

[0236] Reference for RT-qPCR detection method: Bustin SA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611-622.

[0237] (3) Detection of RNA fragment length distribution

[0238] After the reaction, the RNA fragment length distribution was detected using an Agilent Bioanalyzer 2100 as follows:

[0239] First, thoroughly mix the dye concentrate from the RNA 6000 Pico Kit (Agilent Technologies, Model 5067-1513) with the gel matrix to create a gel-dye mixture. Remove bubbles by centrifugation before use. Next, mix 1 μL of RNA standard with 1 μL of denaturing buffer, heat at 65°C for 2 minutes to eliminate RNA secondary structure, and immediately cool on ice.

[0240] Before loading samples onto the chip, add 9 μL of gel-dye mixture to the designated sample wells and 5 μL of ladder standard to the ladder wells. Add 1 μL of denatured RNA standard to each sample well. After loading, seal the chip, insert it into the Bioanalyzer 2100, and run the standard RNA Pico Assay protocol.

[0241] The entire analysis process takes about 30-40 minutes, and the system outputs an electrophoresis peak diagram showing RNA migration and fragment length distribution. Figure 2 As shown. Figure 2It can be seen that the peak is located at 150nt, with a range of 100-200nt; the peak is concentrated, the range is controllable, and the fragment distribution simulates the cfRNA distribution, which is suitable for liquid biopsy.

[0242] (4) RNA standard stability test

[0243] The RNA standards were subjected to a stability test for 7 days.

[0244] Stability testing was performed at various temperatures, including room temperature (25°C), refrigerated at 4°C, and frozen at -20°C. The test lasted for 7 days, with samples collected and tested on days 0, 1, 3, 5, and 7. Throughout the testing process, the RNA standards were protected from repeated freeze-thaw cycles and stored in a dark environment. RNA concentration was measured at each time point using a Qubit assay, and RIN values and fragment length distribution were determined using a Bioanalyzer 2100 to assess the stability of the RNA standards.

[0245] The results of Qubit RNA concentration determination (unit: ng / μL) are shown in Table 7.

[0246] Table 7 RNA standard concentration determination results

[0247]

[0248] The analysis results of Agilent Bioanalyzer 2100 are shown in Table 8.

[0249] Table 8 RNA standards analysis results using Agilent Bioanalyzer 2100

[0250]

[0251] The above results show that at -20°C, RNA concentration and integrity remained essentially unchanged, with no significant shift in the main fragment peak, making it the most suitable condition for long-term storage. At 4°C, RNA concentration decreased slightly, with the RIN value dropping from 8.9 to 8.1, indicating continued stability. Storage at 25°C resulted in rapid RNA degradation, with the RIN value dropping to 6.0 after 7 days, a nearly 40% decrease in concentration. Following the test, RNA concentration and RIN values were measured, showing a concentration of 98% and a RIN of 8.7, indicating that the RNA standard is suitable for bulk transport and storage.

[0252] 5. Design an automated synthesis system

[0253] Microfluidic chip system design:

[0254] Structure: PDMS material, containing 12 independent reaction chambers (each chamber volume 200 nL), integrated temperature control module, accuracy of ±0.1°C.

[0255] The parameter settings are as follows:

[0256] Pressure range: 0-1000 mbar;

[0257] Recommended working pressure: 300-800 mbar, adjusted according to flow resistance and liquid viscosity;

[0258] Response time: <100 ms;

[0259] Pressure stabilization time: ≤500 ms (from set pressure to steady state);

[0260] Pressure resolution: <1 mbar;

[0261] Control accuracy: ±0.5%, full range;

[0262] Liquid exchange cycle switching time: <300 ms, ensuring no residue when switching between reagents.

[0263] The Fluigent MFCS-EZ pressure controller enables precise flow switching, enabling a single chip to produce >1,000 standards per day.

[0264] Example 2

[0265] Preparation of PIK3CA E545K mutation standards

[0266] The preparation method of the standard in this example is as follows:

[0267] Reference type: SEQ ID NO: 3:

[0268] 5'-GAGAGACAAUGAAUUAAGGGAAAGAAGAAA-3';

[0269] Mutant SEQ ID NO: 4:

[0270] 5'-GAGAGACAAUGAAUUAAGGAAAAGAAGAAA-3'.

[0271] To verify the performance and sensitivity of the prepared PIK3CA E545K mutant RNA standard in molecular detection, digital PCR (ddPCR) and real-time quantitative PCR (qPCR) were used to analyze and evaluate the standard.

[0272] In this example, a series of concentration gradient dilutions were prepared using a mutant RNA standard (SEQ ID NO: 3) and its corresponding reference RNA standard (SEQ ID NO: 4). The gradient included: 10 6 , 10 5 , 10 4 , 10³, 10², and 10¹ copies / μL. A fixed concentration of reference RNA standard was added to each concentration to simulate the background interference conditions of mutant alleles in clinical samples.

[0273] The samples were tested sequentially using a digital PCR system and a real-time fluorescence quantitative PCR system, using primers and probes specifically designed for the PIK3CA E545K mutation site. The digital PCR system enables absolute quantification at the single-molecule level, while the real-time fluorescence PCR system employs an allele-specific amplification strategy to differentiate between mutant and wild-type genes.

[0274] The test results show:

[0275] In the digital PCR system, the minimum detectable copy number of the mutant standard is 10 copies / reaction, and the detection signal shows a good linear relationship with the input copy number, with a correlation coefficient (R²) greater than 0.99, demonstrating high quantitative accuracy.

[0276] In the real-time fluorescence PCR system, the mutant primers can be well distinguished from the reference type, with a minimum detection sensitivity of 100 copies / reaction, good signal reproducibility, and a coefficient of variation (CV) of less than 10%.

[0277] Comparative Example 1

[0278] Stability testing

[0279] The stability test of the cfRNA sample prepared by the TRIzol method was performed according to the same test method and test conditions as the RNA standard stability test in Example 1.

[0280] The extraction steps of TRIzol method are as follows:

[0281] (1) Take a healthy human plasma sample and add 3 times the volume of TRIzol reagent (Thermo Fisher). Immediately mix vigorously with a pipette or vortex for 15-30 seconds to ensure sufficient lysis. Let it stand at room temperature for 5-10 minutes.

[0282] (2) Add 0.2 times the volume of the mixture in chloroform, cap the tube tightly, shake vigorously for 15-30 seconds (or vortex) to fully emulsify the solution, let it stand at room temperature for 2-5 minutes; then centrifuge at 12,000×g for 15 minutes at 2-8°C.

[0283] (3) Carefully aspirate the upper aqueous phase and transfer it to a new RNase-free centrifuge tube. Add an equal volume of isopropanol and gently invert several times to mix. Let it stand at room temperature for 10 minutes. Before centrifugation, a flocculent gel-like precipitate can be seen on the side walls and bottom of the tube. This is the RNA precipitate.

[0284] (4) Remove the supernatant, add 1 ml of 75% ethanol to wash the RNA precipitate, mix well on a shaker, and centrifuge at 7,500 × g for 5 min at 2-8°C.

[0285] (5) Remove the supernatant, place in vacuum or in air for 5-10 minutes, dry the RNA precipitate, resuspend the RNA precipitate in RNase-free water after drying, and measure the concentration to obtain the cfRNA sample.

[0286] The cfRNA samples extracted by the above method were stored at 4°C and sampled and tested on days 0, 1, 3, 5, and 7. The test results are shown in Table 9.

[0287] Table 9 Stability test results of cfRNA samples extracted by TRIzol method

[0288]

[0289] The RNA standard of this application is compared with the above cfRNA. Figure 6 The above results show that TRIzol-extracted RNA showed significant degradation after 5 days of storage at 4°C, with concentration dropping by more than 15%. The Bioanalyzer showed significantly abnormal RNA fragment lengths. However, under the same conditions, the concentration of this standard decreased by less than 8%, and the fragment distribution was concentrated, maintaining a high degree of consistency.

[0290] Test Example 1

[0291] Clinical test calibration

[0292] 1. Used for ddPCR threshold setting

[0293] The mutant standard obtained in Example 1 was diluted in a gradient manner (10-10^6 copies / μL) to establish a standard curve. Figure 3The horizontal axis represents the standard concentration (expressed in copies / μL), and the vertical axis represents the positive titer detected in the ddPCR system. As can be seen from the figure, as the standard concentration decreases, the corresponding positive titer decreases linearly, consistent with the expected exponential dilution pattern. The curve fitting results show a correlation coefficient R² of 0.9998, significantly higher than 0.99, indicating good linearity and reproducibility of the detection system. The standard is stable and reliable, and can be used to accurately establish the positive threshold range for ddPCR.

[0294] Furthermore, a standard curve was established using commercially available RNA standards (Thermo Fisher) according to the above steps, e.g. Figure 7 shown.

[0295] The results of comparison between the RNA standards of the present application and commercially available RNA standards are shown in Table 10.

[0296] Table 10 Threshold setting comparison results

[0297]

[0298] This shows that the standard product of this application still maintains better linearity and repeatability in the low copy concentration area (10-100 copies / μL).

[0299] 2. qPCR detection sensitivity determination:

[0300] To detect the EGFR L858R mutation, a TaqMan fluorescent probe was used for fluorescent qPCR. The designed probe is located in the central region of the mutated base (T to G) and has good specificity and thermal stability. The fluorescent probe contains a 5'-fluorophore (FAM) and a 3'-quencher (BHQ1). Reference for the assay method: Livak KJ, et al. (1995). Oligonucleotides with fluorescent dyes for real-time PCR. PCR Methods and Applications, 4(6): 357–362.

[0301] Mutant TaqMan probes:

[0302] SEQ ID NO: 17: 5'-FAM-CAGGACGT ACTCGTCCCAAA-BHQ1-3';

[0303] Reference TaqMan probes:

[0304] SEQ ID NO: 18: 5'-FAM-CAGAACGT ACTCGTCCCAAA-BHQ1-3'.

[0305] First, the RNA mutant standard was reverse transcribed into cDNA, and then 10 4 A serial dilution was performed at 10, 10³, 10², and 10¹ copies / μL, and a standard curve was established. Three replicates were run at each concentration. PCR was performed using the ABI 7500 Real-Time PCR System. After a 2-minute initial denaturation at 95°C, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute were performed. Fluorescence signals were captured using the mutation-specific FAM channel.

[0306] The results of the test are as follows Figure 4 shown.

[0307] Figure 4 The horizontal axis is log10 (copies / μL), and the vertical axis is Ct value. The points are measured data, and the line is the linear fit curve. The fitting equation is: Ct = -3.26 × log10 (copies) + 32.63. The goodness of fit R² = 0.9998, indicating a high linear correlation between Ct value and template concentration. The minimum detectable copy number is 10 copies / μL, and the Ct standard deviation is < 0.5, indicating high reproducibility.

[0308] The determination results show that the standard has excellent sensitivity and quantitative reliability.

[0309] Furthermore, the assay results of the standard of the present application were compared with commercially available RNA standards (Thermo Fisher), and the results are shown in Table 11.

[0310] Table 11 qPCR sensitivity comparison

[0311]

[0312] The above results show that both performed well, but at low concentrations (10 1 When the Ct value of this standard is more stable and the background is lower, the

[0313] 3. Next-generation sequencing (NGS) detection sensitivity determination:

[0314] Using an EGFR L858R mutant RNA standard as a model, cDNA was reverse transcribed and spiked into a healthy individual's cfDNA background at MAF ratios of 0.5%, 0.1%, 0.05%, and 0.02% to create mixed samples. Each sample was prepared using the KAPA HyperPrep kit and sequenced using the Illumina NovaSeq 6000 platform (2 × 150 bp) at an average sequencing depth of 10,000×. Target enrichment was performed using a custom panel. Sequencing data were then subjected to BWA alignment, GATK variant detection, and LoFreq frequency assessment to generate corresponding mutation detection values.

[0315] The results of the test are as follows Figure 5 shown.

[0316] Furthermore, the assay results of the RNA standards of the present application were compared with commercially available RNA standards (Thermo Fisher), and the detection rate results are shown in Table 12.

[0317] Table 12 NGS sensitivity comparison results

[0318]

[0319] As can be seen from the figure, the measured mutation frequency is highly consistent with the expected value, and the minimum detection limit reaches 0.02% MAF, indicating that the standard has extremely high sensitivity and quantitative stability, and is fully suitable for the detection of ultra-low-frequency mutations of ctRNA in tumor liquid biopsies.

[0320] Test Example 2

[0321] Clinical test calibration

[0322] To enhance the specificity and accuracy of the detection system, a reference RNA standard was introduced as a negative control based on Experiment 1. Its use was consistent with the mutant standard described above. This reference standard effectively sets the detection platform's positive threshold, assesses nonspecific signals, and verifies the system's background noise, helping to improve the ability to discriminate low-frequency mutations in clinical applications.

[0323] 1. ddPCR threshold setting:

[0324] Reference standards are used to construct background signal distributions, helping to define positive signal thresholds and negative intervals, and reducing false positives. Simultaneous testing of reference and mutant standards ensures the sensitivity and specificity of the ddPCR system.

[0325] 2. qPCR sensitivity determination:

[0326] The reference standard was prepared at the same concentration gradient as the mutant standard (e.g. 104 The samples were diluted to 10, 10, 10 copies / μL (copies / μL) and tested in parallel as negative controls. The negative threshold was determined by measuring the Ct value, excluding nonspecific amplification or probe hybridization, to ensure the specificity and accuracy of the mutant signal.

[0327] 3. NGS sensitivity verification:

[0328] After reverse transcription of reference RNA standards into cDNA, samples with no mutations or low mutation frequencies are spiked into healthy human cfDNA at varying ratios. These samples are used to assess sequencing error rates, false positive levels, and background noise, and to correct for variant frequency detection accuracy. This negative control sample helps accurately identify low-frequency mutation signals.

[0329] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A tumor circulating nucleic acid standard suitable for liquid biopsy, characterized in that: The standard products include standard product 1 and standard product 2; The standard 1 includes RNA with the sequence shown below: SEQ ID NO: 1: 5'-AUGCGUCUUCACCUGCUGCUGGGUGCGAUG-3'; and / or SEQ ID NO: 2: 5'-AUGCGUCUUCACCUGCAGCUGGGUGCGAUG-3'; The standard 2 includes RNA with the sequence shown below: SEQ ID NO: 3: 5′-GAGAGACAAUGAAUUAAGGGAAAGAAGAAA-3′; and / or SEQ ID NO: 4: 5'-GAGAGACAAUGAAUUAAGGAAAAGAAGAAA-3'.

2. The tumor circulating nucleic acid standard according to claim 1, characterized in that: The standards also include a lysis protectant.

3. The tumor circulating nucleic acid standard according to claim 2, characterized in that: The lysis protection agent includes at least one of RNAlater stabilizing solution, glycerol and DMSO.

4. The tumor circulating nucleic acid standard according to any one of claims 1 to 3, characterized in that The tumor includes at least one of lung cancer, colon cancer and breast cancer.

5. Use of the tumor circulating nucleic acid standard according to any one of claims 1 to 3 in preparing a kit for detecting tumors in a subject.

6. The use according to claim 5, characterized in that The standard 1 is used to detect the EGFR L858R mutation, and the standard 2 is used to detect the PIK3CA E545K mutation.

7. A kit for detecting circulating tumor nucleic acids for liquid biopsy, characterized in that: The invention comprises the tumor circulating nucleic acid standard according to any one of claims 1 to 3.

8. A method for synthesizing a tumor circulating nucleic acid standard according to any one of claims 1 to 3, characterized in that: The steps include: Obtaining a DNA template: selecting a sequence containing a cancer driver gene and a high-frequency mutation site as the original target sequence, optimizing it, and then amplifying the optimized target sequence to obtain the DNA template; In vitro transcription: preparing a transcription system solution and transcribing the DNA template in vitro using the transcription system solution, purifying the transcription product to obtain an RNA product; and RNA fragmentation: The RNA product is fragmented to obtain RNA standards, and the RNA standards are mixed with a lysis protective agent to obtain the tumor circulating nucleic acid standards.

9. The synthesis method according to claim 8, characterized in that The steps of optimizing the original target sequence include: avoiding the homology region and selecting a 15-22 bp region containing the mutation site as the target sequence; adjusting the CG content of the target sequence to less than 60%; eliminating the secondary structure of the target sequence to obtain an optimized target sequence.

10. The synthesis method according to claim 8, characterized in that The method further comprises the step of designing an automated synthesis system, wherein the automated synthesis system comprises a microfluidic chip system or a robotic platform.

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