WO2023280050A1 - Application of reagent for methylation typing of cpg site of eb virus in saliva in preparation of nasopharyngeal carcinoma diagnostic kit - Google Patents

Abstract

Disclosed is an application of a reagent for detecting EB virus methylation in saliva in the preparation of a nasopharyngeal carcinoma diagnostic kit. The present invention establishes a nasopharyngeal carcinoma detection method, and in the method, detection of nasopharyngeal carcinoma is performed by using saliva. The method has high sensitivity and good specificity for the diagnosis of nasopharyngeal carcinoma, and can effectively assist the diagnosis of nasopharyngeal carcinoma. Saliva sampling has the advantages of being non-invasive, non-destructive, free of dependence on medical staff and medical instruments, and simple and easy to operate, so that wide screening can be carried out on site in areas with high nasopharyngeal carcinoma incidence, nasopharyngeal carcinoma can be diagnosed and treated early, the survival rate of patients is further increased, and prognosis is improved.

Description

Application of reagents for methylation typing of Epstein-Barr virus CpG sites in saliva in the preparation of nasopharyngeal carcinoma diagnostic kits technical field

The invention relates to the technical field of nasopharyngeal carcinoma, in particular to the application of a reagent for performing methylation typing of Epstein-Barr virus CpG sites in saliva in the preparation of a nasopharyngeal carcinoma diagnostic kit.

Background technique

Nasopharyngeal carcinoma is a highly invasive and highly metastatic malignancy that is widespread in southern China and Southeast Asia. Although the overall survival rate of patients in the early clinical stage of nasopharyngeal carcinoma reaches about 90%, since the early stage disease basically has no symptoms, more than 70% of the patients with nasopharyngeal carcinoma have progressed to the middle and late stages when they first visit the doctor, and their survival rate is lower than 50%, so promoting early diagnosis and treatment of nasopharyngeal carcinoma is the key to improving the prognosis of nasopharyngeal carcinoma. Therefore, early diagnosis and early treatment of nasopharyngeal carcinoma are the key breakthroughs to improve the survival rate and prognosis of patients. How to find and establish simple and reliable biomarkers for early diagnosis and screening of nasopharyngeal carcinoma in high-incidence areas is an urgent problem to be solved.

The gold standard for clinical diagnosis of nasopharyngeal carcinoma is tissue biopsy under nasopharyngoscope, followed by pathological diagnosis by pathologists. Due to the relatively traumatic nature of the specimens, the small size of the specimens, and the need to observe with the naked eye under a microscope, about 10% of the patients failed to be diagnosed when the specimens were first drawn. Delayed illness. In addition, nasopharyngeal biopsy strictly relies on the clinical experience of clinicians and medical devices such as nasopharyngeal electronic mirrors, making it difficult to carry out extensive screening for nasopharyngeal carcinoma in high-incidence sites. Noninvasive or minimally invasive sampling techniques, combined with the research application of tumor marker detection, are of great significance for the screening and early diagnosis of nasopharyngeal carcinoma.

Current studies have confirmed that the occurrence of nasopharyngeal carcinoma has a certain relationship with Epstein-Barr virus infection. Sufficient research has shown that Epstein-Barr virus infection plays an important role in the pathogenesis of nasopharyngeal carcinoma. Epstein-Barr virus infects more than 90% of the population worldwide. Previous studies have shown that Epstein-Barr virus is mainly spread among the population through saliva, that is, the virus enters B lymphocytes through the oropharyngeal mucosal epithelial cells, and establishes long-term systemic immunity through infection and transformation of B lymphocytes. sexual latent infection. Such EBV-infected B lymphocytes are more likely to accumulate in the oropharyngeal lymphoid tissue. Under certain conditions, EBV latent in B lymphocytes will be activated, cleavage and release EBV, enter the oropharyngeal cells, and after further cleavage and replication, release the virus into saliva and infect new hosts.

In nasopharyngeal carcinoma endemic areas, almost 100% of undifferentiated nasopharyngeal carcinoma (WHO type III) lesions can detect the presence of EBV genome; in addition, nasopharyngeal squamous cell carcinoma (WHO type I) and non-angular The occurrence of nasopharyngeal carcinoma (WHO type II) is also closely related to EBV infection. It can be said that EBV infection of nasopharyngeal epithelial cells is an important feature of nasopharyngeal carcinoma. At present, the markers widely used clinically in the auxiliary diagnosis of nasopharyngeal carcinoma mainly include the following two aspects:

(1) EBV-related serological markers (including VCA-IgA, EA-IgA antibody detection, etc.) have been included in clinical testing, but the biggest disadvantage of these serological antibody indicators is that they cannot reflect the onset and progress of nasopharyngeal carcinoma in real time conditions, and there are problems of insufficient specificity and sensitivity, which can only provide limited reference in clinical practice. At the same time, in the field screening application, the positive predictive value is too low, about 5%, that is, a large number of positive people do not have the incidence of nasopharyngeal carcinoma.

(2) The detection of plasma free EBV copy number is another molecular marker clinically used in the diagnosis of nasopharyngeal carcinoma, but it still lacks sufficient sensitivity for the early diagnosis and early recurrence of nasopharyngeal carcinoma. High-incidence areas have not been widely implemented, and screening studies in Hong Kong, China showed a positive predictive value of approximately 11%.

In general, in addition to the positive predictive value to be further improved, the detection of EBV markers based on blood samples also relies on clinical nurses to take blood samples, which limits its popularization and detection of the whole population in high-incidence sites of nasopharyngeal carcinoma, so it is easy to develop in Sampling techniques and highly efficient diagnostic molecular markers are still of great significance.

Contents of the invention

The object of the present invention is to overcome the above-mentioned deficiencies in the prior art, and to provide the application of the reagent for methylating the CpG site of Epstein-Barr virus in saliva in the preparation of a diagnostic kit for nasopharyngeal carcinoma.

The first object of the present invention is to provide the application of the reagent for methylation typing of Epstein-Barr virus CpG site in saliva in the preparation of nasopharyngeal carcinoma diagnostic kit.

The second object of the present invention is to provide a method for detecting nasopharyngeal carcinoma using saliva.

The third object of the present invention is to provide a set of primers for detecting nasopharyngeal carcinoma.

The fourth object of the present invention is to provide a kit for detecting nasopharyngeal carcinoma.

In order to achieve the above object, the present invention is achieved through the following schemes:

The inventor found that although Epstein-Barr virus infection will release Epstein-Barr virus into saliva, the EBV-DNA in the saliva of nasopharyngeal cancer patients will undergo significant methylation changes, that is, in the saliva of normal people, the Epstein-Barr virus DNA The methylation density of most CpG sites is less than 50%, while in the saliva of patients with nasopharyngeal carcinoma, the density of methylation of most CpG sites on Epstein-Barr virus DNA is greater than 50%. It has not been noticed in the past and is the first discovery. We speculate that EBV in the patient's oropharynx has switched from lytic infection to latent infection. Although the molecular mechanism behind this change and the relationship with nasopharyngeal carcinoma are not very clear, according to this newly discovered rule, The non-invasive diagnosis of nasopharyngeal carcinoma can be realized by liquid biopsy of saliva, that is, the detection of methylation typing of specific CpG sites of Epstein-Barr virus.

Therefore, the present invention claims to protect the application of the reagent for methylating the CpG site of Epstein-Barr virus in saliva in the preparation of a diagnostic kit for nasopharyngeal carcinoma.

Preferably, the reagent performs methylation typing on one or more of the following CpG sites in the Epstein-Barr virus genome:

所述CpG位点位于在EB病毒基因组的11029bp、11044bp、11209bp、11220bp、45849bp、55696bp、55714bp、57945bp、70375bp、108542bp、111401bp、128102bp、137676bp、137698bp和158044bp处，所述EB病毒基因组为GenBank中The LOCUS is NC_007605.

More preferably, the reagent performs methylation typing on the CpG sites located at 11029bp, 11044bp, 11209bp and 11220bp in the Epstein-Barr virus genome.

Further preferably, the reagent is a primer whose nucleotide sequence is shown in SEQ ID NO: 1-4.

Primers for methylated template DNA: forward primer is 5'-TGTTGGCGGGAGAAGGAATAAC-3'(SEQ ID NO:1), reverse primer 5'-CTAAACTTACGTAAACAAAACGT-3'(SEQ ID NO:2); Primers for methylated template DNA: the forward primer is 5'-TTTGTTGGTGGGAGAAGGAATAAT-3' (SEQ ID NO:3), and the reverse primer is 5'-AACTAAACTTACATAAACAAAACAT-3' (SEQ ID NO:4).

A method for detecting nasopharyngeal carcinoma using saliva, performing methylation typing on Epstein-Barr virus CpG sites in saliva, and the CpG sites are one or more of the following:

所述CpG位点位于在EB病毒基因组的11029bp、11044bp、11209bp、11220bp、45849bp、55696bp、55714bp、57945bp、70375bp、108542bp、111401bp、128102bp、137676bp、137698bp和158044bp处，所述EB病毒基因组为GenBank中The LOCUS is NC_007605.

Preferably, methylation typing is performed on the CpG sites located at 11029bp, 11044bp, 11209bp and 11220bp in the Epstein-Barr virus genome in saliva.

Preferably, the methylation typing of the Epstein-Barr virus CpG site in saliva is performed using primers with nucleotide sequences such as SEQ ID NO: 1-4.

Wherein, the primers shown in SEQ ID NOs: 1-2 are primers for methylated template DNA, and the primers shown in SEQ ID NOs: 3-4 are primers for non-methylated template DNA.

The Ct value of primer amplification for methylated template DNA is less than the Ct value of primer amplification for methylated template DNA, indicating that the amount of methylated EBV DNA is large (that is, greater than 50% of the total EBV DNA template quantity). %), the sample source patient is likely to be positive for nasopharyngeal carcinoma; the Ct value of primer amplification for methylated template DNA is greater than the Ct value of primer amplification for methylated template DNA, indicating that the methylated If the amount of EBV DNA is small (that is, less than 50% of the total EBV DNA template), the sample source patient is likely to be negative for nasopharyngeal carcinoma.

The present invention also claims a set of primers for detecting nasopharyngeal carcinoma, the nucleotide sequence of which is shown in SEQ ID NO: 1-4.

And a kit for detecting nasopharyngeal carcinoma, which contains reagents for methylation typing of Epstein-Barr virus CpG sites in saliva.

Preferably, the reagent detects the methylation typing of one or more of the following CpG sites in the Epstein-Barr virus genome:

所述CpG位点位于在EB病毒基因组的11029bp、11044bp、11209bp、11220bp、45849bp、55696bp、55714bp、57945bp、70375bp、108542bp、111401bp、128102bp、137676bp、137698bp和158044bp处，所述EB病毒基因组GenBank中的LOCUS is NC_007605.

More preferably, the reagent performs methylation typing on the CpG sites located at 11029bp, 11044bp, 11209bp and 11220bp in the Epstein-Barr virus genome.

Further preferably, the reagent is a primer whose nucleotide sequence is shown in SEQ ID NO: 1-4.

Preferably, it also contains q-PCR reagents for the dye method.

More preferably, the q-PCR reagents are q-PCR reaction mix and enzyme-free water.

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

The invention establishes a nasopharyngeal carcinoma detection method, which uses saliva to detect nasopharyngeal carcinoma. It has high sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma, and can effectively assist the diagnosis of nasopharyngeal carcinoma. Since saliva sampling is non-invasive and non-destructive, does not need to rely on medical personnel and medical equipment, and is simple and easy to operate, it is possible to carry out extensive on-site screening in areas with a high incidence of nasopharyngeal carcinoma, so as to conduct early diagnosis and early treatment of nasopharyngeal carcinoma, thereby improving the quality of patients. survival rate and improved prognosis.

Description of drawings

Figure 1 shows the calculation of the methylation density of CpG sites on Epstein-Barr virus DNA in 36 cases of saliva samples (including 20 cases of nasopharyngeal carcinoma patients and 16 cases of healthy controls) by capture sequencing.

Fig. 2 is a schematic diagram of the positions of primers for four CpG sites located at 11029bp, 11044bp, 11209bp and 11220bp.

Figure 3 shows the methylation typing of Epstein-Barr virus DNA located at four CpG sites of 11029bp, 11044bp, 11209bp and 11220bp in 36 cases of saliva samples by q-PCR technology.

Figure 4 shows the q-PCR technique for the methylation typing of Epstein-Barr virus DNA at four CpG sites at 11029bp, 11044bp, 11209bp and 11220bp in the saliva samples of verification group 1.

Figure 5 shows the methylation typing of four CpG sites of EB virus DNA located at 11029bp, 11044bp, 11209bp and 11220bp in the saliva samples of verification group 2 by q-PCR technology.

detailed description

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are only used to explain the present invention, and are not intended 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 commercially available reagents and materials unless otherwise specified.

Example 1 Epstein-Barr virus genome-wide methylation capture sequencing

1. Experimental method

1. Sample source

36 cases, of which 20 cases were from nasopharyngeal carcinoma patients & 16 cases were from healthy controls.

2. Collection of saliva samples

Thirty minutes before the saliva sample collection, the subjects were forbidden to eat or drink water. Before saliva collection, the subjects first rinsed their mouths with normal saline, then took the sample collection cup against the chin, waited for about 2-5 mL of saliva to flow out automatically, poured it into a 10 mL centrifuge tube that had been added with 2.5 mL of lysis preservation solution, and sealed it. save. The sample DNA is extracted in advance and can be stored in a -80 degree ultra-low temperature freezer.

3. Extraction of total DNA from saliva

The DNA of saliva samples was extracted using an automated nucleic acid extraction workstation (equipment model Hamilton chemagen-star), and a maximum of 96 samples could be extracted in each batch. The DNA extraction principle adopts the magnetic bead method. After the sample is lysed, the DNA is adsorbed on the magnetic beads, and then eluted after several steps of washing to obtain a pure DNA solution with an elution volume of 150 μl.

4. Capture of EB virus DNA

Since the DNA extracted from saliva samples is mostly DNA from host cells, it is necessary to separate the DNA of Epstein-Barr virus from the total DNA for follow-up experiments. A capture probe covering the entire genome of Epstein-Barr virus was designed and customized, and the DNA extracted from 3 μg saliva samples The DNA molecules of Epstein-Barr virus are captured in the total DNA.

5. Library construction and sequencing

The captured Epstein-Barr virus DNA molecules were converted by bisulfite treatment, and then amplified to establish a library and next-generation sequencing.

6. Analyze the sequencing results

According to the sequencing results, calculate the methylation density of each CpG site (methylation density), that is, for a CpG site at a certain position on the Epstein-Barr virus genome, according to the sequencing results, methylated C and unmethylated According to the number of T, the methylation density of this site is calculated according to the formula C/(C+T).

The methylation of all CpG sites on the Epstein-Barr virus genome was compared and analyzed to find out the methylation sites with significant differences in the salivary Epstein-Barr virus DNA derived from the nasopharyngeal carcinoma group and the control group.

For the above analysis, the Epstein-Barr virus genome was used as LOCUS NC_007605 in GenBank.

2. Experimental results

The W-region repeats on the Epstein-Barr virus genome were removed from the sequencing results, and there were a total of 7531 CpG sites in the remaining regions. In the 36 cases (20 cases from nasopharyngeal carcinoma patients vs 16 cases from healthy controls) salivary Epstein-Barr virus DNA capture In the sequencing experiment, 4 samples detected less than 5% of the CpG sites, which were excluded after quality control. The remaining 32 saliva samples included 18 patients with nasopharyngeal carcinoma and 14 healthy controls.

The Wilcox test was performed on the remaining 32 samples, and there were 5234 sites with statistically significant differences in methylation density (P<0.05). The circles in Figure 1 represent the methylation density of each CpG site on Epstein-Barr virus DNA in the saliva samples of nasopharyngeal carcinoma patients, and the triangles represent the methylation density of EB virus DNA in the saliva samples of healthy controls The methylation density of each CpG site.

It was found that the methylation density of most CpG sites in nasopharyngeal carcinoma was basically above 50%, indicating that the methylated EBV DNA molecules in the sample accounted for the majority (i.e. >50%) of the total EBV DNA molecules ; The methylation density of most CpG sites in the control group was basically below 50%, suggesting that the methylated EBV DNA molecules in the control samples accounted for a minority (ie <50%) of the total EBV DNA molecules.

The present invention firstly found that there is an essential difference in the methylation status of Epstein-Barr virus in the saliva of patients with nasopharyngeal carcinoma and healthy controls, and this regular change has not been noticed in previous studies. The methylation typing of loci provides a clear molecular basis for the diagnosis of nasopharyngeal carcinoma.

Example 2 Screening specific CpG sites to construct a risk prediction model applied to the diagnosis of nasopharyngeal carcinoma

1. Experimental method

1. Screen the sites with significant differences into the risk prediction model:

(1) Inclusion conditions:

CpG sites with significant differences in methylation density between nasopharyngeal carcinoma patients and healthy controls;

Prioritize the selection of hypermethylated sites in special cases (i.e., cases of overall hypomethylation);

(2) Exclusion conditions:

Sites that are hypermethylated in a particular control (ie, an overall hypomethylated control).

According to the inclusion and exclusion conditions, 15 loci were selected (Table 1). The risk model of nasopharyngeal carcinoma was constructed according to the following formula, and the methylation typing score (MTS) for distinguishing between cases and controls was calculated:

Among them, β ₁ refers to the risk prediction coefficient of the hypermethylated site screened in the hypomethylated cases, which is determined to be 10 according to the ratio of the hypomethylated case and the hypermethylated case in the test data set, and c1 is the methylated site of the site methylated C count, t1 is the t count of the site (that is, unmethylated C, converted to T under bisulfite treatment); _β2 is the risk of hypermethylated sites in general hypermethylated cases The prediction coefficient is determined to be 1, ci is the methylated C count of each selected site, ti is the t count of the site, and k is the number of hypermethylated sites included in the model in general hypermethylated cases.

According to the risk prediction model, after substituting the conditions of 15 sites, the methylation typing score (methylation typing score, MTS) of each sample was calculated (Table 2). The value is 10.42-87.95, and the methylation typing score value in the control group is 0-9.72, so the MTS cut-off value is set to 10. According to the MTS, 18 cases of nasopharyngeal carcinoma patients and 14 cases of healthy controls can be effectively distinguished. That is, both sensitivity and specificity are 100%.

Table 1 The positions of 15 CpG sites and their genes on the Epstein-Barr virus genome

serial number	gene	position
1	BFLF1	45849
2	EBNA-1.2, BOLF1, BPLF1	55696
3	EBNA-1.2, BOLF1, BPLF1	55714
4	-	70375
5	BTRF1, BcRF1.2	128102
6	BALF3, BALF4, A73	158044
7	EBNA-1.2, BOLF1, BPLF1	57945
8	BGLF3, BGLF3.5, BGLF4, BGLF5, BGLF1	108542
9	BGLF3, BGLF3.5	111401
10	-	11029
11	-	11044
12	-	11209
13	-	11220
14	BILF2	137676
15	BILF2	137698

Table 2 The 15 CpG sites of Epstein-Barr virus were substituted into the risk prediction model to calculate the methylation typing score of each sample

Example 3 Methylation Degree of Nasopharyngeal Carcinoma Patients and Healthy People

1. Experimental method

In order to further reduce the cost of detecting the methylation of Epstein-Barr virus CpG sites and make the technical scheme easy to be widely promoted on site, on the basis of capturing the sequencing results, q-PCR technology was used to detect the methylation at specific sites of Epstein-Barr virus. Quantitative calculation of the degree of methylation (methylation degree).

According to the distribution of the 15 selected sites, it was noted that the four consecutive CpG sites of 11029bp, 11044bp, 11209bp and 11220bp on the EBV genome are relatively close to each other, which is convenient for designing primers for PCR amplification, so we chose to cover these four sites The region of the point (that is, the 11021-11232bp region of the Epstein-Barr virus DNA C promoter region) was used as a template for q-PCR detection. Two pairs of primers were designed, one pair of primers was used to amplify methylated template EBV DNA, and the other pair of primers was used to amplify unmethylated template EBV DNA.

As shown in Figure 2, the red color in the figure indicates the sequence of the primer position, the primer for the methylated template DNA: the forward primer is 5'-TGTTGGCGGGAGAAGGAATAAC-3'(SEQ ID NO:1), and the reverse primer is 5' -CTAAACTTACGTAAACAAAACGT-3'(SEQ ID NO:2); primers for unmethylated template DNA: the forward primer is 5'-TTTGTTGGTGGGAGAAGGAATAAT-3'(SEQ ID NO:3), and the reverse primer is 5'- AACTAAACTTACATAAACAAAACAT-3' (SEQ ID NO: 4). The forward primer in the two pairs of primers utilizes two CpG sites at 11029 and 11044bp (indicated by shading in the figure), and the reverse primer utilizes two CpG sites at 11209 and 11220bp (indicated by shading in the figure).

Under the same reaction system, q-PCR by dye method was carried out. The q-PCR reaction system is 15 μL, including 7.5 μL of q-PCR reaction mix, 1 μL of primers, 5.5 μL of enzyme-free water, and 1 μL of bisulfite-treated DNA template.

The q-PCR reaction conditions were: 95°C for 10 min, 40 amplification cycles (95°C for 30 s, 60°C for 35 s and 72°C for 30 s), and 72°C for 7 min.

When the amount of methylated EBV DNA in the sample is large (that is, greater than 50% of the total EBV DNA template), the Ct(m) value for the methylated template DNA amplification in the q-PCR reaction will be smaller than that for The Ct(u) value of template amplification without methylation, if the amount of methylated template DNA in the sample is small (ie, less than 50% of the total EBV DNA template amount), the result is opposite. Finally, through the calculation of the relative expression, that is, the calculation method of 2- ^{[Ct(m)-Ct(u)]} is used to obtain the genotype of the four methylation sites 11029bp, 11044bp, 11209bp and 11220bp for each sample Methylation value.

According to the results of whole-genome methylation sequencing of Epstein-Barr virus, most of the EBV DNA in saliva from nasopharyngeal carcinoma patients is methylated, and the degree of methylation is calculated by 2- ^{[Ct(m)-Ct(u)]} It should be >1, while most of the EBV DNA in the saliva from the control group is unmethylated, and the degree of methylation should be <1 after calculation by this formula.

Using this method to detect 36 cases of samples for capture sequencing in Example 1, 20 cases were from nasopharyngeal carcinoma patients, and 16 cases were from healthy controls (exploration group).

2. Experimental results

The results are shown in Table 1 and Figure 3 below. The results showed that the average methylation degree of EBV DNA in the saliva of nasopharyngeal cancer patients was 7.72, and the range of methylation degree was 0.02-53.4. The average value of methylation degree of DNA is only 0.11, and the range of methylation degree is 0.16×10 ^-3 to 0.53.

The degree of methylation = 1 is set as the cut-off value (Cut off value), the sensitivity of this technical system for the diagnosis of nasopharyngeal carcinoma after methylation typing is 90%, and the specificity is 100%.

Table 1 The q-PCR technology system is used to classify the methylation of Epstein-Barr virus, and the calculation statistics of the sensitivity and specificity applied to the diagnosis of nasopharyngeal carcinoma are as follows (selected cut off value = 1)

Embodiment 4 A kind of detection kit of nasopharyngeal carcinoma

1. Composition

The nucleotide sequence is as the primer shown in SEQ ID NO: 1-4, and the q-PCR reagent of the dye method.

Wherein, the primers shown in SEQ ID NOs: 1-2 are primers for methylated template DNA, and the primers shown in SEQ ID NOs: 3-4 are primers for non-methylated template DNA.

2. How to use

Under the same reaction system, the primers shown in SEQ ID NO: 1-2 and the primers shown in SEQ ID NO: 3-4 were used to perform q-PCR by dye method on the DNA of the saliva sample to be tested.

The q-PCR reaction system is 15 μL, including 7.5 μL of q-PCR reaction mix, 1 μL of primers, 5.5 μL of enzyme-free water, and 1 μL of DNA template.

The q-PCR reaction conditions were: 95°C for 10 min, 40 amplification cycles (95°C for 30 s, 60°C for 35 s and 72°C for 30 s), and 72°C for 7 min.

3. Interpretation of results

The Ct value of primer amplification for methylated template DNA is less than the Ct value of primer amplification for methylated template DNA, indicating that the amount of methylated EBV DNA is large (that is, greater than 50% of the total EBV DNA template quantity). %), the sample source patient is likely to be positive for nasopharyngeal carcinoma; the Ct value of primer amplification for methylated template DNA is greater than the Ct value of primer amplification for methylated template DNA, indicating that the methylated If the amount of EBV DNA is small (that is, less than 50% of the total EBV DNA template), the sample source patient is likely to be negative for nasopharyngeal carcinoma.

Example 5 Detection of Resident Samples in Sihui, Guangdong

1. Experimental method

After confirming that the q-PCR technical system of Example 3 can type the methylation situation of Epstein-Barr virus and indicate whether it is suffering from nasopharyngeal carcinoma or not, the kit of Example 4 is used to further test the methylation status of Epstein-Barr virus in 49 cases of nasopharyngeal carcinoma patients. The saliva sample DNA of 65 cases of healthy control personnel was tested and verified, and this group of samples was defined as verification group 1. This sample set is a case-control study based on high-occurrence sites, that is, the cases come from patients with nasopharyngeal carcinoma found through nasal endoscopy during the physical examination of residents in Sihui, Guangdong, and the healthy controls are those who have ruled out nasopharyngeal carcinoma through nasal endoscopy. medical examiner

2. Experimental results

The results are shown in Table 2 and Figure 4 below. The experimental results showed that in the saliva of 49 patients with nasopharyngeal carcinoma, the average methylation degree of Epstein-Barr virus DNA was 68.81, and the expression range was 1.0×10 ^-5 to 754.8, while in In the saliva sample DNA of 65 healthy control persons, the average methylation degree of Epstein-Barr virus DNA was 3.985, and the expression range was 2.1×10 ^-5 to 195.4. Also taking the methylation degree = 1 as the cut-off value, the sensitivity to the diagnosis of nasopharyngeal carcinoma was 83.67% (8/49), and the specificity was 89.23% (7/65).

Table 2:

Example 6 Detection of Sun Yat-sen University Cancer Center Samples

1. Experimental method

On the basis of the above research, use the kit of implementation 4 to further verify in another sample set, which is defined as the verification group 2, and the collected saliva samples are from 60 cases of nasopharyngeal carcinoma patients and 123 cases of controls. This sample set is a hospital-based case-control study, that is, the case samples come from patients with nasopharyngeal carcinoma who visited the Cancer Center of Sun Yat-sen University.

2. Experimental results

The results are shown in Table 3 and Figure 5 below. In the saliva of 60 patients with nasopharyngeal carcinoma, the average methylation degree of Epstein-Barr virus DNA was 42.72, and the expression range was 0.014-474.4, while in the saliva samples of 123 healthy controls Among the DNA, the average value of methylation degree of Epstein-Barr virus DNA is 8.48, and the expression range is 1.71×10 ^-8 ~ 337.8). Also with the methylation degree = 1 as the cut-off value, the sensitivity to the diagnosis of nasopharyngeal carcinoma is 86.67 % (8/60), the specificity was 89.23% (14/123).

table 3

Summary statistics of each group of data in embodiment 7

1. Experimental method

Summarize the data of the above exploration group, verification group 1 and verification group 2.

2. Experimental results

The results are shown in Table 4 below. The results show that the effective diagnosis of nasopharyngeal carcinoma can be achieved by detecting the methylation of the specific CpG sites of salivary Epstein-Barr virus DNA, with a sensitivity of 86.05% and a specificity of 89.71% %.

Table 4:

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the scope of the present invention. For those of ordinary skill in the art, on the basis of the above descriptions and ideas, they can also make There is no need to and cannot exhaustively list all the implementation manners for other changes or changes in different forms. All modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the claims of the present invention.

Claims (14)

1. Application of a reagent for methylation typing of Epstein-Barr virus CpG sites in saliva in the preparation of a diagnostic kit for nasopharyngeal carcinoma.
2. The application according to claim 1, wherein the reagent is methylated to one or more of the following CpG sites in the Epstein-Barr virus genome:

   所述CpG位点位于在EB病毒基因组的11029bp、11044bp、11209bp、11220bp、45849bp、55696bp、55714bp、57945bp、70375bp、108542bp、111401bp、128102bp、137676bp、137698bp和158044bp处，所述EB病毒基因组为GenBank中The LOCUS is NC_007605.
3. The application according to claim 2, characterized in that the reagent performs methylation typing on the CpG sites located at 11029bp, 11044bp, 11209bp and 11220bp in the Epstein-Barr virus genome.
4. The application according to claim 3, wherein the reagent is a primer having a nucleotide sequence as shown in SEQ ID NO: 1-4.
5. A method for detecting nasopharyngeal carcinoma using saliva, characterized in that the methylation typing of Epstein-Barr virus CpG sites in saliva is carried out, and the CpG sites are one or more of the following:

   所述CpG位点位于在EB病毒基因组的11029bp、11044bp、11209bp、11220bp、45849bp、55696bp、55714bp、57945bp、70375bp、108542bp、111401bp、128102bp、137676bp、137698bp和158044bp处，所述EB病毒基因组为GenBank中The LOCUS is NC_007605.
6. The method according to claim 5, characterized in that the methylation typing is performed on the CpG sites located at 11029bp, 11044bp, 11209bp and 11220bp in the Epstein-Barr virus genome in saliva.
7. According to the method of claim 5, it is characterized in that, use nucleotide sequence such as the primer shown in SEQ ID NO: 1～4 to carry out methylation typing to Epstein-Barr virus CpG site in saliva.
8. According to the method according to claim 7, it is characterized in that, the Ct value for primer amplification shown in SEQ ID NO:1～2 of methylated template DNA is less than that shown in SEQ ID NO:3～4 for methylated template DNA The Ct value amplified by the primers indicates that the sample source patient is likely to be positive for nasopharyngeal carcinoma; the Ct value amplified by the primers shown in SEQ ID NO: 1-2 for the methylated template DNA is greater than that for the methylated template DNA If the Ct value amplified by the primers shown in SEQ ID NO: 3-4 is high, the sample source patient is likely to be negative for nasopharyngeal carcinoma.
9. A set of primers for detecting nasopharyngeal carcinoma is characterized in that its nucleotide sequence is as shown in SEQ ID NO: 1-4 primers.
10. A test kit for detecting nasopharyngeal carcinoma is characterized in that it contains a reagent for carrying out methylation typing of Epstein-Barr virus CpG sites in saliva.
11. The kit according to claim 10, wherein the reagent is methylated to one or more of the following CpG sites in the Epstein-Barr virus genome:

    所述CpG位点位于在EB病毒基因组的11029bp、11044bp、11209bp、11220bp、45849bp、55696bp、55714bp、57945bp、70375bp、108542bp、111401bp、128102bp、137676bp、137698bp和158044bp处，所述EB病毒基因组GenBank中的LOCUS is NC_007605.
12. The kit according to claim 11, wherein the reagent performs methylation typing on the CpG sites located at 11029bp, 11044bp, 11209bp and 11220bp in the Epstein-Barr virus genome.
13. The kit according to claim 12, wherein the reagent is a primer having a nucleotide sequence as shown in SEQ ID NO: 1-4.
14. The kit according to claim 13, further comprising q-PCR reagents of the dye method.

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