Methylation Detection Method and Kit for Genomic DNA or Cell-Free DNA Molecules in Biological Sample (2024)

The present invention pertains to the technical field of genetic tests. More particularly, the invention relates to a methylation detection method and kit for genomic DNA or cell-free DNA molecules in a biological sample.

Gene expression can be regulated by DNA methylation. The detection of DNA methylation, therefore, can at least provide some clues as to the regulation of gene expression. Moreover, it has been proved that DNA methylation is involved in a series of important biological processes including early embryonic development, genomic imprinting, inactivation of the X chromosome, the silencing of a repeated sequence, and the development, progression, and metastasis of cancer. Research results have also shown that one major application of DNA methylation is to serve as a biomarker for the early screening and prognosis of tumors.

Humans have approximately 3 billion base pairs, and DNA methylation takes place mainly in the CpG dinucleotides. The human genome has 28 million or so CpG sites, about 60% to 80% of which are methylated. While certain gene areas such as the promoter areas have a sequence rich in CpG sites (also known as a CpG island), CpG islands are seldom methylated. When it comes to tumor cells, however, the overall methylation level is lowered to 20% to 50%, meaning the methylation states of 10% to 60% of the CpG sites in the original normal cells, i.e., about 2.8 to 16.8 million CpG sites, may have changed, especially in the cancer suppressor genes.

The existing DNA methylation detection methods mainly include whole-genome DNA methylation detection and site-specific methylation detection. Whole-genome DNA methylation detection is used primarily in research, to find differential methylated sites, and so on, can be categorized as high-throughput-sequencing-based or chip-based whole-genome DNA methylation detection. Site-specific methylation detection is used chiefly in translational medicine and includes methylation-specific polymerase chain reaction (PCR) (or MSP for short), bisulfite sequencing PCR (BSP), pyrosequencing, and mass spectrometry.

In an actual clinical application or operation, the detection substrate generally has a low DNA content. In early cancer screening, for example, the percentage of circulating tumor DNA (ctDNA) in the circulating free DNA (cfDNA) in a plasma sample is relatively low (can be as low as 0.01%). The concentration of cfDNA in a healthy person's plasma sample ranges approximately from 0 to 100 ng/mL, with the average concentration being about 30 ng/mL, and the length of a ctDNA fragment is generally about 166 bp (when the fragment is produced by lysis of a dead cell) or 150-250 bp (when the fragment is secreted from an extracellular vesicle released by a cancer cell). Under such circumstances, those conventional methods involving capturing DNA through hybridization and performing high-throughput sequencing on the captured DNA have low sensitivity and specificity. As for whole-genome methylation sequencing, a complex process flow is entailed, and subsequent analysis is complicated, not to mention the relatively high capture cost. PCR-based methods such as BSP are capable of detecting methylation but have two deficiencies due to the lack of CpG sites in the primers used. First, as it is not allowed for the primers to include CpG sites, and C is generally converted into T by bisulfite sequencing (BS), the actual operation is relatively difficult, especially in primer design in CpG islands, and the difficult operation is compounded by such issues as low GC contents, low PCR efficiency, and an excessive amount of non-specific product. Second, the signals of a trace amount of methylated fragments tend to be overwhelmed by the signals of unmethylated fragments because of the lack of selectivity of PCR. For example, if BSP is used to detect a sequence that, in 99 out of 100 cases, may not be methylated when secreted from a normal cell but, in 1 out of 100 cases, is methylated when secreted from a cancer cell, the 1% information may get lost when the other 99% cannot be identified as noise or real signals. The use of MSP or a chip can cover only a limited number of CpG sites (the typical target being a single site) and hence has low flexibility. In cases where a common CpG island has a relatively large number of methylated sites, the conventional methods cannot detect the methylation states of the multiple CpG sites in such a sequence.

The present invention in one aspect provides a methylation detection method for genomic DNA or circulating free DNA molecules in a biological sample.

The technical solution to achieving the aforesaid objective includes the following:

The detection method of the present invention can detect DNA methylation in multiple genes in multiple samples at the same time. The entire detection process and the subsequent data analysis step are simple and scientific and require only a small amount of DNA to start with, making the detection method suitable for detecting methylated fragments having a small copy number. The detection method advantageously features high sensitivity, high specificity, a low detection limit, and low detection cost.

The present invention in another aspect provides a methylation detection kit for genomic DNA or circulating free DNA molecules in a biological sample.

The detection method of the present invention uses a methylation-dependent multiplex PCR technique to amplify and enrich a methylated area of interest rapidly and specifically, and the methylation-dependent multiplex PCR technique is optimized as follows: a high-fidelity polymerase is used to perform methylation-dependent multiplex PCR amplification, and the multiplex PCR product is processed with an endonuclease or DNA-fragment-sorting magnetic beads and then purified. The inventor has found that the foregoing optimization is effective in removing non-specific product in the methylation-dependent multiplex PCR amplification product and can therefore prevent the non-specific product from affecting subsequent detection (e.g., from generating noise and occupying the sequencing depth during the detection process), increase the accuracy of the detection result effectively, and lower the detection limit. In particular, the specificity of the methylation-dependent multiplex PCR in the invention can be enhanced by performing the methylation-dependent multiplex PCR with a Phusion U DNA polymerase. The detection method of the invention also uses suitable reference sequences to normalize the detection results of samples with different methylation ratios so as to enable identification of the differences in methylation and further increase the accuracy and reliability of the detection results.

The present invention uses a methylation-dependent multiplex PCR technique and optimizes both the methylation-dependent multiplex PCR amplification method and the product processing method in order to overcome such prior art drawbacks as the existing methylation detection methods requiring a large amount of DNA to start with and the conventional multiplex PCR amplification products not being directly suitable for high-throughput detection. The invention has high sensitivity, high specificity, high accuracy, a low detection limit, and low detection cost as its advantages, can detect a methylation level as low as 0.05%, has a simpler and more scientific detection process and data analysis step than the prior art, and is particularly suitable for detecting methylated fragments having a small copy number.

FIG. 1 schematically shows the process flow of the detection method of the present invention.

FIG. 2 shows the information about the two exemplary CpG sites in the sequence of SEQ ID NO.5 in embodiment 3.

FIG. 3 is a plot showing the limit of quantification (LOQ) detection results, obtained by the detection method of the invention, of the two exemplary CpG sites in the sequence of SEQ ID NO.5 in embodiment 3.

FIG. 4 is plots showing the LOQ detection results, obtained by bisulfite sequencing PCR, of the same two CpG sites in the sequence of SEQ ID NO.5 in embodiment 3.

FIG. 5 is charts showing the detection results, obtained respectively by the detection method of the invention and bisulfite sequencing PCR, of CpG site 1 (ch5_40681550) in the samples in embodiment 4.

FIG. 6 is charts showing the detection results, obtained respectively by the detection method of the invention and bisulfite sequencing PCR, of CpG site 2 (ch5_40681569) in the samples in embodiment 4.

FIG. 7 is a chart showing the detection results of the dimer percentages of products obtained by methylation-dependent multiplex PCR amplification performed respectively with a Q5U enzyme and a Phusion U enzyme.

FIG. 8 is a chart showing the detection results of the dimer percentages and target sequencing percentages of products obtained by methylation-dependent multiplex PCR amplification performed respectively by a conventional PCR method and a touchdown PCR method.

FIG. 9 is a chart showing the detection results of the dimer percentages of methylation-dependent multiplex PCR amplification products purified respectively with XP Beads and Smart Beads.

FIG. 10 is a chart showing the detection results of the dimer percentages and target sequencing percentages of methylation-dependent multiplex PCR amplification products purified respectively with XP Beads and by column purification.

FIG. 11 is a chart comparing the detection results corresponding respectively to methylation-dependent multiplex PCR amplification with different numbers of multiplex PCR amplification cycles.

FIG. 12 shows satisfactory linear regression of signals detected from the standards in embodiment 5, wherein the standards include 17 target sites and have different methylation concentrations respectively.

FIG. 13 shows the receiver operating characteristic (ROC) curve of embodiment 6.

FIG. 14 shows the ROC curve of embodiment 7.

In cases where the conditions of an experiment method used in any of the following embodiments of the present invention are not specified, the conventional conditions (e.g., the conditions stated in Sambrook et al., Molecular Cloning: Cold Spring Harbor Laboratory Press, New York, 1989) or the conditions suggested by the related manufacturer(s) generally apply. All the common chemical reagents used in the embodiments are commercially available.

Unless otherwise defined, each technical or scientific term used in relation to the present invention has the same connotation as generally understood by a person skilled in the art. All such terms used in this specification serve only to expound the embodiments but not to limit the invention.

The terms “comprise” and “have” and their variations are intended to cover non-exclusive inclusions. For example, a process, method, device, product, or apparatus comprising a series of steps or modules is not limited to the specified steps or modules and may further include, in an optional manner, steps or modules that are not specified or are inherent to the process, method, device, product, or apparatus.

As used herein, the terms “a plurality of” and “multiple” refer to two or more than two. The term “and/or” indicates three possible relationships between the objects connected by the term; for example, “A and/or B” may indicate that A exists alone, that A and B coexist, or that B exists alone. The symbol “/”′ generally indicates “or”.

As used herein, the term “converted DNA molecule” may refer to either of the following: 1) A DNA molecule obtained by converting the cytosines (unmethylated) in the DNA molecule into uracils through a deamination reaction while preserving the 5′-methylcytosines in the DNA molecule, wherein the conversion reagent used in some embodiments may be either sodium bisulfite or a ten-eleven translocation (TET) enzyme plus an apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC) enzyme (an enzymatic method). 2) A DNA molecule obtained by converting the 5′-methylcytosines in the DNA molecule into dihydrouracil by a mixed chemical-enzymatic method while preserving the unmethylated cytosine in the DNA molecule, wherein the conversion reagent used in some embodiments may be a TET enzyme plus pyridine borane.

As used herein, the term “methylation-specific primer” refers to a PCR primer designed for a methylated area. Such a primer can bind specifically to a methylated area in a converted DNA molecule.

As used herein, the term “methylated area” refers to a specific genomic area that includes a CpG site.

As used herein, the term “methylation-specific multiplex PCR”, also known as “methylation-dependent multiplex PCR”, refers to PCR amplification of multiple target methylated sites in the same PCR amplification system and concurrently of the adjacent 40-10,000 bp areas by way of methylation-specific primers, wherein the target methylated sites may be located in the primers or the amplicons.

As used herein, the term “methylation level” may refer to: 1) the methylation ratio of a specific CpG site; 2) the average of the methylation ratios of multiple CpG sites in a specific region; or 3) the normalized read of a methylation-amplified fragment.

As used herein, the term “fragment” refers to a fragment whose size ranges from 40 to 10,000 bp.

As used herein, the term “sequencing” includes second-generation sequencing and third-generation sequencing. The principle of second-generation sequencing lies in massive parallel sequencing (MPS). In some embodiments, second-generation sequencing may be: 1) sequencing by synthesis (SBS), i.e., sequencing by DNA polymerase-based synthesis, some representative companies of which are Illumina (reversible terminator sequencing), Thermo Fisher/Life Technologies (Ion Torrent), GenapSys, and Roche Diagnostics (454 pyrosequencing); or 2) sequencing by ligation (SBL), i.e., sequencing by DNA ligase-based ligation, some representative companies of which are BGI Genomics/Complete Genomics (combinatorial probe-anchor ligation (cPAL)) and Thermo Fisher/Applied Biosystems (sequencing by oligonucleotide ligation and detection (SOLID). Third-generation sequencing is single-molecule sequencing. In some embodiments, third-generation sequencing may be: single-molecule real-time fluorescent sequencing (SMRT) (Pacific Biosciences), nanopore sequencing (Oxford Nanopore Technologies (ONT), and Genia Technologies and Stratos Genomics (Roche Diagnostics)), nanogate sequencing (Quantum Biosystems), or sequencing by DNA hydrolysis (e.g., de-synthesis or pyrophosphorolysis) (Base4).

As used herein, the term “universal array” refers to a technique that entails fixing one of a known target DNA fragment (which may be a methylated fragment) and an unknown nucleic acid sequence to a specific vector in an ordered array, allowing sequence-complementary hybridization to take place, and using a fluorescent technique and pattern recognition analysis to detect the hybridization result. In some embodiments, the universal array may be: 1) the microarray technique, some representative companies of which are Affymetrix (Thermo Fisher)/Agilent; 2) the bead array technique, one representative company of which is Illumina; 3) the xMAP (Multi-Analyte Profiling) technique, one representative company of which is Luminex; or 4) the nCounter technique, one representative company of which is NanoString.

In some embodiments, the biological sample includes a tissue sample, a cell sample, or a fluid sample of an organism and is preferably selected from the group consisting of blood, blood serum, blood plasma, a vitreous body, sputum, urine, tear, sweat, and saliva.

In some embodiments, the genomic DNA or circulating free DNA extracted from the biological sample is subjected to chemical conversion.

In some embodiments, the chemical reaction used for DNA conversion includes a chemical method (e.g., a bisulfite method), an enzymatic method (e.g., through a TET enzyme or an APOBEC enzyme), or a mixed chemical-enzymatic method (e.g., through a TET enzyme and pyridine borane).

In some embodiments, the chemical method and/or the enzymatic method converts the unmethylated cytosine in a DNA molecule into uracil and preserves the 5′-methylcytosine in the DNA molecule in order to obtain the converted DNA molecule.

In some embodiments, the mixed chemical-enzymatic method converts the 5′-methylcytosine in a DNA molecule into dihydrouracil and preserves the unmethylated cytosine in the DNA molecule in order to obtain the converted DNA molecule.

In some embodiments, the method further includes processing and purifying the multiplex PCR product by: processing the obtained multiplex PCR product with an endonuclease or DNA-fragment-sorting magnetic beads in order to obtain a processed product, and purifying the processed product.

Here, the term “methylation-dependent multiplex PCR” refers to PCR amplification of multiple methylated sites of interest in the same PCR amplification system and concurrently of the adjacent 50-300 bp areas, wherein the methylated sites of interest may be located in the primers or the amplicons.

Here, the term “specific PCR primer” refers to a primer designed for a methylated site of interest or an adjacent CpG site. Each specific PCR primer includes a methylated CpG site that has been successfully converted with sodium bisulfite so as to ensure specific amplification of the methylated DNA.

The detection method of the present invention can detect the methylation levels of multiple genes in multiple samples at the same time. Different specific PCR primers are used for different genes or different methylated sites, and during the detection process, different specific tag sequences are used for different samples in order to distinguish the samples from one another.

In some embodiments, the reaction conditions of the methylation-dependent multiplex PCR in step S1 are: 98° C. for 30 s to 5 min; followed by 10-25 cycles each starting with 98° C. for 15 s and continuing with 60±10° C. for 15 s to 10 min and then with 68° C.-72° C. for 15 s to 5 min; followed by 68° C.-72° C. for 0 min to 15 min.

In some embodiments, the reaction conditions of the methylation-dependent multiplex PCR in step S1 are: 98° C. for 30 s to 5 min; followed by 10-25 cycles each starting with 98° C. for 15 s and continuing with 60±5° C. for 15 s to 10 min and then with 72° C. for 15 s to 5 min; followed by 72° C. for 0 min to 15 min.

In some embodiments, the reaction conditions of the methylation-dependent multiplex PCR in step S1 are: 98° C. for 30 s to 5 min; followed by 10-25 cycles each starting with 98° C. for 15 s and continuing with 60±5° C. for 15 s to 5 min and then with 72° C. for 15 s to 5 min; followed by 72° C. for 0 min to 15 min.

In some embodiments, the amplification method of the methylation-dependent multiplex PCR in step S1 is a touchdown PCR method. The inventor has found through research that, in the detection system of the present invention, performing the methylation-dependent multiplex PCR by a touchdown PCR method can effectively increase the specificity of the methylation-dependent multiplex PCR and thereby reduce the product of non-specific amplification.

In some embodiments, the reaction conditions of the methylation-dependent multiplex PCR in step S1 are: 98° C. for 30 s; followed by 5-10 cycles each starting with 98° C. for 15 s and continuing with 65±3° C. (or a temperature successively lowered therefrom by 0.2-0.8° C. after each cycle) for 15 s and then with 72° C. for 15 s; followed by 10-25 cycles each starting with 98° ° C. for 15 s and continuing with 60±10° C. for 15 s to 10 min and then with 68° C.-72° C. for 15 s to 5 min; followed by 72° ° C. for 0 min to 15 min.

In some embodiments, the reaction conditions of the methylation-dependent multiplex PCR in step S1 are: 98° C. for 30 s; followed by 5-10 cycles each starting with 98° C. for 15 s and continuing with 65±3° C. (or a temperature successively lowered therefrom by 0.2-0.8° C. after each cycle) for 15 s and then with 72° C. for 15 s; followed by 10-25 cycles each starting with 98° C. for 15 s and continuing with 60±5° C. for 15 s to 10 min and then with 68-72° C. for 15 s to 5 min; followed by 72° C. for 0 min to 15 min.

In some embodiments, the reaction conditions of the methylation-dependent multiplex PCR in step S1 are: 98° C. for 30 s; followed by 5-10 cycles each starting with 98° C. for 15 s and continuing with 65±3° C. (or a temperature successively lowered therefrom by 0.2-0.8° C. after each cycle) for 15 s and then with 72° C. for 15 s; followed by 10-25 cycles each starting with 98° C. for 15 s and continuing with 60±5° C. for 15 s to 5 min and then with 68-72° C. for 15 s to 5 min; followed by 72° C. for 0 min to 15 min.

In some embodiments, the reaction conditions of the methylation-dependent multiplex PCR in step S1 are: 98° C. for 30 s; followed by 5-10 cycles each starting with 98° C. for 15 s and continuing with 65±3° C. (or a temperature successively lowered therefrom by 0.2° ° C.-0.8° ° C. after each cycle) for 15 s and then with 72° C. for 15 s; followed by 15-20 cycles each starting with 98° C. for 15 s and continuing with 60±3° C. for 15 s and then with 72° C. for 15 s; followed by 72° C. for 15 min.

In some embodiments, the reaction conditions of the methylation-dependent multiplex PCR in step S1 are preferably: 98° C. for 30 s; followed by 10 cycles each starting with 98° C. for 15 s and continuing with 65±3° C. (or a temperature successively lowered therefrom by 0.5° C. after each cycle) for 15 s and then with 72° C. for 15 s; followed by 15 cycles each starting with 98° C. for 15 s and continuing with 60±3° ° C. for 15 s and then with 72° C. for 15 s; followed by 72° C. for 15 min. The inventor has found through research that the most target genes can be successfully detected when the multiplex PCR amplification is carried out under these reaction conditions.

In some embodiments, the methylation-dependent multiplex PCR in step S1 uses an enzyme selected from the group consisting of a Phusion DNA polymerase, a Q5 enzyme, a Hieff enzyme, a KAPA DNA polymerase, a Pfu enzyme, a SuperFi enzyme, and a corresponding uracil-compatible polymerase, preferably from the group consisting of a Phusion U enzyme, a KAPA U enzyme, a Q5U enzyme, and a HieffU enzyme.

In some embodiments, the enzyme used in the methylation-dependent multiplex PCR in step S1 is a Phusion DNA polymerase.

In some embodiments, the enzyme used in the methylation-dependent multiplex PCR is preferably the Phusion U Hot Start DNA polymerase. The Phusion U Hot Start DNA polymerase can better increase the specificity of the methylation-dependent multiplex PCR in the detection method of the present invention and thereby reduce the product of non-specific amplification.

In some embodiments, the touchdown PCR method is a nested PCR method.

In some embodiments, the methylation-dependent multiplex PCR in step S1 is performed on one or more different genes and/or one or more different reference sequences simultaneously.

In some embodiments, the one or more different reference sequences are at least two selected from the group consisting of reference sequences 1-4. Using at least two of the reference sequences contributes to more consistent detection results and higher accuracy in comparing the detection results of different samples.

In some embodiments, the one or more different genes are at least one selected from the group consisting of SEQ ID NO.1-SEQ ID NO.8.

In some embodiments, the methylation-dependent multiplex PCR is performed on at least one sequence selected from the group consisting of SEQ ID NO.1-SEQ ID NO.8 under the following reaction conditions: 98° C. for 30 s; followed by 10 cycles each starting with 98° C. for 15 s and continuing with 65±3° C. (or a temperature successively lowered therefrom by 0.2-0.8° C. after each cycle) for 15 s and then with 72° C. for 15 s; followed by 15-20 cycles each starting with 98° ° C. for 15 s and continuing with 60±3° C. for 15 s and then with 72° C. for 15 s; followed by 72° C. for 15 min.

In some embodiments, the methylation-dependent multiplex PCR is performed on at least one sequence selected from the group consisting of SEQ ID NO.1-SEQ ID NO.8 under the following reaction conditions: 98° C. for 30 s; followed by 10 cycles each starting with 98° C. for 15 s and continuing with 65±3° C. (or a temperature successively lowered therefrom by 0.5° C. after each cycle) for 15 s and then with 72° C. for 15 s; followed by 15 cycles each starting with 98° C. for 15 s and continuing with 60±3° ° C. for 15 s and then with 72° C. for 15 s; followed by 72° C. for 15 min.

In some embodiments, the processing with the endonuclease in step S2 is carried out at 37±1° C. for 10 min to 15 min, with the final concentration of the endonuclease in the processing system being 1-10 U/μL.

In some embodiments, the processing with the endonuclease in step S2 is carried out at 37±1° C. for 10 min to 15 min, with the final concentration of the endonuclease in the processing system being 3.5-4.5 U/μL.

In some embodiments, the processing with the endonuclease in step S2 is preferably carried out at 37±1° C. for 10 min, with the final concentration of the endonuclease in the processing system being 4 U/μL.

In some embodiments, the endonuclease is a T4 endonuclease.

In some embodiments, the DNA-fragment-sorting magnetic beads in step S2 are XP magnetic beads.

In some embodiments, the purification in step S2 is purification with magnetic beads.

In some embodiments, the magnetic beads used for the magnetic-bead-based purification are XP magnetic beads. The inventor has found through research that using XP magnetic beads in the detection method of the present invention leads to better purification results.

In some embodiments, the methylation levels of the purified product obtained from step S2 are detected in step S3 by a sequencing method.

In some embodiments, the sequencing method includes second-generation sequencing and third-generation sequencing. Second-generation sequencing is based on massive parallel sequencing (MPS) and may be, for example, sequencing by synthesis (SBS) or sequencing by ligation (SBL). Third-generation sequencing is single-molecule sequencing and may be, for example, single-molecule real-time fluorescent sequencing, nanopore sequencing, nanogate sequencing, or sequencing by DNA hydrolysis.

In some embodiments, the sequencing method in step S3 includes the following steps: (1) repairing, and simultaneously adding a base A to, the 3′ end of the purified product obtained from step S2; (2) ligating a sequencing adapter to the product obtained from step (1); (3) performing PCR amplification, through an adapter primer, on the ligation product obtained from step (2) in order to obtain a sequencing library; and (4) pooling, according to the same mole number, libraries prepared from different samples, and sequencing the pooled libraries.

In some embodiments, step (3) includes 3-7 PCR amplification cycles. Preferably, step (3) includes 7 PCR amplification cycles.

In some embodiments, step (3) further includes purifying, and performing quality inspection on, the sequencing library.

In some embodiments, the sequencing library is purified with XP magnetic beads.

In some embodiments, the sequencing method includes second-generation sequencing and third-generation sequencing. Second-generation sequencing is based on massive parallel sequencing (MPS). Third-generation sequencing is single-molecule sequencing.

In some embodiments, the second-generation sequencing may be sequencing by synthesis (SBS) or sequencing by ligation (SBL).

In some embodiments, the third-generation sequencing may be single-molecule real-time fluorescent sequencing, nanopore sequencing, nanogate sequencing, or sequencing by DNA hydrolysis.

In the present invention, the sequencing method in step S3, i.e., the multi-sample multi-fragment DNA methylation detection method used to detect the methylation levels of the purified product obtained from step S2, is named Methylation-Dependent Amplification and Sequencing (MeDAS).

In some embodiments, a universal array method is used in step S3 to detect the methylation levels of the purified product obtained from step S2. The universal array refers to a technique that entails fixing one of a known target DNA fragment (which may be a methylated fragment) and an unknown nucleic acid sequence to a specific vector in an ordered array, allowing sequence-complementary hybridization to take place, and using a fluorescent technique and pattern recognition analysis to detect the hybridization result. During the detection process, different specific tag sequences are used for different samples in order to distinguish the samples from one another.

In some embodiments, the universal array technique may be the microarray technique, the bead array technique, the xMAP (Multi-Analyte Profiling) technique, or the nCounter technique.

In some embodiments, the sample is a body fluid, cell, or tissue of an organism.

In some embodiments, the body fluid of an organism is a body fluid secreted by an organ or tissue of the human body, and the human body may be in a normal or pathological state.

In some embodiments, the body fluid of an organism is blood, urine, saliva, sweat, cerebrospinal fluid, fluid in the pleural cavity, or ascitic fluid.

In some embodiments, the body fluid of an organism is preferably blood serum, blood plasma, a vitreous body, sputum, urine, tear, sweat, or saliva.

In some embodiments, the kit includes at least two of the foregoing four groups of specific PCR primers.

In some embodiments, the kit includes the foregoing four groups of specific PCR primers.

In some embodiments, the kit further includes a Phusion DNA polymerase, a T4 endonuclease, and/or DNA-purifying magnetic beads.

In some embodiments, the Phusion DNA polymerase is preferably the Phusion U Hot Start DNA polymerase.

In some embodiments, the DNA-purifying magnetic beads are preferably XP magnetic beads.

In some embodiments, the kit further includes a sequencing library construction reagent.

The detection process flow of the detection method provided by the present invention is shown in FIG. 1.

In this embodiment, a multi-sample multi-fragment DNA methylation detection method includes the following steps:

S1. extracting genomic DNA or circulating free DNA from a sample, processing the extracted DNA with the EZDNA Methylation-Gold (ZYMO) kit, and performing methylation-dependent multiplex PCR on the processed DNA through specific primers, wherein the enzyme used in the PCR is a high-fidelity polymerase selected from the group consisting of a Phusion DNA polymerase (e.g., the Phusion Hot Start II DNA Polymerase or the Phusion U Hot Start DNA Polymerase), a Q5 enzyme (e.g., the Q5® Hot Start High-Fidelity DNA Polymerase or the Q5U® Hot Start High-Fidelity DNA Polymerase), a Hieff enzyme (e.g., the Hieff NGS® HG Hot Start Multiplex PCR Enzyme), a KAPA DNA polymerase (e.g., the KAPA HiFi Uracil+ Kit or the KAPA2G Fast Hot Start DNA Polymerase), a Pfu enzyme, and a SuperFi enzyme; wherein the Phusion U Hot Start DNA Polymerase is preferably used in this embodiment; wherein a touchdown PCR method is used as the PCR amplification method; and wherein different specific PCR primers are used for different genes or different methylated sites;

S2. processing and purification of the multiplex PCR product either by applicable method 1, which includes processing the multiplex PCR product obtained from step S1 with a T4 endonuclease, the processing conditions being 37±1° C. for 10-15 min, with the final enzyme concentration in the processing system being 1-10 U/μL (preferably, the processing conditions being 37±1° C. for 10 min, with the final enzyme concentration in the processing system being 4 U/μL), and purifying the enzymatically processed PCR product, the purification method being purification with XP magnetic beads; or by applicable method 2, which includes processing and purifying the multiplex PCR product directly with DNA-fragment-sorting magnetic beads, wherein the magnetic beads are XP magnetic beads; and

S3. detecting, by a sequencing method, the methylation levels of the purified product obtained from step S2: (1) repairing, and simultaneously adding a base A to, the 3′ end of the purified product obtained from step S2; (2) ligating a sequencing adapter to the product obtained from step (1); (3) performing PCR amplification, through an adapter primer, on the ligation product obtained from step (2), with the number of PCR cycles being 3-7 (preferably 7), then purifying the amplification product in order to obtain a sequencing library, and performing quality inspection on and quantifying the sequencing library obtained; and (4) pooling, according to the same mole number, libraries prepared from different samples, and sequencing the pooled libraries with the Illumina MiSeq/MiSeqDx/NextSeq/NextSeqDx platform.

The detection method provided by this embodiment is named Methylation-Dependent Amplification and Sequencing (MeDAS) and can be used to perform DNA methylation detection on multiple genes in multiple samples at the same time. The entire detection process and the subsequent data analysis step are simple and scientific and require only a small initial amount of DNA, making the detection method suitable for detecting methylated fragments having a small copy number. Moreover, the detection method advantageously features high sensitivity, high specificity, a low detection limit, and low detection cost. TABLE 1 compares the features of the MeDAS method of the present invention with those of some existing methylation detection methods:

In the table above, MeDAS stands for Methylation-Dependent Amplification and Sequencing, MSP stands for Methylation-Specific PCR, and BSP stands for Bisulfite Sequencing PCR.

It can be known from TABLE 1 that the MeDAS technique provided by the present invention has such advantages over the prior art as high throughput, high sensitivity, high specificity, a low detection limit, and low detection cost.

In this embodiment, a DNA methylation detection kit includes reagents for performing methylation level detection on the sequences of SEQ ID NO.1-SEQ ID NO.8. The sequences of SEQ ID NO.1-SEQ ID NO.8 are shown in TABLE 2:

More specifically, the kit includes the following constituents:

(1) PCR primers specific to the sequences of SEQ ID NO.1-SEQ ID NO.8, as shown in TABLE 3:

The details of the reference sequences are summarized in TABLE 4:

(3) The Phusion U Hot Start DNA Polymerase, the T4 endonuclease, and the Agencourt AMPure XP Beads.

The PCR primers provided in the kit as specific to the sequences of SEQ ID NO.1-SEQ ID NO.8 were designed and successfully obtained by the inventor after an extensive research and analysis. Those detection primers can ensure the sensitivity and specificity of methylation-dependent multiplex PCR amplification but have no effect on the depth of sequent sequencing. The sequencing depth required for detecting methylated fragments under the detection limit of 2 copies to 0.05% methylation is less than that of an existing method (bisulfite sequencing PCR) by about 2-3 orders of magnitude.

The reference sequences 1-4 (Ref1-4) provided in the kit were obtained through optimization by the inventor, are consistently expressed in different samples, are not subject to the methylation level, and are suitable for use to normalize sequencing data so that the DNA methylation levels of different samples can be compared with one another to produce accurate and reliable comparison results.

In this embodiment, detection results obtained with the DNA methylation detection method of the present invention (i.e., the MeDAS method) were compared with those obtained with bisulfite sequencing PCR (BSP). More specifically, the methylation states of the sequences of SEQ ID NO.1-SEQ ID NO.8 as stated in relation to embodiment 2 were detected separately with the DNA methylation detection method of the invention and BSP. The DNA methylation detection method of the invention used the kit in embodiment 2 and was carried out as follows:

S1. DNA Standards were Obtained and then Processed with a Bisulfite. After that, Methylation-Dependent Multiplex PCR Amplification was Performed on the Processed DNA Standards.

(1) Preparation of a cfDNA mock standard

1) Genomic DNA quantification with Qubit HS

a) Preparation of a Qubit solution:

b) Dilution of the genomic DNA sample: 1 μL of DNA sample+9 μL of EB (elution buffer) were measured out. The DNA sample was diluted 10 times, before 1 μL of the diluted

DNA was quantified.

2) Shearing of the genomic DNA

The genomic DNA was diluted with EB to about 10 ng/μL, and then 300 μL of the diluted DNA was measured out. The three shearing tubes accompanying the kit (each tube having a specification of 130 μL) were each added with 50 μL of the diluted DNA (repeated twice) in order to carry out the following shearing procedure:

3 μL of the sheared DNA was taken out and kept at 4° ° C., waiting for its concentration to be determined and its quality to be inspected with 2100 Bioanalyzer.

3) Purification and storage of the sheared DNA

a) Preparation of 80% ethanol

b) Purification steps: allowing undiluted AMPure magnetic beads to reach equilibrium at room temperature for 30 min→measuring out 240 μL of the magnetic beads, mixing with all the sheared genomic DNA sufficiently in a 1.5 mL LoBind tube with a vortex mixer, and subjecting the mixture to simple centrifugation→incubation at room temperature for 5 min→placing on the magnetic base for 3-4 min, or until the solution becomes clear→transferring the supernatant to a new 1.5 mL LoBind tube→adding 240 μl of undiluted AMPure magnetic beads into the tube→mixing sufficiently with the vortex mixer, followed by simple centrifugation→incubation at room temperature for 5 min→placing on the magnetic base for 4 min, or until the solution becomes clear, followed by removal of the supernatant by suction→adding 1.5 mL of the 80% ethanol in order to carry out washing for 30 s, at the end of which the supernatant is removed→adding 1.5 mL of the 80% ethanol in order to carry out washing for 30 s, at the end of which the supernatant is removed→centrifugation for 1 min, followed by placement on the magnetic base until the liquid becomes clear, followed by removal of excessive liquid by suction→opening the lid and allowing the contents of the tube to dry until the magnetic bead surface is no longer reflective→adding 100 μL of EB for elution, followed by a thorough mix, simple centrifugation, and incubation at room temperature for 2 min→placing on the magnetic base for magnetic attraction for 2 min, or until the liquid becomes clear, followed by collecting the supernatant and transferring the supernatant to a new 1.5 mL LoBind tube; and then repeating the elution step once, with the final volume of the DNA sample being 200 μL.

c) Storage: Storage at −20° C.

4) Quantification and quality inspection of the purified DNA

Quality inspection: 1 μL of the purified cfDNA mock sample was measured out, and the quality of the sample was inspected with 2100 Bioanalyzer. Quantification: 1 μL of the purified sample was measured out and was quantified with Qubit HS.

(2) Preparation of DNA standard substrates with a concentration gradient by a mixing process

1) Preparation of a 100%-methylation control by processing the genomic DNA with a bisulfite

The HG DNA and the cfDNA mock were processed with the EZDNA Methylation-Gold (ZYMO) kit as follows:

a) Preparation of associated buffers:

a. Preparation of the CT Conversion Reagent: 900 μL of water, 50 μL of M-Dissolving Buffer, and 300 μL of M-Dilution Buffer were added into a tube of CT Conversion Reagent in order for the reagent to be dissolved at room temperature. The tube was then placed on a shaker and shaken for 10 min.

b. Preparation of the M-WASH BUFFER: 24 mL of absolute ethanol was added into a flask of M-WASH BUFFER, and the lid of the flask was marked.

b) Adding the following into a PCR tube:

a. 500 ng of DNA, followed by water until the total volume reached 20 μL, wherein the volume of the water added was calculated according to the desired concentration; and

b. 130 μL of the CT Conversion Reagent. The test tube or pipet was then gently tapped to mix the sample.

c) The sample tube was placed in a thermal cycler, which was operated as follows: setting the thermal cycler at 98° ° C. for 10 min and then at 64° C. for 14 h.

d) The column of a collection tube was put into the collection tube, and 600 μL of M-Binding Buffer was added into the column, followed by the sample in step c The column was then covered with its lid and inverted several times to mix the sample.

e) Centrifugation at full speed (>10,000×g) for 30 s, followed by removal of the waste liquid.

f) 200 μL of M-Wash Buffer was added into the column, and then centrifugation was performed at full speed for 30 s.

g) 200 μL of M-Desphonation Buffer was added into the column, and the sample was allowed to rest at room temperature (20-30° C.) for 15-20 min.

h) Centrifugation at full speed for 30 s, followed by removal of the waste liquid.

i) 200 μL of M-Wash Buffer was added into the column, and then centrifugation was performed at full speed for 30 s.

j) 200 μL more of M-Wash Buffer was added, and then centrifugation was performed for 30 s.

k) 25 μL of M-Elution Buffer was added directly to the substrate in the column, and the column was placed into a 1.5 mL tube. DNA elution was carried out by full-speed centrifugation to obtain the final product, i.e., a converted product (which when kept at −20° C. remained good for use for 1 week).

1) The degrees of methylation conversion at 7-10 CpG, CHG, and CHH sites in the standard were determined by a sequencing analysis.

2) Preparation of standards with a concentration gradient of 0%, 0.05%, 0.1%, 0.25%, 0.5%, 1%, and 100% by mixing the 0%-methylation control with the 100%-methylation control

(3) The methylation-dependent multiplex PCR of the present invention and multiplex bisulfide-specific PCR (multiplex BSP) were separately performed on the sequences of SEQ ID NO.1-SEQ ID NO.8 as stated in relation to embodiment 2 and on reference sequences 1-4. The methylation-dependent multiplex PCR primers for use with the sequences of SEQ ID NO.1-SEQ ID NO.8 have been shown above in relation to embodiment 2, and so have the primers for use with reference sequences 1-4. The multiplex BSP primers for use with the sequences of SEQ ID NO.1-SEQ ID NO.8 are shown in TABLE 7.

The methylation-dependent multiplex PCR amplification system of the present invention is shown in TABLE 8, with the amplification procedure shown in TABLE 9:

The BSP detection system is shown in TABLE 10, with the detection procedure shown in TABLE 11:

(1) The foregoing methylation-dependent multiplex PCR products were processed with a T4 endonuclease, the processing conditions being 37±1° C. for 10 min, with the final T4 endonuclease concentration in the processing system being 4 U/μL.

(2) The T4 endonuclease-processed PCR product of each of the different samples was purified by Agencourt AMPure XP Beads as follows:

a) The AMPure XP Beads were allowed to reach equilibrium at room temperature for at least 30 min.

b) 50 ng of the DNA for which a library was to be constructed was added into a 1.5 mL centrifuge tube, followed by 90 μL of (1.8×) AMPure XP Beads. The tube was placed on a vortex mixer, where the sample was mixed at room temperature for 5 min.

c) The centrifuge tube was placed on the magnetic base until the solution became clear (about 3-5 min).

d) The supernatant in the centrifuge tube was carefully removed while the centrifuge tube was kept on the magnetic base.

e) 200 μL of 80% ethanol was added while the centrifuge tube remained on the magnetic base.

f) The tube was allowed to stand for 1 min in order for all the beads to settle. The ethanol was then removed, and e) and f) were repeated once.

g) Simple centrifugation was performed, and then the tube was placed on the magnetic base until the liquid became clear. Excessive liquid was removed by suction. The sample was then dried in a 37° C. heating module for 5 min, or until all the residual ethanol disappeared.

h) 55 μL of EB was added for elution, followed by a thorough mix and incubation at room temperature for 5 min.

i) Simple centrifugation was performed, and then the tube was placed on the magnetic base for 3-5 min, or until the liquid became clear. The supernatant was collected. (A total of 50 μL was collected. 2 μL was taken from each of a number of selected samples in order to carry out a Qubit test, and all such samples were subsequently replenished to 50 μL.)

S3. Detecting, by a Sequencing Method, the Methylation Levels of the Purified Products Obtained from Step S2

Library construction was carried out with the NEB library construction kit Z1901S/L, with specific tag sequences used to distinguish the different samples from one another. The library construction steps are as follows:

(1) The 3′ end of each purified product obtained from step S2 was repaired and was added with a base A at the same time.

An end-repairing and base A addition system was prepared in a 1.5 mL centrifuge tube according to the following table with the DNA purified product obtained from the previous step:

Reaction process: 20° ° C. for 30 min→65° C. for 30 min→held at 4° C., with heated lid: 85° ° C., volume: 60 μL.

(2) A sequencing adapter was ligated to the product having its 3′ end added with the base A, in order to produce a sequencing-adapter-ligated product.

A ligation reaction system was prepared in a 1.5 mL centrifuge tube according to the following table with the product obtained from the previous step:

Reaction process: 20° ° C. for 15 min→held at 4° C., with heated lid closed, volume: 100 μL.

After the reaction, purification was conducted with Agencourt AMPure XP Beads. The DNA was then eluted with 20 μL of EB, and the supernatant (15 μL) was collected.

(3) The sequencing-adapter-ligated product was subjected to PCR amplification, with the number of PCR cycles being 7 in order to obtain a sequencing library. The reaction system is as follows:

Reaction process: 98° C. for 30 s→7 cycles (each starting with 98° C. for 10 s→65° C. for 75 s)→65° ° C. for 5 min→held at 4° C., with heated lid: 105° C., volume: 50 μL.

After the reaction, purification was conducted with Agencourt AMPure XP Beads. The DNA was then eluted with 35 μL of EB, and the supernatant (30 μL) was collected.

Quantification: The library obtained was quantified with Qubit® 2.0 (Invitrogen) and stored at −20° C.

Quality inspection with 2100 Bioanalyzer: Library concentration range for the 2100 Bioanalyzer inspection: 100 pg/μL-10 ng/μL. The inspection was performed according to the quick start guide “5.2100_HighSensitivityDNA_QSG”.

(4) Second-generation high-throughput sequencing

Libraries prepared from the different samples were pooled according to the same mole number. After that, second-generation high-throughput sequencing was performed, and the sequencing steps are as follows:

The mass concentration (ng/μL) of each library was measured with Qubit, and the average length (bp) of the libraries was determined with 2100 Bioanalyzer. Mass concentration was then converted into molar concentration by the following equation. To perform sequencing runs on the sequencing machine, the libraries were mixed, i.e., pooled, according to the ratios of their respective data amounts. After that, the theoretical molar concentrations of the pooled libraries were calculated.

$\begin{array}{c}\mathrm{Molar}\mathrm{concentration}\\ \left(\mathrm{nM}\right)\mathrm{of}\mathrm{library}\end{array}=\frac{\mathrm{Mass}\mathrm{concentration}\left(\mathrm{ng}/\mu L\right)\mathrm{of}\mathrm{library}}{\begin{array}{c}\left(660g/\mathrm{mol}\times \mathrm{average}\mathrm{length}\left(\mathrm{bp}\right)\mathrm{of}\right)\\ \mathrm{library})\times 1000\end{array}}\times {10}^6$

Note: The average length of libraries YG001-047 is 350 bp.

2) Single-end sequencing was performed on the samples according to the procedure of Illumina MiSeq PE-300. The sequencing results produced by MiSeq were DNA sequences in the fastq format. Each sequenced sequence was correlated to the corresponding sample through a sequencing library index and a sample tag sequence (also referred to as a barcode), before the methylation state of each CG site in the detected fragment of each sample was calculated.

The detection results are as follows:

(1) The detection method of the present invention has a lower-LOQ hypothesis than bisulfite sequencing PCR: The detection method of the invention can detect 0.05% methylation (the average sequencing depth required was only 100-200×, and there was a linear trend of the detection signals as the concentration increased), whereas the lower detection limit of bisulfite sequencing PCR is 0.5% methylation (linear relationships seldom occurred even though the sequencing depth was greater than ten thousand X). Take the detection results of two CpG sites in the sequence of SEQ ID NO.5 for example. (Information about the two sites is shown in FIG. 2.) The LOQ of the two CpG sites as detected by the method of the invention is shown in FIG. 3, with the average sequencing depth of each gradient interval being 100-200×. The LOQ of the same two CpG sites as detected by bisulfite sequencing PCR is shown in FIG. 4, with the average sequencing depth of each gradient interval being greater than 9500×.

(2) The detection method of the present invention has a lower-LOD (limit of detection) hypothesis than bisulfite sequencing PCR: Take the two CpG sites ch1_63795447 (−) and chr1_6379497 (−) in the sequence of SEQ ID NO.7 for example. At the LOD gradient of 2 copies, the detection method of the invention can detect the methylation states of the two sites consistently at a sequencing depth of several thousand X, but bisulfite sequencing PCR cannot, even at a sequencing depth of ˜40000× (the detected C was in the interval of sequencing noise), as detailed in TABLE 15:

In this embodiment, the methylation levels of the eight sequences of SEQ ID NO.1-SEQ ID NO.8 in three clinical lung cancer tissue samples and three control tissue samples were detected separately by the detection method of the present invention and bisulfite sequencing PCR. The detection steps are as follows:

S1. Genomic DNA was extracted from the clinical lung cancer tissue samples and the control tissue samples, and the extracted DNA was processed with a bisulfite. After that, methylation-dependent multiplex PCR amplification was performed on the processed DNA.

More specifically, genomic DNA was extracted from the samples with the Qiagen-QIAamp-DNA-FFPE tissue kit (Qiagen, Cat #56404). The extraction steps were carried out according to the kit user guide. The extracted DNA was processed with a bisulfite and subjected to methylation-dependent multiplex PCR amplification as described in relation to embodiment 3.

Steps S2 and S3 were the same as described in relation to embodiment 3.

According to the detection results, the detection method of the present invention can detect, at a sequencing depth of several thousand X, the significant difference in methylation between the lung cancer samples and normal samples, but bisulfite sequencing PCR cannot. Take the detection results of two CpG sites (CpG site 1: ch5_40681550 and CpG site 2: ch5_40681569) in the sequence of SEQ ID NO.5 for example. (The two CpG sites are known to have relatively high methylation levels in lung cancer tissues.) The detection results of CpG site 1 ch5_40681550 are shown in FIG. 5, and those of CpG site 2 ch5_40681569 in FIG. 6. It can be seen in FIG. 5 and FIG. 6 that the method of the invention can find the significant difference of the detected sites in the lung cancer tissue samples at a sequencing depth of 2000×, and that BSP cannot effectively detect the difference between the normal samples and lung cancer samples even at a ten-times-greater sequencing depth, i.e., at 20000×.

The DNA methylation detection method in this comparative example is the same as the DNA methylation detection method in embodiment 3 except that the methylation-dependent multiplex PCR amplification in step S1 used a Q5 enzyme instead of the Phusion DNA polymerase, the Q5 enzyme being used in the same amount as the Phusion DNA polymerase. The methylation-dependent multiplex PCR amplification in the detection method of the present invention uses a high-fidelity enzyme selected from the group consisting of a Phusion DNA polymerase (e.g., the Phusion Hot Start II DNA Polymerase or the Phusion U Hot Start DNA Polymerase), a Q5 enzyme (e.g., the Q5® Hot Start High-Fidelity DNA Polymerase or the Q5U® Hot Start High-Fidelity DNA Polymerase), a Hieff enzyme (e.g., the Hieff NGS® HG Hot Start Multiplex PCR Enzyme), a KAPA DNA polymerase (e.g., the KAPA HiFi Uracil+ Kit or the KAPA2G Fast Hot Start DNA Polymerase), a Pfu enzyme, and a SuperFi enzyme. All the aforesaid high-fidelity enzymes can produce relatively good multiplex amplification results. A Phusion DNA polymerase, in particular, can produce the best multiplex amplification results. This comparative example was intended to compare the multiplex amplification results of a Q5 enzyme (preferably Q5® Hot Start High-Fidelity DNA Polymerase) with those of a Phusion DNA polymerase (preferably the Phusion U Hot Start DNA Polymerase).

Methylation-dependent multiplex PCR amplification was performed on 50 different genes in a 10%-methylation cfDNA mock standard by the methylation-dependent multiplex PCR method used in the DNA methylation detection method of this comparative example and by the methylation-dependent multiplex PCR method used in the DNA methylation detection method of embodiment 3, separately. The PCR detection conditions were the same: 98° C. for 30s; followed by 18 cycles each starting with 98° C. for 15 s and continuing with 60° ° C. for 2 min and then with 72° C. for 1 min; followed by 72° C. for 15 min.

An analysis was conducted with 2100 Bioanalyzer. Dimer percentages were calculated according to the ranges of fragment lengths. The dimer percentages of the amplification products of the two amplification methods were compared.

As shown by the detection results in FIG. 7, using a Phusion DNA polymerase in methylation-dependent multiplex PCR amplification (i.e., the methylation-dependent multiplex PCR method used in the detection method of embodiment 3) can reduce the dimer percentage of the amplification product more effectively than using a Q5 enzyme. A lower dimer percentage makes it easier to carry out subsequent detection on the multiplex PCR amplification product.

The DNA methylation detection method in this comparative example is the same as the DNA methylation detection method in embodiment 3 except that the multiplex PCR amplification in step S1 used a conventional PCR method whose reaction process is shown in TABLE 16:

Methylation-dependent multiplex PCR amplification was performed on 50 different genes in a 100%-methylation cfDNA mock standard by the methylation-dependent multiplex PCR method used in the DNA methylation detection method of this comparative example and by the methylation-dependent multiplex PCR method used in the DNA methylation detection method of embodiment 3, separately. The primer dimer percentages and target sequencing percentages of the amplification products of the two amplification methods were compared.

As shown by the detection results in FIG. 8 (in which “Normal PCR” refers to the conventional PCR method, and “Touchdown PCR” refers to the touchdown PCR method used in the present invention), performing methylation-dependent multiplex PCR amplification by a touchdown PCR method (i.e., the methylation-dependent multiplex PCR method used in the detection method of the invention) can reduce the dimer percentage of the amplification product more effectively (by up to 50 percentage points) than by a conventional PCR method. A lower dimer percentage makes it easier to carry out subsequent detection on the multiplex PCR amplification product and can increase the target gene sequencing percentage (the target gene sequencing percentage was increased by about 40 percentage points).

The DNA methylation detection method in this comparative example is the same as the DNA methylation detection method in embodiment 3 except that the T4 endonuclease-processed methylation-dependent multiplex PCR amplification product was purified in step S2 with Smart Beads instead of the XP Beads. The steps of purifying the multiplex PCR amplification product with Smart Beads are as follows: adding 50 μL of diethyl pyrocarbonate (DEPC) into 50 μL of PCR product→allowing the Smarter magnetic beads to reach equilibrium at room temperature for 30 min→mixing the beads thoroughly, and then mixing 220 μL (2.2×) of the beads with the sample by a vortex mixer→incubation at room temperature for 5 min→simple centrifugation, followed by placement on the magnetic base for magnetic attraction for 5 min, or until the liquid becomes clear, followed by removal of the supernatant by suction→rinsing with 200 μL of 80% ethanol for 30 s, and then removing the supernatant→rinsing with 200 μL of 80% ethanol for 30 s, and then removing the supernatant→simple centrifugation, followed by placement on the magnetic base until the liquid becomes clear, followed by removal of excessive liquid by suction→opening the lid, allowing the contents of the tube to dry until the magnetic bead surface is no longer reflective→adding 55 μL of EB for elution, followed by a thorough mix and then incubation at room temperature for 5 min→simple centrifugation, followed by placement on the magnetic base for magnetic attraction for 3-5 min, or until the liquid becomes clear, followed by collection of the supernatant (A total of 50 μL was collected. 2 μL was taken from each of a number of selected samples in order to carry out a Qubit test, and all such samples were subsequently replenished to 50 μL.).

The methylation-dependent multiplex PCR amplification products of 50 different genes in a 10%-methylation cfDNA mock standard were purified by the purification method used in the DNA methylation detection method of this comparative example and by the purification method used in the DNA methylation detection method of embodiment 3, separately. The dimer percentages of the amplification products corresponding respectively to the two purification methods were compared.

As shown by the detection results in FIG. 9, using XP Beads to purify a methylation-dependent multiplex PCR amplification product (the purification method used in the detection method of the present invention) can reduce the dimer percentage of the amplification product more effectively than using Smart Beads. A lower dimer percentage makes it easier to carry out subsequent detection on the purified multiplex PCR amplification product.

The DNA methylation detection method in this comparative example is the same as the DNA methylation detection method in embodiment 3 except that the T4 endonuclease-processed methylation-dependent multiplex PCR amplification product was purified in step S2 with a purification column instead of the XP Beads. The steps of purifying the multiplex PCR amplification product with a purification column (made by QIAGEN) are as follows: adding a binding solution into the PCR product, and mixing thoroughly by inverting the tube several times, wherein the volume of the binding solution is five times that of the PCR product→transferring into a DNA recovery column, allowing to rest at room temperature for 2 min, followed by centrifugation at 8,000 rpm for Imin→pouring out the waste liquid in the collection tube→repeating once→adding a wash solution to perform elution once, followed by centrifugation at 12,000 rpm for 1 min→adding 50 μL of elution buffer, allowing to rest for 2 min, followed by centrifugation at 12,000 rpm for 1 min.

The methylation-dependent multiplex PCR amplification products of 50 different genes in a 10%-methylation cfDNA mock standard were purified by the purification method used in the DNA methylation detection method of this comparative example and by the purification method used in the DNA methylation detection method of embodiment 3, separately. The dimer percentages and target sequencing percentages of the amplification products corresponding respectively to the two purification methods were compared.

As shown by the detection results in FIG. 10 (in which “Column” refers to purification with a purification column), using XP Beads to purify a methylation-dependent multiplex PCR amplification product (the purification method used in the detection method of the present invention) can reduce the dimer percentage of the amplification product more effectively than using a purification column. A lower dimer percentage makes it easier to carry out subsequent detection on the purified multiplex PCR amplification product and can increase the target gene sequencing percentage.

The DNA methylation detection method in this comparative example is the same as the DNA methylation detection method in embodiment 3 except for the number of methylation-dependent multiplex PCR amplification cycles in step S1. Multiplex PCR amplification in this comparative example was performed under two different conditions: (1) 98° ° C. for 30 s; followed by 10 cycles each starting with 98° ° C. for 15 s and continuing with 65° C. (or a temperature successively lowered therefrom by 0.5° C. after each cycle) for 15 s and then with 72° C. for 15 s; followed by 17 cycles each starting with 98° C. for 15 s and continuing with 60° C. for 15 s and then with 72° C. for 15 s; followed by 72° C. for 15 min; and (2) 98° C. for 30 s; followed by 10 cycles each starting with 98° C. for 15 s and continuing with 65° C. (or a temperature successively lowered therefrom by 0.5° ° C. after each cycle) for 15 s and then with 72° C. for 15 s; followed by 20 cycles each starting with 98° C. for 15 s and continuing with 60° C. for 15 s and then with 72° C. for 15 s; followed by 72° C. for 15 min. The detection method of the present invention can provide detection when the number of methylation-dependent multiplex PCR amplification cycles ranges from 15 to 20. In particular, the most target genes can be successfully detected when the number of methylation-dependent multiplex PCR amplification cycles is 15.

Methylation-dependent multiplex PCR was performed on 153 different genes in differently methylated (i.e., 0.5%, 1%, 5%, and 10%-methylation) cfDNA mock standards by the methylation-dependent multiplex PCR methods used respectively in the two DNA methylation detection methods of this comparative example and the DNA methylation detection method of embodiment 3 of the present invention, followed by sequencing, an analysis, and a comparison. The detection results in FIG. 11 show that, under the conditions of 0.5%, 1%, 5%, and 10% methylation, the number of cycles being 15 leads to successful detection of significantly more target genes than the number of cycles being 17 or 20 and corresponds to the least undetected target genes.

This embodiment was intended to evaluate the detection effect of the DNA methylation detection method of the present invention (i.e., the MeDAS method) on human-cell-line genomic DNA of different methylation levels. The steps of this embodiment are as follows:

(1) Preparation of genomic DNA standards of different methylation levels: The 0% and 100%-methylation human genomic DNA standards were purchased from Zymo (Cat #D5014). The 0%-methylation standard was sourced from the HCT116 [DNMT1 (−/−) DNMT3b (−/−)] cell line, and the 100%-methylation standard was sourced from a methyltransferase-processed product of the 0%-methylation standard and was verified by sequencing. The 25% and 50%-methylation standards were produced by mixing the 0% and 100%-methylation standards in appropriate proportions.

(2) In this embodiment, methylation-dependent multiplex PCR primers were designed for 32 randomly selected CpG sites in the genome, as shown in the following table:

(3) The 0%, 25%, 50%, and 100%-methylation standards were processed with a bisulfite and underwent methylation-dependent multiplex PCR amplification according to the methods used in embodiment 3. Steps S2 and S3 were the same as described in relation to embodiment 3.

As shown by the detection results in FIG. 12, the detection method of the present invention enables satisfactory linear regression (R²>0.8) of signals detected from those standards that, in addition to having different methylation concentrations respectively, included the following 17 target sites: cg16673106, cg25381667, cg24016939, cg22101924, cg21715963, cg16712637, cg15811719, cg14603466, cg14589148, cg13119884, cg12622139, cg12180984, cg07696033, cg06080005, cg04234680, cg03556653, and cg02596331. The detection results show that the detection method of the invention can be used to carry out high-throughput effective detection of the methylation levels of certain target sits in cell-line genomic DNA.

Multiplex methylation amplification was performed on all the conversion product. The reaction constituents included: a combination of primers for 103 gastric cancer differentiation markers obtained by tissue screening, the final concentration of each primer being 50-200 nM; magnesium ions at a concentration of 2-5 mM, or preferably at 3 mM as in this embodiment; and a dNTP mix at a concentration of 100-600 μM, or preferably at 200 μM as in this embodiment. The enzyme used in the reaction was KAPA2G Fast Multiplex PCR Kit (Roche, Cat #KK5802). The reaction conditions were: 95° ° C. for 5 min for initial denaturation, followed by 15-30 or preferably 20 cycles each starting with 95° C. for 15 s for denaturation and continuing with 58-66° ° C., or preferably 63° ° C. as in this embodiment, for 4 min for annealing.

The multiplex reaction system was designed as follows:

Experimental results: All the 103 markers were used in model construction according to the random forest model and were subsequently analyzed. Cleavage in a ratio of 7:3 was repeated 100 times. Given 99% specificity, the test set showed phase-1 detection sensitivity of 87.5%, phase-2 detection sensitivity of 92.9%, phase-3 detection sensitivity of 77.8%, and phase-4 detection sensitivity of 86.7%. The overall detection sensitivity toward all the colorectal cancer samples was 85.3%. The overall AUC (area under the receiver operating characteristic (ROC) curve) was 0.974. The experimental results indicate that the markers can be used together with the method of the present invention in early screening of gastric cancer. The ROC curve is shown in FIG. 13.

This embodiment was intended to evaluate the detection effect of the DNA methylation detection method of the present invention (i.e., the MeDAS method) on markers in breast cancer patients' blood samples, in order to determine whether the markers can be used in conjunction with the detection method in early screening and early diagnosis of breast cancer. For more details about the steps of this embodiment, please refer to embodiment 2 of the invention. The plasma samples of 30 normal people and of 30 breast cancer patients were detected. All the 54 methylation markers obtained by tissue screening were used in model construction according to the random forest model and were subsequently analyzed. Cleavage in a ratio of 7:3 was repeated 100 times. Given 99% specificity, the 11 phase-1 samples in the test set showed 54.5% detection sensitivity, the 15 phase-2 samples showed 26.7% detection sensitivity, and the four phase-3 samples showed 25% detection sensitivity. The overall detection sensitivity toward all the breast cancer samples was 36.7%. The overall AUC was 0.948. The experimental results indicate that the markers can be used together with the method of the invention in early screening of breast cancer. The ROC curve is shown in FIG. 14.

The technical features of the foregoing embodiments can be arbitrarily combined. For the sake of brevity, however, this specification does not describe all the possible combinations of the technical features of the embodiments. All such combinations shall be construed as falling within the scope of the specification, provided that the technical features 5 combined do not conflict with one another.

The embodiments described above disclose only a few modes of implementing the present invention. The relatively specific and detailed description of those embodiments shall not be understood as restrictive of the scope of patent protection for the invention. It should be pointed out that a person of ordinary skill in the art may make various changes and improvements without departing from the concept of the invention, and that all such changes and improvements shall fall within the scope of patent protection for the invention. The scope of patent protection for the invention is defined by the appended claims.