CN116574810A - Kit for diagnosing nasopharyngeal carcinoma by detecting methylation of ZNF671 and ZMYND10 genes - Google Patents

Kit for diagnosing nasopharyngeal carcinoma by detecting methylation of ZNF671 and ZMYND10 genes Download PDF

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CN116574810A
CN116574810A CN202310813663.4A CN202310813663A CN116574810A CN 116574810 A CN116574810 A CN 116574810A CN 202310813663 A CN202310813663 A CN 202310813663A CN 116574810 A CN116574810 A CN 116574810A
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李菲
葛毅媛
黄玄贺
郭奕轩
韩芝斌
许泽仰
胡晓
谢龙旭
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Chaozhou Kaipu Biochemistry Co ltd
Hybribio Ltd
Guangzhou Hybribio Medicine Technology Ltd
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Hybribio Ltd
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Abstract

The invention discloses a kit for diagnosing nasopharyngeal carcinoma by detecting methylation of ZNF671 and ZMYND10 genes. The invention discloses a primer probe combination for detecting methylation of ZNF671 and/or ZMYND10 genes, which is used for determining the type and enzyme cutting site of methylation sensitivity restriction enzyme based on the specific sequence design of the ZNF671 and ZMYND10 genes, realizing rapid enzyme cutting and PCR amplification in the same system, avoiding sulfite treatment, reducing DNA damage, incomplete conversion and the like. The multiplex methylation PCR reaction body constructed by the invention realizes the sensitivity and the accuracy of multi-target detection, is suitable for the auxiliary diagnosis of nasopharyngeal carcinoma, is beneficial to the early diagnosis and the early treatment of the nasopharyngeal carcinoma, and uses the noninvasive safe nasopharyngeal swab as a sample to be detected for detection, thereby reducing the pain and the detection cost of patients.

Description

Kit for diagnosing nasopharyngeal carcinoma by detecting methylation of ZNF671 and ZMYND10 genes
Technical Field
The invention relates to the technical field of molecular diagnosis, in particular to a kit for diagnosing nasopharyngeal carcinoma by detecting ZNF671 and ZMYND10 gene methylation.
Background
Nasopharyngeal carcinoma (nasopharyngeal carcinoma, NPC) originates from nasopharyngeal epithelial cells, a relatively common malignancy of the head and neck. The disease parts of the early nasopharyngeal carcinoma are hidden, the disease symptoms and signs are difficult to identify from other benign diseases, the false diagnosis missing phenomenon easily occurs, the patients already develop to the late stage when diagnosing, the risks of metastasis and recurrence are high, the clinical treatment effect and prognosis are poor, and the five-year survival rate of the patients is only 30% -45%. If the nasopharyngeal carcinoma is found in early stage, the cure rate is up to 80-90%, so that the "early prevention, early discovery and early treatment" is the best means for treating the nasopharyngeal carcinoma. The current clinical diagnosis method of nasopharyngeal carcinoma mainly comprises EBV serological antibody detection, plasma EBV DNA quantitative detection, imaging examination, nasopharyngeal mirror tissue biopsy and the like. The EBV serological antibody detection has the defect of insufficient specificity, and has a lower positive predictive value in nasopharyngeal carcinoma screening. The generation of EBV DNA in plasma mainly comes from the cleavage release after tumor cell apoptosis or necrosis, so the EBV DNA is fragmented, and the EBV DNA released into the plasma in early stage is less, so the sensitivity of the quantitative detection of the EBV DNA in the plasma for diagnosing early stage nasopharyngeal carcinoma is lower. Imaging MRI or CT examinations are safe and noninvasive, but are expensive and have certain false positives. The gold standard for diagnosing nasopharyngeal carcinoma is tissue pathological biopsy under a nasopharyngeal scope, but the materials are invasive and strictly depend on clinical experience of a clinician. Therefore, finding new noninvasive and effective molecular markers for auxiliary diagnosis, efficacy monitoring and prognosis evaluation of nasopharyngeal carcinoma is necessary.
DNA methylation is a common apparent modification that plays an important regulatory role in the growth, development, gene expression pattern, and genomic stability of individuals without altering the DNA sequence. In recent years, a great deal of research shows that abnormal methylation of DNA has a close relation with the occurrence, development and canceration of tumors. Studies have shown that in the course of nasopharyngeal carcinoma, methylation of the methylation regions of a broad range of gene promoters, their alteration is considered an important early event in the course of nasopharyngeal carcinoma. Since nasopharyngeal carcinoma originates from the nasopharyngeal site, detection of tumor molecular markers that are highly efficient and noninvasively available for the nasopharyngeal site can more directly reflect the condition of the lesion site. Therefore, DNA methylation using nasopharyngeal brush sample types is expected to be an effective tumor marker for early diagnosis, efficacy monitoring and prognosis evaluation of nasopharyngeal carcinoma. At present, most methylation detection needs to be performed by performing fluorescence PCR quantitative detection after DNA is converted by adopting traditional sulfite. The traditional sulfite transformation method has the defects of larger damage to nucleic acid, damage to the integrity of the nucleic acid, complicated operation process and longer time consumption. If the conversion is incomplete, false positives may result in the detection.
Methylation restriction enzymes are capable of specifically recognizing and cleaving sites, since 5' methylated cytosines can inhibit certain restriction enzymes from cleaving within their recognition sequences. Under the action of the methylation restriction enzyme, the unmethylated target sequence is cleaved by the enzyme, while the methylated target sequence remains intact, so that the methylated DNA can be distinguished from unmethylated DNA. By methylating the restriction enzyme, bisulfite conversion is avoided and the integrity of the nucleic acid is maintained, but the cleaved product is recovered and purified, which is time consuming, can easily cause contamination and loss of nucleic acid.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a kit for diagnosing nasopharyngeal carcinoma by detecting ZNF671 and ZMYND10 gene methylation.
The first object of the invention is to provide a primer probe combination for detecting methylation of ZNF671 and/or ZMYND10 genes.
The second object of the invention is to provide the application of the primer probe combination in preparing a kit for detecting and diagnosing nasopharyngeal carcinoma.
A third object of the invention is to provide a kit for diagnosing nasopharyngeal carcinoma.
In order to achieve the above object, the present invention is realized by the following means:
primer probe combination for detecting ZNF671 and/or ZMYND10 gene methylation, and nucleotide sequence of primer for detecting ZNF671 gene is shown as SEQ ID NO:1 and 2, the nucleotide sequence of the probe for detecting the ZNF671 gene is shown as SEQ ID NO:3 is shown in the figure; the nucleotide sequence of the primer for detecting the ZMYND10 gene is shown as SEQ ID NO:4 and 5, the nucleotide sequence of the probe for detecting the ZMYND10 gene is shown as SEQ ID NO:6 is shown in the figure;
accession numbers of the ZNF671 gene on NCBI are: NC_000019.10, accession number of the ZMYND10 gene on NCBI is: nc_000003.12.
In the invention, the primer probe combination also comprises a primer and a probe for detecting the reference gene ACTB, and the nucleotide sequence of the primer for detecting the reference gene ACTB is shown as SEQ ID NO:7 and 8, the nucleotide sequence of the probe for detecting the reference gene ACTB is shown as SEQ ID NO: shown at 9.
In the detection process, it is also necessary to label the probe with a fluorescent group and a quenching group. The fluorescent group marked at the 5 'end of the probe for detecting the ZNF671 gene is VIC, and the fluorescent quenching group marked at the 3' end of the probe for detecting the ZNF671 gene is BHQ1; the fluorescent group marked at the 5 'end of the probe for detecting the ZMYND10 gene is FAM, and the fluorescent quenching group marked at the 3' end of the probe for detecting the ZMYND10 gene is MGB.
Further, the fluorescent group marked at the 5 'end of the probe for detecting the reference gene ACTB is CY-5, and the fluorescent quenching group marked at the 3' end of the probe for detecting the reference gene ACTB is BHQ2.
The invention also discloses application of the primer probe combination in preparing a kit for detecting and diagnosing nasopharyngeal carcinoma.
The invention provides a kit for diagnosing nasopharyngeal carcinoma according to the primer probe combination, wherein the kit comprises the primer probe combination.
Further, the kit also contains PCR detection reagents.
In order to avoid the disadvantages of the bisulfite conversion treatment, the kit further comprises a methylation sensitive restriction enzyme comprising one or more of HhaI, bsh1236I and HpaII.
Preferably, the methylation sensitive restriction enzyme is a mixture of three enzymes, hhaI, bsh1236I and HpaII.
Further, three enzymes, hhaI, bsh1236I and HpaII, were mixed in equal volumes.
Further, the PCR detection reagent comprises an enzyme digestion amplification buffer solution, taq DNA polymerase and water without nuclease, wherein the enzyme digestion amplification buffer solution contains Tris-HCl, KCl, mgCl 2 One or more of glycerol, BSA, dimethyl sulfoxide, tween 20, dNTP and dithiothreitol.
Further, the kit also contains positive quality control C666-1 human nasopharyngeal carcinoma cell line DNA and negative quality control NP96 human nasopharyngeal epithelium cell line DNA.
When the kit is used, the method comprises the following steps:
1. taking a nasopharyngeal swab sample of a person to be tested, and extracting sample DNA, namely the DNA template.
2. PCR amplification and detection:
preparing a PCR reaction system comprising the above primer and probe combination, an enzyme-cleaved amplification buffer, a methylation-sensitive restriction enzyme, a DNA polymerase, a DNA template and H 2 O composition.
The concentration of each of the above primers and probes may be 8 to 12. Mu.M, the concentration of the methylation-sensitive restriction enzyme may be 3 to 8U/. Mu.L, and the concentration of the DNA polymerase may be 3 to 8U/. Mu.L.
In the PCR reaction system, the volume of each primer can be 0.3-0.8 mu L, the volume of each probe can be 0.1-0.5 mu L, and the enzyme digestion amplification buffer solution can be prepared from 10-50 mmol/L Tris-HCl, 160-250 mmol/L KCl and 20-50 mmol/L MgCl 2 3 to 5 percent (W/V) of glycerin, 1 to 5 mg/mL of BSA, 0.5 to 1.5 percent (V/V) of dimethyl sulfoxide, 0.1 to 0.4 percent (V/V) of Tween 20, 0.3 to 1 mmol/L of dNTP and 3 to 8 mmol/L of dithiothreitol.
The methylation sensitive restriction enzyme can have a volume of 1-4. Mu.L, the DNA polymerase can have a volume of 0.5-2. Mu.L, the DNA template can have a volume of 3-8. Mu.L, and H 2 The volume of O may be 5 to 15. Mu.L.
In a specific embodiment of the present invention, the concentration of each of the above primers and probes is 10. Mu.M, the concentration of the methylation-sensitive restriction enzyme is 5U/. Mu.L, and the concentration of the DNA polymerase is 5U/. Mu.L.
In the PCR reaction system, the volume of each primer is 0.6 mu L, the volume of each probe is 0.3 mu L, and the enzyme digestion amplification buffer solution consists of 40 mmol/L Tris-HCl, 200 mmol/L KCl and 30 mmol/L MgCl 2 4% (W/V) glycerol, 4 mg/mL BSA, 1% (V/V) dimethyl sulfoxide, 0.2% (V/V) Tween 20, 0.5 mmol/L dNTP and 5mmol/L dithiothreitol.
The methylation sensitive restriction enzyme had a volume of 3. Mu.L, the DNA polymerase had a volume of 1. Mu.L, the DNA template had a volume of 5. Mu.L, H 2 The volume of O was 9. Mu.L.
The PCR amplification procedure was: 37 ℃,15 min,1 cycle; 95 ℃,5 min,1 cycle; fluorescence was collected for a single cycle at 95 ℃,10 sec,60 ℃,30 sec, 45 cycles; 38 ℃,30 sec,1 cycle.
3. Decision criterion
The condition that the result of detecting the sample to be detected is effective is shown as follows: detecting the Ct value of ZNF671 and ZMYND10 genes of the negative quality control product to be more than 40 or no amplification, wherein the Ct value t of an internal reference gene ACTB is less than or equal to 35; and the Ct value of ZNF671 and ZMYND10 genes of the positive quality control product is less than or equal to 30, and the Ct value t of the reference gene ACTB is less than or equal to 35.
The result of detecting the sample to be detected is positive: detecting the ZNF671 or ZMYND10 gene of the nasopharynx swab sample, wherein the Ct is less than or equal to 40, the amplification curve is of an S type, and the Ct value t of the internal reference gene ACTB is less than or equal to 35.
The result of detecting the sample to be detected is negative: detecting the Ct >40 or no amplification of ZNF671 and ZMYND10 genes of the nasopharynx swab sample, wherein the Ct value t of the reference gene ACTB is less than or equal to 35.
If the Ct value of the reference gene ACTB is more than 35, the experiment is invalid, and the added DNA amount is too small, so that the DNA needs to be extracted again for detection.
If the interpretation result is negative, the risk of suffering from nasopharyngeal carcinoma is low, and regular follow-up is recommended; if the interpretation result is positive, the risk of nasopharyngeal carcinoma is high, and microscopic examination or tissue biopsy confirmation is recommended.
Further, the concentration of each of the above primers and probes may be 10. Mu.M, the concentration of the methylation-sensitive restriction enzyme may be 5U/. Mu.L, and the concentration of the DNA polymerase may be 5U/. Mu.L.
In the PCR reaction system, the volume of each of the primers may be 0.6. Mu.L, the volume of each of the probes may be 0.3. Mu.L, the volume of the methylation-sensitive restriction enzyme may be 3. Mu.L, the volume of the DNA polymerase may be 1. Mu.L, the volume of the DNA template may be 5. Mu.L, and H 2 The volume of O was 9. Mu.L.
Compared with the prior art, the invention has the following beneficial effects:
the invention designs a primer and a probe based on the specific sequences of ZNF671 and ZMYND10 genes, determines the type and the enzyme cutting site of methylation sensitive restriction enzyme, realizes rapid enzyme cutting and PCR amplification in the same system, does not need sulfite treatment, and reduces the problems of DNA damage, incomplete conversion and the like.
The multiplex methylation PCR reaction body constructed by the invention realizes the sensitivity and the accuracy of multi-target detection, is suitable for the auxiliary diagnosis of nasopharyngeal carcinoma, is beneficial to the early diagnosis and the early treatment of the nasopharyngeal carcinoma, and uses the noninvasive safe nasopharyngeal swab as a sample to be detected for detection, thereby reducing the pain and the detection cost of patients.
Drawings
FIG. 1 is an amplification plot of methylation detection of nasopharyngeal swab samples of a patient with clinically established nasopharyngeal carcinoma using the kit of example 1 of the present invention.
FIG. 2 is an amplification plot of methylation detection of nasopharyngeal swab samples of a patient clinically diagnosed with non-nasopharyngeal carcinoma using the kit of example 1 of the present invention.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration 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 1A kit for diagnosing nasopharyngeal carcinoma
1. Composition of the composition
1. ZNF671 (Gene accession number: NC_ 000019.10), ZMYND10 (Gene accession number: NC_ 000003.12) and the primer probe combinations of the ACTB genes are shown in Table 1.
Wherein the fluorescent group marked at the 5 'end of the ZNF671-P1 probe is VIC, and the fluorescent quenching group marked at the 3' end is BHQ1; the fluorescent group marked at the 5 'end of the ZMYND10-P1 probe is FAM, and the fluorescence quenching group marked at the 3' end is MGB; the fluorescent group marked at the 5 '-end of the ACTB-P1 probe is CY-5, and the fluorescence quenching group marked at the 3' -end is BHQ2.
TABLE 1 primer probe combinations
In Table 1, the nucleotide sequence of ZNF671 gene is shown as SEQ ID NO:10, designing an upstream primer and a downstream primer and a probe; the nucleotide sequence of the ZMYND10 gene is shown as SEQ ID NO:11, designing an upstream primer and a downstream primer and a probe; the nucleotide sequence of ACTB gene is shown as SEQ ID NO:12, and designing upstream and downstream primers and probes.
2. PCR reaction solution:
(1) Enzyme digestion amplification buffer: from 40 mmol/L Tris-HCl, 200 mmol/L KCl, 30 mmol/L MgCl 2 4% (W/V) glycerol, 4 mg/mL BSA, 1% (V/V) dimethyl sulfoxide (DMSO), 0.2% (V/V) Tween 20, 0.5 mmol/L dNTP and 5mmol/L dithiothreitol.
(2) Taq DNA polymerase.
(3) Methylation sensitive restriction enzyme Mix: hhaI, bsh1236I and HpaII were mixed in equal volumes.
(4) Nuclease-free water.
The above reagents are available from TAKALA Co.
3. Controlling the nature of yang: c666-1 human nasopharyngeal carcinoma cell line DNA; negative quality control: NP96 human nasopharyngeal epithelial cell line DNA.
2. Application method
1. The nasopharyngeal swab of the subject was sampled, and the sample DNA was extracted using the extraction kit Qiagen QIAamp DNA Blood Mini Kit (accession number 51106) of Kaiji Biotechnology Co.
2. PCR amplification and detection
(1) A20. Mu.L PCR reaction system was prepared according to the following Table 2, and the sample DNA was used as a DNA template for detection, and the positive and negative controls were used as controls to replace the DNA template.
TABLE 2 PCR reaction System
(2) And (3) subpackaging the PCR reaction system into PCR eight-joint tubes, and placing the PCR eight-joint tubes into an ABI 7500 fluorescent PCR instrument for reaction. The instrument fluorescence channel settings are shown in table 3 and the amplification procedure settings are shown in table 4.
TABLE 3 fluorescent channel setup
TABLE 4 PCR amplification procedure
3. Decision criterion
As shown in table 5 below, if the interpretation is negative, the risk of having nasopharyngeal carcinoma is low, and periodic follow-up is recommended; if the interpretation result is positive, the risk of nasopharyngeal carcinoma is high, and microscopic examination or tissue biopsy confirmation is recommended.
TABLE 5 criterion
Example 2 example 1 kit detection of nasopharyngeal swab samples
1. Experimental method
Nasopharyngeal swab samples from 60 patients with nasopharyngeal carcinoma and 80 patients with non-nasopharyngeal carcinoma were tested using the kit of example 1.
2. Experimental results
The results are shown in Table 6, with the sensitivity and specificity of the kit of example 1 being 95% (57/60) and 93.75% (75/80), respectively. As shown in FIG. 1, an amplification graph of methylation detection of nasopharyngeal swab samples of a patient clinically diagnosed with nasopharyngeal carcinoma by using a kit according to example 1 of the present invention is shown. As shown in FIG. 2, an amplification plot of a nasopharyngeal swab sample from a patient clinically diagnosed with non-nasopharyngeal carcinoma in accordance with the kit of example 1 of the present invention is shown. Sensitivity (%) =number of positive detected/total number of positive cases, specificity (%) =number of negative detected/total number of negative cases.
Table 6 example 1 kit results for testing nasopharyngeal swab samples
Example 3 detection Effect of restriction enzymes with different methylation sensitivities
1. Experimental method
In methylation sensitive restriction endonucleases, hhaI, bsh1236I, hpaII recognize GCG/C, CG/CG, C/CGG sequences, respectively, where slash "/" indicates cleavage sites.
The present example determines the cleavage site by using the type of methylation-sensitive restriction enzyme.
In the kit of example 1, the methylation restriction enzymes Mix were mixed in equal volumes of HhaI, bsh1236I, hpaII, hhaI and Bsh1236I, hhaI and HpaII, bsh1236I and HpaII, and HhaI, bsh1236I and HpaII, respectively, and negative samples were tested using sterilized water as a control, and experiments were performed in accordance with the method of example 1.
2. Experimental results
As shown in Table 7, the enzyme groups 1-8 can not completely cleave the unmethylated sequence of the negative sample, which may result in false positive results; when three methylation restriction enzymes, hhaI, bsh1236I, hpaII, are used in combination, the negative samples can be completely digested. Thus, the methylation restriction enzyme Mix should be a combination of three methylation restriction enzymes, hhaI, bsh1236I and HpaII.
TABLE 7 negative sample detection results for different methylation-sensitive restriction endonucleases
Example 4 detection Effect of different primer probe sequences
1. Experimental method
As shown in Table 8, the nucleotide sequence of ZNF671 gene is shown as SEQ ID NO:10, the upstream and downstream primers and probes in comparative sets 1 and 2; the nucleotide sequence of the ZMYND10 gene is shown as SEQ ID NO:11, and the upstream and downstream primers and probes in panels 1 and 2.
Wherein, the modification of the fluorescent groups of the probe ZNF671-P2 and the probe ZNF671-P3 is the same as that of the probe ZNF671-P1 in example 1, and the modification of the fluorescent groups of the probe ZMYND10-P2 and the probe ZMYND10-P3 is the same as that of the probe ZMYND10-P1 in example 1.
Other reagents and detection parameters were the same as in example 1, and according to the method of example 1, 10 nasopharyngeal swab samples of patients with nasopharyngeal carcinoma and 10 patients without nasopharyngeal carcinoma were detected, and the primer probe combination with the optimal detection effect was determined.
TABLE 8 design different primer probe sequences for ZNF671 and ZMYND10 Gene specific fragments
2. Experimental results
As shown in table 9, the sensitivity and the specificity of the primer probe combinations of example 1 were 90% and 100%, respectively, the sensitivity and the specificity of the primer probe combinations of comparative group 1 were 70% and 70%, respectively, and the sensitivity and the specificity of the primer probe combinations of comparative group 2 were 50% and 60%, respectively.
The sensitivity of the primer probe combination of example 1 of the present invention is significantly higher than the other two control groups. Sensitivity (%) =number of positive detected/total number of positive cases, specificity (%) =number of negative detected/total number of negative cases.
Table 9 detection results of probe sequences of different primers designed for ZNF671 and ZMYND10 genes
Example 5 detection Effect of different ZNF671 Gene target fragments
1. Experimental method
As shown in Table 10, the nucleotide sequence of ZNF671 gene is shown as SEQ ID NO:25, and the upstream and downstream primers and probes in panels 3 and 4; the nucleotide sequence of the ZMYND10 gene is shown as SEQ ID NO:26, and the upstream and downstream primers and probes in panels 3 and 4.
Wherein, the modification of the fluorescent groups of the probe ZNF671-P4 and the probe ZNF671-P5 is the same as that of the probe ZNF671-P1 in example 1, and the modification of the fluorescent groups of the probe ZMYND10-P4 and the probe ZMYND10-P5 is the same as that of the probe ZMYND10-P1 in example 1.
Other reagents and detection parameters were the same as in example 1, and according to the method of example 1, 10 nasopharyngeal swab samples of patients with nasopharyngeal carcinoma and 10 patients without nasopharyngeal carcinoma were detected, and the primer probe combination with the optimal detection effect was determined.
Table 10 designs different primer probe sequences for different target fragments of ZNF671 and ZMYND10 genes
2. Experimental results
As shown in table 11, the sensitivity and the specificity of the primer probe combinations of example 1 were 90% and 100%, respectively, the sensitivity and the specificity of the primer probe combinations of comparative group 3 were 60% and 40%, respectively, and the sensitivity and the specificity of the primer probe combinations of comparative group 4 were 60% and 60%, respectively.
The sensitivity of the primer probe combination of example 1 of the present invention is significantly higher than the other two control groups. Sensitivity (%) =number of positive detected/total number of positive cases, specificity (%) =number of negative detected/total number of negative cases.
Table 11 results of detection of different target fragments of ZNF671 and ZMYND10 genes
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims (10)

1. A primer probe combination for detecting methylation of ZNF671 and/or ZMYND10 genes is characterized in that the nucleotide sequence of a primer for detecting the ZNF671 genes is shown as SEQ ID NO:1 and 2, the nucleotide sequence of the probe for detecting the ZNF671 gene is shown as SEQ ID NO:3 is shown in the figure; the nucleotide sequence of the primer for detecting the ZMYND10 gene is shown as SEQ ID NO:4 and 5, the nucleotide sequence of the probe for detecting the ZMYND10 gene is shown as SEQ ID NO:6 is shown in the figure;
accession numbers of the ZNF671 gene on NCBI are: NC_000019.10, accession number of the ZMYND10 gene on NCBI is: nc_000003.12.
2. The primer probe combination according to claim 1, wherein the primer probe combination further comprises a primer and a probe for detecting the reference gene ACTB, and the nucleotide sequence of the primer for detecting the reference gene ACTB is shown in SEQ ID NO:7 and 8, the nucleotide sequence of the probe for detecting the reference gene ACTB is shown as SEQ ID NO: shown at 9.
3. The primer probe combination according to claim 1, wherein the fluorescent group marked at the 5 'end of the probe for detecting ZNF671 gene is VIC, and the fluorescent quenching group marked at the 3' end of the probe for detecting ZNF671 gene is BHQ1; the fluorescent group marked at the 5 'end of the probe for detecting the ZMYND10 gene is FAM, and the fluorescent quenching group marked at the 3' end of the probe for detecting the ZMYND10 gene is MGB.
4. The primer probe combination according to claim 2, wherein the fluorescent group marked on the 5 '-end of the probe for detecting the reference gene ACTB is CY-5, and the fluorescent quenching group marked on the 3' -end of the probe for detecting the reference gene ACTB is BHQ2.
5. Use of the primer probe combination according to any one of claims 1 to 4 in the preparation of a kit for detecting and diagnosing nasopharyngeal carcinoma.
6. A kit for diagnosing nasopharyngeal carcinoma, wherein said kit comprises the primer probe combination according to any one of claims 1 to 4.
7. The kit of claim 6, further comprising a PCR detection reagent.
8. The kit of claim 7, further comprising a methylation sensitive restriction enzyme comprising one or more of HhaI, bsh1236I, and HpaII.
9. The kit of claim 7, wherein the PCR detection reagent comprises an enzyme-cleaved amplification buffer, taq DNA polymerase and nuclease-free water, the enzyme-cleaved amplification buffer comprising Tris-HCl, KCl, mgCl 2 One or more of glycerol, BSA, dimethyl sulfoxide, tween 20, dNTP and dithiothreitol.
10. The kit according to claim 7, wherein the kit further comprises a positive quality control C666-1 human nasopharyngeal carcinoma cell line DNA and a negative quality control NP96 human nasopharyngeal epithelium cell line DNA.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110452984A (en) * 2019-07-29 2019-11-15 韩林志 A kind of methylated genes combination for cervical carcinoma DNA methylation assay, primer and probe combination, kit and its application method
WO2022179708A1 (en) * 2021-02-26 2022-09-01 Oncgnostics Gmbh Diagnosis and risk determination for head and neck cancer
CN115851921A (en) * 2021-09-24 2023-03-28 圣湘生物科技股份有限公司 Primer probe combination product, kit and application of primer probe combination product and kit in nasopharyngeal carcinoma methylation detection
CN116334228A (en) * 2023-04-19 2023-06-27 苏州云泰生物医药科技有限公司 Marker for cervical cancer DNA methylation detection and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110452984A (en) * 2019-07-29 2019-11-15 韩林志 A kind of methylated genes combination for cervical carcinoma DNA methylation assay, primer and probe combination, kit and its application method
WO2022179708A1 (en) * 2021-02-26 2022-09-01 Oncgnostics Gmbh Diagnosis and risk determination for head and neck cancer
CN115851921A (en) * 2021-09-24 2023-03-28 圣湘生物科技股份有限公司 Primer probe combination product, kit and application of primer probe combination product and kit in nasopharyngeal carcinoma methylation detection
CN116334228A (en) * 2023-04-19 2023-06-27 苏州云泰生物医药科技有限公司 Marker for cervical cancer DNA methylation detection and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAOAI HAN等: "DNA methylation biomarkers for nasopharyngeal carcinoma", 《PLOS ONE》, pages 1 - 16 *
YIFEI XU等: "Combination of RERG and ZNF671 methylation rates in circulating cell-free DNA: A novel biomarker for screening of nasopharyngeal carcinoma", 《CANCER SCIENCE》, vol. 111, pages 2536 - 2545 *

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