CN116694762A - Kit for detecting nasopharyngeal carcinoma - Google Patents
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Abstract
The invention discloses a kit for detecting nasopharyngeal carcinoma and application of a reagent for methylation typing of CpG sites of an EB virus Wp promoter region in saliva in preparation of a nasopharyngeal carcinoma diagnosis kit. The invention establishes a method and a kit for detecting and diagnosing nasopharyngeal carcinoma aiming at the Wp promoter region of the EB virus, and the method and the kit greatly improve the success rate of detection in the prior art. The nasopharyngeal carcinoma detection method using saliva samples has higher success rate and better application prospect.
Description
Technical Field
The invention relates to the technical field of nasopharyngeal carcinoma detection, in particular to a kit for detecting nasopharyngeal carcinoma.
Background
Nasopharyngeal carcinoma (Nasopharyngeal Carcinoma) is a malignant tumor occurring in the top and back wall of nasopharynx and pharyngeal crypt, and is one of the high malignant tumors in China, and the incidence rate is the first malignant tumor of ear, nose and throat. The five-year survival rate of the early nasopharyngeal carcinoma is higher after the treatment, and the five-year survival rate of the middle-stage and late-stage nasopharyngeal carcinoma is relatively lower, and because the early disease basically has no symptoms, a large number of nasopharyngeal carcinoma patients have progressed to the middle and late stage when first seeing a doctor, thereby promoting the early screening and early diagnosis and early treatment of the nasopharyngeal carcinoma to be the key for improving the prognosis of the nasopharyngeal carcinoma.
Numerous studies have shown that the onset of nasopharyngeal carcinoma is closely related to Epstein-Barr virus (EBV) infection. According to the technical scheme of tumor screening and early diagnosis and early treatment formulated by the Ministry of health of China, plasma EBV antibody screening is recommended for people with high-incidence area of nasopharyngeal carcinoma of 30-59 years old, and nasopharyngeal mirror or MRI clinical examination and further pathological biopsy are recommended for diagnosis. However, research shows that the existing screening strategy based on EB virus antibody still has some limitations, and the positive predictive value is not high, only about 4%.
Single nucleotide polymorphism (single nucleotidepolymorphism, SNP) refers mainly to DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level. It is one of the most common human heritable variants, and a number of studies have shown that it is associated with susceptibility to nasopharyngeal carcinoma: for example, chinese patent (kit for SNP locus detection of susceptibility gene of nasopharyngeal carcinoma), osteopontin functional SNP related to the risk of onset of nasopharyngeal carcinoma and application, kit and gene chip for predicting the risk of onset of nasopharyngeal carcinoma, etc., SNP polymorphism is a genetic search for susceptibility crowd of nasopharyngeal carcinoma, its phenotype is natural, whether an individual has already developed nasopharyngeal carcinoma cannot be reacted, only the probability that an individual with dangerous phenotype will develop in its lifetime can be said to be higher than other individuals. Therefore, the risk prediction based on SNP polymorphism cannot directly detect the occurrence of nasopharyngeal carcinoma, and is applied to early screening and early diagnosis of nasopharyngeal carcinoma.
Previous studies have shown that EBV is transmitted in humans mainly by saliva, i.e. the virus enters B lymphocytes through oropharyngeal mucosal epithelial cells, and long-term systemic latent infection is established by infection of transformed B lymphocytes. Such EBV-infected B lymphocytes are more likely to accumulate in oropharyngeal lymphoid tissues. Under certain conditions, EBV hidden in B lymphocytes can be activated, EBV is released by lysis, and the oropharyngeal cells are released by further lysis and replication to release virus into saliva, so that new host cells are infected.
The EB virus genome is shown to be in a highly methylated state in nasopharyngeal carcinoma, and the expression of the gene is controlled by the highly methylation, so that a long-term latent type II infection is established.
Although EB virus infected persons release EB virus into saliva, in research, it is found that significant methylation modification of EB virus DNA in saliva of nasopharyngeal carcinoma patients occurs, namely, in saliva of normal persons, methylation density of most CpG sites on EB virus DNA is less than 50%, and in saliva of nasopharyngeal carcinoma patients, methylation density of most CpG sites on EB virus DNA is more than 50%, according to the new discovery rule, methylation typing detection is carried out on specific CpG sites of EB virus by carrying out liquid biopsy of saliva, so that noninvasive detection of nasopharyngeal carcinoma can be realized. The currently reported detection sites are located in Cp promoter regions (China patent application of reagent for methylation typing of EB virus CpG sites in saliva in preparation of nasopharyngeal carcinoma diagnostic kit), but the existing literature shows that the saliva EB virus load varies greatly among different individuals, and the problem of low success rate of detection is directly influenced for individuals with low EB virus load, so that development of efficient detection technology is an important premise for application of nasopharyngeal carcinoma saliva biopsy scheme.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a kit for detecting nasopharyngeal carcinoma.
The first object of the invention is to provide the application of the reagent for methylation typing of CpG sites of the Epstein-Barr virus Wp promoter region in saliva in preparing a nasopharyngeal carcinoma diagnosis kit.
It is a second object of the present invention to provide a set of detection primers for detecting nasopharyngeal carcinoma.
It is a third object of the present invention to provide a set of detection probes for detecting nasopharyngeal carcinoma.
The fourth object of the invention is to provide a kit for detecting nasopharyngeal carcinoma.
In order to achieve the above object, the present invention is realized by the following technical scheme:
methylation of the EB virus is a very different form in saliva of nasopharyngeal carcinoma patients and healthy controls, so that the genome of the whole EB virus is presumed to be very different in nasopharyngeal carcinoma patients and healthy controls, and therefore, a region where a Wp promoter is located is proposed as a detection target. The EB virus contains 4 internal repeat regions, namely IR1, IR2, IR3 and IR4, wherein the longest IR1 is the region where the Wp promoter is located, and the region is 12001 bp-35355 bp of the EBV reference genome (NC_ 007605.1), and contains 8 tandem repeat sequences.
The invention claims the application of a reagent for methylation typing of CpG sites of a Wp promoter region of EB virus in saliva in preparing a nasopharyngeal carcinoma diagnosis kit, and specifically, the Wp promoter region is located between 12001bp and 35355bp of an EB virus genome NC_007605.1.
Preferably, the CpG sites are one or more of CpG sites at 12420bp, 12428bp, 12432bp, 15492bp, 15500bp, 15504bp, 18564bp, 18572bp, 18576bp, 21636bp, 21644bp, 21648bp, 24708bp, 24716bp, 24720bp, 27780bp, 27788bp, 27792bp, 30852bp, 30860bp, 30864bp, 33924bp, 33932bp and 33936bp of EB virus genome, and the EB virus genome is NC_007605.1.
Preferably, the reagent comprises a detection primer and/or a detection probe.
Preferably, the nucleotide sequence of the detection primer is shown in SEQ ID NO: 1-2.
The detection primer is as follows:
forward primer: GGGTAGAGATAGGTAGGGT (SEQ ID NO: 1);
reverse primer: CTCTACCTCCCAAACTTACC (SEQ ID NO: 2).
Preferably, the nucleotide sequence of the detection probe is shown in SEQ ID NO: 3-4.
The detection probe comprises:
methylation probe (probe 1): FAM-TTTCGAGGAGGCGTTCGGAGTG-BHQ1 (SEQ ID NO: 3);
unmethylated probe (probe 2): HEX-TTTTGAGGAGGTGTTTGGAGTG-BHQ1 (SEQ ID NO: 4).
As a specific example, the reagents include detection primers and detection probes.
Forward primer: GGGTAGAGATAGGTAGGGT (SEQ ID NO: 1);
reverse primer: CTCTACCTCCCAAACTTACC (SEQ ID NO: 2);
methylation probe (probe 1): FAM-TTTCGAGGAGGCGTTCGGAGTG-BHQ1 (SEQ ID NO: 3);
unmethylated probe (probe 2): HEX-TTTTGAGGAGGTGTTTGGAGTG-BHQ1 (SEQ ID NO: 4).
The above detection primers and detection probes are capable of detecting all the following CpG sites, the positions of each CpG site on the EBV genome (NC_ 007605.1) are:
repeat 1: 12420bp, 12428bp and 12432bp;
repeat 2: 15492bp, 15500bp and 15504bp;
repeat 3: 18564bp, 18572bp and 18576bp;
repeat 4: 21636bp, 21644bp and 21648bp;
repeat 5: 24708bp, 24716bp and 24720bp;
repeat 6: 27780bp, 27788bp and 27792bp;
repeat 7: 30852bp, 30860bp and 30864bp;
repeat 8: 33924bp, 33932bp and 33936bp.
The invention also claims a group of detection primers for detecting nasopharyngeal carcinoma, and the nucleotide sequence of the detection primers is shown in SEQ ID NO: 1-2.
The invention also claims a group of detection probes for detecting nasopharyngeal carcinoma, and the nucleotide sequences of the detection probes are shown in SEQ ID NO: 3-4.
The invention also claims a kit for detecting nasopharyngeal carcinoma, which contains a reagent for methylation typing of CpG sites of the Epstein-Barr virus Wp promoter region in saliva.
Preferably, the CpG sites are one or more of CpG sites at 12420bp, 12428bp, 12432bp, 15492bp, 15500bp, 15504bp, 18564bp, 18572bp, 18576bp, 21636bp, 21644bp, 21648bp, 24708bp, 24716bp, 24720bp, 27780bp, 27788bp, 27792bp, 30852bp, 30860bp, 30864bp, 33924bp, 33932bp and 33936bp of EB virus genome, and the EB virus genome is NC_007605.1.
Preferably, the reagent is the detection primer and/or the detection probe
Preferably, the probe method q-PCR reagent is also included.
The q-PCR reagent of the probe method is q-PCR 2 Xmaster mix and enzyme-free water.
As a specific example, the kit contains the detection primer, the detection probe and q-PCR reagent of the probe method,
wherein, the detection primer is:
forward primer: GGGTAGAGATAGGTAGGGT (SEQ ID NO: 1);
reverse primer: CTCTACCTCCCAAACTTACC (SEQ ID NO: 2);
the detection probe comprises:
methylation probe (probe 1): FAM-TTTCGAGGAGGCGTTCGGAGTG-BHQ1 (SEQ ID NO: 3);
unmethylated probe (probe 2): HEX-TTTTGAGGAGGTGTTTGGAGTG-BHQ1 (SEQ ID NO: 4).
The application method comprises the following steps:
1. PCR reaction
20. Mu.l of a reaction system containing 10. Mu.l of 2 Xmaster mix, 0.75. Mu.l of forward primer, 0.75. Mu.l of reverse primer, 0.8. Mu.l of methylation probe (probe 1), 2.8. Mu.l of methylation probe (probe 1), 4.9. Mu.l of enzyme-free water and 2. Mu.l of bisulfite-treated saliva DNA template.
The reaction conditions were 95℃for 5min followed by 45 cycles, each set at 95℃for 15s,58℃for 60s, and finally 40℃for 30s.
2. Calculating the methylation degree:
after q-PCR is completed, the relative degree of methylation for CpG sites (Methylation Level, ML) is calculated, and the ML value is calculated as: 2 -[Ct(m)-Ct(u)] ，
CT m CT value, CT after amplification of the amplification primer and methylation probe for the site u CT values after amplification for the amplified primer and unmethylated probe for this site.
If only one of the CT values is detected after q-PCR of the sample is completed, the other CT value is defined as 45 substituted into the calculation formula to calculate the relative methylation degree. If neither CT value is detected after the sample q-PCR is completed, the sample is defined to fail detection.
3. Result determination
Setting ml=1 as a cut-off value, namely ML >1, reflecting that the number of the EB virus DNA molecules which are methylated in saliva is more than that of the EB virus DNA molecules which are not methylated, and prompting the occurrence of nasopharyngeal carcinoma; whereas ML <1, suggesting no nasopharyngeal carcinoma; ml=1, suggesting suspected nasopharyngeal carcinoma patients.
Compared with the prior art, the invention has the following beneficial effects:
the invention establishes a method and a kit for detecting and diagnosing nasopharyngeal carcinoma aiming at the Wp promoter region of the EB virus, and the method and the kit greatly improve the success rate of detection in the prior art. The nasopharyngeal carcinoma detection method using saliva samples has higher success rate and better application prospect.
Detailed Description
The invention is further illustrated in detail in the following description and in the detailed examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 method for detecting and diagnosing nasopharyngeal carcinoma aiming at CpG site methylation typing condition of Epstein-Barr virus Wp promoter region in saliva sample
1. Primer and probe sequences designed for Wp promoter region (12001 bp to 35355bp of EB virus genome NC_ 007605.1):
forward primer: GGGTAGAGATAGGTAGGGT (SEQ ID NO: 1);
reverse primer: CTCTACCTCCCAAACTTACC (SEQ ID NO: 2);
methylation probe (probe 1): FAM-TTTCGAGGAGGCGTTCGGAGTG-BHQ1 (SEQ ID NO: 3);
unmethylated probe (probe 2): HEX-TTTTGAGGAGGTGTTTGGAGTG-BHQ1 (SEQ ID NO: 4).
2. Reaction system and reaction conditions for PCR reaction
20. Mu.l of the reaction system, which contained 10. Mu.l of 2 Xmaster mix, 0.75. Mu.l of forward primer, 0.75. Mu.l of reverse primer, 4.9. Mu.l of enzyme-free water and 2. Mu.l of bisulfite-treated saliva DNA.
The reaction conditions were 95℃for 5min followed by 45 cycles, each set at 95℃for 15s,58℃for 60s, and finally 40℃for 30s.
3. Calculating the methylation degree:
after q-PCR is completed, the relative degree of methylation for CpG sites (Methylation Level, ML) is calculated, and the ML value is calculated as: 2 -[Ct(m)-Ct(u)] ，
CT m CT value, CT after amplification of the amplification primer and methylation probe for the site u CT values after amplification for the amplified primer and unmethylated probe for this site. If only one of the CT values is detected after q-PCR of the sample is completed, the other CT value is defined as 45 substituted into the calculation formula to calculate the relative methylation degree. If neither CT value is detected after the sample q-PCR is completed, the sample is defined to fail detection.
4. Result determination
Because the methylation properties of the EB virus in saliva of nasopharyngeal carcinoma patients and healthy control persons are quite different, ML=1 is directly set as a cut-off value, namely ML >1, the quantity of the EB virus DNA molecules which are methylated in saliva is reflected to be more than that of the EB virus DNA molecules which are not methylated, and the nasopharyngeal carcinoma is prompted; whereas ML <1, suggesting no nasopharyngeal carcinoma; ml=1, suggesting suspected nasopharyngeal carcinoma patients
Comparative example 1 method for detecting and diagnosing nasopharyngeal carcinoma aiming at methylation typing of Cp promoter region CpG sites of EBV by saliva sample
1. Primer and probe sequences designed for Cp:
forward primer: GAGTGTTATTTTTGGAATAGTAG the number of the individual pieces of the plastic,
reverse primer: TTAAACTCTCTTATTAACTATAATC the number of the individual pieces of the plastic,
methylation probe (probe 1): FAM-TGAATTTTGTTGGCGGGAGAAGGA-BHQ1, unmethylated probe (probe 2): HEX-TGAATTTTGTTGGTGGGAGAAGGA-BHQ1;
2. reaction system and reaction conditions for PCR reaction
As in example 1.
3. Calculating the methylation degree:
as in example 1.
4. Result determination
As in example 1.
Example 2 comparison of detection limits for methylation detection of the Wp promoter region and Cp promoter region
1. Experimental method
Taking nasopharyngeal carcinoma biopsy DNA as a positive reference. The DNA samples of the biopsies of nasopharyngeal carcinoma were subjected to 10-fold gradient dilution according to the EB virus content in the samples, 6 dilutions were performed consecutively, and the samples were simultaneously tested using the methods of example 1 and comparative example 1. Both methods were performed with 5 biological replicates.
2. Experimental results
The results are shown in Table 1 below, and the experimental results indicate that: (1) The detection method of example 1 has lower CT values than comparative example 1 by about 3 CT values, i.e., by about 8 times; (2) The detection method of example 1 can detect a lower number of methylation of EB virus, which can be increased by almost an order of magnitude relative to the detection method of comparative example 1.
Table 1:
example 3 comparison of detection success Rate and detection failure Rate of methylation detection of the Wp promoter region and Cp promoter region
1. Experimental method
Saliva DNA samples of 137 cases of nasopharyngeal carcinoma patients and 150 cases of non-nasopharyngeal carcinoma controls were examined by the methods of example 1 and comparative example 1, respectively, and these participants were recruited to the center nasopharyngeal clinic for tumor control at the university of Zhongshan, wherein 137 cases of nasopharyngeal carcinoma patients were diagnosed as nasopharyngeal carcinoma patients by nasopharyngeal biopsy, 150 cases of non-nasopharyngeal carcinoma controls were excluded from nasopharyngeal carcinoma by nasopharyngeal biopsy, cases of nasopharyngeal mucositis were diagnosed, and the examination success rate and the examination failure rate were counted.
Wherein success rate of detection = number of samples detected successfully/total number of samples detected,
failure rate of detection = number of samples that failed detection/total number of samples detected.
2. Experimental results
1. Comparison of test success Rate and test failure Rate
The results are shown in the following table 2,
table 2:
in the nasopharyngeal carcinoma patient group, the success rate of detection by the detection method of example 1 is 64.23%, the success rate of detection by the detection method of comparative example 1 is 45.26%, and the success rate of detection by the detection method of example 1 is about 19% higher than that by the detection method of comparative example 1.
In the non-nasopharyngeal carcinoma control group, the success rate of detection by the detection method of example 1 was 76.67%, the success rate of detection by the detection method of comparative example 1 was 65.33%, and the success rate of detection by the detection method of example 1 was about 11% higher than that by the detection method of comparative example 1.
The combined integration of the nasopharyngeal carcinoma patient and the non-nasopharyngeal carcinoma control shows that the detection success rate of the detection method of example 1 is 70.73%, the detection success rate of the detection method of comparative example 1 is 55.75%, and the detection success rate of the detection method of example 1 is about 15% higher than that of the detection method of comparative example 1.
2. Comparison of sensitivity and specificity
The results are shown in the following Table 3,
table 3:
when cov=1 was used as a cutoff value, and when it was greater than 1 and less than 1, and when it was determined as non-nasopharyngeal cancer, the sample successfully tested by the method of example 1 had a sensitivity of 75%, a specificity of 97.39%, an area under the curve (AUC) of 0.8771, and an about log index of 0.7239, both the area under the curve and the about log index were higher, and a better diagnostic effect was shown, compared to the test method of comparative example 1.
Example 5A kit for diagnosing nasopharyngeal carcinoma by detecting methylation of the Epstein-Barr virus Wp promoter region with respect to saliva samples
1. Composition of the composition
Detection probes, detection probes and q-PCR reagents for the probe method,
wherein, the detection primer is:
forward primer: GGGTAGAGATAGGTAGGGT (SEQ ID NO: 1);
reverse primer: CTCTACCTCCCAAACTTACC (SEQ ID NO: 2);
the detection probe comprises:
methylation probe (probe 1): FAM-TTTCGAGGAGGCGTTCGGAGTG-BHQ1 (SEQ ID NO: 3);
unmethylated probe (probe 2): HEX-TTTTGAGGAGGTGTTTGGAGTG-BHQ1 (SEQ ID NO: 4).
The q-PCR reagent of the probe method was 2 Xmaster mix and enzyme-free water.
2. Application method
1. PCR reaction
20. Mu.l of a reaction system containing 10. Mu.l of 2 Xmaster mix, 0.75. Mu.l of forward primer, 0.75. Mu.l of reverse primer, 0.8. Mu.l of methylation probe (probe 1), 2.8. Mu.l of methylation probe (probe 1), 4.9. Mu.l of enzyme-free water and 2. Mu.l of bisulfite-treated saliva DNA.
The reaction conditions were 95℃for 5min followed by 45 cycles, each set at 95℃for 15s,58℃for 60s, and finally 40℃for 30s.
2. Calculating the methylation degree:
after q-PCR is completed, the relative degree of methylation for CpG sites (Methylation Level, ML) is calculated, and the ML value is calculated as: 2 -[Ct(m)-Ct(u)] ，
CT m CT value, CT after amplification of the amplification primer and methylation probe for the site u CT values after amplification for the amplified primer and unmethylated probe for this site.
If only one of the CT values is detected after q-PCR of the sample, the other CT value is defined as 45 substituted into the calculation formula to calculate the relative methylation density. If neither CT value is detected after the sample q-PCR is completed, the sample is defined to fail detection.
3. Result determination
Setting ML=1 as a cut-off value, namely ML >1, wherein the quantity of the EB virus DNA molecules which are methylated in the reaction saliva is more than that of the EB virus DNA molecules which are not methylated, so as to prompt the nasopharyngeal carcinoma; whereas ML <1, suggesting no nasopharyngeal carcinoma; ml=1, suggesting suspected nasopharyngeal carcinoma patients.
Claims (10)
1. The application of the reagent for methylation typing of CpG sites of the EB virus Wp promoter region in saliva in preparing a nasopharyngeal carcinoma diagnosis kit.
2. The use according to claim 1, wherein the CpG sites are one or several of the CpG sites at 12420bp, 12428bp, 12432bp, 15492bp, 15500bp, 15504bp, 18564bp, 18572bp, 18576bp, 21636bp, 21644bp, 21648bp, 24708bp, 24716bp, 24720bp, 27780bp, 27788bp, 27792bp, 30852bp, 30860bp, 30864bp, 33924bp, 33932bp and 33936bp of the epstein barr virus genome, nc_007605.1.
3. The use according to claim 2, wherein the reagent comprises a detection primer and/or a detection probe.
4. The use according to claim 2, wherein the nucleotide sequence of the detection primer is set forth in SEQ ID NO: 1-2.
5. The use according to claim 2, wherein the nucleotide sequence of the detection probe is set forth in SEQ ID NO: 3-4.
6. A group of detection primers for detecting nasopharyngeal carcinoma, which is characterized in that the nucleotide sequence is shown in SEQ ID NO: 1-2.
7. A group of detection probes for detecting nasopharyngeal carcinoma, which are characterized in that the nucleotide sequences are shown in SEQ ID NO: 3-4.
8. A kit for detecting nasopharyngeal carcinoma is characterized by comprising a reagent for methylation typing of CpG sites of a Wp promoter region of Epstein-Barr virus in saliva.
9. The kit of claim 8, wherein the CpG sites are one or more of the CpG sites at 12420bp, 12428bp, 12432bp, 15492bp, 15500bp, 15504bp, 18564bp, 18572bp, 18576bp, 21636bp, 21644bp, 21648bp, 24708bp, 24716bp, 24720bp, 27780bp, 27788bp, 27792bp, 30852bp, 30860bp, 30864bp, 33924bp, 33932bp and 33936bp of the epstein barr virus genome, and the epstein barr virus genome is nc_007605.1.
10. The kit according to claim 7, further comprising a q-PCR reagent according to the probe method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310502182.1A CN116694762A (en) | 2023-05-06 | 2023-05-06 | Kit for detecting nasopharyngeal carcinoma |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310502182.1A CN116694762A (en) | 2023-05-06 | 2023-05-06 | Kit for detecting nasopharyngeal carcinoma |
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| Publication Number | Publication Date |
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| CN116694762A true CN116694762A (en) | 2023-09-05 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310502182.1A Pending CN116694762A (en) | 2023-05-06 | 2023-05-06 | Kit for detecting nasopharyngeal carcinoma |
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| Country | Link |
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2023
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