An analysis detection kit for a high-risk epstein-barr virus strain of nasopharyngeal carcinoma and a method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a nasopharyngeal carcinoma high-risk EB virus strain analysis detection kit and a method and application thereof. The application relates to the technical field of EBV detection. The application reports three unreported nasopharyngeal carcinoma high-risk EB virus SNPs A7001T / A7012G / A7016T, and highly links to the previously reported T6999G highly linked target EBV EBER2. According to the above finding, the application develops a nasopharyngeal carcinoma high-risk EB virus strain rapid typing detection technology, which comprises the following components: a universal primer, a fluorescent probe, NTC, a wild-type quality control product and a mutant quality control product. The technology provided by the application can realize quantitative and typing detection of the nasopharyngeal carcinoma high-risk EB virus strain, and the lower limit of detection is 156 copies / mL of plasma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an analytical detection kit, method and application of a high-risk EB virus strain for nasopharyngeal carcinoma. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is a highly aggressive malignant tumor of the head and neck that occurs in the epithelial mucosa of the nasopharynx. In most countries and regions, the world age-standardized incidence (ASIR) of NPC is usually less than 1 per 100,000 person-years.

[0003] Epstein-Barr virus (EBV) infects most of the world's population in a chronic form, often without clinical symptoms. However, in a minority of individuals, EBV infection is closely associated with the development of a variety of diseases, including infectious mononucleosis and various cancers. Although EBV can infect populations in all regions, the incidence of disease varies considerably. Studies have shown that the occurrence of different diseases or tumors reflects the existence of multiple viral subtypes with different characteristics and prevalence only in limited areas. Sequencing of EBV genes isolated from nasopharyngeal carcinoma (NPC) patients revealed significant differences from the widely prevalent strain (NC_007605.1) in the West. Previous studies have found that EBV typing based on plasma DNA, using the whole EBV genome SNV (single nucleotide variant) profile, can be used for NPC risk scoring. Studies have reported that the BALF2 ​​(I613V, V317M), EBNA1 (V-Val), and BZLF1 promoter (Zp-V3) are associated with NPC risk.

[0004] In the early diagnosis of nasopharyngeal carcinoma (NPC) in endemic areas, serological markers such as Epstein-Barr virus (EBV) viral capsid antigen (VCA) antibody (VCA-IgA) and early antigen (EA) antibody (EA-IgA) are widely used clinically. However, they cannot reflect the tumor status in vivo in a timely manner. In comparison, real-time quantitative polymerase chain reaction (qPCR) detection of cell-free EBV DNA in plasma is considered a more valuable tumor-associated biomarker, with positive significance in NPC diagnosis, treatment efficacy evaluation, and long-term survival prognosis. Therefore, it is of great significance to quantify and genotype EBV nucleic acid in NPC by targeting EBV EBER2 using real-time quantitative PCR technology. Summary of the Invention

[0005] The first objective of this invention is to provide the application of reagents for detecting SNP sites in the preparation of nasopharyngeal carcinoma high-risk subtype classification products.

[0006] A second aspect of the present invention is to provide a primer-probe set.

[0007] A third aspect of the present invention is to provide a detection kit.

[0008] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the invention provides the use of reagents for detecting SNP sites in the preparation of nasopharyngeal carcinoma high-risk subtype classification products; The SNP sites include: 1) The polymorphism of the SNP site at position 7001 from the 5' end to the 3' end of the EBER2 gene is A / T; 2) The polymorphism of the SNP site at position 7012 from the 5' end to the 3' end of the EBER2 gene is A / G; 3) The polymorphism of the SNP site at position 7016 from the 5' end to the 3' end of the EBER2 gene is A / T.

[0009] In some embodiments of the present invention, the sequence of the EBER2 gene is shown in SEQ ID NO: 1.

[0010] In some embodiments of the present invention, when the 7001st position from the 5' end to the 3' end of the EBER2 gene is T (corresponding to the 10th position in SEQ ID NO: 1), the nasopharyngeal carcinoma is a high-risk subtype.

[0011] In some embodiments of the present invention, when the 7012th position from the 5' end to the 3' end of the EBER2 gene is G (corresponding to the 21st position in SEQ ID NO: 1), the nasopharyngeal carcinoma is a high-risk subtype.

[0012] In some embodiments of the present invention, when the 7016th position from the 5' end to the 3' end of the EBER2 gene is T (corresponding to position 25 in SEQ ID NO: 1), the nasopharyngeal carcinoma is a high-risk subtype.

[0013] In some embodiments of the present invention, the reference nucleotide sequence (6992-7016) of the EBER2 gene is shown in SEQ ID NO: 1: CCAACGCTC A GTGCGGTGCT A CCG A In some embodiments of the present invention, the reagent comprises reagents for use in one or more detection techniques or methods selected from the group consisting of: PCR, gene chip method, and nucleic acid sequencing method.

[0014] A second aspect of the present invention provides a primer-probe set, comprising genotyping primers and fluorescent probes for detecting SNP sites.

[0015] In some embodiments of the present invention, the typing primers include EBER2-WT primers and EBER2-Var primers, with sequences shown in SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0016] In some embodiments of the present invention, the fluorescent probe includes an EBER2-WT probe and an EBER2-Var probe, the sequences of which are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0017] In some embodiments of the present invention, the fluorescent probe includes a fluorescent group and a quenching group.

[0018] In some embodiments of the present invention, the fluorescent probe includes VIC, FAM, and Q670.

[0019] In some embodiments of the present invention, the quenching groups include BHQ1 and BHQ2.

[0020] In some embodiments of the present invention, the fluorescent probe includes a TaqMan fluorescent probe, wherein a fluorescent group is carried at the 5' end and a quenching group is carried at the 3' end.

[0021] A third aspect of the present invention provides a detection kit comprising the reagents for detecting SNP sites according to the first aspect of the present invention.

[0022] In some embodiments of the present invention, the reagent for detecting SNP sites includes the primer-probe set described in the second aspect of the present invention.

[0023] In some embodiments of the present invention, the detection kit further includes an enzyme, a reaction buffer, a negative control, and a positive control.

[0024] In some embodiments of the present invention, the enzyme includes a DNA polymerase with high specificity and 5'→3' exonuclease activity, specifically a hot-start Taq DNA polymerase.

[0025] A fourth aspect of the present invention provides the application of the primer and probe set of the second aspect of the present invention in the preparation of a product for rapid detection of high-risk EB virus subtypes in nasopharyngeal carcinoma.

[0026] A fifth aspect of the present invention provides a rapid real-time fluorescence quantitative detection method for high-risk Epstein-Barr virus subtypes in nasopharyngeal carcinoma. This method utilizes the primer-probe set or the reagent kit described above, and includes the following steps: Step 1: Extract EB virus DNA from plasma from NPC patients; Step 2: Using the DNA extracted in Step 1 as a template, prepare the corresponding qPCR reaction system using the above kit, and perform qPCR amplification in a real-time PCR instrument capable of collecting data from the FAM and Q670 channels to obtain the amplification curves and cycle thresholds (Cq values) of EB virus DNA in the sample in the FAM and Q670 fluorescence channels. Step 3: Obtain amplification curves for different fluorescence channels using the qPCR reaction from Step 2, and analyze the subtypes of EB virus in the sample; Step 4: Fit the relationship between the copy number and Cq value obtained from the wild-type and mutant quality control samples provided in the kit to obtain a standard curve. Substitute the Cq value obtained from the qPCR reaction in Step 2 into the standard curve to calculate the EB virus DNA copy number in the sample. In some embodiments of the present invention, the standard curves are obtained by diluting inactivated EB virus DNA containing wild-type and mutant EBER2 target sequences with known copy numbers, and collecting quantified standard DNA copy numbers, amplification curves of different fluorescence channels, and Cq values ​​through steps 1-4. The amplification curves of different fluorescence channels are classified according to the collected data, and linear fitting is performed based on the collected Cq values ​​to obtain two standard curves with Cq values ​​on the ordinate and EB virus DNA copy number on the abscissa. The FAM channel amplification curve represents the wild type, and the Q670 channel amplification curve represents the mutant type.

[0027] In some embodiments of the present invention, the sample is plasma or saliva.

[0028] In a sixth aspect, the present invention provides the application of the method described above in improving the positive predictive rate of early screening for nasopharyngeal carcinoma.

[0029] The beneficial effects of this invention are: This invention provides a rapid detection kit for specifically differentiating high-risk Epstein-Barr virus (EBV) subtypes in nasopharyngeal carcinoma (NPC). Based on real-time quantitative PCR (qPCR), it utilizes an EBER2-Var probe targeting the EBER2 sequence of high-risk EBV subtypes in NPC and an EBER2-WT probe targeting the wild-type EBER2 sequence to achieve rapid typing and quantitative detection of high-risk EBV subtypes in NPC. This method possesses the sensitivity and stability of PCR detection. Furthermore, it does not require in vitro culture, has low laboratory safety requirements, high safety, is simple to operate, and requires minimal labor, making it more suitable for clinical use. This invention, by screening for EBER2-Var sequences specifically present in NPC, serves as a potential tumor biomarker for early screening, early diagnosis, optimal treatment decision-making, prognosis prediction, and recurrence prediction in NPC, possessing extremely important clinical significance. Attached Figure Description

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The Manhattanplot results are based on 2919 SNPs from the EBV genomes of 62 NPC patients and 142 healthy individuals. The red line represents the cutoff value. P =5×10 -8 .

[0031] Figure 2 For linkage disequilibrium analysis of 23 SNP loci in EBV genome EBERs from 62 NPC patients and 142 healthy individuals, * represents the loci used in this invention.

[0032] Figure 3 The results of EBV-EBER2 PCR amplification in different cell models.

[0033] Figure 4 Comparison of EBV-EBER2 Sanger sequencing results in different cell models.

[0034] Figure 5 Amplification curve for quantifying EBV DNA copy number in the C666-1 cell model.

[0035] Figure 6 Amplification curves for sensitivity detection of probes EBER2-WT and EBER2-Var.

[0036] Figure 7 Standard curves fitted to the sensitivity detection of probes EBER2-WT and EBER2-Var.

[0037] Figure 8Amplification curves for specific detection of probes EBER2-WT and EBER2-Var.

[0038] Figure 9 Linear range plots for probes EBER2-WT (A) and EBER2-Var (B).

[0039] Figure 10 Standard curves fitted to probes EBER2-WT and EBER2-Var standards.

[0040] Figure 11 A curve showing the amplification of EB virus DNA in plasma from NPC-derived cells.

[0041] Figure 12 This study compares the detection performance of probes EBER2-WT and EBER2-Var with that of a commercial EB virus nucleic acid quantitative detection kit. Detailed Implementation

[0042] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0043] Example 1: Discovery of key SNP sites in high-risk EBV subtypes of nasopharyngeal carcinoma To screen for SNPs in high-risk EBV genomes from endemic nasopharyngeal carcinoma (NPC) regions, this study included 493 publicly available EBV genome sequences derived from plasma, tissue, and saliva samples from 257 NPC patients and 236 healthy individuals. All EBV genome sequence information was obtained from the NCBI database (GenBank IDs MH590370-MH590579, RDD ID RDDA2021659371 (www.researchdata.org.cn) and Bioproject ID PRJNA522388). Using NC_007605.1 as the reference genome, this study calculated 11,127 SNPs. Most SNPs were found in NPC-derived EBV and healthy NPC-derived EBV, primarily distributed in latent genes EBNA2, EBNA3, and LMP1, and cleavage genes BZLF1, BRFF2, BDLF3, and BKRF2. Latent genes showed a higher prevalence of nonsynonymous mutations compared to cleavage genes.

[0044] To investigate EBV SNPs associated with NPC risk, this embodiment performed a meta-analysis of the previously disclosed EBV genomes to identify EBV risk genes in nasopharyngeal carcinoma in high-prevalence areas. A total of 3025 SNPs associated with NPC risk were identified in this embodiment, and a cutoff value was set after Bonferroni correction. P= 1.56×10 -5 A total of 55 SNPs were screened, including the risk gene EBER2 SNPs (T6999G / A7001T / A7012G / A7016T) Figure 1 The reference nucleotide sequence (6992-7016) of the EBER2 gene is shown in SEQ ID NO: 1: CCAACGCTC A GTGCGGTGCT A CCG A .

[0045] To investigate the distribution characteristics of EBV SNPs associated with NPC risk, this study used sequence information from 1592 samples from NPCs and healthy individuals published by Hui KF and Xu (Int J Cancer. 2019 Jun 15). After Bonferroni correction for sex, age, and population structure, the prevalence frequencies of risk genes EBER2 SNPs T6999G, A7001T, A7012G, A7016T and BALF2 ​​SNPs A162215C, T162476C, C163364T in NPCs and healthy individuals were analyzed. The results showed that the prevalence of high-risk EBER2 haplotypes (T6999G / A7001T / A7012G / A7016T) was 91.44% in NPC-derived patient samples and 45.34% in healthy human samples; the prevalence of high-risk BALF2 ​​haplotypes (A162215C, T162476C, C163364T and A162215C, T162476C, C163364) was 85.02% in NPC-derived patient samples and 51.76% in healthy human samples (Table 1). This embodiment further performed linkage disequilibrium analysis on EBER2 SNPs T6999G, A7001T, A7012G, and A7016T to investigate whether the presence of these four SNPs indicates high linkage. The results showed that EBER2 SNPs T6999G, A7001T, A7012G, and A7016 exhibited a high degree of linkage imbalance (R). 2 >0.95), indicating that the occurrence of these 4 SNPs is highly correlated. Figure 2 ).

[0046]

[0047] To further verify the correlation between EBER2 polymorphism and high-incidence areas of nasopharyngeal carcinoma, this embodiment designed primers specifically to amplify the EBER2 target sequence and performed a BLAST search to ensure cross-reactivity.

[0048] Subjects for testing: subclonal cell lines C666-1 and NPC43 of nasopharyngeal carcinoma xenografts from southern China; other cell lines NOK(EBV-), NOK(EBV+), AGS(EBV-), AGS(EBV+), SNU719, N5(EBV+), NPC53, HONE1, HK1(EBV-), CNE2, Akata(EBV+), CIR, Raji, P3HR1, YT, NKYS, SNK1, and SNK6.

[0049] Genomic DNA was extracted from the above cell lines to amplify the EB virus EBER2 sequence for Sanger sequencing.

[0050] Table 2 Primer Sequences

[0051] The PCR system used was the commercially available 2 × Taq PCR StarMix (GenStar, A012-01), with a total volume of 50 μL. The preparation method is as follows: Table 3

[0052] Amplification was performed using the T100™ PCR instrument (Bio-rad). The procedure is as follows: Table 4

[0053] PCR products were subjected to 1% agarose gel electrophoresis at 160V for 20 min. The imaging results of the ChemiDoc™ Imaging System (Bio-rad) are as follows: Figure 3As shown in the image, the PCR products of samples exhibiting distinct bands were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for Sanger sequencing. Multiple sequence alignment analysis of the sequencing results showed that the EBER2 sequences of EBV in NOK(EBV+), AGS(EBV+), SNU719, N5, HONE1, CNE2, Akata(EBV+), CIR, Raji, P3HR1, YT, NKYS, SNK1, and SNK6 cell models were identical to the wild-type EBER2 sequence (EBER2-WT, GenBank: NC007605), exhibiting a wild-type pattern of T6999 / A7001 / A7012 / A7016 (14 / 14, 100%). The EBER2 sequences of EBV in C666-1 and NPC43 cell models were identical to the mutant EBER2 sequence (EBER2-Var, GenBank: KF373730), exhibiting a mutant pattern of T6999G / A7001T / A7012G / A7016T (2 / 2, 100%). Figure 4 The results indicate that, in cell models, EBER2 (SNP GTGT) achieved 100% effectiveness in subtyping nasopharyngeal carcinoma in endemic areas.

[0054] Example 2: Design and Detection Performance Verification of High-Risk Viral Subtype Typing Probes for Nasopharyngeal Carcinoma 1. DNA extraction In this embodiment of the invention, the nucleic acid extraction method used for cell samples was from a commercially available kit (FastPureBlood / Cell / Tissue DNA Isolation Mini Kit (Vazyme, DC112-01)). This kit uses magnetic beads and a buffer system to isolate and purify high-quality viral nucleic acids from cells / tissues. The nucleic acid extraction method used for plasma samples was from a commercially available kit (Magnetic Blood DNA Extraction Kit (Vazyme, DM102-01)) used in conjunction with a fully automated nucleic acid extractor. During the nucleic acid extraction process, the kit's procedures were followed, and a 1×10⁻⁶ tbsp solution was used in the experiment. 6 Cells or 1 mL of plasma are used to extract DNA for better nucleic acid enrichment. Additionally, an appropriate volume of nuclease-free water needs to be selected for the final nucleic acid elution process.

[0055] 2. Detection Method Genomic DNA was purified from cell cultures using the nucleic acid extraction method described above, and then used for qPCR quantitative detection of high-risk EBV subtypes as described below. A known copy number of EBV standard dilution buffer (containing EBV genomic DNA from cells) was added to a separate well of the reaction. The copy number of EBV DNA in the C666-1 cell standard was quantified, cycle threshold (Cq value) data were collected, and a standard curve was created.

[0056] According to Example 1, this invention designs primer sets capable of simultaneously targeting different EB virus subtypes EBER2 and probe sets specifically distinguishing different EB virus subtypes EBER2, and performs BLAST search to ensure cross-reactivity. The primer and probe sequences are as follows: Table 5

[0057] The PCR system used was based on the commercially available TIANGEN (FP206) 2× SuperReal qPCR PreMix (Probe), with a total volume of 20 μL, prepared as follows: Table 6

[0058] Quantitative detection was performed using the CFX Maestro Software for CFX Real-Time PCR Instruments 96-well real-time PCR detection system (Bio-rad). The procedure is as follows: Table 7

[0059] EBER2-WT / EBER2-Var probe performance testing 3. Establishment of standard products In this embodiment of the invention, Raji cells (hereinafter referred to as Raji), a human Burkitt's lymphoma cell model latently infected with Epstein-Barr virus (EBV), were used as wild-type standards, and C666-1 (hereinafter referred to as C666-1), a nasopharyngeal carcinoma xenograft cell model derived from southern China latently infected with EBV, were used as mutant standards. Raji cells, as determined by FISH, showed an EBV load of approximately 50 copies / cell. Using Raji cells as standards, quantitative PCR detection of EBV copy number in C666-1 cells was performed using the BamHI-W probe. The amplification curves are shown below. Figure 5 As shown, the EBV DNA copy number in C666-1 cells was 21 copies / cell. DNA extraction and detection methods were as described above.

[0060] 4. Probe sensitivity detection 1) Raji cell standards and C666-1 cell standards were serially diluted according to a gradient concentration (1×10⁻⁶). 9 copies / mL, 1×10 8 copies / mL, 1×10 7 copies / mL, 1×10 6 copies / mL, 1×10 5 copies / mL, 1×10 4 copies / mL, 1×10 3 copies / mL, 1×10 2 copies / mL, 1×10 1 copies / mL, 1×10 0 (Copies / mL), add 2 μL of template to each well, and set up nuclease-free water as a negative control. Each concentration is repeated 3 times.

[0061] Amplification curves as follows Figure 6 As shown, the fitted standard curve is as follows: Figure 7 As shown.

[0062] Table 8 Linear detection range of probe EBER2-WT / Var

[0063] Std - standard; SD - standard deviation; CV - coefficient ofvariation 2) Raji cell standards and C666-1 cell standards were serially diluted according to a gradient concentration (100 copies / well, 50 copies / well, 25 copies / well, 12.5 copies / well, 6.25 copies / well, 3.13 copies / well, 1.56 copies / well, 0.78 copies / well). Nuclease-free water was set up as a negative control. Each concentration was repeated 10 times.

[0064] The above-described PCR system and PCR quantitative detection procedure were used.

[0065] After PCR, adjust the amplification curves for those with baseline anomalies and adjust the thresholds. For example, to detect the EBER2-Var mutant genotype, the threshold line needs to be set above the wild-type standard. When detecting the mutant genotype (EBER2-Var), the threshold is set above the wild-type standard. The mutant genotype standard (C666-1) has a Cq value and an amplification curve, while sterile enzyme-free water and the wild-type standard have no Cq value (negative). If a Cq value appears, the value is determined as negative. When detecting the wild-type genotype (EBER2-WT), the threshold is set above the mutant genotype standard. The wild-type genotype (Raji) has a Cq value and an amplification curve, while sterile enzyme-free water and the mutant standard have no Cq value (negative) and no amplification curve.

[0066] Based on the test results, the detection limit for EBER2 was determined. A sample was considered positive if the Cq value was <40 and a clear amplification curve was observed. A sample was included in the detection range if 9 or more out of 10 repeated tests yielded a positive result. The lowest detection range was recorded as the detection limit. The statistics for the detection limits are shown in Table 9.

[0067] Table 9. Detection Limit Analysis of EBER2-WT / Var

[0068] All statistical analyses were performed using Microsoft Excel and Graphpad Prism 6.0 (Graphpad Software, Inc., San Diego, CA).

[0069] 5. Probe specificity detection Test specimens: To test the detection specificity of the rapid detection technology for high-risk subtypes of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma, genomic DNA from two EBV-positive nasopharyngeal carcinoma cell lines, C666-1 and NPC43, derived from NPC patients in southern China, and 13 other cell lines, including AGS (EBV+), SNU719, N5, HONE1, CNE2, Akata (EBV+), CIR, Raji, P3HR1, YT, NKYS, SNK1, and SNK6, was used as the detection targets. The DNA extraction method described in Experiment 1 above was employed.

[0070] The above-described PCR system and PCR quantitative detection procedure were used.

[0071] After PCR, adjustments are made to the amplification curves with baseline anomalies and to adjust the thresholds. For example, when detecting the EBER2-Var mutant genotype, the threshold line needs to be set above the wild-type standard. When detecting the mutant genotype (EBER2-Var), the threshold is set above the wild-type standard. The mutant genotype standard (C666-1) has a Cq value and an amplification curve, while sterile enzyme-free water and the wild-type standard have no Cq value (negative). If a Cq value appears, it is considered negative. When detecting the wild-type genotype (EBER2-WT), the threshold is set above the mutant genotype standard. The wild-type genotype (Raji) has a Cq value and an amplification curve, while sterile enzyme-free water and the mutant standard have no Cq value (negative) and no amplification curve. The amplification curve is as follows. Figure 8 As shown.

[0072] Real-time quantitative PCR results: EBER2 of EBV in AGS (EBV+), SNU719, N5, HONE1, CNE2, Akata (EBV+), CIR, Raji, P3HR1, YT, NKYS, SNK1, and SNK6 cell models was specifically recognized by the EBER2-WT probe. EBER2 of EBV in C666-1 and NPC43 cell models was specifically recognized by the EBER2-Var probe. There was no cross-recognition between the EBER2-WT and EBER2-Var probes. When the template was the EBV-negative cell model Akata (EBV-), no obvious amplification curve was observed in qPCR amplification.

[0073] 6. Plotting the probe standard curve Using the test results (Table 1), a standard curve equation was fitted, with EB virus DNA copy number as the x-axis and Cq value as the y-axis. The fitted standard curve is shown below. Figure 9 As shown.

[0074] Example 3: Validation of the Nasopharyngeal Carcinoma High-Risk Subtype Classification Detection Kit 1. Nasopharyngeal carcinoma high-risk subtype typing probe detection cell line model Referring to the probe specificity detection method in Example 2, copy number quantification and typing of EBV in two EBV-positive nasopharyngeal carcinoma cell lines C666-1 and NPC43 derived from NPC patients in southern China, and 13 other cell lines AGS (EBV+), SNU719, N5, HONE1, CNE2, Akata (EBV+), CIR, Raji, P3HR1, YT, NKYS, SNK1, and SNK6 were performed.

[0075] Each cell contains 2 copies of the β-globin gene. Genomic DNA was extracted from the cells and converted using a conversion factor of 6.6 pg / cell. The calculated β-globin gene copy number in 100 ng of cell DNA was 3 × 10⁻⁶. 4 The number of EBV DNA copies per cell was estimated by analyzing the relationship between the β-globin gene and cell number. Standard curves for EBER2-WT and EBER2-Var standards are shown below. Figure 10 As shown.

[0076] Table 10

[0077] 2. Validation of NPC-derived plasma samples for high-risk subtype typing probes in nasopharyngeal carcinoma. To validate the detection efficacy of the newly developed rapid detection technology for high-risk subtypes of nasopharyngeal carcinoma (NPC), plasma samples from 50 NPC cases (collected from June 2020 to February 2022) and 50 cases of extranodal nasal NKT lymphoma (collected from June 2022 to April 2024) were used as the detection subjects. Information is shown in Table 11. The sample collection for this invention was approved by the Ethics Committee of Sun Yat-sen University Cancer Center (Approval Letter No.: B2026-265-01), complying with the principles of the ICH-GCP review opinion and relevant regulations / guidelines.

[0078] All samples were clinically positive for EBV DNA via qPCR targeting the BamHI-W region. In this experiment, 200 μL of plasma sample was used for subsequent DNA extraction. Elution was performed using 50 μL of TE buffer, with 2 μL of the elution buffer used as a template for subsequent qPCR reactions. Water was used as a negative control, and amplification curves and thresholds were adjusted. (Positive criteria: Cq value > 40 was considered negative; Cq value < 40 with a clear amplification curve was considered positive). The Vazyme Magnetic Blood DNA Extraction Kit (Vazyme, DM102-01) was used with an automated nucleic acid extractor to extract cell-free EBV DNA from 200 μL of plasma. The DNA was ultimately dissolved in 50 μL of TE buffer.

[0079] The extracted DNA was amplified by qPCR, and the free EB virus in the plasma was quantitatively detected and genotyped.

[0080] Amplification curves of NPC and NKT-derived plasma samples are shown below. Figure 11 As shown.

[0081]

[0082] The results are shown in Tables 12 and 13. The EBV DNA detection rate of plasma samples from NPC sources was 96%, with the EBER2-Var detection rate at 100%. For plasma samples from ENKTL sources, the EBV detection rate was 94%, with EBER2-WT accounting for 93.62% and mixed infection accounting for 6.38%. The reason for the undetectable copy number in some samples may be due to excessively low EBV viral load.

[0083]

[0084] Table 13

[0085] Table 14. qPCR test results of clinical samples

[0086] 3. Performance Comparison Results of Nasopharyngeal Carcinoma High-Risk Subtype Typing Kit and Commercial EB Virus Nucleic Acid Quantitative Detection Kit To test the detection performance of the newly developed nucleic acid detection kit compared to commercially available EB virus nucleic acid quantitative detection kits, we used 200 μL of 8 NPC-derived plasma samples stored in the laboratory for subsequent DNA extraction. All samples were eluted with 50 μL of TE buffer, and 2 μL of the elution buffer was used as a template for subsequent qPCR reactions. Water was used as a negative control, and the amplification curve and threshold were adjusted. The extraction of DNA was amplified by qPCR as described in (2) above, and the free EB virus in the plasma was quantitatively detected and genotyped.

[0087] The results showed that, when the same four samples were tested, the copy number quantification results of kit 1 were 3.01 log10 copies / mL, 3.73 log10 copies / mL, 2.53 log10 copies / mL, and 3.01 log10 copies / mL, respectively; the copy number quantification results of kit 2 were 3.43 log10 copies / mL, 4.07 log10 copies / mL, 3.28 log10 copies / mL, and 3.34 log10 copies / mL, respectively; and the copy number quantification results of probe EBER2-Var were 2.67 log10 copies / mL, 3.33 log10 copies / mL, 2.72 log10 copies / mL, and 2.48 log10 copies / mL, respectively. Figure 12 The EBER2-WT / EBER2-Var probe detection kit has the advantage of being able to perform genotyping detection on NPC-derived samples.

[0088] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of reagents for detecting SNP sites in the preparation of nasopharyngeal carcinoma high-risk subtype classification products; The SNP sites include: 1) The polymorphism of the SNP site at position 7001 from the 5' end to the 3' end of the EBER2 gene is A / T; 2) The polymorphism of the SNP site at position 7012 from the 5' end to the 3' end of the EBER2 gene is A / G; 3) The polymorphism of the SNP site at position 7016 from the 5' end to the 3' end of the EBER2 gene is A / T.

2. The application according to claim 1, characterized in that: The sequence of the EBER2 gene is shown in SEQ ID NO:

1.

3. The application according to claim 2, characterized in that: When the EBER2 gene has a T position at position 7001 from the 5' end to the 3' end, the nasopharyngeal carcinoma is a high-risk subtype; or When the EBER2 gene has a G at position 7012 from the 5' end to the 3' end, the nasopharyngeal carcinoma is a high-risk subtype; or When the EBER2 gene has a T value at position 7016 from the 5' end to the 3' end, the nasopharyngeal carcinoma is a high-risk subtype.

4. The application according to claim 1, characterized in that: The reagents comprise reagents for use in one or more detection techniques or methods selected from the group consisting of: PCR, gene chip method, and nucleic acid sequencing method.

5. A primer-probe set, characterized in that: The primer-probe set includes genotyping primers and fluorescent probes for detecting SNP sites.

6. The primer-probe set according to claim 5, characterized in that: The typing primers include EBER2-WT primers and EBER2-Var primers, with sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

7. The primer-probe set according to claim 6, characterized in that: The fluorescent probes include the EBER2-WT probe and the EBER2-Var probe, with sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

8. The primer-probe set according to claim 7, characterized in that: The fluorescent probe includes a fluorescent group and a quenching group.

9. A test kit, characterized in that: The detection kit includes the reagent for detecting SNP sites in the application described in any one of claims 1 to 4; or The detection kit includes the primer and probe set as described in any one of claims 5 to 8.

10. The detection kit according to claim 9, characterized in that: The test kit also includes enzymes, reaction buffer, negative control, and positive control.

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