A group of diagnostic markers for nasopharyngeal carcinoma and their detection reagents

Abstract

The present invention discloses a group of nasopharyngeal carcinoma diagnostic markers and detection reagents thereof. The method discloses the use of differentially methylated gene regions as nasopharyngeal carcinoma diagnostic markers and quantitative methylation-specific PCR reagents for detecting differentially methylated gene regions in preparing a nasopharyngeal carcinoma diagnostic kit, wherein the genes are one or more of IMPA2, ITPKB, ITGA4, PI9, AMIGO2, or VAV3. A quantitative methylation-specific PCR detection method is established using differentially methylated regions of IMPA2, ITPKB, ITGA4, PI9, AMIGO, and VAV3 for the diagnosis of nasopharyngeal carcinoma, and the detection rate, sensitivity, and specificity are significantly improved compared to the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of diagnosis of nasopharyngeal carcinoma, and more specifically, to a group of nasopharyngeal carcinoma diagnostic markers and detection reagents thereof. Background Art

[0002] Nasopharyngeal carcinoma is a malignant tumor that is prone to occur in the posterior wall of the nasopharynx and the pharyngeal recess. Common clinical symptoms include nasal congestion, blood in mucus, stuffy ears, hearing loss, diplopia and headache. Although the site of nasopharyngeal carcinoma is connected to the outside world through the nasal cavity and oral cavity, there are basically no symptoms in the early stage. Therefore, when nasopharyngeal carcinoma patients first visit the doctor, less than 30% of them are in the early stage of the disease. Not only does the five-year survival rate of patients with early nasopharyngeal carcinoma after treatment is high, but the cost of treatment is also significantly lower than that of mid- and late-stage nasopharyngeal carcinoma. Therefore, early screening and early diagnosis are the key to solving the problem of nasopharyngeal carcinoma.

[0003] Chinese Patent 202111251797.9 "A Diagnostic Marker for Nasopharyngeal Carcinoma" discloses that the detection of Epstein-Barr virus methylated CpG sites can be used as a diagnostic marker for nasopharyngeal carcinoma. It has the advantages of high sensitivity and high specificity in nasopharyngeal brush or nasopharyngeal swab (blind nasopharyngeal examination) samples that do not rely on the guidance of clinicians and nasopharyngeal endoscopes. It is conducive to the promotion and application of nasopharyngeal brush or nasopharyngeal swab (blind nasopharyngeal examination) technical solutions that do not rely on the guidance of clinicians and nasopharyngeal endoscopes, and promotes the early screening and diagnosis of nasopharyngeal carcinoma.

[0004] Nasal NK / T cell lymphoma is a rapidly growing type of non-Hodgkin lymphoma that is common in Asian populations. This lymphoma usually occurs in organs or tissues other than lymph nodes, most commonly in the nasal cavity, and can also affect the sinuses or upper throat. The most common initial symptoms of patients with this disease are nasal congestion, runny nose, and blood in the nose, followed by edema and ulcerative necrotic lesions, mainly affecting the nasal mucosa, nasal septum, paranasal sinuses, and nasopharynx. Clinically, imaging and pathological biopsy are usually used for diagnosis. The occurrence and development of both nasopharyngeal carcinoma and nasal NK / T cell lymphoma are closely related to Epstein-Barr virus infection. At the same time, due to the same site of onset and similar clinical manifestations, it is difficult to distinguish between the two diseases by only testing the Epstein-Barr virus load through blind nasopharyngeal swab sampling. Although the exact mechanism of the disease is still unclear, there are differences in the methylation pattern of Epstein-Barr virus in Epstein-Barr virus-related tumors, so nasopharyngeal carcinoma and nasal NK / T cell lymphoma can be differentially diagnosed from the perspective of Epstein-Barr virus methylation.

[0005] Therefore, Chinese Patent 202310539250.1 "Diagnostic markers for distinguishing nasopharyngeal carcinoma from nasal NK / T-cell lymphoma and their applications" further discloses that the detection of 138059 / 138073bp CpG sites to replace the original 11029bp / 11044bp CpG sites can achieve differential diagnosis of nasal NK / T-cell lymphoma, so as to further improve the diagnostic efficiency of the technical solution for nasopharyngeal carcinoma.

[0006] Although the detection of Epstein-Barr virus DNA methylation based on blind nasopharyngeal swab sampling has shown good results in the diagnosis of nasopharyngeal carcinoma, since the technical solution of blind nasopharyngeal swab sampling is completely dependent on the detection of Epstein-Barr virus, there is theoretically insufficient sampling of tumor cells infected with Epstein-Barr virus. Therefore, there may be a small number of cases that are judged as negative due to weak Epstein-Barr virus methylation detection signals, and there is a possibility of missed detection. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a group of nasopharyngeal carcinoma diagnostic markers and detection reagents thereof.

[0008] The first object of the present invention is to provide a quantitative methylation-specific PCR reagent for detecting differentially methylated regions of genes for use in preparing a diagnostic kit for nasopharyngeal carcinoma

[0009] The second object of the present invention is to provide a quantitative methylation-specific PCR reagent for detecting differentially methylated regions of the gene.

[0010] The third object of the present invention is to provide a kit for diagnosing nasopharyngeal carcinoma.

[0011] The premise for EB virus to infect and enter nasopharyngeal epithelial cells is that some dysplasia or atypical hyperplasia occurs in nasopharyngeal epithelial cells, and there will be some epigenetic changes at the molecular level. After infecting and entering nasopharyngeal epithelial cells, EB virus will further aggravate epigenetic changes. Therefore, host DNA methylation can be further used for auxiliary diagnosis of nasopharyngeal carcinoma.

[0012] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0013] The present invention claims the use of a quantitative methylation-specific PCR reagent for detecting differentially methylated regions of genes in preparing a diagnostic kit for nasopharyngeal carcinoma, wherein the genes are one or more of IMPA2, ITPKB, ITGA4, PI9, AMIGO2, or VAV3;

[0014] The differentially methylated region of the gene IMPA2 is NC_000018.9: 11,980,559 to 11,980,832 bp;

[0015] The differentially methylated region of the gene ITPKB is NC_000001.10: 226,925,108 to 226,925,381 bp;

[0016] The differentially methylated region of the gene ITGA4 is NC_000002.11:182,321,912~

[0017] 182,322,163 bp;

[0018] The differentially methylated region of the gene PI9 is ​​NC_000006.11: 2,903,436 to 2,903,756 bp;

[0019] The differentially methylated region of the gene AMIGO2 is NC_000012.11: 47,473,928 to 47,474,281 bp;

[0020] The differentially methylated region of the gene VAV3 is NC_000001.10:108,507,448~

[0021] 108,507,823bp.

[0022] Preferably, the quantitative methylation-specific PCR reagents for examining the differentially methylated regions of the gene IMPA2 are primers having nucleotide sequences as shown in SEQ ID NOs: 4 and 5, and a probe.

[0023] More preferably, the nucleotide sequence of the probe is as shown in SEQ ID NO:6.

[0024] Preferably, the quantitative methylation-specific PCR reagents for the differentially methylated region of the gene ITPKB are primers with nucleotide sequences as shown in SEQ ID NOs: 7 and 8, and probes.

[0025] More preferably, the nucleotide sequence of the probe is as shown in SEQ ID NO:9.

[0026] Preferably, the quantitative methylation-specific PCR reagents for the differentially methylated region of gene ITGA4 are primers with nucleotide sequences as shown in SEQ ID NOs: 1 and 2, and probes.

[0027] More preferably, the nucleotide sequence of the probe is as shown in SEQ ID NO:3.

[0028] Preferably, the quantitative methylation-specific PCR reagents for the differentially methylated region of the gene PI9 are primers with nucleotide sequences as shown in SEQ ID NOs: 10 and 11, and probes.

[0029] More preferably, the nucleotide sequence of the probe is as shown in SEQ ID NO:12.

[0030] Preferably, the quantitative methylation-specific PCR reagents for the differentially methylated region of the AMIGO2 gene are primers having nucleotide sequences as shown in SEQ ID NOs: 13 and 14, and a probe.

[0031] More preferably, the nucleotide sequence of the probe is as shown in SEQ ID NO:15.

[0032] Preferably, the quantitative methylation-specific PCR reagents for the differentially methylated region of the gene VAV3 are primers having nucleotide sequences as shown in SEQ ID NOs: 16 and 17, and a probe.

[0033] More preferably, the nucleotide sequence of the probe is the probe shown in SEQ ID NO:18.

[0034] Preferably, the gene is one or more of IMPA2, ITPKB or AMIGO2.

[0035] Most preferably, the gene is AMIGO2.

[0036] Preferably, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quencher group.

[0037] More preferably, the fluorescent group is FAM.

[0038] More preferably, the quencher group is BHQ1.

[0039] The present invention also claims a quantitative methylation-specific PCR reagent for detecting the differentially methylated regions of the gene.

[0040] Preferably, the quantitative methylation-specific PCR reagents are primers and probes.

[0041] The present invention also claims a kit for diagnosing nasopharyngeal carcinoma, which contains the quantitative methylation-specific PCR reagent for detecting differentially methylated regions of genes.

[0042] Preferably, it also contains primers with nucleotide sequences as shown in SEQ ID NOs: 16 and 17 for the internal reference, and a probe with a nucleotide sequence as shown in SEQ ID NO: 18.

[0043] Preferably, it also contains quantitative PCR reagents.

[0044] Preferably, a bisulfate conversion agent is also contained.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The present invention utilizes the differentially methylated regions of IMPA2, ITPKB, ITGA4, PI9, AMIGO and VAV3 to establish a quantitative methylation-specific PCR detection method for the diagnosis of nasopharyngeal carcinoma, and the detection rate, sensitivity and specificity are significantly improved compared with the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a scatter plot comparing the methylation levels of CpG sites in the differentially methylated regions of ITGA4, WIF1, and SHISA3 genes in the GSE52068 and GSE62336 databases between groups.

[0048] Figure 2 This is a scatter plot comparing the methylation levels of CpG sites in the differentially methylated regions of AMIGO2, ITPKB, VAV3, PI9, and IMPA2 genes in the GSE52068 and GSE62336 databases between groups.

[0049] Figure 3 Agarose gel electrophoresis results of PCR amplification of ITGA4, WIF1, SHISA3, IMPA2, ITPKB, PI9, AMIGO2, and VAV3 target regions using bisulfite-converted C666 cell DNA.

[0050] Figure 4 This is the Sanger sequencing result of ITGA4 amplification product (partial sequence example).

[0051] Figure 5 This is the Sanger sequencing result of SHISA3 amplification product (partial sequence example).

[0052] Figure 6 This is the Sanger sequencing result of IMPA2 amplification product (partial sequence example).

[0053] Figure 7 This is the Sanger sequencing result of the ITPKB amplification product (partial sequence example).

[0054] Figure 8 This is the Sanger sequencing result of PI9 amplification product (partial sequence example).

[0055] Fig. 9 This is the Sanger sequencing result of AMIGO2 amplification product (partial sequence example).

[0056] Fig.10 This is the Sanger sequencing result of VAV3 amplification product (partial sequence example).

[0057] Fig.11 This is a scatter plot comparing the relative expression levels of ITGA4, WIF1, IMPA2, ITPKB, PI9, AMIGO2, and VAV3 genes detected by qMSP among the groups. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0059] Example 1 Acquisition of differentially methylated regions

[0060] 1. Experimental Methods

[0061] Two NPC methylation-related data sets (GSE52068 and GSE62336) in public databases were used to obtain differentially methylated regions associated with NPC. Both databases are Illumina 450k methylation chip detection data, and the probes on the Illumina 450k methylation chip can detect the methylation levels of 485,577 CpG sites on the human genome.

[0062] Among them, the database GSE52068 contains Illumina 450k methylation chip data of 24 NPC tissue samples and 24 non-NPC tissue samples; GSE62336 contains Illumina 450k methylation chip data of 25 NPC tumor tissue samples and corresponding adjacent control tissue samples.

[0063] 1. Acquisition of differentially methylated regions of 16 common genes

[0064] The methylation data of 16 common genes in the GSE52068 and GSE62336 databases were analyzed to obtain regions with different methylation levels in NPC and non-cancer control samples (i.e., differentially methylated regions).

[0065] Among them, 16 common genes are: RASSF1A (Gene ID: 11186), DAPK1 (Gene ID: 1612), CDKN2A (Gene ID: 1029), CDH1 (Gene ID: 999), RARB (Gene ID: 5915), CDKN2B (Gene ID: 1030), ITGA4 (Gene ID: 3676), ZNF671 (Gene ID: 79891), WIF1 (Gene ID: 11197), ITGA9 (Gene ID: 3680), SHISA3 (Gene ID: 152573), SEPTIN9 (Gene ID: 10801), DACT1 (Gene ID: 51339), NFATC2 (Gene ID: 4773), SFRP5 (Gene ID: 6425), ZMYND10 (Gene ID:51364).

[0066] The differentially methylated region is composed of multiple CpG sites with different methylation levels between the NPC group and the non-cancer control group samples (i.e., differentially methylated CpG sites), among which the differentially methylated CpG sites with higher methylation levels in the NPC group samples than in the non-cancer control group samples are regarded as high methylated CpG sites.

[0067] Therefore, we first obtained the highly methylated CpG sites among the CpG sites successfully detected by methylation chips in 16 common gene sequences based on the Illumina 450k methylation chip data of the two datasets; and then merged the obtained methylated CpG sites to obtain differentially methylated regions.

[0068] Among them, the highly methylated CpG sites were: the statistical test corrected P value of the difference in methylation levels between the NPC group and the control group was <0.05, and the average methylation level in the NPC group was higher than that in the control group;

[0069] Furthermore, based on the annotation information of the Illumina 450k methylation chip, the chromosome and base position of each successfully detected CpG site were extracted and sorted in order of chromosome and base position: if two or more highly methylated CpG sites were located on the same chromosome and had adjacent base positions in the sorting, they were merged into one differentially methylated region.

[0070] 2. Obtaining differentially methylated regions based on non-NPC data in TCGA and GEO and GSE52068 and GSE62336

[0071] The GSE52068 and GSE62336 databases were combined with the methylation data of tonsils (13 samples, derived from databases GSE178219 and GSE50192), oropharynx (4 samples, derived from database GSE98811), hypopharynx (27 samples, derived from databases GSE178218 and TCGA-HNSC) normal tissues and healthy human lymphocytes (148 samples, derived from database GSE40279) samples in the TCGA and GEO public databases as the reference data of normal nasopharyngeal tissues, and the highly methylated CpG sites were obtained according to the following standards; the obtained methylated CpG sites were further merged to obtain differentially methylated regions.

[0072] Among them, the highly methylated CpG sites need to meet the following requirements: (1) In GSE52068, the statistic of the statistical test of the difference in methylation levels between the NPC sample group and the non-cancer control sample group of the CpG site is ≥10; (2) (i) the average methylation level of each sample in the control group of the CpG site in GSE52068 is <0.01; (ii) or the average methylation level of the CpG site in the control group in GSE52068 is ≥0.01 but <0.03, and the average methylation level of the control group in GSE62336 is <0.1; (3) The average methylation level of the CpG site in the tonsil, oropharynx, hypopharynx normal tissue and healthy human lymphocyte samples is <0.11.

[0073] Furthermore, the hypermethylated CpG sites were obtained by the above criteria, and the chromosome and base position data of each hypermethylated CpG site were extracted and sorted in sequence according to the annotation information of the Illumina 450k methylation chip. Among the CpG sites successfully detected in the Illumina 450k methylation chip, two or more hypermethylated CpG sites on the same chromosome and adjacent in base position in the above sorting were merged into one differentially methylated region.

[0074] 2. Experimental Results

[0075] (1) The results showed that among the above 16 genes, only seven genes, namely RASSF1, ITGA4, SEPTIN9, WIF1, SHISA3, ZNF671 and NFATC2, had continuous differentially methylated regions in their sequences ( Figure 1 ).

[0076] Since the samples used in the GSE52068 database were NPC and non-NPC controls, and the samples used in the GSE62336 database were NPC and adjacent controls, the GSE52068 database contained 5 or more CpG sites with inter-group methylation level differences ≥ 0.2 and inter-group difference test-corrected P values ​​< 0.05 in the GSE62336 database as the standard, and it was found that the methylation level differences of the three genes ITGA4, WIF1 and SHISA3 were the most significant.

[0077] Among them, the differentially methylated region of ITGA4 was NC_000002.11: 182,321,912 to 182,322,163 bp;

[0078] The differentially methylated regions of WIF1 are NC_000012.11:65,514,821 to 65,515,670;

[0079] The differentially methylated region of SHISA3 is NC_000004.11:42,399,499~42,400,158.

[0080] (2) In addition, 21 genes were found to have differentially methylated regions, among which AMIGO2, IMPA2, ITPKB, PI9, and VAV3 genes contained highly methylated CpG sites with large differences in methylation levels between groups (Δβ≥0.3). Figure 2 ).

[0081] Among them, the differentially methylated region of AMIGO2 was NC_000012.11: 47,473,928 to 47,474,281 bp;

[0082] The differentially methylated region of IMPA2 is NC_000018.9: 11,980,559 to 11,980,832 bp;

[0083] The differentially methylated region of ITPKB is NC_000001.10: 226,925,108~226,925,381bp;

[0084] The differentially methylated region of PI9 is ​​NC_000006.11: 2,903,436 to 2,903,756 bp;

[0085] The differentially methylated region of VAV3 is NC_000001.10: 108,507,448 to 108,507,823 bp.

[0086] Example 2 Sanger sequencing detection of target gene methylation status in clinical samples

[0087] 1. Experimental Methods

[0088] Using nasopharyngeal brush DNA samples from 8 nasopharyngeal carcinoma cases and 8 non-cancer controls, 8 genes were obtained for Example 1: ITGA4, WIF1, SHISA3, AMIGO2, IMPA2, ITPKB, PI9 and VAV3. The differentially methylated regions within each gene sequence were amplified by PCR, and the amplified products were subjected to Sanger sequencing.

[0089] NPC and non-cancer control samples were collected from the outpatient clinic of the Department of Nasopharyngeal Surgery, Cancer Center, Sun Yat-sen University and grouped according to the results of nasopharyngeal pathology.

[0090] The PCR amplification primer sequences are as follows in Table 1:

[0091] Table 1 PCR amplification primers for differentially methylated regions

[0092]

[0093] The PCR reaction system is as shown in Table 2:

[0094] Table 2 PCR reaction system for differentially methylated regions

[0095]

[0096] PCR reaction conditions:

[0097] Table 3 PCR reaction conditions for differentially methylated regions

[0098]

[0099] 2. Experimental Results

[0100] (1) PCR amplification of differentially methylated regions

[0101] PCR primers were designed for the differentially methylated region sequences, and the DNA samples of C666 cells infected with EB virus were used as templates to detect the primer amplification effect. The results are shown in Figure 3 The differentially methylated region of ITGA4 was too long and was divided into 4 short fragments for PCR. The 1st to 3rd fragments could amplify the target product, but the 4th fragment could not be amplified. The differentially methylated region of WIF1 was divided into 2 short fragments for PCR amplification, but the target product could not be amplified in both fragments. The differentially methylated regions of SHISA3, IMPA2, ITPKB, PI9, AMIGO2, and VAV3 could all be amplified.

[0102] (2) Sanger sequencing results of differentially methylated regions

[0103] For ITGA4 gene ( Figure 4), in the successfully detected samples, most of the CpG sites in the amplified product sequences of NPC samples were methylated, while those in the control samples were all unmethylated.

[0104] For SHISA3 gene ( Figure 5 ), in the successfully detected samples, the sequences of the amplified products of NPC and control samples were almost all unmethylated TpG, and there was no significant difference between the groups.

[0105] For IMPA2 gene ( Figure 6 ), in the successfully detected samples, most of the CpG sites in the amplified product sequences of NPC samples were methylated, while those in the control samples were all unmethylated.

[0106] For ITPKB gene ( Figure 7 ), in the successfully detected samples, most of the CpG sites in the amplified product sequences of NPC samples were methylated, while those in the control samples were all unmethylated.

[0107] For PI9 gene ( Figure 8 ), the control sample amplified with methylated primers had weak bands and failed Sanger sequencing. All samples that were successfully sequenced were NPC samples, and it was suggested that all the CpG sites in the amplified product region were methylated.

[0108] For AMIGO2 gene ( Fig. 9 ), in this region, the control sample amplified with methylated primers had weak bands and Sanger sequencing failed. All the samples that were successfully sequenced were NPC samples, and it was suggested that the amplified product region was dominated by methylated CpG sites.

[0109] For VAV3 gene ( Fig.10 ), in this region, the control sample amplified with methylated primers had weak bands and Sanger sequencing failed. All the samples that were successfully sequenced were NPC samples, and it was suggested that all the CpG sites in the amplified product region were methylated.

[0110] The above results suggest that there are differences in methylation levels of six genes, ITGA4, IMPA2, ITPKB, PI9, AMIGO2 and VAV3, between NPC and non-cancer control samples, while there is no obvious difference in the SHISA3 gene.

[0111] Example 3 Quantitative methylation-specific PCR (qMSP) detection of methylation status in differentially methylated regions of clinical samples

[0112] 1. Experimental Methods

[0113] 139 nasopharyngeal swab clinical samples were collected from the outpatient clinic of the Department of Nasopharyngeal Surgery, Cancer Center of Sun Yat-sen University. They were grouped according to the results of nasopharyngeal pathology sampling of patients, and were derived from 80 nasopharyngeal carcinoma patients and 59 non-cancer control patients. DNA in the samples was extracted and converted with bisulfite. The differentially methylated regions of the 6 genes ITGA4, IMPA2, ITPKB, PI9, AMIGO2 and VAV3 in Example 2 were relatively quantitatively detected by real-time fluorescence quantitative methylation specific PCR (qMSP), and the internal reference gene ACTB was detected at the same time. The primer and probe sequences used are shown in Table 4 below.

[0114] Table 4qMSP primer probe sequences

[0115]

[0116]

[0117]

[0118] The qMSP reaction system is shown in Table 5:

[0119] Table 5qMSP reaction system

[0120]

[0121] The qMSP reaction conditions are shown in Table 6:

[0122] Table 6qMSP reaction conditions

[0123]

[0124] 2. Experimental Results

[0125] The qMSP test results of nasopharyngeal swab DNA samples are shown in Table 7.

[0126] Table 7 Ct values ​​of qMSP detection of DNA from 139 nasopharyngeal swab samples

[0127]

[0128]

[0129]

[0130]

[0131]

[0132] After filling the undetected Ct values ​​with 45, the 2^[-(Ct 目标 -Ct 内参 )] formula to calculate the relative expression of each gene in each sample. The intergroup comparison of each gene among samples is as follows Fig.11 shown.

[0133] According to the internal reference ACTB amplification Ct value, samples with low sample quality (Ct ≥ 33) were deleted, and 138 samples (80 NPC and 58 non-cancer controls) remained. Each gene was used to predict the samples. If the gene can be detected in the sample, it is judged as positive, otherwise it is negative. The detection sensitivity and specificity of each gene are shown in Table 8 below:

[0134] Table 8 Single indicator evaluation results

[0135]

[0136]

[0137] The methylation products of the six genes were almost not detected in the control samples, but could be detected in most case samples. Using single gene detection, IMPA2 (sensitivity = 0.7000, specificity = 0.9828), AMIGO2 (sensitivity = 0.7500, specificity = 0.9310), and ITPKB (sensitivity = 0.6875, specificity = 0.9483) were relatively better.

[0138] Example 4 A kit for diagnosing nasopharyngeal carcinoma

[0139] 1. Composition

[0140] The nucleotide sequences used for quantitative methylation-specific PCR are shown in SEQ ID NOs: 1 to 18, and the primer-probe combination is specifically:

[0141] Primers for detecting the differentially methylated region of gene ITGA4 with nucleotide sequences as shown in SEQ ID NOs: 1 and 2, and a probe with a nucleotide sequence as shown in SEQ ID NO: 3;

[0142] Primers for detecting the differentially methylated region of the gene IMPA2 with nucleotide sequences as shown in SEQ ID NOs: 4 and 5, and a probe with a nucleotide sequence as shown in SEQ ID NO: 6;

[0143] The nucleotide sequences of the primers for detecting the differentially methylated region of the gene ITPKB are as shown in SEQ ID NOs: 7 and 8, and the nucleotide sequence of the probe is as shown in SEQ ID NO: 9;

[0144] The nucleotide sequences of the primers for detecting the differentially methylated region of the gene PI9 are shown in SEQ ID NOs: 10 and 11, and the nucleotide sequence of the probe is shown in SEQ ID NO: 12.

[0145] Primers for detecting the differentially methylated region of the gene AMIGO2 with nucleotide sequences as shown in SEQ ID NOs: 13 and 14, and a probe with a nucleotide sequence as shown in SEQ ID NO: 15;

[0146] Primers for detecting the differentially methylated region of the gene VAV3 with nucleotide sequences as shown in SEQ ID NOs: 16 and 17, and a probe with a nucleotide sequence as shown in SEQ ID NO: 18;

[0147] Primers with nucleotide sequences as shown in SEQ ID NOs: 16 and 17 for the internal reference ACTB, and a probe with a nucleotide sequence as shown in SEQ ID NO: 18;

[0148] The 5' end of the above probes is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ1.

[0149] Also contains Luna Universal Probe qPCR Mix and bisulfite conversion reagent.

[0150] 2. Usage

[0151] The qMSP reaction system is shown in Table 5, and the qMSP reaction conditions are shown in Table 6.

[0152] 3. Result judgment

[0153] First, the sample quality was judged by the Ct value of the internal reference gene ACTB amplification, and the sample quality was considered qualified when the ACTB amplification Ct value was < 33. The detection was considered positive when the Ct value obtained by the qMSP experiment of any one or more of the ITGA4, IMPA2, ITPKB, PI9, AMIGO2, and VAV3 genes was greater than 33, otherwise it was considered negative.

[0154] Example 5 Quantitative Methylation-Specific PCR (qMSP) Detection of Clinical Nasopharyngeal Carcinoma Missed Samples

[0155] 1. Experimental samples: DNA from 4 nasopharyngeal swab samples of nasopharyngeal carcinoma that were missed. The CpG sites at 138059 and 138073 bp of the Epstein-Barr virus (NC_007605.1) were detected for the diagnosis of nasopharyngeal carcinoma. The missed samples were judged to be negative due to weak detection signals. For specific detection methods, see: 202310539250.1 "Diagnostic markers for distinguishing nasopharyngeal carcinoma from nasopharyngeal NK / T-cell lymphoma and their applications".

[0156] 2. Experimental Methods

[0157] The methylation of IMPA2, ITPKB, ITGA4, PI9, AMIGO2 and VAV3 of the host was detected in the sample, and the specific inspection method was the same as that in Example 4.

[0158] 3. Experimental Results

[0159] The results are shown in Table 9. According to the detection statistics, at least one gene in these four samples tested positive, which is a significant improvement over the existing technology. Among them, the detection effect of IMPA2 index is the best, AMIGO2, ITPKB, and ITGA4 each detected one case, and PI9 and VAV3 were not detected, which is similar to the effect in Table 8.

[0160] Table 9 Ct values ​​of qMSP detection of missed samples of clinical nasopharyngeal carcinoma

[0161]

Claims

1. Use of a quantitative methylation-specific PCR reagent for detecting differentially methylated regions of genes in the preparation of a diagnostic kit for nasopharyngeal carcinoma, characterized in that: The gene is one or more of IMPA2, ITPKB or AMIGO2. The differentially methylated region of the gene IMPA2 is NC_000018.9: 11,980,559 to 11,980,832 bp; The differentially methylated region of the gene ITPKB is NC_000001.10: 226,925,108 to 226,925,381 bp; The differentially methylated region of the gene AMIGO2 is NC_000012.11: 47,473,928 to 47,474,281 bp; The PCR reagents are primers and probes for PCR.

2. The use according to claim 1, characterized in that: The quantitative methylation-specific PCR reagents for examining the differentially methylated regions of the gene IMPA2 are primers with nucleotide sequences as shown in SEQ ID NOs: 4 and 5, and a probe.

3. The use according to claim 1, characterized in that: The quantitative methylation-specific PCR reagents for the differentially methylated region of the gene ITPKB are primers with nucleotide sequences as shown in SEQ ID NOs: 7 and 8, and probes.

4. The use according to claim 1, characterized in that: The quantitative methylation-specific PCR reagents for the differentially methylated region of the gene AMIGO2 are primers with nucleotide sequences as shown in SEQ ID NOs: 13 and 14, and a probe.

5. A kit for diagnosing nasopharyngeal carcinoma, characterized in that: A quantitative methylation-specific PCR reagent for detecting differentially methylated regions of genes as described in claim 1.

6. The kit according to claim 5, characterized in that It also contains primers for the nucleotide sequences of the internal reference as shown in SEQ ID NO: 16 and 17, and a probe.

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