Marker for lung cancer screening, probe composition and use thereof

Abstract

The application discloses a marker for lung cancer screening, a probe composition and application thereof, and the marker is selected from any one of three markers. The marker is used to sensitively and specifically detect the methylation state of the gene, so that the marker can be used for detection of peripheral blood free DNA, and the composition has the advantages of non-invasive screening of asymptomatic people, reduced harm caused by invasive detection, higher sensitivity and accuracy, and real-time monitoring.

Description

[0001] This case is a divisional application of the application filed on December 31, 2021, entitled "Biomarkers, probe compositions and their applications for lung cancer screening", with application number 202111674532.X. Technical Field

[0002] This invention relates to the field of biotechnology, and more particularly to a biomarker, probe composition and its application for lung cancer screening. Background Technology

[0003] Lung cancer is one of the cancers with the highest incidence and mortality rates worldwide. In my country, it also ranks first in both incidence and mortality. Routine screening methods for lung cancer include low-dose spiral CT (LDCT) and protein biomarkers such as carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), and neuron-specific enolase (NSE). However, these methods vary in sensitivity and specificity. Currently, DNA methylation has been proven to be tissue-specific and can be used for early cancer detection. Furthermore, the methylation characteristics of circulating tumor DNA (ctDNA) can be used to trace the primary tumor site. Summary of the Invention

[0004] The purpose of this invention is to provide a biomarker and probe composition for detecting lung cancer, which can be used for lung cancer screening. The biomarker is used in a non-invasive manner for screening asymptomatic individuals and for prognostic detection of cancer patients, reducing the harm caused by invasive detection and having higher sensitivity and accuracy.

[0005] The specific technical solution of this invention is as follows:

[0006] 1. A biomarker for detecting lung cancer, characterized in that the biomarker is selected from one of the following: USP33, CHRNB2, and TMEM219.

[0007] 2. The marker according to claim 1, characterized in that the nucleotide sequence of the marker is selected from one shown in SEQ ID NO: 1-3, preferably the marker is a methylated marker.

[0008] 3. A probe composition, characterized in that the probe composition comprises a probe targeting the methylation of the marker described in item 1 or 2.

[0009] 4. The probe composition according to claim 3, characterized in that the probe composition comprises a first probe composition with high methylation and a second probe composition with low methylation, the first probe composition being used for hybridization with a region of CG with high methylation after bisulfite conversion, and the second probe composition being used for hybridization with a region of CG with low methylation after bisulfite conversion;

[0010] Preferably, the first probe composition comprises n probes that hybridize with each nucleotide of the sense and antisense strands of the CG hypermethylated region converted by bisulfite.

[0011] Preferably, the second probe composition comprises m probes that hybridize to each nucleotide of the sense and antisense strands of the hypomethylated region of CG converted by bisulfite.

[0012] Preferably, n and m are both any integers from 1 to 10;

[0013] Preferably, there is an overlap of x1 nucleotides between the (n-1)th probe and the nth probe, where x1 is any integer from 0 to 100;

[0014] Preferably, there is an overlap of x2 nucleotides between the (m-1)th probe and the mth probe, where x2 is any integer from 0 to 100;

[0015] More preferably, the first probe composition comprises one or two nucleotide sequences as shown in SEQ ID NO:4-9, and the second probe composition comprises one or two nucleotide sequences as shown in SEQ ID NO:10-15.

[0016] 5. Use of a biomarker in the preparation of a kit for detecting lung cancer, characterized in that the biomarker is selected from one of the following: USP33, CHRNB2 and TMEM219.

[0017] 6. The use according to item 5, characterized in that the nucleotide sequence of the marker is selected from one shown in SEQ ID NO:1-3, preferably the marker is a methylated marker;

[0018] Preferably, the probe composition is used to target methylated biomarkers in lung cancer;

[0019] Preferably, the probe composition is the probe composition described in item 3 or 4.

[0020] 7. A composition for lung cancer detection, characterized in that the composition comprises a nucleic acid for detecting methylation selected from any of the following biomarkers: USP33, CHRNB2, and TMEM219.

[0021] 8. The composition according to claim 7, characterized in that the nucleotide sequence of the marker is selected from one shown in SEQ ID NO: 1-3.

[0022] 9. The composition according to item 7 or 8, characterized in that the nucleic acid comprises the probe composition according to item 3 or 4;

[0023] Preferably, the nucleic acid comprises:

[0024] Primers, wherein the primers are fragments of at least 9 nucleotides in the target sequence of the marker, the fragments containing at least one CpG dinucleotide sequence;

[0025] Preferably, the nucleic acid further includes:

[0026] A probe that hybridizes to at least 15 nucleotide fragments in the target sequence of the marker under moderately or strictly controlled conditions, the fragments containing at least one CpG dinucleotide sequence;

[0027] Preferably, the composition further includes an agent for converting the 5-position unmethylated cytosine base of the target sequence of the marker into uracil;

[0028] Preferably, the nucleic acid used to detect the methylation of the target sequence of the biomarker further includes:

[0029] Blockers that preferentially bind to target sequences in an unmethylated state.

[0030] 10. A kit comprising the marker described in item 1 or 2, or the probe composition described in item 3 or 4, or the composition described in any one of items 7-9.

[0031] 11. A chip comprising the marker described in item 1 or 2, the probe composition described in item 3 or 4, or the composition described in item 7 or 8.

[0032] The effects of the invention

[0033] This invention utilizes epigenomics and bioinformatics techniques to analyze genomic methylation data of lung cancer, identify multiple methylation genes associated with lung cancer, and determine the target sequences for abnormal methylation of lung cancer methylation genes. Furthermore, by using the target sequences of these methylation genes, the methylation status of the genes can be detected sensitively and specifically, which can then be used for the detection of cell-free DNA in peripheral blood.

[0034] The composition described in this invention is used for screening asymptomatic individuals in a non-invasive manner, reducing the harm caused by invasive testing. The composition has higher sensitivity and accuracy, enabling real-time monitoring. Detailed Implementation

[0035] The present invention will now be described in detail. While specific embodiments of the invention have been shown, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0036] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions are preferred embodiments for carrying out the invention; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of the invention. The scope of protection of this invention is determined by the appended claims.

[0037] The present invention provides a biomarker for detecting lung cancer, the biomarker being selected from one of the following: USP33, CHRNB2 and TMEM219.

[0038] In one specific embodiment, the nucleotide sequence of the marker is selected from one shown in SEQ ID NO:1-3, and preferably, the marker is a methylated marker.

[0039] The nucleotide sequence of USP33 is shown in SEQ ID NO:1; the nucleotide sequence of CHRNB2 is shown in SEQ ID NO:2; and the nucleotide sequence of TMEM219 is shown in SEQ ID NO:3.

[0040] The sequences of the above markers are all sequences that have not undergone bisulfite conversion.

[0041] The present invention provides a probe composition comprising a probe that targets the methylation of the marker.

[0042] Methylation refers to the methylation process occurring at the 5th carbon atom of cytosine in CpG dinucleotides. As a stable modified state, it can be inherited by newly generated daughter DNA during DNA replication under the action of DNA methyltransferases, making it an important epigenetic mechanism. During DNA methylation, methylation of the gene promoter region can lead to transcriptional silencing of tumor suppressor genes, thus it is closely related to tumorigenesis. Abnormal methylation includes hypermethylation of tumor suppressor genes and DNA repair genes, hypomethylation of repetitive DNA sequences, and loss of imprinting of certain genes, all of which are associated with the development of various tumors.

[0043] The methylation described in this invention can be methylation level, methylation degree, or methylation state. When analyzing the methylation of such target sequences, those skilled in the art can use quantitative determination methods to determine the methylation.

[0044] The probes are single-stranded or double-stranded DNA molecules ranging in length from tens to hundreds or even thousands of base pairs. Utilizing the denaturation, renaturation, and high precision of base pairing, they can bind (hybridize) with complementary unlabeled single-stranded DNA or RNA in the test sample via hydrogen bonds, forming a double-stranded complex (hybrid). After washing away the unpaired probes, the hybridization reaction results can be detected using autoradiography or enzyme-linked reactions. In this application, the region complementary to the probe or undergoing hybridization is the specific target region, and multiple probes are combined to form a probe composition.

[0045] In one specific embodiment, the probe composition comprises a hypermethylated first probe composition and a hypomethylated second probe composition, the first probe composition being used to hybridize with hypermethylated regions of CG converted from bisulfite, and the second probe composition being used to hybridize with hypomethylated regions of CG converted from bisulfite.

[0046] The hypermethylation refers to the conversion of the marker by bisulfite, where the base C is changed to the base T, but if it is the base CG, the base C remains unchanged.

[0047] The term "hypomethylation" refers to the fact that after the marker is converted by bisulfite, all the bases CG are not methylated, and all C bases are converted to T bases.

[0048] Because the methylation state varies from person to person, the sequences obtained from bisulfite conversion of the markers also differ. An extreme case for each marker is shown here, where all CGs in the region are in a hypermethylated state, along with the hypermethylated sequence of its complementary strand:

[0049] An extreme case of the sequence of SEQ ID NO:1 is shown in SEQ ID NO:16;

[0050] The complementary strand of the sequence in the extreme case is shown in SEQ ID NO:17;

[0051] An extreme case of the sequence of SEQ ID NO:2 is shown in SEQ ID NO:18;

[0052] The complementary strand of the sequence in the extreme case is shown in SEQ ID NO:19;

[0053] An extreme case of the sequence SEQ ID NO:3 is shown in SEQ ID NO:20;

[0054] The complementary strand of the sequence in the extreme case is shown in SEQ ID NO:21.

[0055] Similarly, since the methylation state varies from person to person, an extreme case is shown here, in which all CGs are in a hypomethylated state, and the hypomethylated sequence of their complementary strand is also shown:

[0056] An extreme case of the sequence of SEQ ID NO:1 is shown in SEQ ID NO:22;

[0057] The complementary strand of the sequence in the extreme case is shown in SEQ ID NO:23;

[0058] An extreme case of the sequence of SEQ ID NO:2 is shown in SEQ ID NO:24;

[0059] The complementary strand of the sequence in the extreme case is shown in SEQ ID NO:25;

[0060] An extreme case of the sequence of SEQ ID NO:3 is shown in SEQ ID NO:26;

[0061] The complementary strand of the sequence in the extreme case is shown in SEQ ID NO:27.

[0062] In one specific embodiment, the first probe composition comprises n probes that hybridize with each nucleotide of the sense and antisense strands of the CG hypermethylated region converted from bisulfite.

[0063] The second probe composition comprises m probes that hybridize to each nucleotide of the sense and antisense strands of the hypomethylated region of CG converted by bisulfite.

[0064] The present invention does not impose any limitation on the number of probes in the first probe composition and the second probe composition. Those skilled in the art can select them as needed. For example, m and n can be any integer from 1 to 10, and m and n can be the same or different.

[0065] For example, m and n can be any integers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, preferably m = n = 2.

[0066] In one specific implementation, there is an x1 nucleotide overlap between the (n-1)th probe and the nth probe, preferably, x1 is any integer from 0 to 100;

[0067] Preferably, there is an overlap of x2 nucleotides between the (m-1)th probe and the mth probe, where x2 is any integer from 0 to 100.

[0068] x1 and x2 can be the same or different. When x1 is 0, it means that the tail of the (n-1)th probe is connected to the head of the nth probe. Similarly, when x2 is 0, it means that the tail of the (m-1)th probe is connected to the head of the mth probe.

[0069] This invention hybridizes a probe composition with a biomarker that has undergone bisulfite conversion, wherein a first probe composition with high methylation hybridizes with a region of high methylation of CG, and a second probe composition with low methylation hybridizes with a region of low methylation of CG, thereby enabling efficient and accurate detection of the methylation level of the target sequence, and thus enabling its use in lung cancer screening.

[0070] In one specific embodiment, the highly methylated first probe composition comprises one or two of the following: SEQ ID NO:4-9.

[0071] The hypomethylated second probe composition includes one or both of the following: SEQ ID NO:10-15.

[0072] The first probe composition for hybridization with the USP33 methylated sequence comprises a nucleotide sequence as shown in SEQ ID NO:4-5;

[0073] The first probe composition for hybridization with the CHRNB2 methylated sequence comprises the nucleotide sequence shown in SEQ ID NO:6-7;

[0074] The first probe composition for hybridization with the TMEM219 methylated sequence comprises a nucleotide sequence as shown in SEQ ID NO:8-9;

[0075] The second probe composition for hybridization with the methylated sequence of USP33 comprises the nucleotide sequence shown in SEQ ID NO:10-11;

[0076] The second probe composition for hybridization with the CHRNB2 methylated sequence comprises the nucleotide sequence shown in SEQ ID NO:12-13;

[0077] The second probe composition for hybridization with the TMEM219 methylated sequence comprises the nucleotide sequence shown in SEQ ID NO:14-15.

[0078] This invention provides the use of biomarkers in the preparation of kits for the detection of lung cancer, said biomarkers being selected from one of the following: USP33, CHRNB2, and TMEM219.

[0079] In one specific embodiment, the nucleotide sequence of the marker is selected from one shown in SEQ ID NO:1-3, and preferably the marker is a methylated marker.

[0080] This invention provides the use of a probe composition in the preparation of a kit for detecting lung cancer, the probe composition being used to target biomarkers of lung cancer methylation.

[0081] In one specific embodiment, the probe composition is the probe composition described above.

[0082] The present invention provides a composition for lung cancer detection, the composition comprising a nucleic acid methylated from any of the following biomarkers: USP33, CHRNB2 and TMEM219, preferably, the nucleotide sequence of the biomarker is selected from one shown in SEQ ID NO:1-3.

[0083] In one specific embodiment, the nucleic acid comprises the probe composition described above.

[0084] In one specific implementation, the nucleic acid includes:

[0085] Primers, wherein the primers are fragments of at least 9 nucleotides in the target sequence of the marker, the fragments containing at least one CpG dinucleotide sequence.

[0086] If bisulfite is used to convert the NDA of the test sample, the nucleic acid used to detect the methylation of the target sequence of the marker includes a fragment of at least 9 nucleotides in the sequence after bisulfite conversion of the target sequence of the marker, the fragment containing at least one CpG dinucleotide sequence.

[0087] In one specific implementation, the nucleic acid further includes:

[0088] A probe that hybridizes to at least 15 nucleotide fragments in the target sequence of the marker under moderately or strictly controlled conditions, the fragments containing at least one CpG dinucleotide sequence.

[0089] In one specific embodiment, the composition further includes a reagent for converting the 5-position unmethylated cytosine base of the target sequence of the marker into uracil, such as a bisulfite; preferably, the nucleic acid for detecting the methylation of the target sequence of the marker further includes:

[0090] Blockers that preferentially bind to target sequences in an unmethylated state.

[0091] The blocking agent is designed to improve the amplification specificity of PCR primers. The 5' end of the blocking agent's nucleotide sequence overlaps with the 3' end of the forward or reverse primer by 5 nucleotides or more. The blocking agent is complementary to the target DNA strand of the forward or reverse primer. The melting temperature of the blocking agent is 5°C higher than that of the forward or reverse primer. The nucleotide sequence of the blocking agent contains at least one CpG dinucleotide sequence and is complementary to the unmethylated target DNA sequence after bisulfite conversion. Therefore, when the genomic DNA of the biological sample to be tested is a mixture of methylated and unmethylated states, especially when the methylated DNA is far less than the unmethylated DNA, the unmethylated DNA, after bisulfite conversion, will preferentially bind to the blocking agent, thus binding to the DNA template and the PCR primer, preventing PCR amplification. The methylated DNA, however, does not bind to the blocking agent and instead binds to the primer, resulting in PCR amplification. The fragments obtained through amplification can then be detected directly or indirectly.

[0092] The present invention provides a kit comprising the above-described marker, probe composition, or composition.

[0093] In one specific embodiment, the kit also includes a container for containing biological samples from the subject.

[0094] In one specific implementation, the kit also includes instructions for using and interpreting the test results.

[0095] The biological sample may be, for example, peripheral blood, whole blood, plasma, or serum.

[0096] This invention does not impose any limitations on the method for detecting target sequence methylation levels using the above-described kit. Those skilled in the art can choose the appropriate method as needed. For example, this invention provides a method for detecting biomarker target sequence methylation levels using the above-described kit, which includes the following steps:

[0097] Collect samples from test subjects;

[0098] Extract and purify DNA from the sample;

[0099] Construct DNA libraries for sequencing from purified DNA samples;

[0100] The constructed DNA library was transformed with bisulfite;

[0101] The bisulfite-converted DNA library was amplified by pre-PCR.

[0102] Hybridization capture of pre-PCR amplified samples was performed using probe compositions;

[0103] The product captured by hybridization was amplified by PCR.

[0104] High-throughput next-generation sequencing was performed on the PCR-amplified and hybridized capture products.

[0105] Sequencing data are analyzed to determine the methylation level of the samples;

[0106] The threshold for each biomarker is calculated based on the methylation status of existing samples. The patient's disease status is determined based on the methylation level of a certain biomarker in the sample. If the methylation level of a certain biomarker in the sample exceeds the threshold, it is a cancer sample; if it is below the threshold, it is a healthy sample.

[0107] For example, the present invention provides a method for detecting the target sequence methylation level of a biomarker using the above-described kit, comprising the following steps:

[0108] (1) Collect peripheral blood from the subject and separate plasma or serum;

[0109] (2) Extracting cell-free DNA from plasma or serum;

[0110] (3) Use reagents to treat the free DNA obtained in step (2) to convert the 5-position unmethylated cytosine base to uracil or other bases. That is, the 5-position unmethylated cytosine base of the target sequence of the marker is converted to uracil or other bases. The converted bases are different from the 5-position unmethylated cytosine bases in terms of hybridization performance and are detectable.

[0111] (4) The free DNA treated in step (3) is contacted with DNA polymerase and primers for the target sequence of the marker, so that the target sequence of the treated marker is amplified to produce an amplification product or is not amplified; if the target sequence of the treated marker undergoes DNA polymerization, an amplification product will be produced; if the target sequence of the treated marker does not undergo DNA polymerization, it will not be amplified.

[0112] (5) Detect the amplification products using probes;

[0113] (6) Based on the presence or absence of the amplification product, determine the methylation status of at least one CpG dinucleotide of the target sequence of the marker, thereby determining the methylation level of the target sequence of the marker.

[0114] The present invention provides a chip comprising the above-described marker or the above-described probe composition or the above-described composition.

[0115] The chip, also known as a gene chip, uses a hybridization sequencing method. This method involves determining the nucleic acid sequence by hybridizing with a set of nucleic acid probes with known sequences. Probes with known target nucleotide sequences are immobilized on the surface of a substrate. When a fluorescently labeled nucleic acid sequence in solution achieves complementary matching with the corresponding nucleic acid probe on the gene chip, the position of the probe with the strongest fluorescence intensity is determined to obtain a set of probe sequences that are completely complementary.

[0116] The chip is mainly fabricated using glass or silicon wafers as carriers, and oligonucleotide fragments or cDNA are arranged sequentially on the carriers using in-situ synthesis and micro-matrix methods.

[0117] The chip described in this invention is based on signal detection of DNA sequence hybridization after bisulfite treatment. Bisulfite treatment converts unmethylated cytosine into uracil, while methylated cytosine remains unchanged. Then, uracil is converted into thymine, and finally, chip hybridization is performed. Finally, the type of added base is determined based on the fluorescence color, thereby determining whether the site is methylated.

[0118] This invention provides a method for lung cancer screening, comprising:

[0119] The methylation level of the detection marker, and

[0120] The risk of a subject developing lung cancer is determined based on the methylation level, and the biomarker is selected from one of the following: USP33, CHRNB2, and TMEM219.

[0121] Example

[0122] This invention provides a general and / or specific description of the materials and methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0123] Example 1: Screening Markers

[0124] 1) Sample Collection: Downloaded 450k methylation chip cancer tissue data from TCGA, involving 7769 cancer tissue samples from 26 types of tumors, including adrenocortical carcinoma (80), bladder urothelial carcinoma (409), acute myeloid leukemia (140), low-grade glioma (654), breast cancer (740), cervical cancer (286), colorectal cancer (348), esophageal cancer (183), uveal melanoma (80), and head and neck squamous cell carcinoma (…). 527), renal cell carcinoma (660), liver cancer (377), lung adenocarcinoma (425), lung squamous cell carcinoma (372), diffuse large B-cell lymphoma (29), ovarian serous cystadenocarcinoma (10), pancreatic cancer (184), mesothelioma (116), prostate cancer (488), skin melanoma (104), sarcoma (117), gastric cancer (397), testicular cancer (134), thymic carcinoma (94), thyroid cancer (506), endometrial cancer (309). For healthy individuals, Bocheng collected plasma from 38 healthy individuals and performed whole-genome bisulfite sequencing (WGBS).

[0125] 2) Candidate biomarker screening: For healthy plasma samples, the third quartile (Q3), also known as the "larger quartile," of the β value for each probe corresponding to the 450K region was calculated. Sites with Q3 < 0.02 were screened, resulting in List1. For 450K microarray tissue data, the first quartile (Q1), also known as the "smaller quartile," of the β value for each probe corresponding to the 450K region was calculated. Sites with Q1 > 0.1 were screened, resulting in List2. The intersection of List1 and List2 yielded 65,739 differentially methylated regions.

[0126] 3) Biomarker selection: Lung cancer-specific biomarkers were selected from the above biomarkers, resulting in 29 biomarkers. Simultaneously, the difference in methylation levels between lung cancer (including adenocarcinoma and squamous cell carcinoma) tissues (797) and adjacent normal tissues (73) in the TCGA 450k chip was required to be greater than 0.2, ultimately yielding 17 differentially methylated regions.

[0127] 4) Biomarker Validation: Probes were designed to capture the 17 differentially methylated regions mentioned above. Validation was performed using data from Borcheng plasma samples (24 lung cancer samples and 38 healthy individuals), ultimately yielding three biomarkers capable of distinguishing between lung cancer and healthy individuals. Their sequences are shown in SEQ ID NO: 1-3.

[0128] Based on the obtained target sequence region, a custom probe composition (panel) is prepared, comprising a highly methylated first probe composition and a low-methylated second probe composition, wherein, for each marker, the first probe composition comprises two probes, and for SEQ ID NO:1, its first probe composition comprises nucleotide sequences as shown in SEQ ID NO:4-5;

[0129] For SEQ ID NO:2, the first probe composition comprises the nucleotide sequence shown in SEQ ID NO:6-7; for SEQ ID NO:3, the first probe composition comprises the nucleotide sequence shown in SEQ ID NO:8-9.

[0130] The second probe composition comprises two probes, wherein, for SEQ ID NO:1, the second probe composition comprises the nucleotide sequence shown in SEQ ID NO:10-11; for SEQ ID NO:2, the second probe composition comprises the nucleotide sequence shown in SEQ ID NO:12-13; and for SEQ ID NO:3, the second probe composition comprises the nucleotide sequence shown in SEQ ID NO:14-15.

[0131] Then, it is verified in plasma samples, and the experimental detection method is as follows:

[0132] 1.1.cfDNA Extraction and Purification

[0133] 1.1.1. Plasma sample preparation:

[0134] Centrifuge the blood sample at 2000g for 10 minutes at 4℃, and transfer the plasma to a new centrifuge tube. Centrifuge the plasma sample at 16000g for 10 minutes at 4℃. Proceed to the next step depending on the type of collection tube used. In this experiment, the type of collection tube used was other.

[0135] Table 1

[0136]

[0137] 1.1.2. Fracturing and Binding

[0138] 1.1.2.1. Prepare the binding solution / bead mixture according to the table below, and then mix thoroughly.

[0139] Table 2

[0140]

[0141] Add an appropriate volume of plasma sample.

[0142] 1.1.2.2. Thoroughly mix the plasma sample and the binding solution / bead mixture.

[0143] 1.1.2.3. Mix thoroughly on a rotary mixer for 10 minutes to allow the cfDNA to bind to the magnetic beads.

[0144] 1.1.2.4. Place the binding tube on the magnetic rack for 5 minutes until the solution becomes clear and the magnetic beads are completely adsorbed on the magnetic rack.

[0145] 1.1.2.5. Carefully discard the supernatant with a pipette, and continue to keep the tube on the magnetic rack for a few minutes. Remove any remaining supernatant with a pipette.

[0146] 1.1.3. Washing

[0147] 1.1.3.1. Resuspend the beads in 1 ml of washing solution.

[0148] 1.1.3.2. Transfer the resuspension to a new, non-adsorbed 1.5 ml centrifuge tube. Retain the binding tube.

[0149] 1.1.3.3. Place the centrifuge tube containing the bead resuspension on a magnetic rack for 20 seconds.

[0150] 1.1.3.4. Aspirate the supernatant obtained from the separation and wash the binding tube. Collect the residual beads after washing back into the resuspension and discard the lysis / binding tube.

[0151] 1.1.3.5. Place the tube on the magnetic rack for 2 minutes, until the solution becomes clear and the beads gather on the magnetic rack. Remove the supernatant with a 1 ml pipette.

[0152] 1.1.3.6. Leave the tube on the magnetic rack and remove as much residual liquid as possible using a 200μL pipette.

[0153] 1.1.3.7. Remove the tube from the magnetic rack, add 1 ml of washing solution, and vortex for 30 seconds.

[0154] 1.1.3.8. Place on a magnetic rack for 2 minutes until the solution is clear and the beads gather on the magnetic rack. Remove the supernatant with a 1 ml pipette.

[0155] 1.1.3.9. Leave the tube on the magnetic rack and use a 200 μL pipette to completely remove any remaining liquid.

[0156] 1.1.3.10. Remove the tube from the magnetic rack, add 1 ml of 80% ethanol, and vortex for 30 seconds.

[0157] 1.1.3.11. Place on a magnetic rack for 2 minutes until the solution becomes clear, then remove the supernatant with a 1 ml pipette.

[0158] 1.1.3.12. Leave the tube on the magnetic rack and remove any remaining liquid using a 200 μL pipette.

[0159] 1.1.3.13. Repeat steps 1.1.3.10-1.1.3.12 once with 80% ethanol to remove the supernatant as much as possible.

[0160] 1.1.3.14. Leave the tube on the magnetic rack and let the beads dry in the air for 3-5 minutes.

[0161] 1.1.4. Elution of cfDNA

[0162] 1.1.4.1. Add the elution buffer according to the table below.

[0163] Table 3

[0164]

[0165] 1.1.4.2. Vortex for 5 minutes, place on a magnetic rack for 2 minutes, the solution becomes clear, and aspirate the cfDNA from the supernatant.

[0166] 1.1.4.3. The purified cfDNA can be used immediately, or the supernatant can be transferred to a new centrifuge tube and stored at -20°C.

[0167] 1.2.gDNA fragmentation and purification:

[0168] 1.2.1. According to the Qubit concentration, take 2 μg gDNA, add water to make up to 125 μl, add to a 130 μl Covaris fragmentation tube, and set the program: 50W, 20%, 200 cycles, 250s.

[0169] 1.2.2. After the fragmentation is completed, take 1 μl of sample and use Agilent 2100 to detect the fragment. After normal fragmentation, the main peak of the sample is about 150bp-200bp.

[0170] For cfDNA samples, Agilent 2100 was used for fragment detection, and the Qubit was directly used for subsequent experiments.

[0171] 1.3. End repair, adding "A" at the 3' end:

[0172] 1.3.1. Take 20 ng of fragmented gDNA or cfDNA into a PCR tube, add nuclease-free water to a final volume of 50 μl, add the following reagents, and vortex to mix:

[0173] Table 4

[0174] gDNA / cfDNA 50μl Termination of repair and A-tailed buffer 7μl Termination of repair and A-tailed enzyme mixture 3μl Total volume 60μl

[0175] 1.3.2. Set the following program to perform the reaction on the PCR instrument: hot lid temperature 85℃.

[0176] Table 5

[0177] 20℃ 30min 65℃ 30min 4℃ ∞

[0178] 1.4. Connector connection and purification:

[0179] 1.4.1. Refer to the table below to dilute the connector to a suitable concentration in advance:

[0180] Table 6

[0181]

[0182]

[0183] 1.4.2. Prepare the following reagents according to the table below, gently pipette and mix well, then briefly centrifuge:

[0184] Table 7

[0185] End-stage repair, addition of "A" reaction product 60μl connector 5μl Nuclease-free water 5μl Ligation buffer 30μl DNA ligase 10μl Total volume 110μl

[0186] 1.4.3. Set the following program to perform the reaction on the PCR instrument: without a heat cap.

[0187] Table 8

[0188] 20℃ 30min 4℃ ∞

[0189] 1.4.4. Add purified magnetic beads to the following system for the experiment (Agencourt AMPure XP magnetic beads should be brought to room temperature and mixed thoroughly beforehand):

[0190] Table 9

[0191] Connector products 110μl Agencourt AMPure XP beads 110μl Total volume 220μl

[0192] 1.4.4.1. Gently whisk and mix 6 times.

[0193] 1.4.4.2. Incubate at room temperature for 5-15 minutes, then place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clarify.

[0194] 1.4.4.3. Remove the supernatant, keep the PCR tube on the magnetic rack, add 200 μl of 80% ethanol solution to the PCR tube, and let it stand for 30 seconds.

[0195] 1.4.4.4. Remove the supernatant, then add 200 μl of 80% ethanol solution to the PCR tube, let it stand for 30 seconds, and then completely remove the supernatant (it is recommended to use a 10 μl pipette to remove any residual ethanol solution at the bottom).

[0196] 1.4.4.5. Let stand at room temperature for 3-5 minutes to allow the residual ethanol to evaporate completely.

[0197] 1.4.4.6. Add 22 μl of nuclease-free water, remove the PCR tube from the magnetic rack, gently aspirate and resuspend the magnetic beads to avoid generating air bubbles, and let stand at room temperature for 2 minutes.

[0198] 1.4.4.7. Place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clarify.

[0199] 1.4.4.8. Use a pipette to draw 20 μl of supernatant and transfer it to a new PCR tube.

[0200] 1.5 Treatment and purification of bisulfite:

[0201] 1.5.1. Prepare the required reagents in advance and dissolve them. Add the reagents according to the table below:

[0202] Table 10

[0203] Connector to purified product 20μl 40μl bisulfite solution 85μl 85μl DNA protection buffer 35μl 15μl Total volume 140μl 140μl

[0204] 1.5.2. DNA protection buffer turns the liquid blue upon addition. Gently pipette to mix, then divide into two tubes and place them on the PCR instrument.

[0205] 1.5.3. Set the following program and run it: Heat cover 105℃.

[0206] Table 11

[0207] 95℃ 5min 60℃ 10min 95℃ 5min 60℃ 10min 4℃ ∞

[0208] 1.5.4. Brief centrifugation: Combine the two identical samples into a single clean 1.5 ml centrifuge tube.

[0209] 1.5.5. Add 310 μl of buffer BL to each sample (add 1 μl of vector RNA (1 μg / μl) for sample volumes less than 100 ng), vortex to mix, and briefly centrifuge.

[0210] 1.5.6. Add 250 μl of anhydrous ethanol to each sample, vortex to mix for 15 s, centrifuge briefly, and add the mixture to the corresponding prepared centrifuge column.

[0211] 1.5.7. Let stand for 1 minute, centrifuge for 1 minute, transfer the liquid in the collection tube back to the centrifuge column, centrifuge for 1 minute, and discard the liquid in the centrifuge tube.

[0212] 1.5.8. Add 500 μl of buffer BW (note whether to add anhydrous ethanol), centrifuge for 1 min, and discard the waste liquid.

[0213] 1.5.9. Add 500 μl of buffer BD (note whether to add anhydrous ethanol), cap the tube, and incubate at room temperature for 15 min. Centrifuge for 1 min and discard the liquid.

[0214] 1.5.10. Add 500 μl of buffer BW (note whether to add anhydrous ethanol), centrifuge for 1 min, discard the liquid, and repeat once, for a total of 2 times.

[0215] 1.5.11. Add 250 μl of anhydrous ethanol, centrifuge for 1 min, transfer the centrifuge column to a new 2 ml collection tube, and discard all remaining liquid.

[0216] 1.5.12. Place the centrifuge column into a clean 1.5ml centrifuge tube, add 20μl of nuclease-free water to the center of the centrifuge column membrane, gently cap the tube, incubate at room temperature for 1 min, and centrifuge for 1 min.

[0217] 1.5.13. Transfer the liquid in the collection tube back to the centrifuge column, let it stand at room temperature for 1 min, and then centrifuge for 1 min.

[0218] 1.6. Pre-amplification and purification before hybridization:

[0219] 1.6.1. Prepare the reaction system according to the table below, mix by pipetting, and briefly centrifuge:

[0220] Table 12

[0221]

[0222] 1.6.2. Set the following program and start the PCR program: Heat lid 105℃

[0223] Table 13

[0224]

[0225] 1.6.3. The number of PCR cycles should be adjusted according to the amount of DNA used. Reference data is shown below:

[0226] Table 14

[0227]

[0228]

[0229] 1.6.4. Add 50 μl of Agencourt AMPure XP magnetic beads to the PCR tube after the reaction is complete, and mix well by pipetting to avoid generating air bubbles (Agencourt AMPure XP should be mixed and equilibrated at room temperature beforehand).

[0230] 1.6.5. Incubate at room temperature for 5-15 minutes, then place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clarify.

[0231] 1.6.6. Remove the supernatant, keep the PCR tube on the magnetic rack, add 200 μl of 80% ethanol solution to the PCR tube, and let it stand for 30 seconds.

[0232] 1.6.7. Remove the supernatant, then add 200 μl of 80% ethanol solution to the PCR tube, let it stand for 30 seconds, and then completely remove the supernatant (it is recommended to use a 10 μl pipette to remove any residual ethanol solution at the bottom).

[0233] 1.6.8. Let stand at room temperature for 5 minutes to allow the residual ethanol to evaporate completely.

[0234] 1.6.9. Add 30 μl of nuclease-free water, remove the centrifuge tube from the magnetic rack, and use a pipette to gently aspirate and resuspend the magnetic beads.

[0235] 1.6.10. Let stand at room temperature for 2 min, then place the 200 μl PCR tube on a magnetic rack for 2 min to allow the solution to clarify.

[0236] 1.6.11. Use a pipette to transfer the supernatant to a new 200 μl PCR tube (place on an ice box), label the sample number on the reaction tube, and prepare for the next reaction.

[0237] 1.6.12. Take 1 μl of sample and use Qubit to determine the library concentration, and record the library concentration.

[0238] 1.6.13. Take 1 μl of sample and use Agilent 2100 to determine the length of the library fragments. The library length is approximately between 270 bp and 320 bp.

[0239] 1.7. Sample-probe hybridization:

[0240] 1.7.1. Mix the sample library with various Hyb blockers according to the following system, labeled as B:

[0241] Table 15

[0242] Preamplification products 750ng corresponding volume Hyb human blocking device 5μl Connector blocking material 6μl enhancer 5μl

[0243] 1.7.2. Place the prepared sample and Hyb blocking agent mixture into a vacuum concentration centrifuge, open the PCR tube cap, start the centrifuge, turn on the vacuum pump switch, and begin concentration.

[0244] 1.7.3. Redissolve the dried sample in approximately 9 μl of nuclease-free water, for a total volume of 10 μl. Gently pipette and mix well. After a short centrifugation, place on ice for later use and label as B.

[0245] 1.7.4. Melt the Hyb buffer at room temperature. After melting, a precipitate will appear. Mix well and preheat in a 65°C water bath. After complete dissolution (without precipitate or turbidity), take 20 μl of Hyb buffer and place it in a new 200 μl PCR tube. Cap the tube and label it A. Continue to incubate in a 65°C water bath until ready for use.

[0246] 1.7.5. The previously described methylation probe sequence was synthesized by Aijitaikang Biotechnology (Beijing) Co., Ltd.:

[0247] 1.7.6. Take 5 μl of RNase blocking agent and 2 μl of probe composition and place them in a 200 μl PCR tube. Gently pipette and mix well. After a short centrifugation, place on ice and set aside for use. Label as C.

[0248] 1.7.7. Set the PCR instrument parameters: heat the lid at 100℃, 95℃ for 5 minutes; then maintain at 65℃.

[0249] 1.7.8. Place PCR tube B on the PCR instrument and run the above procedure.

[0250] 1.7.9. When the temperature of the PCR instrument drops to 65℃, place PCR tube A on the PCR instrument for incubation and cover it with the PCR instrument's heat-sensitive lid.

[0251] 1.7.10.5 min later, place C on the PCR machine for incubation and cover with the PCR instrument's hot lid.

[0252] 1.7.11. After placing PCR tube C into the PCR instrument for 2 minutes, adjust the pipette to 13 μl, transfer 13 μl of Hyb buffer from PCR tube A to PCR tube C, and transfer all the sample from PCR tube B to PCR tube C. Gently pipette 10 times to mix thoroughly, avoiding the generation of a large number of air bubbles. Seal the tube cap, cover with the PCR instrument's heating cap, and incubate at 65°C overnight (16-24 h).

[0253] 1.8. Capture the target region DNA library:

[0254] 1.8.1. Preparation for capturing magnetic beads

[0255] 1.8.1.1. Remove the magnetic bead (Dynabeads MyOne Streptavidin T1 magnetic bead) from 4℃ and vortex oscillate it for re-suspending.

[0256] 1.8.1.2. Place 50 μl of magnetic beads into a new PCR tube, place it on a magnetic rack for 1 min to allow the solution to clarify, and remove the supernatant.

[0257] 1.8.1.3. Remove the PCR tube from the magnetic rack, add 200 μL of binding buffer, gently aspirate and mix several times, then resuspend the magnetic beads.

[0258] 1.8.1.4. Place on the magnetic rack for 1 minute, then remove the supernatant.

[0259] 1.8.1.5. Repeat steps 3-4 twice, cleaning the magnetic beads a total of 3 times.

[0260] 1.8.1.6. Remove the PCR tube from the magnetic rack, add 200 μL of binding buffer, gently aspirate and shake 6 times to resuspend the magnetic beads for later use.

[0261] 1.8.2. Capturing the target DNA library

[0262] 1.8.2.1. Keep the hybridization product PCR tube C on the PCR instrument, add the prepared 200 μL of capture magnetic beads to the hybridization product PCR tube C, mix with a pipette 6 times, and place on a rotary mixer to bind at room temperature for 30 min (the rotation speed should preferably not exceed 10 rpm).

[0263] 1.8.2.2. Place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clarify, then remove the supernatant.

[0264] 1.8.2.3. Add 200 μL of washing buffer 1 (23.5 ml nuclease-free water, 1.25 ml 20×SSC, 250 μl 10% SDS) to PCR tube C, gently pipette and mix 6 times, place on a rotary mixer and wash for 15 min (the rotation speed should preferably not exceed 10 rpm), then briefly centrifuge, place the PCR tube on a magnetic rack for 2 min to allow the solution to clarify, and remove the supernatant.

[0265] 1.8.2.4. Add 200 μl of preheated (65℃) washing buffer 2 (24.6 ml nuclease-free water, 125 μl 20×SSC, 250 μl 10% SDS), gently aspirate and mix 6 times, incubate at 65℃ for 10 min on a mixer, and wash at 800 rpm.

[0266] 1.8.2.5. Briefly centrifuge, place the PCR tube on a magnetic rack for 2 minutes, and remove the supernatant. Repeat the washing process twice more using Wash Buffer 2, for a total of 3 times. Finally, thoroughly remove Wash Buffer 2.

[0267] 1.8.2.6. Keep the PCR tube on the magnetic rack, add 200 μl of 80% ethanol to the PCR tube, let it stand for 30 seconds, then completely remove the ethanol solution and let it air dry at room temperature for 2 minutes.

[0268] 1.8.2.7. Add 30 μL of nuclease-free water to the PCR tube, remove the PCR tube from the magnetic rack, and gently aspirate and shake 6 times to resuspend the magnetic beads for later use.

[0269] 1.9. Post-capture amplification and purification

[0270] 1.9.1. Prepare the reaction system according to the table below for enrichment of the capture library. After gently mixing by pipetting, briefly centrifuge:

[0271] Table 16

[0272]

[0273] 1.9.2. Set the following program, place the sample in the PCR instrument, and run the program: hot lid 105℃.

[0274] Table 17

[0275]

[0276] 1.9.3. After PCR, add 55 μl of Agencourt AMPure XP magnetic beads to the sample and gently mix with a pipette.

[0277] 1.9.4. Incubate at room temperature for 5 min, then place the PCR tube on a magnetic rack for 3 min to allow the solution to clarify.

[0278] 1.9.5. Remove the supernatant, keep the PCR tube on the magnetic rack, add 200 μl of 80% anhydrous ethanol, and let stand for 30 seconds.

[0279] 1.9.6. Remove the supernatant, then add 200 μl of 80% anhydrous ethanol to the PCR tube, let stand for 30 seconds, and then completely remove the supernatant.

[0280] 1.9.7. Let stand at room temperature for 5 minutes to allow the residual ethanol to evaporate completely.

[0281] 1.9.8. Add 25 μl of nuclease-free water, remove the PCR tube from the magnetic rack, gently pipette to mix and resuspend the magnetic beads, and incubate at room temperature for 2 min.

[0282] 1.9.9. Place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clarify.

[0283] 1.9.10. Use a pipette to transfer 23 μl of supernatant to a 1.5 ml centrifuge tube and label the sample information.

[0284] 1.9.11. Take 1 μl of the library and use Qubit to quantify it, and record the library concentration.

[0285] 1.9.12. Take 1 μl of sample and use an Agilent 2100 to determine the length of the library fragment.

[0286] 1.9.13. Sequencing was performed using the Illumina high-throughput sequencing platform.

[0287] 1.10. Methylation Bioinformatics Analysis Workflow. The general process is as follows: Use FASTP quality control software to check sequencing quality, remove low-quality reads, then use Bismark alignment software to align the clean, quality-controlled data to the reference genome, use Bismark_methylation_extractor software to extract the corresponding methylation sites, and finally calculate the methylation level of each marker.

[0288] Example 2

[0289] Based on 24 clinically diagnosed lung cancer samples collected from Beijing and 38 healthy human samples collected from Beijing, the methylation library construction method described in Example 1 was used to calculate the methylation levels of the three screened methylation biomarkers. The thresholds (hereinafter referred to as sites or markers) and independently distinguishable AUC values ​​of these three methylation biomarkers in the lung cancer sample and normal human sample datasets were calculated and are shown in Table 18.

[0290] The methylation level threshold is calculated as follows: An ROC curve is plotted using the pROC package in R based on the dataset (containing the type and methylation level of each sample). The confusion matrix corresponding to the optimal threshold point on the ROC curve will be the basis for calculating sensitivity, specificity, and accuracy. Typically, the Youden index is used for selection. The Youden index, also known as the correctness index, is the sum of sensitivity and specificity minus 1: Youden index = Sensitivity + Specificity - 1. The Youden index ranges from 0 to 1, representing the classification model's overall ability to distinguish between true patients and non-patients. A higher Youden index indicates better classification model performance. The threshold, sensitivity, and specificity for each marker are shown in Table 18.

[0291] As can be seen from Table 18, the AUC value of the marker described in this invention is relatively high.

[0292] Table 18 Specific performance data of the three methylation markers

[0293] SEQ ID NO.1 0.11 0.94 0.89 0.93 SEQ ID NO.2 0.11 0.87 0.91 0.90 SEQ ID NO.3 0.11 0.95 0.84 0.92

[0294] Example 3

[0295] Six human samples (S1-3 were healthy samples, and S4-6 were lung cancer patient samples) were collected using the methylation biomarker detection method of this application, following the method described in Example 1. A library was constructed, and the samples were sequenced using the Illumina platform. The sequencing data were analyzed using the aforementioned bioinformatics process to obtain the methylation level of each biomarker. Based on the threshold for each biomarker, the disease status of the patients was predicted. Samples exceeding the threshold were considered cancer samples, while those below the threshold were considered healthy samples. Specific results are shown in Table 19.

[0296] In the interpretation results, 0 represents the classification as normal, i.e., healthy; 1 represents the classification as abnormal, i.e., tumor.

[0297] Table 19 shows the methylation values ​​and interpretation results of the samples.

[0298]

[0299] In summary, the inventors of this invention have obtained methylation genes associated with lung cancer and identified target sequences for abnormal methylation of lung cancer methylation genes. Furthermore, through these target sequences, the methylation status of these genes can be detected sensitively and specifically, thereby enabling the detection of cell-free DNA in peripheral blood. Moreover, the composition described in this invention enables real-time monitoring with higher sensitivity and accuracy.

[0300] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

[0301] The sequence list is shown in Table 20:

[0302] Table 20

[0303]

[0304]

Claims

1. The use of a reagent for detecting biomarkers in the preparation of a kit for detecting lung cancer, characterized in that, The biomarker is the CHRNB2 gene, the nucleotide sequence of which is selected from the sequence shown in SEQ ID NO:2, and the biomarker is a methylated biomarker.

2. The use according to claim 1, characterized in that, The biomarkers also include the TMEM219 gene.

3. A composition for lung cancer detection, characterized in that, The composition includes nucleic acids for detecting methylation of the CHRNB2 gene, the nucleotide sequence of which is shown in SEQ ID NO:2; The nucleic acid includes a probe composition for targeting the methylated CHRNB2 gene in liver cancer. The probe composition comprises a hypermethylated first probe composition and a hypomethylated second probe composition, wherein the first probe composition is used to hybridize with hypermethylated regions of bisulfite-converted CG, and the second probe composition is used to hybridize with hypomethylated regions of bisulfite-converted CG. The first probe composition comprises the nucleotide sequence shown in SEQ ID NO:6-7; the second probe composition comprises the nucleotide sequence shown in SEQ ID NO:12-13.

4. The composition according to claim 3, characterized in that, The composition also includes nucleic acids for detecting methylation of the TMEM219 gene.

5. The composition according to claim 3 or 4, characterized in that, The composition further includes a reagent that converts the 5-position unmethylated cytosine base of the target sequence of the marker into uracil.

6. The composition according to claim 3 or 4, characterized in that, The nucleic acid used for detecting target sequence methylation of biomarkers also includes: Blockers that preferentially bind to target sequences in an unmethylated state.

7. A kit comprising the composition of any one of claims 3-6.

8. A chip comprising the composition of any one of claims 3-6.

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