Combinations and applications of methylation biomarkers for the diagnosis or prediction of nasopharyngeal carcinoma, kits and computer-readable storage media

Abstract

This invention discloses a combination of methylation molecular markers for the diagnosis or prediction of nasopharyngeal carcinoma, consisting of three methylation molecular markers located in the human ZNF804A, FOXL1, and CASZ1 genes: chr2:184598880-184599080, chr16:86578884-86579084, and chr1:10642015-10642215, specifically located using hg38. This invention also discloses the application of this combination of methylation molecular markers, related kits, and analytical processing procedures recorded on a computer-readable storage medium. The methylation levels of the ZNF804A, FOXL1, and CASZ1 genes, as biomarkers for the early diagnosis or prediction of nasopharyngeal carcinoma, enrich the options available to those skilled in the art.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and gene detection, specifically relating to combinations and applications of methylation biomarkers for the diagnosis or prediction of nasopharyngeal carcinoma, reagent kits, and computer-readable storage media. Background Technology

[0002] In recent years, with the rapid development of diagnostic and treatment technologies, the five-year survival rate for patients with early-stage nasopharyngeal carcinoma has reached over 90%. However, due to the relatively hidden nature of the primary lesion and the lack of obvious early symptoms, about 80% of patients are diagnosed at an intermediate or advanced stage, resulting in a high mortality rate.

[0003] According to the "Technical Guidelines for Early Diagnosis and Treatment of Cancer," it is recommended that residents aged 30-59 in high-incidence areas undergo screening for Epstein-Barr virus (EBV) antibodies. Currently, research on nasopharyngeal carcinoma screening in my country has made phased progress, significantly improving early diagnosis rates and reducing mortality. However, existing screening programs have two pressing issues: First, while current EBV antibody testing methods have high sensitivity, their specificity is insufficient, with a positive predictive value of <5%, meaning that over 95% of positive cases are false positives, leading to significant psychological burdens for many antibody-positive individuals. Second, current screening programs lack individualized screening strategies tailored to different risk levels. A precise cancer screening strategy should comprehensively consider multiple risk factors, including genetics and environment, to identify different risk groups for cancer, and then develop practical and effective screening, early diagnosis, and early treatment plans for each risk group to improve screening efficiency.

[0004] Professor Xia Ningshao's team, through a systematic study of the anti-EBV antibody profile in the serum of nasopharyngeal carcinoma (NPC) patients and healthy controls, discovered a novel serological biomarker for NPC: total antibody recognizing the peptide encoded by the BNLF2b gene (referred to as "P85-Ab"). They also collaborated with Wantai Biopharmaceutical to develop a high-throughput automated detection kit. From 2020 to 2021, Professor Xia Ningshao's team, in collaboration with Professor Ji Mingfang's team at Zhongshan People's Hospital, conducted a prospective cohort study of approximately 25,000 participants in Zhongshan City. The study compared the efficacy of P85-Ab detection with traditional dual-antibody protocols in NPC screening head-to-head. P85-Ab showed higher sensitivity for early-stage NPC, with a positive predictive value more than twice that of dual-antibody screening (10.0% vs. 4.3%). (Li T, LiF, Guo X, Hong C, Yu X, Wu B, Lian S, Song L, Tang J, Wen S, Gao K, Hao M, Cheng W, Su Y, Zhang S, Huang S, Fang M, Wang) Y,Ng MH,Chen H,Luo W,Ge S,Zhang J,Xia N,JiM.Anti-Epstein-Barr Virus BNLF2b for Mass Screening for NasopharyngealCancer.N Engl J Med.2023Aug 31;389(9):808-819.doi:10.1056 / NEJMoa2301496.PMID:37646678.).

[0005] However, the positive predictive value of a single screening method is still below 20%. Therefore, there is an urgent need to discover new biomarkers for nasopharyngeal carcinoma screening to improve screening efficiency.

[0006] Almost all tumors are caused and promoted by a combination of genetic alterations and epigenetic variations. By comparing tumor cells with normal cells, a large number of epigenetic abnormalities have been reported, among which DNA methylation is the most common epigenetic effect.

[0007] Cytosine methylation on DNA is a covalent, "acquired" modification of DNA. DNA methylation is carried out by DNA cytosine methyltransferases (DNMTs). DNMTs can transfer a methyl group from an S-adenosylmethionine residue to the C-5 position of cytosine. DNA methylation occurs almost specifically at CpG dimer sites. CpG dimers are unevenly distributed in the human genome, with concentrated enriched regions generally called CpG islands. Such CpGs are present in repetitive sequences of the human genome and in the 5' regulatory regions of many genes. Abnormal DNA methylation in tumors includes both hypomethylation (or demethylation) and hypermethylation. Hypermethylation occurs, but is not limited to, tumor suppressor genes, while hypomethylation occurs, but is not limited to, proto-oncogenes.

[0008] All aspects of tumor development and progression may be related to changes in DNA methylation, involving cell cycle regulation, DNA damage repair, biochemical metabolism of carcinogenic compounds, apoptosis, and angiogenesis. Different types of tumors may have a specific group of tumor suppressor genes with high methylation and proto-oncogenes with low methylation, i.e., a specific methylation profile of the cancer type, which may be used to identify the type of cancer.

[0009] Methylation enrichment is an analytical method used to study methylation modifications on DNA. DNA methylation is an important epigenetic modification involving the addition of methyl groups to the cytosine ring in the DNA molecule. This modification plays a crucial role in biological processes such as gene expression, cell differentiation, and genome stability. Therefore, understanding the state of DNA methylation is essential for comprehending biological processes and the development of diseases.

[0010] Common methylation enrichment techniques include methylation-specific PCR (MSP), methylation-sensitive restriction enzyme digestion, methylated DNA immunoprecipitation sequencing (MeDIP), methylated DNA immunoprecipitation sequencing (MeDIP-Seq), and MBD-Seq (Methyl-CpGBinding domain sequencing), among others.

[0011] MSP uses methylation-specific primers to selectively amplify methylated DNA fragments via PCR. It is simple, rapid, and suitable for analyzing specific CpG sites, but it cannot provide genome-wide methylation information and is only applicable to pre-defined target regions.

[0012] Methylation-sensitive restriction enzyme cleavage utilizes the difference in sensitivity of restriction enzymes to DNA sequences to distinguish between methylated and unmethylated DNA regions. This method is based on the principle that DNA methylation affects the sensitivity of bases on the cytosine ring to restriction enzymes. This technique does not require expensive sequencing technology and can be analyzed using methods such as gel electrophoresis. However, it cannot provide high-resolution information on individual CpG sites, typically providing the methylation status of the entire region. Furthermore, it is limited by the specificity of the selected restriction enzyme, which may result in missed or overdetected methylation sites. Additionally, it cannot directly distinguish between 5-methylcytosine and other forms of DNA modification.

[0013] MeDIP-Seq uses methylated DNA antibodies to selectively enrich methylated DNA fragments, which are then analyzed using high-throughput sequencing. It can enrich entire methylated genomic regions, making it suitable for whole-genome methylation analysis. However, it cannot provide high-resolution information on individual CpG sites.

[0014] MBD-Seq uses methylated DNA-binding proteins (such as MBD2 or MBD3) to enrich methylated DNA fragments, which are then analyzed by sequencing. It offers high enrichment efficiency and is suitable for genome-wide methylation analysis. However, similar to MeDIP-Seq, it cannot provide high-resolution information on individual CpG sites.

[0015] Methylation is also known as methylation conversion. Common methylation sequencing techniques include bisulfite sequencing (BS-seq). BS-seq uses bisulfite to treat DNA, converting unmethylated cytosine to uracil, while leaving methylated cytosine unaffected, and then analyzes the DNA through sequencing. It can provide high-resolution information on individual CpG sites and can perform methylation analysis across the entire genome. However, the experimental procedures are relatively complex.

[0016] Studies on early cancer screening have found that circulating cell-free DNA (cfDNA) methylation detection serves as a non-invasive, low-cost, sensitive, and accurate basis for early tumor detection, and can be used for cancer screening and various cancer classifications. Circulating tumor DNA (ctDNA) methylation is an important epigenetic modification. Gene methylation occurs before cancer develops and is a crucial mechanism in cancer development, acting as a "switch" regulating gene expression. Its stability and consistency are good, making it an ideal method for early cancer screening.

[0017] Currently, screening methods for ctDNA methylation markers have achieved significant breakthroughs in colorectal cancer, gastric cancer, bladder cancer, liver cancer, and other cancers, with as many as 21 methylation detection kits approved by the National Medical Products Administration. However, there are currently no commercially available DNA methylation markers related to nasopharyngeal carcinoma (NPC). With the maturation of methylation technology, more and more NPC-related methylation markers are being discovered and studied. Tian et al., through biopsies of NPC patients, found that DNA methylation of four genes (RASSF1A, WIF1, DAPK1, and RARβ2) showed higher sensitivity and specificity than EBV DNA markers in the early stages (I and II) of NPC. The "Expert Consensus on the Clinical Application of Nasopharyngeal Carcinoma Markers" points out that promoter methylation markers can be combined with EBV testing as a supplement to NPC risk assessment, and applied to the early detection and diagnosis of NPC.

[0018] By detecting changes in nasopharyngeal carcinoma-specific methylation gene levels in ctDNA, we can discover new nasopharyngeal carcinoma methylation biomarkers with higher sensitivity and specificity, and construct an early screening and diagnosis model to better meet the clinical needs for early screening and diagnosis of nasopharyngeal carcinoma. Summary of the Invention

[0019] One of the technical problems to be solved by this invention is to provide a combination of methylation molecular markers for the diagnosis or prediction of nasopharyngeal carcinoma, which consists of the following three methylation molecular markers located in the human ZNF804A (Zinc Finger Protein 804A), FOXL1 (Forkhead Box L1), and CASZ1 (Castor Zinc Finger 1) genes: chr2:184598880-184599080, chr16:86578884-86579084, and chr1:10642015-10642215, respectively, and specifically located by hg38.

[0020] The second technical problem to be solved by the present invention is to provide a reagent for detecting the combination of methylated molecular markers as described above in the preparation of products for diagnosing or predicting nasopharyngeal carcinoma.

[0021] In some embodiments, the reagents for detecting the combination of methylation molecular markers as described above include reagents used by any one or more of the following methods, which include at least one of methylated DNA immunoprecipitation real-time PCR, bisulfite conversion real-time PCR, bisulfite conversion sequencing, methylation microarray sequencing, and methylation-specific PCR.

[0022] In some embodiments, the reagent for detecting the combination of methylation molecular markers as described above includes primer and probe combination a and / or primer and probe combination b;

[0023] Primer and probe combinations a: primer pairs as shown in SEQ ID No. 1 and SEQ ID No. 2 and Taqman MGB probe as shown in SEQ ID No. 3, for quantitative real-time PCR amplification of chr2:184598880-184599080; primer pairs as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6, for quantitative real-time PCR amplification of chr16:86578884-86579084; primer pairs as shown in SEQ ID No. 7 and SEQ ID No. 8 and Taqman MGB probe as shown in SEQ ID No. 9, for quantitative real-time PCR amplification of chr1:10642015-10642215;

[0024] Primer and probe combination b: Primer pairs as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12, for quantitative real-time PCR amplification of chr2:184598880-184599080; primer pairs as shown in SEQ ID No. 13 and SEQ ID No. 14 and Taqman MGB probe as shown in SEQ ID No. 15, for quantitative real-time PCR amplification of chr16:86578884-86579084; primer pairs as shown in SEQ ID No. 16 and SEQ ID No. 17 and Taqman MGB probe as shown in SEQ ID No. 18, for quantitative real-time PCR amplification of chr1:10642015-10642215.

[0025] The third technical problem to be solved by this invention is to provide a kit for the diagnosis or prediction of nasopharyngeal carcinoma. The kit is used to detect a combination of methylation molecular markers; the combination of methylation molecular markers consists of the following three methylation molecular markers located in the human ZNF804A, FOXL1, and CASZ1 genes: chr2:184598880-184599080, chr16:86578884-86579084, and chr1:10642015-10642215, respectively, specifically located using hg38.

[0026] The kit includes primer and probe combination a and / or primer and probe combination b;

[0027] Primer and probe combinations a: primer pairs as shown in SEQ ID No. 1 and SEQ ID No. 2 and Taqman MGB probe as shown in SEQ ID No. 3, for quantitative real-time PCR amplification of chr2:184598880-184599080; primer pairs as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6, for quantitative real-time PCR amplification of chr16:86578884-86579084; primer pairs as shown in SEQ ID No. 7 and SEQ ID No. 8 and Taqman MGB probe as shown in SEQ ID No. 9, for quantitative real-time PCR amplification of chr1:10642015-10642215;

[0028] Primer and probe combination b: Primer pairs as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12, for quantitative real-time PCR amplification of chr2:184598880-184599080; primer pairs as shown in SEQ ID No. 13 and SEQ ID No. 14 and Taqman MGB probe as shown in SEQ ID No. 15, for quantitative real-time PCR amplification of chr16:86578884-86579084; primer pairs as shown in SEQ ID No. 16 and SEQ ID No. 17 and Taqman MGB probe as shown in SEQ ID No. 18, for quantitative real-time PCR amplification of chr1:10642015-10642215.

[0029] In some embodiments, methylated fragments are enriched using methylated DNA immunoprecipitation (MeDIP) technology, and then quantitative real-time PCR is performed using the enriched DNA as a template under primer and probe combination a conditions; the kit also includes a methylated DNA antibody selected from one of 5-methylcytidine antibody, 5-methylcytosine (5-mC) antibody, 5-hydroxymethylcytosine (5-hmC) antibody, 5-formylcytosine (5-fC) antibody, and 5-carboxycytosine (5-caC) antibody.

[0030] In some implementations, it also includes one or more of the following: Rapid Taq Master Mix, methylation enrichment reagents based on the 5-methylcytosine antibody principle, and methylation conversion reagents based on the bisulfite conversion principle.

[0031] In some embodiments, the 3' end of the Taqman MGB probe has an MGB and a fluorescence quenching group, and the 5' end has a fluorescent group; the combination of the fluorescence quenching group and the fluorescent group is selected from BHQ1 or NFQ with FAM, BHQ2 with VIC or HEX, BHQ2 with Cy3, and BHQ2 with Cy5.

[0032] In some implementations, the three Taqman MGB probes in primer and probe combination a carry different combinations of fluorescence quenching groups and fluorescent groups, enabling the reading of corresponding Ct values ​​based on fluorescence color differences in a real-time PCR amplification reaction with three Taqman MGB probes.

[0033] In some implementations, the three Taqman MGB probes in primer and probe combination b carry different combinations of fluorescence quenching groups and fluorescent groups, enabling the reading of corresponding Ct values ​​based on fluorescence color differences in a real-time PCR amplification reaction with three Taqman MGB probes.

[0034] In some implementations, the three Taqman MGB probes of primer and probe combination a carry the same combination of fluorescence quenching groups and fluorescent groups.

[0035] In some implementations, the three Taqman MGB probes of primer and probe combination b carry the same combination of fluorescence quenching and fluorescent groups.

[0036] The fourth technical problem to be solved by the present invention is to provide a computer-readable storage medium, including a program, which can be executed by a processor to analyze and process the fluorescence quantitative PCR detection data of the methylation molecular marker combination as described above, and obtain the nasopharyngeal carcinoma diagnosis result, including the following steps:

[0037] Ct was obtained by real-time PCR using DNA enriched by methylated DNA immunoprecipitation (cfDNA) as a template, under primer and probe combination a. ZNF804A 、Ct FOXL1 、Ct CASZ1 Formulas I and II are used to process and judge the detection results;

[0038] Formula I: logistic scores=e k / (1+e k )

[0039] Formula II: k = -0.847 × Ct ZNF804A -0.494×Ct FOXL1 -0.893×Ct CASZ1 +79.432

[0040] A logistic scores ≤ 700 indicate a negative nasopharyngeal carcinoma, while a logistic scores > 700 indicate a positive nasopharyngeal carcinoma.

[0041] For Ct values ​​> 45 or no Ct value detected by real-time PCR, the Ct value is counted as 45 for substitution into Formula II.

[0042] Primer and probe combination a: Primer pairs as shown in SEQ ID No. 1 and SEQ ID No. 2 and Taqman MGB probe as shown in SEQ ID No. 3, used for real-time quantitative PCR amplification of chr2:184598880-184599080 to obtain Ct. ZNF804A Primer pairs as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6 were used for real-time quantitative PCR amplification of chr16:86578884-86579084 to obtain Ct. FOXL1 Primer pairs as shown in SEQ ID No. 7 and SEQ ID No. 8 and Taqman MGB probe as shown in SEQ ID No. 9 were used for real-time quantitative PCR amplification of chr1:10642015-10642215 to obtain Ct. CASZ1 .

[0043] This invention also provides another computer-readable storage medium, including a program executable by a processor to analyze and process real-time quantitative PCR detection data of the methylation molecular marker combination as described above, to obtain a nasopharyngeal carcinoma diagnosis result, including the following steps:

[0044] Ct was obtained by real-time PCR using bisulfite-converted DNA as a template under primer and probe combination b conditions. ZNF804A 、Ct FOXL1 、Ct CASZ1 Formulas I and III are used to process and judge the detection results;

[0045] Formula I: logistic scores=e k / (1+e k )

[0046] Formula III: k = -0.388 × Ct ZNF804A -0.199×Ct FOXL1 -0.659×Ct CASZ1 +44.324

[0047] A logistic scores ≤ 700 indicate a negative nasopharyngeal carcinoma, while a logistic scores > 700 indicate a positive nasopharyngeal carcinoma.

[0048] For Ct values ​​> 45 or no Ct value detected by real-time PCR, the Ct value is counted as 45 for substitution into Formula III.

[0049] Primer and probe combination b: Primer pairs as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12, used for real-time quantitative PCR amplification of chr2:184598880-184599080 to obtain Ct. ZNF804A Primer pairs as shown in SEQ ID No. 13 and SEQ ID No. 14 and Taqman MGB probe as shown in SEQ ID No. 15 were used for real-time quantitative PCR amplification of chr16:86578884-86579084 to obtain Ct. FOXL1 Primer pairs as shown in SEQ ID No. 16 and SEQ ID No. 17 and Taqman MGB probe as shown in SEQ ID No. 18 were used for real-time quantitative PCR amplification of chr1:10642015-10642215 to obtain Ct. CASZ1 .

[0050] In some implementations, the quantitative PCR detection data processed by the computer-readable storage medium are derived from the following quantitative PCR reaction: the reaction volume is 35 μL; the reaction reagent used is 2×Rapid TaqMaster Mix; the reaction conditions are 95℃ for 5 minutes, 95℃ for 15 seconds, 60℃ for 40 seconds, and 45 cycles of amplification.

[0051] Compared with the prior art, the present invention has the following technical effects:

[0052] This invention provides methylation molecular markers for the ZNF804A, FOXL1, and CASZ1 gene methylation regions (chr2:184598880-184599080, chr16:86578884-86579084 and chr1:10642015-10642215, specifically located using hg38) as methylation markers for the diagnosis (including early diagnosis and auxiliary diagnosis) or prediction of nasopharyngeal carcinoma, thus enriching the options available to those skilled in the art.

[0053] This invention uses methylated DNA immunoprecipitation combined with quantitative real-time PCR to detect the methylation level of methylated regions of the ZNF804A, FOXL1, and CASZ1 genes. It has high sensitivity and strong specificity and has very important clinical application value.

[0054] Based on the methylation levels of specific regions of the ZNF804A, FOXL1, and CASZ1 genes, this invention enables early detection of nasopharyngeal carcinoma, providing more biomarker (methylation molecular markers are a type of biomarker) options for nasopharyngeal carcinoma screening and early diagnosis.

[0055] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0056] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0057] Figure 1 The procedure for detecting ZNF804A, FOXL1, and CASZ1 methylation genes in Example 2 of this invention is shown.

[0058] Figures 2A-2F An amplification curve of a sample detected by qPCR based on the methylated DNA immunoprecipitation enrichment method is shown (e.g.) Figure 2A , 2B (as shown in Figure 2C) and amplification curves based on qPCR detection using bisulfite conversion treatment (as shown in Figure 2C). Figure 2D , 2E (as shown in 2F).

[0059] Figure 3A The ROC curves of 83 samples were shown for detection by qPCR based on the methylated DNA immunoprecipitation enrichment method.

[0060] Figure 3B The ROC curves of qPCR detection based on bisulfite conversion treatment for 83 samples are shown. Detailed Implementation

[0061] To facilitate understanding by those skilled in the art, some terms appearing in this document are explained and clarified.

[0062] In this document, the singular forms “an,” “an,” and “the” include their plural forms unless the context otherwise requires. Thus, for example, “an agent” can be understood to include multiple agent components.

[0063] In this document, unless otherwise stated, the terms “comprising,” “including,” or “containing” mean that the listed values, steps, or ingredients are included, but do not exclude the inclusion of other values, steps, or ingredients.

[0064] In this document, the terms "individual" or "patient" are used interchangeably and refer to a vertebrate, preferably a mammal. A mammal may be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples.

[0065] In this article, “normal healthy” samples refer to samples of the same type isolated from individuals known to be free of the cancer, tumor, polyp, or adenoma described herein.

[0066] The term "AUC" is an abbreviation for "area under the curve." Specifically, it refers to the area under the receiver operating characteristic (ROC) curve. An ROC curve is a plot of the true positive rate versus the false positive rate for different possible cutoff points in a diagnostic test. It shows the balance between sensitivity and specificity based on the selected cutoff point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under the ROC curve (AUC) is a measure of a diagnostic test (the larger the area, the better; optimal is 1; randomized trials will have an ROC curve with an area of ​​0.5 on the diagonal; see: J. Pegan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0067] To detect changes in nasopharyngeal carcinoma-specific methylation gene levels in ctDNA, discover new nasopharyngeal carcinoma methylation biomarkers with higher sensitivity and specificity, and construct early screening and diagnosis models, the inventors of this invention have devoted considerable effort to developing effective nasopharyngeal carcinoma methylation biomarkers in cfDNA, enabling early diagnosis of cancer and carcinogenesis risk. Unexpectedly, specific regions of the ZNF804A, FOXL1, and CASZ1 genes were found to be methylated in nasopharyngeal carcinoma cancer cells. This invention utilizes these specific regions of genes as methylation biomarkers.

[0068] In this invention, through long-term exploration and verification with a large number of clinical samples, the inventors unexpectedly discovered that the methylation levels of the ZNF804A, FOXL1, and CASZ1 genes differ significantly between nasopharyngeal carcinoma and non-nasopharyngeal carcinoma. The protein encoded by the ZNF804A gene is a zinc finger binding protein, and its polymorphism, particularly at the rs1344706 site, is considered to be associated with increased susceptibility to schizophrenia, bipolar disorder, and heroin. The FOXL1 gene is a member of the Forkhead transcription factor family, which plays an important role in regulating physiological processes such as cell proliferation, differentiation, metabolism, and organ development. The CASZ1 gene, or castor zinc finger 1 gene, is a transcription factor with a zinc finger structure. It plays an important role in various biological processes, including cell proliferation, differentiation, apoptosis, and tumorigenesis and development. The expression level of the CASZ1 gene is regulated by multiple factors, among which methylation is an important regulatory mechanism.

[0069] Early detection of cancer may be achieved through methylated DNA analysis of specific types of cancer. Currently, the mainstream method for methylation analysis is bisulfite treatment, a process involving denaturation, deamination, and desulfonation. The DNA is first denatured into single strands, then subjected to extreme temperatures, salt, acidity, and alkalinity. The resulting transformed DNA is predominantly single-stranded, with a mixture of double strands, fragment nicks, gaps, and uracil-state nucleotides. This process typically results in the loss of 90% of the DNA template, leaving a significant amount of methylation information undetectable in subsequent processes. Furthermore, incomplete or over-conversion during base transformation introduces human bias, which is further amplified by subsequent PCR amplification, leading to inaccurate signals. Therefore, current methylation markers obtained through bisulfite treatment generally suffer from low sensitivity, especially in blood samples where the already limited number of free DNA fragments becomes significantly more difficult to detect after bisulfite treatment.

[0070] An amplification curve is a graphical representation of product accumulation during polymerase chain reaction (PCR). It is generated by monitoring the increase in fluorescence signal in the reaction solution. The following are characteristics of a typical PCR amplification curve:

[0071] Initial stage:

[0072] Threshold Cycle (Ct) value: In the early stages of a PCR reaction, the fluorescence signal may be low, but it gradually increases as PCR products accumulate. The Ct value refers to the number of cycles required in a PCR reaction for the fluorescence signal to rise above a pre-set threshold. A lower Ct value indicates a higher initial amount of target DNA in the sample.

[0073] Exponential growth phase:

[0074] Exponential phase: In the middle of the PCR reaction, the PCR products grow exponentially. At this time, the rate of increase in the Ct value accelerates, reflecting the exponential growth of the target DNA in the PCR reaction.

[0075] Platform Phase:

[0076] Plateau stage: In the later stages of the PCR reaction, the accumulation of PCR products reaches saturation and no longer increases exponentially. During this stage, the PCR amplification curve forms a plateau, and the increase in the Ct value becomes slow.

[0077] In some embodiments of the present invention, the diagnosis is an early diagnosis, specifically, the early stage is nasopharyngeal carcinoma stage 0 to I or stage II.

[0078] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0079] Example 1: Discovery of Specific Methylation Gene Loci in Nasopharyngeal Carcinoma

[0080] To screen for biomarkers of specific methylation in nasopharyngeal carcinoma, this study collected blood samples and corresponding homologous carcinoma tissues (paraffin-embedded tissue specimens) from 155 patients clinically diagnosed with nasopharyngeal carcinoma (also known as nasopharyngeal carcinoma-positive samples), as well as blood samples from 208 patients clinically diagnosed with nasopharyngeal carcinoma-negative samples. Differentially methylated genes or regions screened in blood samples were required to be validated in tissue samples to be considered as more reliable clinically significant methylation markers. In this article, "clinically diagnosed" refers to patients diagnosed with nasopharyngeal carcinoma according to the guidelines for the diagnosis and treatment of nasopharyngeal carcinoma (2023) (also known as nasopharyngeal carcinoma-positive samples) and patients without nasopharyngeal carcinoma (also known as nasopharyngeal carcinoma-negative samples or normal healthy individuals).

[0081] 1. Prepare a methylated DNA sample library

[0082] (1) DNA extraction

[0083] cfDNA was extracted using a commercially available extraction kit, following the instructions in the manufacturer's manual.

[0084] Quality control of nucleic acid concentration and fragment distribution was performed using Qubit 4.0 and Qsep100, respectively. The yield of cfDNA extracted from 2 mL of human plasma should be greater than 5 ng, with an enrichment peak at or near 167 bp. When the yield was greater than 50 ng, fragmentation quality control was performed using Qsep100 capillary electrophoresis. If large fragments were found, magnetic beads were used for fragment screening to remove them.

[0085] Genomic DNA was extracted from samples of cancerous tissue, adjacent normal tissue, and normal tissue using standard commercial kits or self-prepared reagents, following the instructions. Genomic DNA can be fragmented by sonication or enzyme digestion to obtain DNA samples approximately 200 bp in length, facilitating library construction.

[0086] (2) Library Construction

[0087] Commercially available library construction kits can be used following the instructions. Kits such as the Rapid Plus DNA LibPrep Kit for Illumina (Cat. No. RK20208, ABclonal) or the VAHTS Universal Pro DNA Library Prep Kit for Illumina Vazyme (Cat. No. ND608-02, Novizan) can be used for end repair, adding an "A" tail, and ligation to adapters. This example uses the VAHTS Universal Pro DNA Library Prep Kit for Illumina Vazyme (Cat. No. ND608-02, Novizan) for library construction.

[0088] (3) Immunoprecipitation of methylated DNA

[0089] Methylation enrichment of cfDNA and tissue genomic DNA was performed using different reactions. Each methylation enrichment reaction could simultaneously complete 12–100 cfDNA library mixtures (approximately 10 ng of cfDNA library per sample) and 10–24 genomic DNA library mixtures (approximately 100 ng of genomic DNA library per sample). Methylation enrichment based on the 5-methylcytosine (5mC) antibody principle was performed using commercially available methylation enrichment kits or self-prepared reagents according to the instructions. This example used the zymoMeDIP kit (catalog number D5101-A). After the methylation enrichment reaction, the DNA was purified according to the instructions and subjected to 10–12 rounds of conventional PCR amplification using universal sequencing primers to obtain methylated DNA fragment libraries with a yield of over 500 ng per reaction.

[0090] 2. Prepare the DNA probe library

[0091] (1) Probe Design

[0092] The inventors selected the longest ZNF804A, FOXL1, and CASZ1 gene transcripts from the NCBI database to confirm their gene locations. Sequence information was obtained from the promoter region, 5' UTR, first exon 1, and a 1kb range upstream of the start codon.

[0093] Probes were designed to target the methylated regions of the ZNF804A, FOXL1, and CASZ1 genes.

[0094] Probe design principles:

[0095] (1) The target area is fully covered without any gaps;

[0096] (2) No overlap is provided;

[0097] (3) Each probe is 120nt in length.

[0098] Table 1 details the specific genomic locations corresponding to the target regions and the specific genomic locations corresponding to the regions covered by the probes designed accordingly.

[0099] (2) Probe Synthesis

[0100] Using the probe design principles described above, a total of 64 probes were designed targeting the methylation regions of the ZNF804A, FOXL1, and CASZ1 genes, covering all possible CpG sites. The probe coverage information is shown in Table 1.

[0101] Table 1. Probe Coverage Information

[0102]

[0103]

[0104] 3. DNA capture probe hybridization

[0105] Liquid-phase hybridization capture was performed using NadPrep hybridization capture reagent (Cat. No. REF1005101, NadPrep). Hybridization capture reactions could be single-hybrid or multi-hybrid, and the total amount of MeDIP amplified library added for each hybridization capture reaction should range from 300 ng to 8 μg. After purification, 500 ng of library (or all if less than 500 ng) was added, along with Human Cot DNA and NadPrep reagent. Nano Blockers were dried in a vacuum concentrator preheated to 42°C at a speed of 1000 rpm. After drying, the prepared hybridization reaction solution (containing the probe panel) was added, and the mixture was vortexed and centrifuged briefly. Hybridization was carried out for 4-16 hours at the following hybridization programs: 95°C / 30 sec; 65°C / Hold (100°C hot lid). Then, the washed streptavidin magnetic beads were added to the hybridization system and incubated for 40 minutes, vortexing every 10 minutes to ensure complete resuspension of the magnetic beads. It is worth noting that the hybridization capture reaction temperature was the conventional 65°C, not the 63°C required for methylation probes designed based on bisulfite conversion.

[0106] After the hybridization capture reaction is complete, wash the bound magnetic beads with the four washing solutions provided in the kit, discarding any residual solution at each step; finally, add 20 μL of nuclease-free water and gently vortex to mix.

[0107] 4. PCR amplification and purification after hybridization capture

[0108] The hybridization capture product was amplified by PCR using the amplification reagents from the VAHTS Universal Pro DNA Library Prep Kit for Illumina (Cat. No. ND608-02, Vazyme), with 12-13 cycles. After amplification, the product was purified using an equal volume of VAHTS DNA Clean Beads (Cat. No. N411-03, Vazyme) to obtain a relatively pure hybridization capture library. The library concentration was quantified using Qubit 4.0, and fragment size was determined using a Qsep100 fully automated nucleic acid and protein analyzer.

[0109] 5. Library sequencing and bioinformatics analysis

[0110] Dilute the library to be used to 4 nM and mix it according to the required data volume. The total data volume should not exceed 120 G. After mixing, take out 5 μL of the library, add 5 μL of 0.2 N NaOH, mix by pipetting, and denature for 5 minutes. Immediately after denaturation, add 990 μL of HT1 Buffer (REF: 15058251, Illumina), vortex to mix, take out 105 μL and add 1295 μL of HT1 Buffer, vortex to mix, and the resulting library is 1.5 pM.

[0111] The sequencer used was an Illumina NextSeq 550Dx. Reagents used included High Output Reagent Cartridge v2 (REF:15057929, Illumina) (300 cycles), High Output Flow Cell Cartridge v2.5 (REF:20022408, Illumina), and Buffer Cartridge v2 (REF:15057941, Illumina). 1300 μL of the library was added to the sample space of the High Output Reagent Cartridge v2, and each reagent was added sequentially. Sequencing could then begin. This example used paired-end sequencing, with a total sequencing time of approximately 30 hours.

[0112] 6. Quality control of sequencing data

[0113] Fastp (version 0.22.0) was used for quality control of the sequencing data to remove low-quality bases. The overall Q20 of the clean data was above 90%, and the Q30 was above 85%. The average sequencing depth was around 300×. The average target hit rate of the probes in the above probe combination was above 80%, demonstrating that this embodiment, based on the combination of methylation immunoprecipitation and liquid hybridization to capture probes, is feasible and effective for detecting cancer-related methylation regions.

[0114] 7. Analysis of differentially expressed methylation regions of ZNF804A, FOXL1, and CASZ1 genes associated with nasopharyngeal carcinoma

[0115] The DiffBind tool (version 3.8.4) was used to screen for differential peaks between tumor and non-tumor groups. Two algorithms, DESeq and EdgeR, were used, prioritizing regions that intersect within the panel. The screening criteria were: 1) False Discovery Rate (FDR) < 0.01, and 2) Fold change < -1. The most significant characteristic methylation regions meeting these criteria were then identified as differentially methylated regions between the nasopharyngeal carcinoma and non-nasopharyngeal carcinoma groups.

[0116] In this embodiment, the characteristic methylation regions with the most significant differences between the nasopharyngeal carcinoma group and the non-nasopharyngeal carcinoma group were screened from the regions of the ZNF804A, FOXL1, and CASZ1 genes covered by 64 probes as methylation differential regions. The methylation CpG sites of these methylation differential regions are shown in Table 2.

[0117] Table 2. Methylated CpG sites in regions of methylation difference

[0118]

[0119]

[0120] Analysis of the RPM (Reads per million mapped reads) of methylated differentially methylated regions (reads covering CpG sites in Table 2) in 155 nasopharyngeal carcinoma-positive samples and 208 healthy controls showed a significant difference (P < 0.005). Furthermore, the reads covering CpG sites in Table 2 from nasopharyngeal carcinoma cfDNA samples were also validated in nasopharyngeal carcinoma tissue samples, with P values ​​all less than 0.005, indicating a significant difference.

[0121] Example 2: Detection of ZNF804A, FOXL1, and CASZ1 methylation genes in clinical samples and comparison of different treatment regimens

[0122] To further verify the clinical efficacy of differentially methylated regions of the ZNF804A, FOXL1, and CASZ1 genes related to nasopharyngeal carcinoma in plasma samples, the inventors used qPCR to test two additional groups of samples (37 plasma samples clinically diagnosed with nasopharyngeal carcinoma and 46 plasma control samples with negative nasopharyngoscopy results). Among the 37 clinically diagnosed plasma samples, 8 were from stage 0-I, 9 from stage II, 8 from stage III, and 12 from stage IV.

[0123] The flowchart for ZNF804A, FOXL1, and CASZ1 methylation gene detection is as follows: Figure 1 As shown, specifically:

[0124] (1) DNA extraction

[0125] cfDNA was extracted using a commercially available extraction kit, following the instructions in the manufacturer's manual.

[0126] Quality control of nucleic acid concentration and fragment distribution was performed using Qubit 4.0 and Qsep100, respectively. The yield of cfDNA extracted from 4 mL of human plasma should be greater than 10 ng, with an enrichment peak at or near 167 bp. When the yield exceeds 50 ng, Qsep100 capillary electrophoresis is used for fragmentation quality control. For large fragment contamination, magnetic beads are used for fragment screening to remove large fragments.

[0127] (2) Treatment of methylated DNA

[0128] ①Methylated DNA Immunoprecipitation

[0129] Take half of the total extracted nucleic acid and perform cfDNA methylation enrichment. Different reactions are carried out. Methylation enrichment based on the 5mC antibody principle is performed using the zymoMeDIP kit (catalog number D5101-A). After the methylation enrichment reaction is carried out according to the instructions, the elution volume is 50 μL.

[0130] ②Methylated DNA bisulfite conversion

[0131] Half of the total extracted nucleic acid was subjected to cfDNA methylation and bisulfite treatment. This was carried out in different reactions. Based on the principle of bisulfite conversion, the methylated DNA treatment was performed using the ZYMO RESEARCH DNA Transformation Kit (EZ DNA Methylation Kit, D5002). The elution volume was 50 μL.

[0132] (3) qPCR detection

[0133] Primer and probe synthesis was performed at Shanghai Bailige Biotechnology Co., Ltd., and the specific sequence information is as follows:

[0134] The sequences of Taqman MGB probe primer pairs enriched by immunoprecipitation of methylated DNA are shown in Table 3.

[0135] Table 3. Immunoprecipitation enrichment of methylated DNA using Taqman MGB probe primer pairs

[0136]

[0137] The probe is labeled with MGB at the 3' end, with a fluorescent group (FAM) at the 5' end and a quencher group (BHQ1) at the 3' end.

[0138] Using methylated DNA enriched by immunoprecipitation as a template, PCR amplification was performed. The final concentration of each primer was 10 μM, and each gene was amplified in a singlet reaction. The PCR reaction system consisted of 5 μL of enriched template DNA, 2.5 μL of premixed solution containing the primers, 17.5 μL of PCR reagent (2×Rapid Taq Master Mix), and water to a final volume of 35 μL. The PCR reaction conditions were as follows: 95℃ for 5 minutes, 95℃ for 15 seconds, 60℃ for 40 seconds, for 45 cycles.

[0139] The primer pairs for the Taqman MGB probe after bisulfite conversion are shown in Table 4.

[0140] Table 4. Primer pairs for Taqman MGB probes after bisulfite conversion

[0141]

[0142] The probe is labeled with MGB at the 3' end, with a fluorescent group (FAM) at the 5' end and a quencher group (BHQ1) at the 3' end.

[0143] Using bisulfite-converted DNA as a template, PCR amplification was performed. Each primer had a final concentration of 10 μM, and each gene was amplified in a singlet reaction. The PCR reaction system consisted of 5 μL of enriched template DNA, 2.5 μL of premixed solution containing the primers, 17.5 μL of PCR reagent (2×Rapid Taq Master Mix), and water to a final volume of 35 μL. The PCR reaction conditions were as follows: 95℃ for 5 minutes, 95℃ for 15 seconds, 60℃ for 40 seconds, for 45 cycles.

[0144] (4) Analysis of clinical sample test results

[0145] The data from the test were analyzed, and 83 samples were enriched by methylated DNA immunoprecipitation and DNA treatment by bisulfite, and then detected by qPCR.

[0146] Figures 2A-2F This section shows examples of qPCR amplification curves from samples of the same nasopharyngeal carcinoma patient (sample number: NPC101275) obtained using both methylated DNA immunoprecipitation enrichment and bisulfite-treated DNA methods. Figure 2A-2C The figures represent the qPCR amplification curves after immunoprecipitation enrichment of methylated DNA from the ZNF804A, FOXL1, and CASZ1 genes, respectively, with Ct values ​​of 32.94, 34.62, and 34.29. Figure 2D-2E The qPCR amplification curves for bisulfite-treated DNA are shown, with Ct values ​​of 35.7, 45, and 35.54. The method of enriching methylated DNA through immunoprecipitation followed by qPCR detection has significant advantages.

[0147] For samples with a Ct value > 45 or no Ct value detected (Undetermined), the Ct value was set to 45. Logistic regression was used to calculate the Ct value, and ROC curves were plotted based on the calculation results. For example... Figure 3A and Figure 3BAs shown, the areas under the curves (AUCs) of the ROC curves obtained by the two different methods were 0.991 and 0.942, respectively. Based on the ROC curves, thresholds (cut-off values) were set for different methods: for qPCR detection based on methylated DNA immunoprecipitation enrichment (i.e., methylated DNA immunoprecipitation real-time PCR), the cut-off value was set to logistic scores = 700; for qPCR detection based on bisulfite conversion treatment (i.e., bisulfite conversion real-time PCR), the cut-off value was set to logistic scores = 700. If the logistic scores of the three genes ZNF804A, FOXL1, and CASZ1 amplified in the tested sample were equal to or lower than the set cut-off value, the sample was judged as negative; otherwise, it was judged as positive. Therefore, the test results of 83 samples were statistically analyzed. The formula is as follows: Formula I: logistic scores = e k / (1+e k )

[0148] Immunoprecipitation of methylated DNA (Formula II): k = -0.847 × Ct ZNF804A -0.494×Ct FOXL1 -0.893×Ct CASZ1 +79.432

[0149] Bisulfite conversion (Formula III): k = -0.388 × Ct ZNF804A -0.199×Ct FOXL1 -0.659×Ct CASZ1 +44.324

[0150] Table 5 shows a comparison between qPCR detection based on methylated DNA immunoprecipitation enrichment method and nasopharyngoscopy results (gold standard). Table 6 shows a comparison between qPCR detection results based on bisulfite conversion treatment and nasopharyngoscopy results. Table 7 shows a comparison between qPCR detection results based on methylated DNA immunoprecipitation enrichment method and qPCR detection results based on bisulfite conversion treatment.

[0151] Table 5. Comparison of qPCR detection results and nasopharyngoscopy results based on methylated DNA immunoprecipitation enrichment method.

[0152]

[0153] Table 6. Comparison of qPCR detection results and nasopharyngoscopy results based on bisulfite conversion treatment.

[0154]

[0155] Table 7. Comparison of methylated DNA-based immunoprecipitation enrichment methods with those based on bisulfite conversion treatment

[0156]

[0157] As shown in Tables 5-7, the differentially methylated regions of the ZNF804A, FOXL1, and CASZ1 genes, when validated on the qPCR detection platform based on the methylated DNA immunoprecipitation enrichment method, exhibited higher sensitivity (86.5%) for nasopharyngeal carcinoma and maintained high specificity (93.5%) for non-nasopharyngeal carcinoma samples, with an accuracy of 90.4%. Overall, the performance was superior to that of qPCR detection using bisulfite conversion treatment.

[0158] (5) Analysis of clinical nasopharyngeal carcinoma staging sample test results

[0159] Of the 37 plasma samples clinically diagnosed with nasopharyngeal carcinoma, 8 were from stage 0-I, 9 from stage II, 8 from stage III, and 12 from stage IV. Tables 8-9 show the detection and statistical analysis of samples from different pathological stages of nasopharyngeal carcinoma, comparing the results of qPCR detection based on methylated DNA immunoprecipitation enrichment with qPCR with those based on bisulfite conversion treatment.

[0160] Table 8. Detection of nasopharyngeal carcinoma samples at different pathological stages

[0161]

[0162] Table 9. Statistical analysis of sensitivity of different pathological stages of nasopharyngeal carcinoma

[0163]

[0164] As shown in Tables 8 and 9, the differentially methylated regions of the ZNF804A, FOXL1, and CASZ1 genes maintained high sensitivity (75.0% and 88.9%) for nasopharyngeal carcinoma (NPC) stages 0-1 and 2, respectively, when validated on a qPCR detection platform based on the methylated DNA immunoprecipitation enrichment method. This demonstrated superior performance for early NPC detection compared to bisulfite-treated qPCR, providing a novel potential marker for early NPC detection. Due to the high sensitivity of this invention in early NPC detection, it can be used for NPC prediction. This invention uses cfDNA as the analysis sample, which is readily available. Furthermore, because cfDNA contains a wealth of information, it is not limited to the diagnosis (including auxiliary diagnosis) and / or prediction of NPC, but can also be used for the prediction and / or diagnosis of other diseases, exhibiting advantages over single tissue samples in the context of high-throughput sequencing analysis. For example, nasopharyngeal swabs cannot be used as samples for the diagnosis and / or prediction of other diseases such as gastric cancer and colon cancer.

[0165] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Modifications and improvements to the present invention are possible without exceeding the concept and scope defined by the claims. Therefore, the content of the embodiments in this specification should not be construed as a limitation of the present invention.

Claims

1. The application of a reagent for detecting combinations of methylated molecular markers in the preparation of products for diagnosing or predicting nasopharyngeal carcinoma, characterized in that, The reagents for detecting combinations of methylated molecular markers for the diagnosis or prediction of nasopharyngeal carcinoma include primer and probe combinations a and / or primer and probe combinations b. Primer and probe combinations a: primer pairs as shown in SEQ ID No. 1 and SEQ ID No. 2 and Taqman MGB probe as shown in SEQ ID No. 3; primer pairs as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6; primer pairs as shown in SEQ ID No. 7 and SEQ ID No. 8 and Taqman MGB probe as shown in SEQ ID No. 9; Primer and probe combination a uses DNA enriched by immunoprecipitation of methylated DNA cf DNA as a template; Primer and probe combinations b: primer pairs as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12; primer pairs as shown in SEQ ID No. 13 and SEQ ID No. 14 and Taqman MGB probe as shown in SEQ ID No. 15; primer pairs as shown in SEQ ID No. 16 and SEQ ID No. 17 and Taqman MGB probe as shown in SEQ ID No. 18; Primer and probe combination b uses bisulfite-converted DNA as a template.

2. The application as described in claim 1, characterized in that, The reagents for detecting combinations of methylated molecular markers include reagents used in any one or more of the following methods, wherein the methods include at least one of methylated DNA immunoprecipitation fluorescence quantitative PCR and bisulfite conversion fluorescence quantitative PCR.

3. A reagent kit for diagnosing or predicting nasopharyngeal carcinoma, characterized in that, The kit includes one or more of the following: primer and probe combination a, and reagents required for methylation enrichment based on the principle of 5-methylcytosine antibody; or primer and probe combination b; or primer and probe combination a, one or more of the following: reagents required for methylation enrichment based on the principle of 5-methylcytosine antibody, and primer and probe combination b. Primer and probe combinations a: primer pairs as shown in SEQ ID No. 1 and SEQ ID No. 2 and Taqman MGB probe as shown in SEQ ID No. 3; primer pairs as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6; primer pairs as shown in SEQ ID No. 7 and SEQ ID No. 8 and Taqman MGB probe as shown in SEQ ID No. 9; Primer and probe combinations b: primer pairs as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12; primer pairs as shown in SEQ ID No. 13 and SEQ ID No. 14 and Taqman MGB probe as shown in SEQ ID No. 15; primer pairs as shown in SEQ ID No. 16 and SEQ ID No. 17 and Taqman MGB probe as shown in SEQ ID No.

18.

4. The kit according to claim 3, characterized in that, It also includes one or more of the Rapid Taq Master Mix and reagents required for methylation conversion based on the principle of bisulfite conversion.

5. The reagent kit as described in claim 3, characterized in that, The Taqman MGB probe has MGB and BHQ1 or MGB and NFQ at its 3' end and FAM at its 5' end; or the Taqman MGB probe has MGB and BHQ2 at its 3' end and VIC or HEX at its 5' end; or the Taqman MGB probe has MGB and BHQ2 at its 3' end and Cy3 at its 5' end; or the Taqman MGB probe has MGB and BHQ2 at its 3' end and Cy5 at its 5' end.

6. A computer-readable storage medium, characterized in that, The program includes a process executable by a processor to analyze and process real-time quantitative PCR detection data of methylation molecular marker combinations for the diagnosis or prediction of nasopharyngeal carcinoma, and to obtain a nasopharyngeal carcinoma determination result, including the following steps: Ct was obtained by real-time PCR using DNA enriched by methylated DNA immunoprecipitation (cfDNA) as a template, under primer and probe combination a. ZNF804A 、Ct FOXL1 、Ct CASZ1 Formulas I and II are used to process and judge the detection results; Formula I: logistic score=e k / (1+e k ) Formula II: k = -0.847 × Ct ZNF804A -0.494×Ct FOXL1 -0.893×Ct CASZ1 +79.432 A logistic scores ≤ 700 indicate a negative nasopharyngeal carcinoma, while a logistic scores > 700 indicate a positive nasopharyngeal carcinoma. For Ct values ​​> 45 or no Ct value detected by real-time PCR, the Ct value is counted as 45 for substitution into Formula II. Primer and probe combination a: Primer pairs as shown in SEQ ID No. 1 and SEQ ID No. 2 and Taqman MGB probe as shown in SEQ ID No. 3, used for real-time PCR amplification to obtain Ct ZNF804A Primer pairs as shown in SEQ ID No. 4 and SEQ ID No. 5, and the Taqman MGB probe as shown in SEQ ID No. 6, were used for real-time PCR amplification to obtain Ct. FOXL1 Primer pairs as shown in SEQ ID No. 7 and SEQ ID No. 8, and the Taqman MGB probe as shown in SEQ ID No. 9, were used for real-time PCR amplification to obtain Ct. CASZ1 .

7. A computer-readable storage medium, characterized in that, The program includes a process executable by a processor to analyze and process real-time quantitative PCR detection data of methylation molecular marker combinations for the diagnosis or prediction of nasopharyngeal carcinoma, and to obtain a nasopharyngeal carcinoma determination result, including the following steps: Ct was obtained by real-time PCR using bisulfite-converted DNA as a template under primer and probe combination b conditions. ZNF804A 、Ct FOXL1 、Ct CASZ1 Formulas I and III are used to process and judge the detection results; Formula I: logistic score=e k / (1+e k ) Formula III: k = -0.388 × Ct ZNF804A -0.199×Ct FOXL1 -0.659×Ct CASZ1 +44.324 A logistic scores ≤ 700 indicate a negative nasopharyngeal carcinoma, while a logistic scores > 700 indicate a positive nasopharyngeal carcinoma. For Ct values ​​> 45 or no Ct value detected by real-time PCR, the Ct value is counted as 45 for substitution into Formula III. Primer and probe combination b: Primer pairs as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12, used for real-time PCR amplification to obtain Ct ZNF804A Primer pairs as shown in SEQ ID No. 13 and SEQ ID No. 14, and the Taqman MGB probe as shown in SEQ ID No. 15, are used for quantitative real-time PCR amplification to obtain Ct. FOXL1 Primer pairs as shown in SEQ ID No. 16 and SEQ ID No. 17, and the Taqman MGB probe as shown in SEQ ID No. 18, are used for real-time PCR amplification to obtain Ct. CASZ1 .

8. The computer-readable storage medium as claimed in claim 6 or 7, characterized in that, The quantitative PCR detection data analyzed by the program came from the following quantitative PCR reaction: the reaction system was 35 μL; the reaction reagent used was 2×Rapid Taq Master Mix; the reaction conditions were 95℃ for 5 minutes, 95℃ for 15 seconds, 60℃ for 40 seconds, and 45 cycles of amplification.

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