CN109777889B - Application of EB virus genome variation site and kit - Google Patents
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Abstract
The invention belongs to the field of PCR detection kits, and relates to an application of a combination of variation sites selected from EB virus genomes in preparation of a nasopharyngeal carcinoma onset risk prediction kit, wherein the variation sites are as follows: loc162215, loc162476 and loc163364. The invention also provides a nasopharyngeal carcinoma onset risk prediction kit based on the variation sites of the EB virus genome, which comprises a PCR amplification primer and a single base extension primer for detecting the combination of 3 variation sites of the EB virus genome, wherein the variation sites are as follows: loc162215, loc162476 and loc163364. The kit can establish a nasopharyngeal carcinoma onset risk prediction kit based on the combination of 3 variation sites, estimates the relative risk degree of individuals suffering from nasopharyngeal carcinoma, is suitable for screening of people in a nasopharyngeal carcinoma high incidence area, and has positive guiding significance and important application value for tumor prevention, early diagnosis and early treatment of tumors and prognosis improvement.
Description
The present application is a divisional application of an invention patent application having an application number of 201511004514.5, an application date of 2015, 12/25, and an invention name of "a kit for detecting a disease-related EB virus mutation site".
Technical Field
The invention belongs to the field of PCR detection kits, and relates to application of a combination of variation sites selected from EB virus genomes in preparation of a nasopharyngeal carcinoma onset risk prediction kit, and the nasopharyngeal carcinoma onset risk prediction kit based on the variation sites of the EB virus genomes.
Background
With the extension of the average life span of human beings and the change of life behavior patterns and the like, malignant tumors have become important diseases seriously threatening human health and serious public health problems worldwide. In recent years, chinese malignant tumors are in a high incidence, new cases account for more than 20% of new cases in the world every year, and according to statistics, the number of new cancer cases in China is about 350 ten thousand every year, and the number of death cases exceeds 200 ten thousand every year, so that the cancer cases are important causes of death of people in China. Cancer can be reduced and controlled by implementing evidence-based cancer prevention, early discovery, and cancer patient management strategies.
Nasopharyngeal carcinoma is one of common malignant tumors in China, and new cases account for about 80% of the world every year, and are mostly seen in provinces such as Guangdong, guangxi, hainan, fujian, hunan and Jiangxi in the south China. The etiological research of nasopharyngeal carcinoma shows that genetic, environmental and EB virus infection is the main cause of the disease. Because the nasopharyngeal cavity is hidden and the early symptoms are not obvious, the early diagnosis rate is low, the survival rate of the nasopharyngeal cancer patients is greatly influenced, and the screening is a better method for improving the early diagnosis rate and reducing the death rate of the nasopharyngeal cancer patients so far.
EB virus is the first virus found to be related to human tumorigenesis, is widely infected in global population, has an infection rate of 90 percent and is latent in B lymphocytes of individuals in an asymptomatic infection mode; in addition to nasopharyngeal carcinoma, EB virus infection is also associated with a variety of tumorigenesis, including Burkitt's lymphoma, hodgkin's lymphoma, NK/T cell lymphoma, and some gastric cancers. The mechanisms of EB virus evolution and specific subtype selection, which finally lead to local high incidence of diseases such as nasopharyngeal carcinoma, and the like, are still to be explained. However, EB virus typing is the basis for identifying differences in viral gene sequences and for discovering disease-specific virus subtypes. A great deal of research work in the past mainly classifies EB viruses according to gene sequence information of individual sites or regions of EB virus genomes, some types of the EB viruses are not specific for nasopharyngeal carcinoma, the infection rate of EB virus subtypes close to that of nasopharyngeal carcinoma patients exists in normal control populations in the same region, and compared with EB virus genomes of about 172Kb, the exhaustion strategy greatly reduces the possibility of detecting EB virus subtypes related to nasopharyngeal carcinoma.
EB virus has been identified as a class I carcinogen by the International agency for research on cancer (IARC), and nasopharyngeal carcinoma has been closely associated with EB virus. But an effective nasopharyngeal carcinoma onset risk prediction product is lacked at present. Therefore, EB virus sequences are analyzed at the whole genome level to identify nasopharyngeal carcinoma related virus variation sites and virus subtypes, and prediction of nasopharyngeal carcinoma onset risks based on EB virus variation sites is necessary. The risk prediction kit can be used for screening of people in a nasopharynx cancer high-risk area, early diagnosis and early treatment of high-risk people with nasopharynx cancer and disease treatment research, and finally enables nasopharynx cancer patients to be diagnosed and effectively treated in time, so that prognosis of the patients is improved, and tumor burden of individuals, families and society is relieved.
Disclosure of Invention
The first purpose of the invention is to provide the application of the combination of variation sites selected from EB virus genome in preparing a kit for predicting the onset risk of nasopharyngeal carcinoma.
The invention discloses an application of a combination of 3 variation sites selected from EB virus genomes in preparation of a nasopharyngeal carcinoma onset risk prediction kit, wherein the variation sites are as follows: loc162215, loc162476 and loc163364.
The second purpose of the invention is to provide a nasopharyngeal carcinoma onset risk prediction kit based on the variation site of EB virus genome.
The nasopharyngeal carcinoma onset risk prediction reagent kit comprises a PCR amplification primer and a single base extension primer which are used for detecting the combination of 3 variation sites of an EB virus genome, wherein the variation sites are as follows: loc162215, loc162476 and loc163364.
According to the further characteristics of the nasopharyngeal carcinoma onset risk prediction kit, the kit further comprises a DNA extraction reagent and a PCR reaction reagent.
According to the further characteristics of the kit for predicting the onset risk of nasopharyngeal carcinoma, the sequences of the PCR amplification primers for detecting loc162215 are SEQ ID NO:94 and SEQ ID NO:95 respectively, and the sequence of the single base extension primer is SEQ ID NO:96; the sequences of the PCR amplification primers for detecting loc162476 are respectively SEQ ID NO 97 and SEQ ID NO 98, and the sequence of the single base extension primer is SEQ ID NO 99; the sequences of the PCR amplification primers for detecting loc163364 are SEQ ID NO 103 and SEQ ID NO 104 respectively, and the sequence of the single base extension primer is SEQ ID NO 105.
Based on the research on the EB virus whole genome association of nasopharyngeal carcinoma population and normal control population based on large-scale sample size, 36 EB virus variation sites (see the attached sequence table) are identified for the first time, so that the kit for detecting the EB virus variation sites related to diseases is prepared. By detecting 3 EB virus variation sites, namely loc162215, loc162476 and loc163364, EB virus subtype classification standards can be established, the relative risk degree of an individual suffering from nasopharyngeal carcinoma is estimated, the method is suitable for screening of people in a high-incidence area of the nasopharyngeal carcinoma, and the method has positive guiding significance and important application value for tumor prevention, early diagnosis and early treatment of the tumor and improvement of prognosis.
The inventor adopts a method of virus whole genome targeted capture sequencing and case control research, and successfully verifies the 36 EB virus variation sites in a large-scale independent population sample, wherein the 36 EB virus variation sites comprise 3 EB virus variation sites, namely loc162215, loc162476 and loc163364. By utilizing the kit, genotyping detection can be carried out on genomic DNA (deoxyribonucleic acid) of tissues, peripheral blood or saliva of a detected person, and EB virus subtypes at high risk and low risk of nasopharyngeal carcinoma can be effectively identified by further combining with EB virus subtype classification standards of 3 variation site combinations created by the inventor, so that the risk degree of the detected person suffering from the nasopharyngeal carcinoma is evaluated.
The kit is simple and convenient to operate, stable in system and accurate in result, estimates the relative risk degree of an individual suffering from nasopharyngeal carcinoma through the EB virus subtype classification standard, utilizes a risk scoring model to conjecture the risk degree of the person suffering from nasopharyngeal carcinoma, overcomes the defect that the existing risk judgment of the nasopharyngeal carcinoma is carried out only by depending on results of few susceptibility gene indexes and the like, can detect the person suffering from nasopharyngeal carcinoma at any age stage, is suitable for screening of people in a nasopharyngeal carcinoma high-incidence area, and has positive guiding significance and important application value for tumor prevention, early diagnosis and early treatment of tumors and improvement of prognosis.
Detailed Description
Identification of disease-associated EB virus variation sites
Taking the EB virus related disease-nasopharyngeal carcinoma as an example, EB virus variation sites are determined.
Through the EB virus whole genome association research of 157 nasopharyngeal carcinoma patients and 47 normal control populations from a high incidence area, EB virus variation sites which are obviously related to the incidence of the nasopharyngeal carcinoma are identified, and 36 variation sites to be researched are determined by combining the biological significance and the success rate of a site typing experiment.
In the cohort studied by the inventors, mutation site association analysis was performed to screen the following 36 sites of the epstein barr virus genome: loc613, loc629, loc632, loc2773, loc6210, loc6584, loc7048, loc40607, loc44531, loc60527, loc62500, loc63009, loc67746, loc84414, loc108347, loc115575, loc122141, loc126187, loc128269, loc132225, loc137316, loc139684, loc145737, loc145919, loc146628, loc150022, loc 3828 xzft 3828, loc 3925 zxft 3983, loc 54545427, loxft 349678, and loxft 349635, and loxft 349696, and loxft 349635, and loxft 3435, and 3446, and loxft 3296.
GenBank accession number is NC _007605.1.
The correlation degree of the 36 variable sites and the nasopharyngeal carcinoma is shown in the table 1.
Table 1: degree of association of 36 sites of EB virus genome with nasopharyngeal carcinoma (157 nasopharyngeal carcinoma patients and 47 normal control population)
(II) kit for detecting EB virus variation sites related to diseases
The kit of the invention comprises: PCR amplification primers and single base extension primers for detecting 36 variation sites of EB virus genome, wherein the 36 variation sites are loc613, loc629, loc632, loc2773, loc6210, loc6584, loc7048, loc40607, loc44531, loc60527, loc62500, loc 009, loc67746, loc84414, loc108347, loc115575, loc 141, loc126187, loc128269, loc132225, loc137316, loc 57 zxft 3757, loc145737, loc145919, loc146628, loc 3826 zxft 3726, 54zxft 3726, lox 3896, lox 3978, lox 3296, lox 3896, lox 3224 zxft 3296, lox 3226, lox 3296, lox 3224 zxft. GenBank accession number is NC _007605.1.
1. Preparation of the kit
1. PCR amplification primer and single base extension primer for designing and synthesizing mutation site
According to the sequence of 200bp on the upstream and downstream of 36 variation sites of EB virus, PCR amplification primers and single base extension primers of the variation sites are designed and synthesized.
Table 2: the sequence of 200bp on the upstream and downstream of 36 variation sites of EB virus determined by the invention
The sequences of PCR amplification primers (PCRP) and single base extension primers (UEP) designed according to the sequences in Table 2 are shown in Table 3 below. It should be noted that the sequences of the PCR amplification primer (PCRP) and the single base extension primer (UEP) that are varied by one or more bases based on the sequences in Table 3 and still can amplify the sequences in Table 2 are within the scope of the present invention.
Table 3: sequences of PCR amplification primer (PCRP) and single base extension primer (UEP) for detecting mutation site
2. Construction of the kit
Other components of the kit include: PCR enzyme, dNTP mixed solution, buffer solution, diluent and the like, and the details are shown in the following detection method.
2. Detection method
1. DNA extraction
DNA in tissues, blood and saliva is extracted using QIAGEN DNeasy Blood & Tissue Kit, QIAGEN QIAamp DNA Blood Midi Kit, QIAGEN QIAamp DNA Midi Kit or the like. The total amount of DNA is determined by a spectrophotometer, and the agarose gel electrophoresis quality is checked and the electrophoresis band is usually not less than 20kb. After quality control, the DNA concentration was adjusted to 50 ng/. Mu.l and transferred to 384 well plates and stored at-20 ℃ until use.
2. PCR amplification
PCR amplification was performed in 384-well plates using multiplex PCR, with a total volume of 5. Mu.l per reaction system.
(1) PCR MasterMix solution was prepared in 1.5ml EP tubes.
| PCR Master Mix | For each reaction, μ l |
| 10 XPCR buffer | 0.5 |
| MgCl2(25mM) | 0.4 |
| dNTP mix(25mM) | 0.1 |
| HotStar Taq(5U/μl) | 0.1 |
| Water (W) | 1.9 |
| PCR primer mixture | 1 |
| Total volume | 4 |
(2) Mu.l of PCR Master Mix solution was added to each well of the 384-well plate using a 24-channel applicator. The 384-well plate is a PCR reaction plate.
(3) The prepared DNA sample 384-well plate was removed, and 1. Mu.l of the DNA sample was added to each well of the PCR reaction plate using a 24-channel sample applicator, and each 5. Mu.l of the PCR reaction system contained 20 to 50ng of template DNA, 0.5U of Hotstart Taq, 0.5pmol each of the two amplification primers, and 0.1. Mu.l of 25mM dNTPs.
(4) The PCR reaction conditions were set on a 384-well compatible PCR instrument as follows: 4 minutes at 94 ℃; 20 seconds at 94 ℃,30 seconds at 56 ℃, 1 minute at 72 ℃ and 45 cycles; 3 minutes at 72 ℃; keeping at 4 ℃. The 384-well PCR reaction plate was placed on a PCR instrument, and the PCR reaction was initiated.
3. PCR product shrimp alkaline phosphatase treatment
(1) After the PCR reaction was completed, the reaction product was treated with SAP (shrimp alkaline phosphatase) to remove free dNTPs in the system.
(2) A shrimp alkaline phosphatase treatment reaction solution (SAP Mix) was prepared.
| SAP Mix | For each reaction, μ l |
| Water (W) | 1.53 |
| SAP buffer (10X) | 0.17 |
| SAP enzyme (1.7U/. Mu.l) | 0.3 |
| Total volume | 2 |
(3) Using a 24-channel applicator, 2. Mu.l of SAP Mix were added to each well of a 384-well PCR reaction plate. Each Shrimp Alkaline Phosphatase (SAP) treated well had a total volume of 7. Mu.l, 5. Mu.l of PCR product, and 2. Mu.l of SAP mixture.
(4) The 384-well plate was placed on a 384-well plate compatible PCR instrument, and the PCR reaction conditions were set: 40 minutes at 36 ℃; 5 minutes at 85 ℃; the temperature is maintained at 4 ℃, and a PCR instrument is started to perform shrimp alkaline phosphatase treatment.
4. Single base extension
(1) After the completion of the shrimp alkaline phosphatase treatment reaction, single-base extension reaction was carried out in a total volume of 9. Mu.l.
(2) A single-base extension reaction solution (EXTEND Mix) was prepared.
| EXTEND Mix | For each reaction, μ l |
| Water (W) | 0.619 |
| Extension primer mixture 1 or 2 | 0.94 |
| iPLEX buffer plus | 0.2 |
| iPLEX stop solution | 0.2 |
| iPLEX enzyme | 0.041 |
| Total volume | 2 |
(3) Using a 24-channel applicator, 2. Mu.l of EXTEND Mix was added to each reaction well of a 384-well reaction plate. For each reaction well, the single base extension reaction system contained 7. Mu.l of SAP-treated product and 2. Mu.l of EXTEND Mix reaction solution.
(4) Placing the 384-well plate on a PCR instrument compatible with the 384-well plate, and setting the PCR reaction conditions as follows:
i.94 ℃ for 30 seconds
II.94 ℃ for 5 seconds
III.52 ℃ for 5 seconds
IV.80 deg.C, 5 seconds
V. back to III,4 times or more
VI, back to II,39 times or more
VII.72 ℃ for 3 minutes
VIII.4 ℃ Retention
And starting a PCR instrument to perform single base extension reaction.
5. Resin purification
(1) Lay Clean Resin flat into 6mg Resin preparation board;
(2) Add 16. Mu.l of water to each reaction well containing the extension product;
(3) Pouring the dried resin into a reaction plate for the extension products, sealing the membrane, and vertically rotating at a low speed for 30 minutes to ensure that the resin is fully contacted with reactants;
(4) The resin was allowed to sink to the bottom of the reaction well by centrifugation at 4000rpm for 2 minutes.
6. Chip sample application
The SEQUENOM MassARRAY Nanodispenser RS1000 spotter was started and the resin purified extension product was transferred to 384-well SpectroCHIP (SEQUENOM) chips.
7. Mass spectrometric detection
The spotted SpectroCHIP chip is detected by using MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time of flight-mass spectrometry), and the detection result is typed by using TYPER 4.0 Software (SEQUENOM) and output.
3. Analysis of results
For the effect of the kit of the present invention, the inventors analyzed the P values and the ratio (OR) of the 36 EB virus variation sites by independent validation of 513 nasopharyngeal carcinoma patients and 640 normal control populations with large sample size using logistic regression model, and the OR value of each variation site was evaluated by logistic regression analysis after adjusting age and gender. The analytical results are shown in the following table.
Table 4: correlation analysis of nasopharyngeal carcinoma onset risk and 36 EB virus variation sites (513 nasopharyngeal carcinoma patients and 640 normal control population)
As shown in Table 4, correlation analysis of logistic regression revealed that 36 EB virus variation sites were significantly associated with the risk of nasopharyngeal carcinoma, wherein the variation sites loc162215, loc162476 and loc163364 were highly correlated with nasopharyngeal carcinoma, OR (95% confidence interval, 95% CI) was 0.15 (0.10-0.24), 8.21 (5.50-12.24) and 6.48 (4.85-8.66), and the test P values were all less than 0.001.
Claims (4)
1. Use of a combination of 3 variant sites selected from the group consisting of EB virus genome, wherein the variant sites are: loc162215C/A, loc162476T/C and loc163364C/T.
2. A nasopharyngeal carcinoma onset risk prediction kit, characterized in that: the kit comprises a PCR amplification primer and a single base extension primer for detecting the combination of 3 variation sites of an EB virus genome, wherein the variation sites are as follows: loc162215C/A, loc162476T/C and loc163364C/T.
3. The nasopharyngeal cancer onset risk prediction kit according to claim 2, wherein: also comprises a DNA extraction reagent and a PCR reaction reagent.
4. The nasopharyngeal carcinoma onset risk prediction kit according to claim 2, wherein:
the sequences of the PCR amplification primers for detecting loc162215 are respectively SEQ ID NO:94 and SEQ ID NO:95 and the sequence of the single base extension primer is SEQ ID NO:96;
the sequences of the PCR amplification primers for detecting loc162476 are respectively SEQ ID NO:97 and SEQ ID NO:98, and the sequence of the single base extension primer is SEQ ID NO:99;
the sequences of the PCR amplification primers for detecting loc163364 are respectively SEQ ID NO:103 and SEQ ID NO:104 and the sequence of the single base extension primer is SEQ ID NO:105.
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| CN201511004514.5A CN105506180B (en) | 2015-12-25 | 2015-12-25 | Detect the kit of the relevant Epstein-Barr virus variant sites of disease |
| CN201811205535.7A CN109777889B (en) | 2015-12-25 | 2015-12-25 | Application of EB virus genome variation site and kit |
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| CN111690056B (en) * | 2020-04-30 | 2021-12-21 | 清华大学 | Monoclonal antibody for neutralizing EB virus and application thereof |
| CN115786509A (en) * | 2022-10-21 | 2023-03-14 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Nasopharynx cancer risk prediction kit |
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| CN103045743A (en) * | 2012-12-28 | 2013-04-17 | 中山大学肿瘤防治中心 | Kit for detecting susceptibility gene SNP locus of nasopharynx cancer |
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| CN103045743A (en) * | 2012-12-28 | 2013-04-17 | 中山大学肿瘤防治中心 | Kit for detecting susceptibility gene SNP locus of nasopharynx cancer |
Non-Patent Citations (4)
| Title |
|---|
| A single nucleotide polymorphism in the Epstein-Barr virus genome is strongly associated with a high risk of nasopharyngeal carcinoma;Fu-Tuo Feng等;《Chin J Cancer》;20151216;第34卷(第12期);摘要、"Result"以及表3 * |
| Epstein–Barr Virus Genetic Variation in Lymphoblastoid Cell Lines Derived from Kenyan Pediatric Population;Kenneth O Simbiri等;《PLoS One》;20150501;第10卷(第5期);摘要 * |
| Genome Diversity of Epstein-Barr Virus from Multiple Tumor Types and Normal Infection;Anne L. Palser等;《Journal of Virology》;20150318;第89卷(第10期);摘要、材料和方法 * |
| Genome-Wide Analysis of Epstein-Barr virus（EBV） Isolated from EBV-associated gastric carcinoma（EBVaGC）;Ying Liu等;《Oncotarget》;20151224;第7卷(第4期);摘要、图6、补充材料表4-5 * |
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