Nucleic acid molecule library and application thereof in gastric cancer detection

Abstract

The invention belongs to the field of biological medicine, and relates to a nucleic acid molecule library and application thereof in gastric cancer detection. The nucleic acid molecule library is prepared by a method comprising the following steps: (1) obtaining a nucleic acid molecule with an anchor primer at one end, wherein one single chain in the nucleic acid molecule is complementary with a treated target nucleic acid molecule; (2) carrying out PCR (Polymerase Chain Reaction) amplification by using an anchor primer or an inner side primer of the anchor primer and a primer of a 5'end upstream sequence of the target gastric cancer CpG short tandem marker by taking a product of the step (1) as a template; (3) carrying out PCR (Polymerase Chain Reaction) amplification by using a CpG short tandem primer and adding an anchoring sequence by taking a product in the step (2) as a template; and (4) taking the product in the step (3) as a template, and carrying out PCR (Polymerase Chain Reaction) amplification by using a linker primer capable of annealing with the anchoring sequence. The nucleic acid molecule library provided by the invention can effectively detect or diagnose gastric cancer, and has good sensitivity and specificity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a nucleic acid molecule library and its use for gastric cancer detection. The present application also relates to a gastric cancer detection method based on peripheral blood multi-target DNA methylation. BACKGROUND

[0002] Gastric cancer is a serious disease that endangers human health. There are more than 350,000 new cases of gastric cancer in China each year, and more than 260,000 deaths. Current clinical detection methods for gastric cancer mainly include gastroscopy, imaging examination, and tumor marker detection. However, gastroscopy has invasiveness and complication risks, and patient compliance is poor. For patients with complex lesion locations, it may be difficult to obtain biopsy specimens. Imaging examination has a risk of radiation exposure, and the recognition rate of early lesions is low. The detection sensitivity of traditional blood tumor markers such as CEA, CA19-9, and CA125 is low.

[0003] During the development of gastric cancer, methylation abnormalities affect the expression of related genes. Researchers have found that the occurrence of cancer can be detected by detecting the methylation level of specific sites. Circulating cell-free DNA (ccfDNA) refers to DNA in the circulating blood that is in an extracellular free state. In recent years, the "liquid biopsy" technology for ccfDNA has rapidly developed, showing good prospects for application in clinical cancer screening and monitoring.

[0004] A DNA methylation detection method for gastric cancer has been reported in the literature (2022 Clinical Chemistry, Genome-Scale Methylation Analysis of Circulating Cell-Free DNA in Gastric Cancer Patients, PMID: 34791072). This method uses methylation CpG short tandem amplification and sequencing (MCTA-Seq) technology, and detects gastric cancer based on peripheral blood using CGCGCGG short tandem primers (targeting 20525 CGCGCGG, 9373 CpG islands). The results show that a panel consisting of 153 methylated CGCGCGG-CpGs (where all 3 Cs of CGCGCGG are methylated, and the first to nth CpGs downstream are also all methylated) can effectively detect gastric cancer. However, this method has limitations such as complicated purification steps, low CpG short tandem capture efficiency, and coverage of non-target sites, resulting in time-consuming detection and low sensitivity, which is not conducive to commercialization.

[0005] DNA methylation refers to the chemical modification of DNA cytosine (C) by adding a methyl group to form methylcytosine. It is the main epigenetic modification on mammalian genomic DNA, and almost all of it occurs on CpG dinucleotides. CpG dinucleotides are not randomly distributed in the genome: in the genome of normal human adult cells, on the one hand, CpG sites scattered throughout the genome are mostly in a methylated state, while on the other hand, CpG sites highly concentrated in genomic regions called CpG islands (CGIs) are mostly in a demethylated state. CpG islands are mainly located in the promoter and exon regions of genes. They are regions rich in CpG dinucleotides and are generally 300-3000bp in length.

[0006] DNA methylation plays a crucial role in the transcriptional regulation of CpG islands. Demethylation is a prerequisite for active transcription at CpG island promoters. Once methylated, these regions recruit a series of factors, including methylated DNA-binding proteins and histone deacetylases, leading to transcriptional silencing. CpG island hypermethylation and genome-wide hypomethylation are common phenomena in human tumors and contribute to tumor development and progression. Furthermore, DNA methylation is involved in the regulation of numerous physiological processes, including X chromosome inactivation, transposon silencing, genomic imprinting, embryonic stem cell differentiation, germ cell development, and learning and memory. For a review, see Deaton and Bird, 2011.

[0007] With existing technologies, whole-genome DNA methylation testing is very expensive, and single-site DNA methylation testing provides very little information. Therefore, the development of effective targeted detection methods is needed. If targeted capture is performed before bisulfite treatment, the bisulfite treatment results in significant sample loss. If capture is performed after bisulfite treatment, the steps are cumbersome and require addressing methylation heterogeneity. Furthermore, capturing highly fragmented DNA samples is a technical challenge. Many clinical samples, such as circulating free DNA and formaldehyde-paraffin-fixed samples, are already highly fragmented, making the technical challenge even greater.

[0008] Therefore, it is necessary to develop a gastric cancer detection method based on multi-target DNA methylation in peripheral blood with higher targeting performance and simpler operation. Summary of the Invention

[0009] After in-depth research and creative work, the present inventors established a targeted amplification method based on a combination of 68 gastric cancer mCGCGCGG-CpG markers.

[0010] In the examples, the inventors used the targeted detection method of the 68 marker combination to detect ccfDNA in plasma samples from gastric cancer patients (n=101) and control group participants (n=177). The classifier formed by the 68 primer combinations had a sensitivity of 63.3% (19 / 30) for stage I, 76.6% (23 / 30) for stage II, and 92.7% (38 / 41) for stage III for gastric cancer, with a specificity of 89.3% (158 / 177). The following invention is provided:

[0011] One aspect of the present invention relates to a nucleic acid molecule library, which is prepared by a method comprising the following steps:

[0012] (1) obtaining a nucleic acid molecule having an anchor primer at one end, wherein a single strand of the nucleic acid molecule is complementary to a treated target nucleic acid molecule; wherein the treated target nucleic acid molecule is obtained by subjecting the target nucleic acid molecule to the following treatment: converting unmethylated cytosine in the target nucleic acid molecule into uracil, while not converting methylated cytosine into uracil;

[0013] (2) using the product of step (1) as a template, performing PCR amplification using an anchor primer or an inner primer of the anchor primer, and a primer targeting the 5' upstream sequence of the gastric cancer CpG short tandem marker (gastric cancer-specific CpG short tandem) (CpG short tandem outer targeting primer);

[0014] (3) using the product of step (2) as a template, performing PCR amplification using CpG short tandem primers and adding an anchor sequence;

[0015] (4) using the product of step (3) as a template, and performing PCR amplification using an adapter primer that can anneal to the anchor sequence (the second anchor sequence, preferably corresponding to the i7 portion of the Illumina True-Seq sequencing adapter);

[0016] Wherein, the PCR amplification in step (3) and step (4) is performed in one reaction system, or in two reaction systems in sequence.

[0017] Without being bound by theory, the product of step (1) is double-stranded. In one embodiment, only the complementary strand has an anchor primer, and the template strand does not. Therefore, the phrase "complementary to the treated target nucleic acid molecule" in step (1) means that the complementary strand in the product of step (1) is "complementary to the treated target nucleic acid molecule."

[0018] In some embodiments of the present invention, the nucleic acid molecule library is a DNA library.

[0019] In some embodiments of the present application, the nucleic acid molecule library, wherein step (1) comprises step 1A or step 1B as follows:

[0020] 1A. converting unmethylated cytosine in the target nucleic acid molecule into uracil while methylated cytosine is not converted, and then using a DNA polymerase to amplify using random primers or semi-random primers with unique molecular identifiers (UMI sequences) attached to obtain nucleic acid molecules complementary to the treated target nucleic acid molecules; preferably, the DNA polymerase is Klenow (exo-);

[0021] 1B. performing end repair, A-tailing and anchoring primer ligation on the target nucleic acid molecule, and then converting unmethylated cytosine therein into uracil while methylated cytosine is not converted, and then using a DNA polymerase to amplify to obtain nucleic acid molecules complementary to the treated target nucleic acid molecules; or converting unmethylated cytosine therein into uracil while methylated cytosine is not converted, and then anchoring primer ligation is performed on the treated target nucleic acid molecule, and then using a polymerase to amplify to obtain nucleic acid molecules complementary to the treated target nucleic acid molecules.

[0022] In the latter method of step 1B, methods known in the art can be referred to, for example, the method described in the document Non-invasive early detection of cancer four years before conventional diagnosis using a blood test PMID:32694610. Without being limited by theory, this method adds an anchoring primer to the template molecule, and then amplifies using the anchoring primer sequence, so it has an anchoring primer on both the template strand and the complementary strand.

[0023] In some embodiments of the present application, the nucleic acid molecule library, wherein in step 1A or step 1B, the unmethylated cytosine is converted into uracil (methylated cytosine is not converted) by treating the target nucleic acid molecule with bisulfite or treating the target nucleic acid molecule with cytosine deaminase (e.g., TET methylcytosine dioxygenase 2 and APOBEC3A enzyme).

[0024] In some embodiments of the present application, the unmethylated cytosine is converted into uracil by treating the target nucleic acid molecule with TET methylcytosine dioxygenase 2 (TET2) and APOBEC3A enzyme.

[0025] The treatment using TET methylcytosine dioxygenase 2 and APOBEC3A enzyme includes the steps of: using TET methylcytosine dioxygenase 2 to catalyze the oxidation of 5-methylcytosine (5mC) to generate 5-carboxylcytosine (5caC), and then using APOBEC3A enzyme to specifically deaminate, converting unmethylated cytosine (C) into uracil (U) (5caC is not deaminated due to structural differences). Without being limited by theory, in subsequent amplification, the recognition of 5-carboxylcytosine by DNA polymerase is similar to cytosine, not uracil. DNA polymerase recognizes uracil as thymine (T), which directly pairs with guanine (A), so that uracil will be converted to thymine after amplification. And it still recognizes 5-carboxylcytosine as cytosine, which pairs with adenine (G), and is still cytosine after amplification.

[0026] In some embodiments of the present application, the nucleic acid molecule library, wherein in step 1A, the semi-random primer comprises, from 5' end to 3' end: an anchor sequence (a first anchor sequence, preferably corresponding to the i5 part of the illumina true-seq sequencing adapter), a UMI sequence, and a semi-random sequence comprising a CpG site;

[0027] Preferably, the semi-random primer comprises a sequence as shown in SEQ ID NO: 69 to SEQ ID NO: 72.

[0028] In some embodiments of the present application, the nucleic acid molecule library, wherein in step (1), the nucleic acid molecule having an anchor primer at one end and complementary to the target nucleic acid molecule after treatment is obtained by amplification (polymerase chain reaction); preferably, dUTP is added during amplification, so that uracil is incorporated into the complementary nucleic acid molecule. In some embodiments of the present application, the number of moles of dUTP added is 1-3 times, for example 1.5-2.5 times, 1.8-2.2 times or 2 times the number of moles of dNTP (dATP, dCTP or dGTP).

[0029] In some embodiments of the present application, the nucleic acid molecule library, wherein in step (2), the step of removing uracil residues is further included.

[0030] Preferably, uracil-DNA glycosylase (UDG) is used to remove uracil residues.

[0031] In some embodiments of the present application, the nucleic acid molecule library, wherein in step (2), no dUTP is added in the PCR amplification.

[0032] In some embodiments of the present application, the nucleic acid molecule library, wherein in step (2),

[0033] the sequence of the inner primer of the anchor primer is set forth in SEQ ID NO: 73; and / or

[0034] the sequence of the CpG short tandem is selected from the group consisting of CGCGCGG, CGGCGGCGG, CGCGCGA, CGCGCGT and CGACGACGA;

[0035] Preferably, the length of the primer targeting the sequence upstream of the 5' end of the gastric cancer CpG short tandem marker is 16-32 bp or 18-28 bp, and / or the distance between the 3' end of the primer and the 5' end of the CpG short tandem is within 25 bp or within 20 bp.

[0036] Preferably, the sequence of the primer targeting the sequence upstream of the 5' end of the gastric cancer CpG short tandem marker is selected from any 10, any 20, any 30, any 40, any 50, any 60, all 68, 30-68, 40-68, 50-68 or 60-68 sequences of SEQ ID NO: 1 to SEQ ID NO: 68.

[0037] Without being bound by theory, preferably, the length of the primer targeting the sequence upstream of the 5' end of the gastric cancer CpG short tandem marker is in principle not more than 32 bp or 28 bp (except for individual cases), and the distance between the 3' end of the primer and the 5' end of the CpG short tandem (e.g. CGCGCGG) sequence is in principle within 25 bp or within 20 bp (except for individual cases), that is, the quantification range of the "sequence upstream of CGCGCGG" is preferably within 32+25 bp, 32+20 bp, 28+25 bp or 28+20 bp, i.e. about 48-57 bp. The "sequence upstream of CGCGCGG" is different for different CpG short tandem. After bisulfite or enzyme treatment, the CGCGCGG upstream sequence has a commonality, for example, after bisulfite treatment, the unmethylated C (C of the di-nucleotide) of the sequence is converted to U, so that the C content in the primer is very small, mostly G / A / T.

[0038] In some embodiments of the present application, the gastric cancer CpG short tandem marker is a gastric cancer marker nucleic acid molecule comprising or complementary to a CpG short tandem. In some embodiments of the present application, the gastric cancer CpG short tandem marker is a gastric cancer CpG short tandem marker reported in the prior art. In some embodiments of the present application, the gastric cancer CpG short tandem marker is as shown in Table 1.

[0039] Without being bound by theory, there are several benefits to designing primers upstream: 1) Increasing the number of original molecules captured. Because CGCGCGG is relatively short, the capture efficiency of original molecules is low (approximately 5-10% of molecules are captured). However, using targeted primers for enrichment can greatly increase the capture efficiency of original molecules (estimated to increase to 50-100%). 2) Bioinformatics analysis is very convenient. Since the targeted primer is upstream, all reads begin with CGCGCGG, exactly the same as the original MCTA-Seq, so analysis is very convenient. If it is designed downstream, each site will have a different sequence, and the workload of analysis is huge.

[0040] In some embodiments of the present invention, the nucleic acid molecule library, wherein in step (3),

[0041] The sequence of the short tandem CpG was selected from CGCGCGG, CGGCGGCGG, CGCGCGA, CGCGCGT, and CGACGACGA;

[0042] Preferably, the sequence of the CpG short tandem primer is shown as SEQ ID NO: 74.

[0043] In some embodiments of the present invention, the nucleic acid molecule library, wherein in step (4),

[0044] The sequences of the linker primers are shown in SEQ ID NO:75 and SEQ ID NO:76.

[0045] In some embodiments of the present invention, the nucleic acid molecule library, wherein the PCR in step 1A is performed for one or more rounds (e.g., two or three rounds);

[0046] Preferably, when multiple rounds are performed, Klenow (exo-) is supplemented to the reaction system.

[0047] In some embodiments of the present invention, the nucleic acid molecule library further comprises the step of purifying the products obtained by PCR amplification in step 1A, step (2), step (3) and / or step (4).

[0048] In some embodiments of the present invention, the nucleic acid molecule library, wherein,

[0049] The target nucleic acid molecule is a DNA molecule;

[0050] Preferably, the target nucleic acid molecule is derived from mammalian cells, tissues, blood or urine, such as human, preferably from human plasma or serum.

[0051] Another aspect of the present invention relates to a primer combination comprising:

[0052] Primer A, sequences shown in SEQ ID NO:69 to SEQ ID NO:72;

[0053] Primer B, the sequence is shown in SEQ ID NO:73;

[0054] Sequence C, which is selected from any 10, any 20, any 30, any 40, any 50, any 60, all 68, 30-68, 40-68, 50-68 or 60-68 sequences of SEQ ID NO: 1 to SEQ ID NO: 68;

[0055] Sequence D, the sequence is shown in SEQ ID NO:74;

[0056] Sequence E, the sequence of which is shown in SEQ ID NO:75; and

[0057] Sequence F, the sequence is shown in SEQ ID NO:76.

[0058] Another aspect of the present invention relates to use of any one of the nucleic acid molecule libraries or primer combinations of the present invention in preparing a drug or a kit for detecting gastric cancer.

[0059] Another aspect of the present invention relates to a kit for detecting gastric cancer, comprising the primer combination of the present invention and a PCR reagent.

[0060] Another aspect of the present invention relates to a method for detecting or diagnosing gastric cancer, comprising the step of sequencing the nucleic acid molecule library described in any one of the present invention; preferably,

[0061] When the total number of marker molecules (i.e., the total number of UMIs) is greater than or equal to 9 (e.g., the 68 markers in the present invention), the result is judged to be positive or the risk of gastric cancer is high (further screening or diagnosis is required);

[0062] When the total number of marker molecules (eg, the 68 markers in the present invention) is less than 9, the result is judged to be negative or the risk of gastric cancer is low.

[0063] In this application, the term "MCTA-Seq" (methylated CpG short tandem amplification sequencing) refers to a technology that selectively amplifies short tandem CGCGCGG sequences of methylated CpGs to enrich for methylated CpG islands, thereby enabling non-invasive detection of tumors and other diseases. MCTA-Seq technology performs three reactions in a single tube: in the first stage, linear amplification of bisulfite-treated DNA using random or semi-random primers (attached with unique molecular identifier (UMI) sequences) is performed to obtain CG-rich genomic regions; in the second stage, primers ending with CGCGCGG sequences are added to selectively amplify CpG tandem sites; and in the third stage, PCR amplification is performed using index primers with anchor sequences to obtain the final library.

[0064] As used herein, the term "short CpG tandem" refers to a sequence greater than or equal to 7 nucleotides in length, with 2 or 3 CpGs present within the first 7 nucleotides from the 3' end. In preferred embodiments, the sum of Cs and Gs within the first 10 nucleotides from the 3' end is greater than or equal to 7. Examples of short CpG tandems include, but are not limited to, CGCGCGG, CGGCGGCGG, CGCGCGA, CGCGCGT, and CGACGACGA.

[0065] In the present invention, the term "CpG short tandem primer" refers to a primer comprising the above-mentioned CpG short tandem or complementary to the above-mentioned CpG short tandem.

[0066] In the present invention, the term "CGCGCGG-CpG" refers to a genomic region between a short CpG sequence CGCGCGG and a specific CG downstream thereof. In some embodiments of the present invention, "CGCGCGG-CpG" is as shown in Table 1.

[0067] In the present invention, the term "targeting" a nucleic acid sequence means that the primer comprises a sequence that is at least partially complementary to the nucleic acid sequence (also referred to as a target sequence or target nucleic acid sequence) and is thus capable of hybridizing to the nucleic acid sequence. As used herein, the "targeting sequence" of a primer means a sequence comprised by the primer that is at least partially complementary to the target nucleic acid sequence.

[0068] In the present invention, "random primers" means primers having random sequences (see, for example, US Pat. Nos. 5,043,272 and 5,106,727, which are incorporated herein by reference).

[0069] Random primers can be generated using available oligonucleotide synthesis procedures; randomness in sequence can be introduced by providing a mixture of nucleotide residues in the reaction mixture in one or more addition steps (to produce a mixture of oligonucleotides having a random sequence). Thus, random primers can be generated by sequentially incorporating nucleotide residues from a 25% mixture of dATP, dCTP, dGTP, and dTTP to form oligonucleotides. Other ratios of dNTPs can be used (e.g., increasing or decreasing the ratio of any one or more dNTPs and adjusting the ratios of the other dNTPs so that the total amount is 100%).

[0070] The term "random primers" specifically includes a collection of individual oligonucleotides of varying sequence, which can be represented, for example, by the general formula 5'-XXX...XX-3', where X represents a nucleotide residue from a mixture of dNTPs with a custom percentage added to the oligonucleotide. For example, if the mixture contains 25% each of dATP, dCTP, dGTP, and dTTP, the random primers shown will contain a mixture of oligonucleotides with approximately a 25% probability of having an A, C, G, or T at each position.

[0071] In the present invention, the term "semi-random primer" refers to a primer in which a portion of the sequence is fixed (known) and the other portion is random. As actually employed in the examples of the present invention, the primer contains C, while the other positions are H (A, T, and C have equal probability of appearing in H) and are degenerate. The length of the semi-random primer is greater than or equal to 4 nucleotides and can bind to the nucleic acid molecule that has undergone cytosine to uracil conversion. The second nucleotide at the 3' end of the semi-random primer is C, and the first 7 nucleotides from the 3' end of the primer contain 1 CpG. Apart from this, the primer only contains C, A, and T. In a preferred embodiment, the semi-random primer is a mixture of equal proportions of four primer molecules: 5'-HHHCGCH-3', 5'-HHCGHCH-3', 5'-HCGHHCH-3', and 5'-CGHHHCH-3'.

[0072] As used herein, the term "anchor sequence" refers to a sequence that is at least partially complementary to a portion of a sequencing adapter or a portion of a vector in a DNA library and is capable of hybridizing to the sequencing adapter or the portion of the vector. The length of the anchor sequence is generally greater than or equal to 10 bases, for example, 10-200, 20-100, 30-90, 40-80, 50-70, or 50-60 bases.

[0073] In the present invention, the term "anchor primer" refers to a primer comprising an anchor sequence, which can hybridize with a partial sequence of a sequencing adapter or a partial sequence of a vector in a DNA library.

[0074] In the present invention, when "primer" is mentioned, unless otherwise specified, it may refer to a primer or a pair of primers, or a set of primers or a mixture of several sets of primers.

[0075] In the present invention, unless otherwise specified, A, B, C, D, E, and F in "primer A", "primer B", "primer C", "primer D", "primer E", and "primer F" are mainly for reference distinction and do not have a typical order meaning.

[0076] In the present invention, unless otherwise specified, the "first anchor sequence" and the "second anchor sequence" are mainly used for reference distinction and do not have a typical order meaning.

[0077] Advantageous Effects of the Invention

[0078] The present invention achieves one or more of the following technical effects (1) to (3):

[0079] (1) Improved the amplification efficiency of short tandem CpG markers for gastric cancer, such as gastric cancer-specific mCGCGCGG-CpG;

[0080] (2) The present invention can effectively detect / diagnose gastric cancer with high sensitivity and / or specificity;

[0081] (3) The present invention can be applied to non-invasive or minimally invasive detection of gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figures 1 to 6 : Detection of 68 mCGCGCGG-CpG sites in plasma in gastric cancer patients and control participants.

[0083] Figure 1 and Figure 3 : Total UMI counts of 68 gastric cancer markers in gastric cancer patients and control subjects, respectively. *P < 0.05, **P < 0.01, **P < 0.0001, two-tailed MWW test.

[0084] Figure 2 、 Figure 4 and Figure 5 : are the AUC values ​​of the training set, test set and merged cohort, respectively.

[0085] Figure 6 :Detection sensitivity of stage I-III gastric cancer in the training and test sets with specificity of 88.7% and 90.6%, respectively. DETAILED DESCRIPTION

[0086] Embodiments of the present application will be described in detail below with reference to Examples, but those skilled in the art will understand that the following Examples are for illustrative purposes only and should not be considered limiting of the scope of the present application. Unless otherwise indicated, the specific conditions used in the following Examples were as follows: unless otherwise indicated, conventional conditions or manufacturer's recommended conditions were used. Unless otherwise indicated, the reagents or instruments used were conventional products available on the market.

[0087] Example 1: Targeted amplification of 68 mCGCGCGG-CpG markers using primer combinations for plasma detection of gastric cancer

[0088] (I) Experimental materials and instruments

[0089] The human samples involved in this example were from the Department of General Surgery, Peking University Third Hospital. All subjects read and signed the informed consent form before enrollment. The collection and use of all samples were strictly in accordance with the approved protocol by the Medical Ethics Committee of Peking University Third Hospital, project number LM2020108, LM2023660. A total of 101 patients with clinically diagnosed gastric cancer and 177 control participants without malignant tumors were included.

[0090] Peripheral blood collection was performed by nursing staff according to standard blood collection procedures. Peripheral blood was collected from the median cubital vein using a vacuum blood collection tube containing potassium ethylenediaminetetraacetate (K2EDTA) and gently inverting the blood collection tube 10 times. The blood collection tube was stored at 4°C. Centrifugation was performed within 6 hours. The specific steps are as follows:

[0091] 1) Set the low-speed centrifuge to slow rise and slow fall. The blood collection tube was centrifuged at 1,600g for 10 minutes at 4°C;

[0092] 2) Gently remove the blood collection tube. At this time, the blood is divided into three layers: the upper layer is plasma, the middle layer is white blood cells and platelets, and the lower layer is red blood cells;

[0093] 3) Compare the color card to check the degree of hemolysis in the upper layer. Hemolysis less than 1g / L is a qualified sample;

[0094] 4) Carefully transfer the upper layer of plasma to a 2mL sterile centrifuge tube, taking care not to aspirate the middle layer. Centrifuge the plasma at 16,000g for 10 minutes at 4°C;

[0095] 5) After the plasma centrifugation is completed, carefully aspirate the supernatant without touching the bottom cell pellet and divide it into new 2mL sterile centrifuge tubes;

[0096] 6) Label and record the plasma samples and store them at -80°C or directly proceed to ccfDNA extraction.

[0097] ccfDNA was extracted from 2mL of plasma using the VAHTS Free-Circulating DNA Maxi Kit. The extraction steps are as follows:

[0098] 1) Transfer the thawed plasma sample to a 15 mL centrifuge tube;

[0099] 2) Add 100 μL Proteinase K, 3.2 mL Buffer L / B, and 60 μL of thoroughly mixed VAHTS Particles G to the sample;

[0100] 3) Vortex to mix for 5 seconds and incubate at room temperature for 5 minutes, inverting 2-3 times to mix;

[0101] 4) Place the centrifuge tube on a magnetic stand and let it sit for about 3-5 minutes. Once the solution is clear, keep the magnetic beads adsorbed and carefully remove the supernatant.

[0102] 5) Remove the magnetic stand and add 1 mL of Buffer WA (with anhydrous ethanol). Use a pipette to disperse the magnetic beads and pipette to mix thoroughly. Transfer the resuspended magnetic beads to a new 1.5 mL low-binding centrifuge tube.

[0103] 6) If there are residual magnetic beads on the wall of the centrifuge tube, add 100 μL of Buffer WA to rinse and transfer them to the centrifuge tube;

[0104] 7) Place the centrifuge tube on a magnetic rack and carefully remove the supernatant after the solution has clarified.

[0105] 8) Remove the magnetic stand, add 1 mL of Buffer WB (with anhydrous ethanol), and vortex to mix for 5 seconds;

[0106] 9) After brief centrifugation, place the tube on a magnetic rack and carefully remove the supernatant after the solution has clarified.

[0107] 10) Repeat steps 8-9 and carefully aspirate the supernatant.

[0108] 11) Open the lid and let it air-dry at room temperature for 2-5 minutes until there is no liquid left in the tube and the surface of the magnetic beads is not reflective (to avoid over-drying that may affect the final yield);

[0109] 12) Remove the magnetic stand, add 25 μL of double-distilled water, and vortex for 1 minute;

[0110] 13) After standing at room temperature for 5 minutes, place the sample back on the magnetic rack and let it stand for 1-3 minutes;

[0111] 14) Pipette the supernatant into a new low-adsorption centrifuge tube;

[0112] 15) Pipette 1 μL of sample and use Qubit to detect dsDNA concentration. Mark the sample information and concentration on the tube wall and store at -80°C.

[0113] The instrument is a conventional PCR instrument and the like.

[0114] (2) Experimental methods

[0115] The present inventors selected 68 gastric cancer mCGCGCGG-CpG markers (Table 1) from reported literature (PMID: 31010820 and PMID: 34791072) to design primers. Targeted amplification primers were designed outside the 5' end of these mCGCGCGG-CpG sites. The primer design principles were as follows:

[0116] 1) Calculate the annealing temperature using the NEB Tm calculator (https: / / tmcalculator.neb.com / #! / main), Taq 20 nM, 60°C;

[0117] 2) GC content of 50-60%;

[0118] 3) Primer length should be 20-28 bp in principle;

[0119] 4) The 3' end is preferably CGC (i.e., its 3' end is CGC at the 5' end of CGCGCGG, but GGCGC is avoided), followed by starting with C (i.e., its 3' end is C at the 5' end of CGCGCGG), and thirdly, the distance between the 3' end of the primer and the 5' end of the CGCGCGG sequence should be within 20 bp in principle.

[0120] The sequences of the designed primers are shown in Table 1 below.

[0121] Table 1: mCGCGCGG-CpG tag and targeting primer sequences

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] In the present invention, the gastric cancer detection method based on peripheral blood multi-target DNA methylation refers to the following steps 1 to 5.

[0128] 1. Bisulfite treatment of DNA samples

[0129] EZ DNA Methylation-Lightning kit TMKit (ZYMO RESEARCH) and follow the instructions provided by the manufacturer. The specific steps are as follows:

[0130] 1.1 Add 20 μl of plasma-extracted ccfDNA sample into a PCR tube;

[0131] 1.2 Add 130 μl of CT Conversion Reagent into the DNA sample, mix by flicking or blowing with a gun head;

[0132] 1.3 Add 100 ng of carrier RNA into the mixture to reduce the loss caused by non-specific binding of DNA to the filter membrane;

[0133] 1.4 Place the PCR tube in a thermal cycler and perform the following program: 98°C for 8 minutes, 54°C for 1 hour, and 4°C for storage (not more than 20 hours);

[0134] 1.5 Add 600 μL of M-Binding Buffer into a 1.5 mL PCR tube, and add the conversion product of the DNA sample in step 1.4, mix by inverting;

[0135] 1.6 Centrifuge at maximum speed (>12,000 g) for 30 seconds, and discard the supernatant;

[0136] 1.7 Add 100 μl of M-Wash Buffer (with ethanol added), centrifuge at maximum speed for 30 seconds, and discard the supernatant;

[0137] 1.8 Add 200 μl of Desulphonation Buffer, and let the purification column stand at room temperature for 15-20 minutes;

[0138] 1.9 Centrifuge at maximum speed for 30 seconds, and discard the supernatant;

[0139] 1.10 Add 200 μl of M-Wash Buffer (with ethanol added), centrifuge at maximum speed for 30 seconds, and discard the supernatant;

[0140] 1.11 Repeat step 1.10 once, and place the purification column into a new 1.5 mL centrifuge tube;

[0141] 1.12 Add 20 μl of double-distilled water, and centrifuge at maximum speed for 30 seconds to elute the DNA.

[0142] 2. Primer A and DNA polymerase for linear amplification

[0143] 2.1 Prepare the eluted DNA obtained in step 1.12 in a PCR tube according to the following amplification reaction system 1 (Table 2).

[0144] Table 2: Amplification reaction system 1

[0145] content volume DNA samples and water 21 μL NEBuffer 2 3μL dNTP / dUTP Mix 3μL Primers A1-A4 (5 μM)* 2μL Klenow(exo-)** 1μL (add later) Total 30 μL

[0146] dNTP / dUTP Mix (dATPdCTP / dGTP 2mM each, dUTP 4mM)

[0147] In Table 2, *Primers A1-A4 are an equimolar mixture of the following four primers, with a total concentration of 5 μM:

[0148] A1(H=A / T / C):

[0149]

[0150] A2:

[0151]

[0152] A3:

[0153]

[0154] A4:

[0155]

[0156] in:

[0157] H=A / T / C (A, T, and C have equal probability of appearing in H). In fact, A1 itself is a combination of several specific primers, and A2, A3, and A4 can be understood similarly.

[0158] The underlined wavy line portion is the 3' end portion of the primer, the underlined straight line portion is the 5' end portion of the primer (anchor sequence), and the underlined double straight line portion is the unique molecular identification tag (UMI) sequence portion of the primer.

[0159] In Table 2, **Klenow (exo-) was added in step 2.3.

[0160] 2.2 Place the PCR tube in a PCR thermal cycler and perform the following program: 95°C for 1 minute, then store at 4°C.

[0161] 2.3 Add 1 μl Klenow fragment (exo-) (NEB catalog number M0212S), mix well, and centrifuge briefly;

[0162] 2.4 Perform the following program in a PCR thermal cycler: 4°C for 50 seconds, 10°C for 1 minute, 20°C for 4 minutes, 30°C for 4 minutes, 37°C for 4 minutes, 95°C for 30 seconds, and 4°C for a pause.

[0163] 2.5 Prepare the following amplification reaction system 2 (Table 3) in a PCR tube;

[0164] Table 3: Amplification reaction system 2

[0165] content volume water 1.15 μL NEBuffer 2 0.25 μL dNTP / dUTP Mix 0.1μL Klenow(exo-) 1 μL Total 2.5 μL

[0166] 2.6 Add the above reaction system 2 to the above reaction system 1, mix well, and centrifuge instantaneously;

[0167] 2.7 Perform the following program in a PCR thermal cycler: 4°C for 50 seconds, 10°C for 1 minute, 20°C for 4 minutes, 30°C for 4 minutes, 37°C for 4 minutes, and 4°C for pause.

[0168] 2.8 Centrifuge at 4°C for 1 minute and place on ice.

[0169] 3. AMPure XP magnetic beads (BECKMAN COULTER) purification

[0170] 3.1 Prepare 80% ethanol using double distilled water and anhydrous ethanol in a ratio of 2:8;

[0171] 3.2 Vortex and mix AMPure XP beads and let them stand at room temperature for 30 minutes.

[0172] 3.3 Add 130 μl of 4×AMPure magnetic beads to each tube of product from 2.8, vortex to mix, centrifuge briefly, and incubate at room temperature for 10 minutes.

[0173] 3.4 Place the PCR tube on a magnetic rack and let it sit for 2 minutes until the magnetic beads are adsorbed to one side. Aspirate and discard the supernatant.

[0174] 3.5 Add 200 μl of 80% ethanol to each tube, let it stand at room temperature for 30 seconds, aspirate and discard the supernatant;

[0175] 3.6 Repeat 3.5 once, remove all ethanol, and dry the magnetic beads at room temperature for 10 minutes. Do not allow the beads to dry out too much;

[0176] 3.7 Add 21 μl double-distilled water to each tube for eluate, remove the PCR tube from the magnetic rack, shake to mix, and incubate at room temperature for 10 minutes;

[0177] 3.8 Place the PCR tube on the magnetic rack and let it stand for 2 minutes until the magnetic beads are adsorbed to one side. Pipette 19 μl of eluted product and place it in a new PCR tube.

[0178] 4. Amplification and purification using targeted primers and DNA polymerase

[0179] 4.1 Prepare the following amplification reaction system 3 (Table 4) in a PCR tube.

[0180] Table 4: Amplification reaction system 3

[0181]

[0182] *Primer B: where the underlined part is the 3' end part of the primer, and the straight line part is the 5' end part of the primer. Primer B corresponds to the partial anchor sequence in primer A, acting as an inner primer.

[0183] **: Primer C: equimolar mixture of 68 primers (see Table 1 above), the concentration of each primer is 1 μM:

[0184] 4.2 The following procedure is carried out in a PCR thermal cycler: 98°C for 45 seconds, then 98°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds, for a total of 10 cycles, then 72°C for 1 minute, 4°C pause.

[0185] 4.3 4°C centrifugation for 1 minute, ice on standby.

[0186] 4.4 Add Uracil-DNA Glycosylase (UDG, 1 U / μL, Thermo Scientific, EN0362) 1 μL, 37°C for 30 minutes.

[0187] 4.5 4×AMPure XP magnetic bead purification, elute with 37 μL water.

[0188] 5. Amplification and purification using primers D / E / F and DNA polymerase

[0189] 5.1 The following amplification reaction system 4 (Table 5) is configured in a PCR tube.

[0190] Table 5: Amplification reaction system 4

[0191] content volume DNA sample from 4.5 35.5μl Takara Ex Taq Buffer 5μl Primer D (10 μM)* 1 μl Primer E (10 μM)** 1.25 μl Primer F (10 μM)** 1.25 μl dNTP (2.5 mM) 5μl Takara HS Ex Taq 1 μl Total 50 μL

[0192] *Primer D:

[0193] (D = A / T / G) (here DDDD is any combination of ATGG, AAGT, etc., random base synthesis is used when synthesizing the primer, which can synthesize such random primers, primer D does not constitute a primer pair), designed according to the inner targeting site, where the underlined part is the 3' end part of the primer, and the straight line part is the 5' end part of the primer.

[0194] **Primer E:

[0195]

[0196] The underlined part is the same as the primer A part, the double underlined part is the index sequence (NovaSeqindex), and the ununderlined part is the sequencing adapter;

[0197] **Primer F:

[0198] The underlined part is the same as the primer D part, the double underlined part is the index sequence (NovaSeqindex); the ununderlined part is the sequencing adapter;

[0199] Primers E and F are adapter primers that can be used to construct libraries for Illumina TruSeq. Primer F can anneal to its complementary sequence (i.e., the product of a single amplification with primer D), resulting in exponential amplification, and the resulting product is a library with a complete structure.

[0200] 5.1 Perform the following program in a PCR thermocycler: 95°C for 3 minutes, 50°C for 30 seconds, 72°C for 1 minute, followed by 20 cycles of amplification: 95°C for 30 seconds, 64°C for 30 seconds, 72°C for 1 minute, and finally 72°C for 5 minutes, pause at 4°C.

[0201] 5.2 Purify with 1.2× AMPure XP magnetic beads, add 21 μL of water for elution, and place 20 μL of the eluted product in a new PCR tube to obtain a high-throughput sequencing library.

[0202] 6. Sequencing and preliminary data processing

[0203] 6.1 Fragment Analyzer (Advanced Analytical) system detects the size of high-throughput sequencing library inserts and performs absolute quantitative analysis of library concentration using QPCR;

[0204] 6.2 Perform high-throughput sequencing analysis on the library using an Illumina NovaSeq 6000 sequencer using paired-end sequencing with a read length of 150 bp, detecting 2 Gb per sample to obtain raw sequencing data.

[0205] 7. Data Analysis

[0206] Adapter sequences were trimmed using Cutadapt (v3.4). Primer sequences were subsequently removed using a custom script, and low-quality reads were filtered using Fastp (v0.23.0) with the parameters '-Aq 20-u 50-n5-l 37'. To improve analysis accuracy, reads containing more than three unmethylated CH sites (CC, CA, CT) were further excluded. High-quality reads were aligned to the human reference genome (hg19) using Bismark (v0.16.3) and subsequently sorted by coordinates using Samtools (v1.3.1). Only aligned reads containing ≥2 CpG sites within ±3 bp of the 5' end were retained to eliminate nonspecific PCR amplification products. The PCR deduplication process was as follows: First, 5 bp unique molecular identifier (UMI) sequences were extracted from the 5' end of the R1 reads, and reads with all five UMI base quality values ​​greater than 20 were retained. All UMI sequences were supported by ≥2 reads to ensure the reliability of the results. For each sample, the UMI numbers of the 68 mCGCGCGG-CpGs markers were summed to obtain the total UMI number.

[0207] Data from gastric cancer patients (n=101) and control participants (n=177) were randomly assigned to two groups: a training set consisting of 71 patients and 124 controls, and a test set consisting of 30 patients and 53 controls. Analysis was performed based on the total number of UMIs in each sample. Receiver operating characteristic (ROC) curves, heat maps, boxplots, and scatterplots were generated using custom R scripts and R packages.

[0208] (3) Experimental results

[0209] Gastric cancer patient test results Figures 1 to 6 shown.

[0210] In the training set, the total UMI counts in gastric cancer patients were significantly higher than those in control participants (median counts were 10, 15, and 38 in patients with stage I, II, and III gastric cancer (according to the TNM staging system, AJCC eighth edition), respectively, compared with 3 in controls; Figure 1 The AUC values ​​were 0.782 for stage I, 0.846 for stage II, 0.949 for stage III, and 0.869 for stage I-III ( Figure 2 The total UMI count here refers to the sum of the number of molecules detected for all 68 mCGCGCGG-CpG sites.

[0211] In the test set, total UMI counts were also significantly increased in patients with gastric cancer compared with control participants (median counts were 12, 21, and 94.5 in patients with stage I, II, and III gastric cancer, respectively, compared with 3 in the control group; Figure 3The AUC values ​​were 0.812 for stage I, 0.969 for stage II, 0.984 for stage III, and 0.928 for stage I-III ( Figure 4 ).

[0212] When the training set and test set were combined, the AUC values ​​were 0.792 for stage I, 0.883 for stage II, 0.959 for stage III, and 0.887 for stage I-III ( Figure 5 ).

[0213] The cutoff point (Cutoff) was determined to be 9 based on the upper left corner of the ROC curve of the training set. If a total UMI greater than or equal to 9 is used to distinguish between gastric cancer patients and control participants, that is, a total UMI greater than or equal to 9 is considered positive, indicating gastric cancer patients, and less than 9 is considered negative, indicating non-gastric cancer controls, the sensitivity of gastric cancer detection in the training set (true positive rate, that is, the number of gastric cancer samples judged as positive by the test results / the total number of gastric cancer samples in this group) is 61.9% (13 / 21) for stage I, 71.4% (15 / 21) for stage II, and 93.1% (27 / 29) for stage III. The specificity (true negative rate, that is, the number of control samples judged as negative by the test results / the total number of control samples in this group) is 88.7% (110 / 124, Figure 6 ). The test set was then tested using this threshold point. The sensitivity of gastric cancer detection in the test set was 66.7% (6 / 9) for stage I, 88.9% (8 / 9) for stage II, and 91.7% (11 / 12) for stage III. The specificity was 90.6% (48 / 53, Figure 6 When the training and test sets were combined, the sensitivity of gastric cancer was 63.3% (19 / 30) for stage I, 76.6% (23 / 30) for stage II, and 92.7% (38 / 41) for stage III, respectively. The overall sensitivity was 79.2% (80 / 101) and the specificity was 89.3% (158 / 177).

[0214] It is reported that the MCTA-Seq method (PMID: 34791072) has a sensitivity of 44% (10 / 23) in stage I, 59% (10 / 17) in stage II, 78% (38 / 49) in stage III / IV, and a specificity of 92% (75 / 82); the SEPT9 / RNF180 fluorescence quantitative PCR method has a sensitivity of 50.9% in stage I, 61.8% in stage II, 67.3% in stage III, and a specificity of 84.8% in detecting gastric cancer (Cancer Communications 2023, Combining methylated SEPTIN9and RNF180 plasma markers for diagnosis and early detection of gastric cancer).

[0215] The results show that the sensitivity and / or specificity of the present invention for gastric cancer detection are better than those reported above.

[0216] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A nucleic acid molecule library, prepared by a method comprising the following steps: (1) obtaining a nucleic acid molecule having an anchor primer at one end, wherein a single strand in the nucleic acid molecule is complementary to the treated target nucleic acid molecule; wherein, The treated target nucleic acid molecule is obtained by subjecting the target nucleic acid molecule to the following treatment: unmethylated cytosine in the target nucleic acid molecule is converted into uracil, and methylated cytosine is not converted into uracil; (2) using the product of step (1) as a template, performing PCR amplification using an anchor primer or an inner primer of the anchor primer, and a primer targeting the 5' upstream sequence of the gastric cancer CpG short tandem marker (CpG short tandem outer targeting primer); (3) using the product of step (2) as a template, performing PCR amplification using CpG short tandem primers and adding an anchor sequence; (4) using the product of step (3) as a template, and performing PCR amplification using an adapter primer that can anneal to the anchor sequence; Wherein, the PCR amplification in step (3) and step (4) is performed in one reaction system, or in two reaction systems in sequence. The nucleic acid molecule library according to claim 1 , which is a DNA library.

3. The nucleic acid molecule library according to any one of claims 1 to 2, wherein Step (1) includes the following step 1A or step 1B: 1A. Converting unmethylated cytosine in a target nucleic acid molecule to uracil, while not converting methylated cytosine, and then amplifying the nucleotide sequence using random primers or semi-random primers linked to a unique molecular identifier (UMI) sequence using a DNA polymerase to obtain a nucleic acid molecule complementary to the treated target nucleic acid molecule; preferably, the DNA polymerase is Klenow (exo-); 1B. The target nucleic acid molecule is end-repaired, filled with A, and ligated with an anchor primer. Unmethylated cytosine is then converted to uracil, while methylated cytosine is not converted. Amplification is then performed using a DNA polymerase to obtain a nucleic acid molecule complementary to the treated target nucleic acid molecule; alternatively, unmethylated cytosine is converted to uracil, while methylated cytosine is not converted. The treated target nucleic acid molecule is then ligated with an anchor primer and amplified using a polymerase to obtain a nucleic acid molecule complementary to the treated target nucleic acid molecule.

4. The nucleic acid molecule library according to claim 3, wherein In step 1A or step 1B, the target nucleic acid molecule is treated with bisulfite or cytosine deaminase (such as TET methylcytosine dioxygenase 2 and APOBEC3A enzyme) to convert unmethylated cytosine into uracil.

5. The nucleic acid molecule library according to claim 3, wherein In step 1A, the semi-random primer comprises, from the 5' end to the 3' end, an anchor sequence, a UMI sequence, and a semi-random sequence comprising a CpG site; Preferably, the semi-random primers comprise the sequences shown in SEQ ID NO:69 to SEQ ID NO:

72.

6. The nucleic acid molecule library according to any one of claims 1 to 5, wherein In step (1), a nucleic acid molecule having an anchor primer at one end and complementary to the treated target nucleic acid molecule is obtained by polymerase amplification; preferably, dUTP is added during polymerase amplification to incorporate uracil into the complementary nucleic acid molecule.

7. The nucleic acid molecule library according to any one of claims 1 to 6, wherein Step (2) further includes the step of removing uracil residues; Preferably, uracil residues are removed using Uracil-DNA glycosylase (UDG); Preferably, dUTP is not added during the PCR amplification in step (2).

8. The nucleic acid molecule library according to any one of claims 1 to 7, wherein In step (2), The sequence of the inner primer of the anchor primer is shown in SEQ ID NO: 73; and / or The sequence of the short tandem CpG was selected from CGCGCGG, CGGCGGCGG, CGCGCGA, CGCGCGT, and CGACGACGA; Preferably, the length of the primer targeting the 5' upstream sequence of the gastric cancer CpG short tandem marker is 16-32 bp or 18-28 bp, and / or the distance between the 3' end of the primer and the 5' end of the CpG short tandem marker is within 25 bp or within 20 bp; Preferably, the sequence of the primer targeting the 5' upstream sequence of the gastric cancer CpG short tandem marker is selected from any 10, any 20, any 30, any 40, any 50, any 60, all 68, 30-68, 40-68, 50-68 or 60-68 sequences of SEQ ID NO: 1 to SEQ ID NO:

68.

9. The nucleic acid molecule library according to any one of claims 1 to 8, wherein In step (3), The sequence of the short tandem CpG was selected from CGCGCGG, CGGCGGCGG, CGCGCGA, CGCGCGT, and CGACGACGA; Preferably, the sequence of the CpG short tandem primer is shown as SEQ ID NO:

74.

10. The nucleic acid molecule library according to any one of claims 1 to 9, wherein In step (4), The sequences of the linker primers are shown in SEQ ID NO:75 and SEQ ID NO:

76.

11. The nucleic acid molecule library according to any one of claims 1 to 10, wherein The PCR in step 1A is performed for one or more rounds (e.g., two or three rounds); Preferably, when multiple rounds are performed, Klenow (exo-) is supplemented to the reaction system.

12. The nucleic acid molecule library according to any one of claims 1 to 11, wherein The method may further include purifying the product obtained by PCR amplification in step 1A, step (2), step (3) and / or step (4).

13. The nucleic acid molecule library according to any one of claims 1 to 12, wherein The target nucleic acid molecule is a DNA molecule; Preferably, the target nucleic acid molecule is derived from mammalian cells, tissues, blood or urine, such as human, preferably from human plasma or serum.

14. Primer combination, comprising: Primer A, sequences shown in SEQ ID NO:69 to SEQ ID NO:72; Primer B, the sequence is shown in SEQ ID NO:73; Sequence C, which is selected from any 10, any 20, any 30, any 40, any 50, any 60, all 68, 30-68, 40-68, 50-68 or 60-68 sequences of SEQ ID NO: 1 to SEQ ID NO: 68; Sequence D, the sequence is shown in SEQ ID NO:74; Sequence E, the sequence of which is shown in SEQ ID NO:75; and Sequence F, the sequence is shown in SEQ ID NO:

76.

15. Use of the nucleic acid molecule library according to any one of claims 1 to 13 or the primer combination according to claim 14 in the preparation of a drug or a kit for detecting gastric cancer.

16. A kit for detecting gastric cancer, comprising the primer combination according to claim 14 and a PCR reagent.

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