Nucleic acid molecule library and application thereof in colorectal cancer detection

Abstract

The invention belongs to the field of biological medicine, and relates to a nucleic acid molecule library and application thereof in colorectal 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 primer of the anchor primer and a primer of a 5'end upstream sequence of a targeting colorectal 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 can effectively detect or diagnose colorectal cancer, and has good sensitivity and specificity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a nucleic acid molecule library and its use in colorectal cancer detection. The present invention also relates to a colorectal cancer detection method based on multi-target DNA methylation in peripheral blood. Background Art

[0002] Colorectal cancer is a serious disease that endangers human health. In China, colorectal cancer ranks second in the incidence of malignant tumors, with more than 510,000 new cases and 240,000 deaths each year. Currently, clinical detection methods for colorectal cancer mainly include colonoscopy, imaging examinations, stool and tumor marker testing. However, colonoscopy is invasive and has the risk of complications, patient compliance is poor, and it may be difficult to obtain biopsy specimens for patients with complex lesions. Imaging examinations have the risk of radiation exposure and the recognition rate of early lesions is relatively low. Traditional blood tumor markers such as CEA, CA19-9 and CA125 have low detection sensitivity. Stool-based screening methods such as FIT and FOBT have suboptimal sensitivity for colorectal cancer detection and poor patient acceptance. Studies have shown that most people suitable for screening tend to choose blood tests.

[0003] During the development and progression of colorectal cancer, abnormal methylation affects the expression of related genes. Researchers have discovered that detecting methylation levels at specific sites can be used to detect the onset of cancer. Circulating cell-free DNA (ccfDNA) refers to DNA that circulates free outside of cells in the blood. In recent years, "liquid biopsy" technology targeting ccfDNA has rapidly developed, showing promising applications in clinical cancer screening and monitoring.

[0004] An existing literature (2019 Clinical Chemistry, Detection of Colorectal Cancer inCirculating Cell-Free DNA by Methylated CpG Tandem Amplification and Sequencing, PMID: 31010820) reported a DNA methylation detection method for colorectal cancer. This method uses methylated CpG short tandem amplification and sequencing (MCTA-Seq) technology to target more than 9,000 CpG islands through CGCGCGG short tandem primers for colorectal cancer detection based on peripheral blood. The results showed that a panel consisting of 80 methylated CGCGCGG-CpGs (mCGCGCGG-CpGs, in which all 3 Cs of CGCGCGG are methylated, and the 1st to nth CpGs downstream are also all methylated) can effectively detect colorectal cancer. However, this method has the limitations of cumbersome purification steps, low CpG short tandem capture efficiency, and coverage of non-interest 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 colorectal 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 inventors established a targeted amplification method for 72 colorectal cancer mCGCGCGG-CpG marker combinations.

[0010] In the examples, the inventors used a combination of 72 colorectal cancer mCGCGCGG-CpG marker-targeted primers to detect ccfDNA in plasma samples from colorectal cancer patients (n=170) and control group participants (n=168). The classifier formed by these 72 primer combinations had a sensitivity of 71.7% (33 / 46) for stage I, 90.9% (50 / 55) for stage II, and 92.8% (64 / 69) for stage III, with a specificity of 89.9% (151 / 168). The results showed that this method had better sensitivity for colorectal cancer detection than the previously reported MCTA-Seq method (PMID: 31010820), which had a sensitivity of 63.3% (19 / 30) for stage I, 80% (52 / 65) for stage II, 83% (39 / 47) for stage III / IV, and a specificity of 90% (119 / 132) for colorectal cancer detection. This provides the following invention:

[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 colorectal cancer CpG short tandem marker (colorectal 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 invention, the nucleic acid molecule library, wherein step (1) comprises the following step 1A or step 1B:

[0020] 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-);

[0021] 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.

[0022] In some embodiments of the present application, the nucleic acid molecule library, wherein in step 1A or step 1B, the unmethylated cytosine is converted to uracil by treating the target nucleic acid molecule with bisulfite or treating the target nucleic acid molecule with cytosine deaminases (e.g. TET methylcytosine dioxygenase 2 and APOBEC3A enzyme).

[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 to uracil by treating the target nucleic acid molecule with bisulfite or treating the target nucleic acid molecule with cytosine deaminases (e.g. TET methylcytosine dioxygenase 2 and APOBEC3A enzyme).

[0024] In some embodiments of the present application, the nucleic acid molecule library, wherein in step 1A or step 1B, the unmethylated cytosine is converted to uracil by treating the target nucleic acid molecule with bisulfite or treating the target nucleic acid molecule with cytosine deaminases (e.g. TET methylcytosine dioxygenase 2 and APOBEC3A enzyme).

[0025] The treatment with TET methylcytosine dioxygenase 2 and APOBEC3A enzyme includes the steps of: using TET methylcytosine dioxygenase 2 to catalyze 5-methylcytosine (5mC) to oxidize 5-carboxylcytosine (5caC), and then using APOBEC3A enzyme to specifically deaminate, converting the unmethylated cytosine (C) to uracil (U) (5caC is not deaminated due to structural differences). Without being limited by theory, in subsequent amplification, the DNA polymerase recognizes 5-carboxylcytosine similar to cytosine, not uracil. The DNA polymerase recognizes uracil as thymine (T) and directly pairs with guanine (A), so that after amplification, uracil will be converted to thymine. 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 primers comprise the sequences shown in SEQ ID NO: 73 to SEQ ID NO: 76.

[0028] In some embodiments of the present invention, the nucleic acid molecule library, 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 amplification (polymerase amplification); preferably, dUTP is added during amplification to incorporate uracil into the complementary nucleic acid molecule. In some embodiments of the present invention, the molar number of dUTP added is 1-3 times that of dNTP (dATP, dCTP or dGTP), for example, 1.5-2.5 times, 1.8-2.2 times or 2 times.

[0029] In some embodiments of the present invention, the nucleic acid molecule library, wherein step (2) further comprises the step of removing uracil residues;

[0030] Preferably, uracil residues are removed using Uracil-DNA glycosylase (UDG).

[0031] In some embodiments of the present invention, the nucleic acid molecule library, wherein, in step (2), dUTP is not added during the PCR amplification.

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

[0033] The sequence of the inner primer of the anchor primer is shown in SEQ ID NO: 77; and / or

[0034] The sequence of the short tandem CpG was selected from CGCGCGG, CGGCGGCGG, CGCGCGA, CGCGCGT, and CGACGACGA;

[0035] Preferably, the length of the primer targeting the 5' upstream sequence of the colorectal 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;

[0036] Preferably, the sequence of the primer targeting the 5' upstream sequence of the colorectal cancer CpG short tandem marker is selected from any 10, any 20, any 30, any 40, any 50, any 60, any 65, any 70, all 72, 30-72, 40-72, 50-72, 60-72, 65-72 or 70-72 sequences of SEQ ID NO:1 to SEQ ID NO:72.

[0037] Without being bound by theory, preferably, the length of the primer targeting the 5' upstream sequence of the colorectal cancer CpG short tandem marker does not exceed 32 bp or 28 bp (except for some individual cases) in principle, 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 some individual cases). That is to say, the quantitative range of the "sequence upstream of CGCGCGG" is preferably 32+25 bp, 32+20 bp, 28+25 bp or 28+20 bp, i.e., within the range of approximately 48-57 bp. The "sequence upstream of CGCGCGG" of different CpG short tandems is different. After bisulfite or enzymatic treatment, the CGCGCGG upstream sequence has commonalities. For example, after bisulfite treatment, the unmethylated C (C of CH dinucleotide) of the sequence is converted to U, so that the C content in the primer is very low, and most of it is G / A / T.

[0038] In some embodiments of the present invention, the colorectal cancer CpG short tandem marker is a colorectal cancer marker nucleic acid molecule comprising a CpG short tandem or complementary to a CpG short tandem. In some embodiments of the present invention, the colorectal cancer CpG short tandem marker is a colorectal cancer CpG short tandem marker reported in the prior art. In some embodiments of the present invention, the colorectal 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: 78.

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

[0044] The sequence of the adaptor primer is shown in SEQ ID NO: 79 and SEQ ID NO: 80.

[0045] In some embodiments of the present application, the nucleic acid molecule library, wherein the PCR in step 1A is performed for one or more rounds (e.g. 2 rounds or 3 rounds).

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

[0047] In some embodiments of the present application, the nucleic acid molecule library, wherein the step of purifying the products amplified by PCR in step 1A, step (2), step (3) and / or step (4) will be further included.

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

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

[0050] Preferably, the target nucleic acid molecule is from a cell, tissue, blood or urine of a mammal, e.g. a human, preferably from the plasma or serum of a human.

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

[0052] Primer A, the sequence of which is shown in any one of SEQ ID NO: 73 to SEQ ID NO: 76;

[0053] Primer B, the sequence of which is shown in SEQ ID NO: 77;

[0054] Sequence C, the sequence of which is selected from any 10, any 20, any 30, any 40, any 50, any 60, any 65, any 70, all 72, 30-72, 40-72, 50-72, 60-72, 65-72 or 70-72 sequences of SEQ ID NO: 1 to SEQ ID NO: 72;

[0055] Sequence D, the sequence of which is shown in SEQ ID NO: 78;

[0056] Sequence E, the sequence of which is shown in SEQ ID NO: 79; and

[0057] Sequence F, the sequence of which is shown in SEQ ID NO: 80.

[0058] Still another aspect of the present application relates to the use of any one of the nucleic acid molecule libraries or primer combinations of the present application in the preparation of a medicament or kit for detecting colorectal cancer.

[0059] Another aspect of the present invention relates to a kit for detecting colorectal 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 colorectal 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 8 (e.g., the 72 markers in the present invention), the result is positive or the risk of colorectal cancer is high (further screening or diagnosis is required);

[0062] When the total number of marker molecules (i.e., the total number of UMIs) is less than 8, the result is negative or the risk of colorectal 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 colorectal cancer short tandem CpG markers, such as colorectal cancer-specific mCGCGCGG-CpG;

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

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

[0082] Figures 1 to 6 : Detection of 72 colorectal cancer mCGCGCGG-CpG markers in plasma in colorectal cancer patients and control participants.

[0083] Figure 1 and Figure 4: Total UMI counts of 72 colorectal cancer mCGCGCGG-CpG markers in colorectal cancer and control participants, respectively. ***P<0.0001, two-sided Mann-Whitney-Wilcoxon test.

[0084] Figure 2 、 Figure 5 and Figure 6 : are the AUC values ​​of the training set, test set and merged cohort, respectively.

[0085] Figure 3 : Assay sensitivity for detecting stages I to III colorectal cancer in different cohorts. DETAILED DESCRIPTION

[0086] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0087] Example 1: Targeted amplification of 72 mCGCGCGG-CpG markers using primer combinations for plasma detection of colorectal cancer

[0088] (1) Experimental materials and instruments

[0089] The human samples used in this example were obtained from the Department of General Surgery at Peking University Third Hospital. All subjects read and signed informed consent forms before enrollment. All sample collection and use were conducted in strict accordance with protocols approved by the Medical Ethics Committee of Peking University Third Hospital, project numbers LM2020108 and LM2023660. The study included 170 patients with clinically confirmed colorectal cancer and 168 controls without malignant tumors.

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

[0091] 1) Centrifuge the blood collection tube at 1,600 g for 10 minutes at 4°C in a low-speed centrifuge set to slow ascent and slow descent.

[0092] 2) Gently remove the blood collection tube. The blood will now separate into three layers: plasma on the top, white blood cells and platelets in the middle, and red blood cells on the bottom.

[0093] 3) Check the hemolysis degree of the upper layer against the colorimetric card. The sample is qualified if the hemolysis is less than 1g / L.

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

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

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

[0097] ccfDNA was extracted from 2 mL of plasma using the VAHTS Free-Circulating DNA Maxi Kit. The extraction steps were 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 cap and let it stand at room temperature for 2-5 minutes until no liquid remains in the tube and no reflection is observed on the surface of the magnetic beads (avoiding excessive drying affecting 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 stand and stand for 1-3 minutes;

[0111] 14) Absorb the supernatant into a new low-absorption centrifuge tube;

[0112] 15) Absorb 1 μL of sample to detect dsDNA concentration using Qubit, and mark sample information, concentration, etc. on the tube wall, and store at -80℃.

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

[0114] (ii) Experimental method

[0115] The inventors selected 72 colorectal cancer mCGCGCGG-CpG markers (Table 1) from the reported literature (PMID: 31010820 and PMID: 34791072) to design primers. The 5' end of these mCGCGCGG-CpG sites is designed to target the outer primers, and the design principles of the primers are as follows:

[0116] 1) The annealing temperature was calculated using NEB Tm caculator (https: / / tmcalculator.neb.com / #! / main) with Taq 20nM, 60℃;

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

[0118] 3) The length of the primer is 20-28bp in principle;

[0119] 4) The 3' end is preferably CGC (i.e. the 3' end of CGC at the 5' end of CGCGCGG, but avoiding GGCGC), followed by C (i.e. the 3' end of C at the 5' end of CGCGCGG), and then the 3' end of the primer is within 20bp of the 5' end of the CGCGCGG sequence.

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

[0121] Table 1: mCGCGCGG-CpG markers and target primer sequences

[0122]

[0123]

[0124]

[0125]

[0126]

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

[0128] 1. Bisulfite treatment of DNA samples

[0129] EZ DNA Methylation-Lightning kit TM Kit (ZYMO RESEARCH) and follow the manufacturer's instructions. The specific steps are as follows:

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

[0131] 1.2 Add 130 μl of CT Conversion Reagent to the DNA sample and mix thoroughly by flicking or pipetting.

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

[0133] 1.4 Place the PCR tubes in a thermal cycler and perform the following program: 98°C for 8 minutes, 54°C for 1 hour, and store at 4°C (for no more than 20 hours) until use.

[0134] 1.5 Add 600 μL of M-Binding Buffer to a 1.5 mL PCR tube and add the conversion product of the DNA sample from step 1.4. Invert to mix.

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

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

[0137] 1.8 Add 200 μl of Desulphonation buffer and incubate the column at room temperature for 15-20 minutes.

[0138] 1.9 Centrifuge at maximum speed for 30 seconds and discard the column liquid;

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

[0140] 1.11 Repeat 1.10 once and place the purification column into a new 1.5ml 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 following amplification reaction system 1 (Table 2) in a PCR tube using the eluted DNA obtained in step 1.12.

[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]

[0183] *: Primer B: The wavy underlined portion is the 3' end of the primer, and the straight underlined portion is the 5' end of the primer. Primer B corresponds to the partial anchor sequence in primer A and serves as an inner primer.

[0184] **: Primer C: an equimolar mixture of 72 primers (see Table 1 above), each primer at a concentration of 1 μM:

[0185] 4.2 Perform the following program in a PCR thermal cycler: 98°C for 45 seconds, then 98°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, for a total of 10 cycles, followed by 72°C for 1 minute and a pause at 4°C.

[0186] 4.3 Centrifuge at 4°C for 1 minute and place on ice.

[0187] 4.4 Add 1 μL of Uracil-DNA Glycosylase (UDG, Uracil-DNA Glycosylase, 1 U / μL, ThermoScientific, EN0362) and incubate at 37°C for 30 minutes.

[0188] 4.5 Purify with 4×AMPure XP magnetic beads and elute with 37 μL of water.

[0189] 5. Amplification and purification using primers D / E / F and DNA polymerase

[0190] 5.1 Prepare the following amplification reaction system 4 (Table 5) in a PCR tube.

[0191] Table 5: Amplification reaction system 4

[0192] 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

[0193] *: Primer D:

[0194] (D=A / T / G) (here DDDD is any combination of ATGG, AAGT, etc., and this type of random primer can be synthesized using random base synthesis during primer synthesis. Primer D does not constitute a primer pair), designed according to the inner target site, wherein the underlined wavy portion is the 3' end portion of the primer, and the underlined straight portion is the 5' end portion of the primer.

[0195] **: Primer E:

[0196]

[0197] 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;

[0198] **Primer F:

[0199] 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;

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 6. Sequencing and Analysis

[0204] 6.1 Fragment Analyzer (Advanced Analytical) system detects the size of insert fragments in high-throughput sequencing libraries and performs absolute quantitative analysis of library concentration using QPCR;

[0205] 6.2 The library was subjected to high-throughput sequencing analysis on an Illumina NovaSeq 6000 sequencer using paired-end sequencing with a read length of 150 bp to obtain 2 Gb of raw sequencing data.

[0206] 7. Data Analysis

[0207] 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 72 mCGCGCGG-CpGs markers were summed to obtain the total UMI number.

[0208] Colorectal cancer and control sample data were randomly assigned to two groups: a training set consisting of 84 colorectal cancer patients and 84 control participants; and a test set consisting of 86 colorectal cancer patients and 84 control participants. 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.

[0209] (3) Experimental results

[0210] Colorectal cancer patient test results Figures 1 to 6 shown.

[0211] In the training set, the results showed that the total UMI counts in the plasma samples of patients with colorectal cancer were significantly higher than those in the control group (the median UMI counts in patients with stage I, II, and III colorectal cancer (according to the TNM staging, AJCC eighth edition) were 14, 71, and 47, respectively, while the median UMI counts in the control group were 3; Figure 1). ROC curve analysis showed that the area under the curve (AUC) value was 0.868 for stage I, 0.983 for stage II, 0.966 for stage III, and 0.945 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.

[0212] The cutoff point (Cutoff) was determined to be 8 based on the upper left corner of the ROC curve of the training set. If a total UMI greater than or equal to 8 is used to distinguish between colorectal cancer patients and control participants, that is, a total UMI greater than or equal to 8 is considered positive, indicating colorectal cancer patients, and less than 8 is considered negative, indicating normal controls, the sensitivity of colorectal cancer detection in the training set (true positive rate, that is, the number of colorectal cancer samples judged as positive by the test results / the total number of colorectal cancer samples in this group) is 78.3% (18 / 23) for stage I, 92.6% (25 / 27) for stage II, and 91.2% (31 / 34) for stage III. Figure 3 ). The specificity was 90.5% (76 / 84).

[0213] In the test set, total UMI counts were also significantly increased in colorectal cancer patients compared to control participants ( Figure 4 ); AUC values ​​were 0.880 for stage I, 0.950 for stage II, 0.978 for stage III, and 0.943 for stage I-III ( Figure 5 When a threshold of UMI greater than or equal to 8 was used to distinguish between colorectal cancer patients and control participants, the sensitivity of colorectal cancer detection in the test set was 65.2% (15 / 23) for stage I, 89.3% (25 / 28) for stage II, and 94.3% (33 / 35) for stage III. Figure 3 ). The specificity was 89.3% (75 / 84).

[0214] Combined with the training and test sets, the AUC values ​​for distinguishing colorectal cancer from control participants were 0.870 for stage I, 0.966 for stage II, 0.973 for stage III, and 0.943 for stage I-III ( Figure 6 The sensitivity of colorectal cancer detection was 71.7% (33 / 46) for stage I, 90.9% (50 / 55) for stage II, and 92.8% (64 / 69) for stage III. The overall sensitivity was 86.5% (147 / 170) and the specificity was 89.9% (151 / 168).

[0215] 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 colorectal 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: 73 to SEQ ID NO:

76.

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: 77; 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 colorectal 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 colorectal cancer CpG short tandem marker is selected from any 10, any 20, any 30, any 40, any 50, any 60, any 65, any 70, all 72, 30-72, 40-72, 50-72, 60-72, 65-72 or 70-72 sequences of SEQ ID NO: 1 to SEQ ID NO:

72.

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:

78.

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: 79 and SEQ ID NO:

80.

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:73 to SEQ ID NO:76; Primer B, the sequence is shown in SEQ ID NO: 77; Sequence C, which is selected from any 10, any 20, any 30, any 40, any 50, any 60, any 65, any 70, all 72, 30-72, 40-72, 50-72, 60-72, 65-72, or 70-72 sequences of SEQ ID NO: 1 to SEQ ID NO: 72; Sequence D, the sequence is shown in SEQ ID NO:78; Sequence E, the sequence of which is shown in SEQ ID NO: 79; and Sequence F, the sequence is shown in SEQ ID NO:

80.

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 colorectal cancer.

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

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