CN117757945A - Reagent and kit for detecting methylation of SOX1-SEPTIN9-ZIC1 gene of cervical cancer - Google Patents
Reagent and kit for detecting methylation of SOX1-SEPTIN9-ZIC1 gene of cervical cancer Download PDFInfo
- Publication number
- CN117757945A CN117757945A CN202410160371.XA CN202410160371A CN117757945A CN 117757945 A CN117757945 A CN 117757945A CN 202410160371 A CN202410160371 A CN 202410160371A CN 117757945 A CN117757945 A CN 117757945A
- Authority
- CN
- China
- Prior art keywords
- seq
- detection
- gene
- sox1
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 61
- 206010008342 Cervix carcinoma Diseases 0.000 title claims abstract description 52
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 title claims abstract description 52
- 201000010881 cervical cancer Diseases 0.000 title claims abstract description 52
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 52
- 230000011987 methylation Effects 0.000 title claims abstract description 25
- 238000007069 methylation reaction Methods 0.000 title claims abstract description 25
- 238000001514 detection method Methods 0.000 claims abstract description 140
- 239000000523 sample Substances 0.000 claims abstract description 108
- 101150042012 SEPTIN9 gene Proteins 0.000 claims abstract description 13
- 101150016852 Zic1 gene Proteins 0.000 claims abstract description 13
- 101150099498 SOX1 gene Proteins 0.000 claims abstract description 12
- 238000003745 diagnosis Methods 0.000 claims abstract description 6
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 108010085238 Actins Proteins 0.000 claims description 12
- 108020004414 DNA Proteins 0.000 claims description 9
- 230000007067 DNA methylation Effects 0.000 claims description 7
- 150000002429 hydrazines Chemical class 0.000 claims description 7
- 102000007469 Actins Human genes 0.000 claims description 6
- 201000010099 disease Diseases 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 238000010791 quenching Methods 0.000 claims description 6
- 230000000171 quenching effect Effects 0.000 claims description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 4
- 230000000295 complement effect Effects 0.000 claims description 4
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 2
- 102100029136 Collagen alpha-1(II) chain Human genes 0.000 claims description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 2
- 101000771163 Homo sapiens Collagen alpha-1(II) chain Proteins 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 22
- 238000012216 screening Methods 0.000 abstract description 3
- 239000000439 tumor marker Substances 0.000 abstract description 3
- 101000632056 Homo sapiens Septin-9 Proteins 0.000 description 23
- 101000652332 Homo sapiens Transcription factor SOX-1 Proteins 0.000 description 23
- 101000976653 Homo sapiens Zinc finger protein ZIC 1 Proteins 0.000 description 23
- 102100028024 Septin-9 Human genes 0.000 description 19
- 208000032124 Squamous Intraepithelial Lesions Diseases 0.000 description 19
- 102100030248 Transcription factor SOX-1 Human genes 0.000 description 19
- 206010028980 Neoplasm Diseases 0.000 description 10
- 239000012188 paraffin wax Substances 0.000 description 10
- 230000003321 amplification Effects 0.000 description 9
- 238000003199 nucleic acid amplification method Methods 0.000 description 9
- 102100023497 Zinc finger protein ZIC 1 Human genes 0.000 description 8
- 238000011160 research Methods 0.000 description 8
- 239000003550 marker Substances 0.000 description 7
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 6
- 230000003902 lesion Effects 0.000 description 6
- 101150005988 cin2 gene Proteins 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 208000019065 cervical carcinoma Diseases 0.000 description 4
- -1 etc.) Chemical compound 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 208000034254 Squamous cell carcinoma of the cervix uteri Diseases 0.000 description 3
- 201000006612 cervical squamous cell carcinoma Diseases 0.000 description 3
- 238000013399 early diagnosis Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 206010041823 squamous cell carcinoma Diseases 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- AOSFMYBATFLTAQ-UHFFFAOYSA-N 1-amino-3-(benzimidazol-1-yl)propan-2-ol Chemical compound C1=CC=C2N(CC(O)CN)C=NC2=C1 AOSFMYBATFLTAQ-UHFFFAOYSA-N 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 230000008836 DNA modification Effects 0.000 description 2
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- YQRQRUBEHCXCPR-UHFFFAOYSA-M cesium hydrogen sulfite Chemical compound [Cs+].OS([O-])=O YQRQRUBEHCXCPR-UHFFFAOYSA-M 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- DJEHXEMURTVAOE-UHFFFAOYSA-M potassium bisulfite Chemical compound [K+].OS([O-])=O DJEHXEMURTVAOE-UHFFFAOYSA-M 0.000 description 2
- 229940099427 potassium bisulfite Drugs 0.000 description 2
- 235000010259 potassium hydrogen sulphite Nutrition 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 2
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 2
- 229940001584 sodium metabisulfite Drugs 0.000 description 2
- 235000010262 sodium metabisulphite Nutrition 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 206010000084 Abdominal pain lower Diseases 0.000 description 1
- 206010001197 Adenocarcinoma of the cervix Diseases 0.000 description 1
- 208000034246 Adenocarcinoma of the cervix uteri Diseases 0.000 description 1
- 208000037157 Azotemia Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010008263 Cervical dysplasia Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 101100310656 Homo sapiens SOX1 gene Proteins 0.000 description 1
- 206010020524 Hydronephrosis Diseases 0.000 description 1
- 208000004608 Ureteral Obstruction Diseases 0.000 description 1
- 206010046788 Uterine haemorrhage Diseases 0.000 description 1
- 206010046910 Vaginal haemorrhage Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 201000008395 adenosquamous carcinoma Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000001369 bisulfite sequencing Methods 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 201000006662 cervical adenocarcinoma Diseases 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 208000000649 small cell carcinoma Diseases 0.000 description 1
- 208000009852 uremia Diseases 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of gene diagnosis, in particular to a detection reagent and a kit for methylation of cervical cancer SOX1-SEPTIN9-ZIC1 genes; the detection primer, the probe, the detection reagent and the kit for detecting methylation of the SOX1 gene, the SEPTIN9 gene and the ZIC1 gene of cervical cancer are designed, so that the detection primer, the probe, the detection reagent and the kit are used for detecting methylation degree of the SOX1 gene, the SEPTIN9 gene and the ZIC1 gene, have high sensitivity and strong specificity, and are suitable for large-scale crowd screening and conventional molecular diagnosis; the sensitivity of detection by adopting the detection reagent and the kit designed by the application is higher than that of the cervical cancer marker reported in the prior art, the sensitivity is greatly improved, and the kit has great application value for diagnosis of cervical cancer.
Description
Technical Field
The invention relates to the technical field of gene diagnosis, in particular to a detection reagent and a kit for methylation of cervical cancer SOX1-SEPTIN9-ZIC1 genes.
Background
Cervical cancer is a characteristic malignancy in women, the incidence of which in women is inferior to breast cancer, and it is counted that about 20 or more tens of thousands of women die annually. Cervical cancer is classified into 3 kinds of cervical squamous cell carcinoma, cervical adenocarcinoma and cervical squamous cell carcinoma according to pathological classification, wherein cervical squamous cell carcinoma is the most common, cervical cancer patients commonly have irregular vaginal bleeding, contact bleeding, abnormal leucorrhea, lower abdominal pain and other symptoms (early insignificant), and most patients also combine with ureteral obstruction, hydronephrosis, uremia and other complications along with the extension of the course of the disease. Cervical intraepithelial lesions are the most representative manifestations of precancerous lesions, and have symptoms similar to those of early cervical cancer, and the difficulty in identifying the two is relatively high.
The methylation tumor marker is taken as an innovative detection technology and is an ideal marker for early screening of cancers. Although some methylated tumor markers have been found in the prior art, they are limited by detection reagents or detection means of methylated tumor markers, resulting in insufficient sensitivity and specificity of tumor markers.
Therefore, how to improve the sensitivity and specificity of tumor marker detection is a urgent problem to be solved.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a reagent and a kit for detecting methylation of SOX1-SEPTIN9-ZIC1 genes of cervical cancer.
The invention discovers that the SOX1-SEPTIN9-ZIC1 gene is subjected to methylation joint detection for the first time, and can realize detection of cervical cancer and precancerous lesions thereof with high specificity and high sensitivity.
In a first aspect, the present application provides a primer, which adopts the following technical scheme:
a primer selected from the group consisting of SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO: 32. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 49. SEQ ID NO:50, or an amino acid sequence having at least 80% or more homology thereto.
Preferably, the primer is selected from the group consisting of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO: 32. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 49. SEQ ID NO:50 or a complement thereof.
Preferably, the primer is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2. SEQ ID NO:4 and SEQ ID NO: 5. SEQ ID NO:7 and SEQ ID NO: 8. SEQ ID NO:22 and SEQ ID NO: 23. SEQ ID NO:25 and SEQ ID NO: 26. SEQ ID NO:28 and SEQ ID NO: 29. SEQ ID NO:31 and SEQ ID NO: 32. SEQ ID NO:43 and SEQ ID NO: 44. SEQ ID NO:46 and SEQ ID NO: 47. SEQ ID NO:49 and SEQ ID NO:50, and at least one primer pair shown in FIG.
More preferably, the primer is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2. SEQ ID NO:22 and SEQ ID NO: 23. SEQ ID NO:43 and SEQ ID NO: 44.
In a second aspect, the present application provides a probe, which adopts the following technical scheme:
a probe comprising a probe for detecting SOX1 gene, SEPTIN9 gene, ZIC1 gene;
the probe is selected from SEQ ID NO:3. SEQ ID NO: 6. SEQ ID NO:9. SEQ ID NO:24. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NO:33. SEQ ID NO:45. SEQ ID NO: 48. SEQ ID NO:51 or a complement thereof.
Preferably, the probe is selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO:24. SEQ ID NO:45.
Preferably, a fluorescence quenching group is attached to the 3 '-end of the probe, and a fluorescence reporting group is attached to the 5' -end.
Preferably, the fluorescence quenching group is BHQ, BHQ1, BHQ2, TAMRA or MGB, and the fluorescence reporting group is FAM, CY3, CY5, HEX, ROX or TET.
More preferably, the fluorescence quenching group is BHQ1 and the fluorescence reporting group is FAM.
In a third aspect, the application provides an application of the primer and/or the probe in preparing a cervical cancer detection reagent or a kit, and the application adopts the following technical scheme:
the primer and the probe are applied to the preparation of cervical cancer detection reagents or kits.
Preferably, the detection reagent or the kit is used for detecting the sequence of the SOX1-SEPTIN9-ZIC1 gene modified by the conversion reagent.
Preferably, the conversion reagent is one or more of bisulfite, hydrazine salt or bisulphite. The conversion reagent includes a hydrazine salt, bisulfite (e.g., sodium bisulfite, etc.), bisulfite (e.g., sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, etc.), or a reagent that can produce one or more of a hydrazine salt, bisulfite compound under appropriate reaction conditions.
More preferably, the conversion reagent is a bisulfite.
In a fourth aspect, the present application provides a cervical cancer detection reagent, which adopts the following technical scheme:
a reagent for detecting cervical cancer comprises the primer and the probe.
Preferably, the detection reagent further comprises one or more of dNTPs, DNA polymerase, buffer solution and nuclease-free water.
Preferably, the detection reagent further comprises a detection reagent for a reference gene.
Preferably, the internal reference is beta-actin or COL2A1; furthermore, other methylation-detected reference genes of the prior art can be used as reference genes.
Preferably, the reference gene is beta-actin.
Preferably, the reference gene beta-actin detection reagent contains a primer pair and a probe for detecting the reference gene.
Preferably, the primer pair of the reference gene beta-actin detection reagent is SEQ ID NO:58 and SEQ ID NO:59, the probe is SEQ ID NO:60.
preferably, the detection region of the detection reagent is a SOX1 gene genome or a promoter region thereof, a SEPTIN9 gene genome or a promoter region thereof, a ZIC1 gene genome or a promoter region thereof, specifically comprising the following steps:
the detection region of the detection reagent aiming at the SOX1 gene is SEQ ID NO:61 or SEQ ID NO: 62; more preferably, the detection region of the detection reagent against SOX1 gene is SEQ ID NO: 61.
Preferably, the detection region of the detection reagent against the SEPTIN9 gene is SEQ ID NO:63 or SEQ ID NO:64, a sequence shown in seq id no; more preferably, the detection region of the detection reagent against the SEPTIN9 gene is SEQ ID NO: 63.
Preferably, the detection region of the detection reagent against the ZIC1 gene is SEQ ID NO:65 or SEQ ID NO:66, a sequence shown in seq id no; more preferably, the detection region of the detection reagent against the ZIC1 gene is SEQ ID NO: 65.
In a specific embodiment, the application provides a cervical cancer detection reagent, which comprises a primer probe combination and adopts the following technical scheme:
a reagent for detecting cervical cancer comprises a primer pair and a probe for detecting SOX1 gene, SEPTIN9 gene and ZIC1 gene;
the sequences of the primer pair and probe for SOX1 gene detection are shown in any one of the following groups:
group one:
the primer pair is SEQ ID NO:1 and SEQ ID NO:2, the probe is SEQ ID NO:3, a step of;
group II:
the primer pair is SEQ ID NO:4 and SEQ ID NO:5, the probe is SEQ ID NO:6, preparing a base material;
group III:
the primer pair is SEQ ID NO:7 and SEQ ID NO:8, the probe is SEQ ID NO:9.
preferably, the primer pair and probe for SOX1 gene detection are:
group one: the primer pair is SEQ ID NO:1 and SEQ ID NO:2, the probe is SEQ ID NO:3.
the sequences of the primer pair and the probe for SEPTIN9 gene detection are shown in any one of the following groups:
group four:
the primer pair is SEQ ID NO:22 and SEQ ID NO:23, the probe is SEQ ID NO:24, a step of detecting the position of the base;
group five:
the primer pair is SEQ ID NO:25 and SEQ ID NO:26, the probe is SEQ ID NO:27;
group six:
the primer pair is SEQ ID NO:28 and SEQ ID NO:29, the probe is SEQ ID NO:30;
group seven:
the primer pair is SEQ ID NO:31 and SEQ ID NO:32, the probe is SEQ ID NO:33.
preferably, the primer pair and probe for detecting SEPTIN9 gene detection:
group four: the primer pair is SEQ ID NO:22 and SEQ ID NO:23, the probe is SEQ ID NO:24.
the sequences of the primer pair and the probe for ZIC1 gene detection are shown in any one of the following groups:
group eight:
the primer pair is SEQ ID NO:43 and SEQ ID NO:44, the probe is SEQ ID NO:45;
group nine:
the primer pair is SEQ ID NO:46 and SEQ ID NO:47, the probe is SEQ ID NO:48;
group ten:
the primer pair is SEQ ID NO:49 and SEQ ID NO:50, the probe is SEQ ID NO:51.
preferably, the primer pair and probe for ZIC1 gene detection:
group eight: the primer pair is SEQ ID NO:43 and SEQ ID NO:44, the probe is SEQ ID NO:45.
in a fifth aspect, the present application provides a kit for detecting cervical cancer, which adopts the following technical scheme:
a kit for detecting cervical cancer, comprising the above primer or the above probe or the above detection reagent.
In a sixth aspect, the present application provides a method for detecting methylation of a SOX1-SEPTIN9-ZIC1 gene, which adopts the following technical scheme:
a method for detecting methylation of SOX1-SEPTIN9-ZIC1 gene, which comprises detection using the above-described primer, the above-described probe, or the above-described detection reagent or the above-described kit, which is not used for disease diagnosis and treatment purposes.
Preferably, a method for detecting methylation of SOX1-SEPTIN9-ZIC1 gene comprises the following steps:
s1, extracting DNA from a sample to be detected, and then treating the sample with a conversion reagent to obtain a modified sample to be detected;
s2, detecting methylation of the SOX1-SEPTIN9-ZIC1 of the sample to be detected after modification of the conversion reagent by using the primer, the probe or the detection reagent or the kit.
Preferably, the detected Δct value obtained in step S2 is compared with a threshold value, and if the Δct value is less than the threshold value, the sample is positive, which indicates that the collected sample to be tested contains at least one of methylated SOX1 gene, SEPTIN9 gene, and ZIC1 gene.
Preferably, the conversion reagent is one or more of bisulfite, hydrazine salt or bisulphite. The conversion reagent includes a hydrazine salt, bisulfite (e.g., sodium bisulfite, etc.), bisulfite (e.g., sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, etc.), or a reagent that can produce one or more of a hydrazine salt, bisulfite compound under appropriate reaction conditions.
More preferably, the conversion reagent is a bisulfite.
In a seventh aspect, the present application provides a diagnostic system for cervical cancer, which adopts the following technical scheme:
a diagnostic system for cervical cancer, the diagnostic system comprising:
a. a detection member: a detection means for quantitatively detecting the degree of DNA methylation of the SOX1-SEPTIN9-ZIC1 gene;
b. and a result judgment means.
Preferably, the detection means is used for quantitatively detecting the DNA methylation degree result of the SOX1-SEPTIN9-ZIC1 gene, and outputting the possibility or risk value of cervical cancer.
Preferably, the detection component for quantitatively detecting the DNA methylation degree of the SOX1-SEPTIN9-ZIC1 gene comprises the primer, the probe or the detection reagent or the kit.
Preferably, the risk of illness is that methylation results of the sample to be detected and the normal sample are compared according to the passing results, and when methylation of the sample to be detected and the normal sample has a significant difference or extremely significant difference, the result judges that the risk of illness of the sample to be detected is high.
The inventor has conducted intensive researches and for the first time revealed a reagent and a kit for detecting methylation of SOX1-SEPTIN9-ZIC1 genes of cervical cancer, wherein the methylated DNA is located in a human SOX1 gene and a promoter region thereof, a SEPTIN9 gene and a promoter region thereof, and a ZIC1 gene and a promoter region thereof. The inventor not only verifies the detection rate of SOX1, SEPTIN9 and ZIC1 genes in a tissue sample, but also verifies that SOX1, SEPTIN9 and ZIC1 genes have the same high specificity and sensitivity in a TCT sample, and can obviously improve the detection rate of cervical cancer through combination of SOX1, SEPTIN9 and ZIC1 genes. The inventor further improves the detection performance by optimizing the primer detection of SOX1, SEPTIN9 and ZIC1 genes. The present invention has been completed on the basis of this finding.
The inventor searches and refers to a large number of documents and analyzes data of a The Cancer Genome Atlas (TCGA) platform, and based on Reduced Representation Bisulfite Sequencing (RRBS) high-throughput second-generation sequencing methylation DNA detection technology, cervical paraffin samples are detected and deeply analyzed to obtain markers with clinical diagnosis significance for cervical cancer and precancerous lesions, and meanwhile, qPCR (quantitative polymerase chain reaction) is adopted to sequentially pass through the cervical paraffin samples and TCT cervical exfoliated cell samples for verification, and finally, 3 genes, namely SOX1 genes, SEPTIN9 genes and ZIC1 genes, are detected in a combined mode, so that cervical cancer and precancerous lesion patients are identified with high specificity and high sensitivity.
In summary, the present application includes at least one of the following beneficial technical effects:
the application discloses a reagent and a kit for detecting methylation of SOX1-SEPTIN9-ZIC1 genes of cervical cancer, which not only verify detection rates of SOX1 genes, SEPTIN9 genes and ZIC1 genes in tissue samples, but also verify that the SOX1 genes, SEPTIN9 genes and ZIC1 genes have the same high specificity and sensitivity in TCT samples, and can obviously improve the detection rate of cervical cancer by combining the SOX1 genes, SEPTIN9 genes and ZIC1 genes; particularly, the high sensitivity detection of squamous intraepithelial lesions (HSIL) is of great significance for early diagnosis and early discovery of cervical cancer.
Detailed Description
The technical scheme of the invention is further described by the following specific examples, which do not represent limitations on the scope of the invention; some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.
Example 1: design of primer probe combination
Various research data show that methylation state and distribution of the same gene are not uniform, so that methylation primers and probe detection systems designed by different regions are selected for the same gene, diagnostic detection efficacy of the same tumor is different for the same sample, even if the selected regions are unsuitable, so that the diagnostic effect on the tumor is completely absent, and the inventor selects SOX1, SEPT9 and ZIC1 preferable regions after repeated research and comparison as follows:
the preferred regions of SOX1, SEPTIN9, ZIC1 genes are as follows: the detection region of the SOX1 gene is SEQ ID NO:61, a sequence shown in seq id no; the detection region of the SEPTIN9 gene is SEQ ID NO: 63; the detection region of the ZIC1 gene is SEQ ID NO: 65.
In order to complete the invention, the inventor screens a large number of genes, finally confirms SOX1, SEPTIN9 and ZIC1 genes as preferable genes to be detected, takes beta-actin genes as reference genes, researches the distribution condition of methylation sites of the genes, and designs detection primer probes for detection.
The detection primer probes of each gene are as follows:
TABLE 1 primer pairs and probe combinations
Example 2: detection of SOX1, SEPTIN9, ZIC1 genes in Paraffin samples
In order to complete the invention, the inventor screens a large number of genes, finally confirms SOX1, SEPTIN9 and ZIC1 genes as preferable genes to be detected, takes beta-actin genes as reference genes, researches the distribution condition of methylation sites of the genes, and designs detection primer probes for detection.
The detection primer probes of each gene in this example are as follows:
the detection primers and probes of SOX1 are:
SOX1-MF2: SEQ ID NO:1, a sequence shown in seq id no;
SOX1-MR2: SEQ ID NO:2, a sequence shown in seq id no;
SOX1-P2: SEQ ID NO:3, a sequence shown in 3;
the detection primers and probes of SEPTIN9 are:
SEPT9-MF1: SEQ ID NO:22, a sequence shown in seq id no;
SEPT9-MR1: SEQ ID NO:23, a sequence shown in seq id no;
SEPT9-P1: SEQ ID NO:24, a sequence shown in seq id no;
the detection primers and probes of ZIC1 are as follows:
ZIC1-MF3: SEQ ID NO: 43.
ZIC1-MR3: SEQ ID NO:44, a sequence shown in seq id no;
ZIC1-P3: SEQ ID NO:45;
the detection primers and probes of the beta-actin are as follows:
ACTB-MF SEQ ID NO:58, and a sequence shown in seq id no;
ACTB-MR SEQ ID NO: 59;
ACTB-P, SEQ ID NO:60.
Sample detection:
sample information: a total of 50 cervical paraffin samples were tested, of which 20 samples were tested in the control group; 30 positive samples (pathology is larger than or equal to CIN2, and is abbreviated as CIN2+) and comprises 10 HSIL samples and 20 squamous carcinoma samples (14 cervical carcinoma samples in stage I/II and 6 cervical carcinoma samples in stage III/IV).
The test process comprises the following steps:
1. extraction of DNA
Specimens of cervical cancer and high-grade squamous intraepithelial lesions (HSIL) patients and paraffin tissue specimens of non-tumor patients were collected, and DNA extraction was performed according to the protocol of Meiy Bio-company kit HiPureFFPE DNA Kit (D3126-03).
2. DNA modification
Bisulfite modification was performed with the instructions of ZYMO rest biological company kit EZ DNA MethylationTM KIT (D5002).
3. Amplification and detection
Preparing a liquid system:
TABLE 2 liquid distribution System
Amplification procedure:
TABLE 3 amplification procedure
4. Detection result
Taking a delta Ct value (delta Ct=marker Ct value-ACTB Ct value) as a sample positive judgment standard, wherein the positive judgment value delta Ct of SOX1 is 7.5, the positive judgment value delta Ct of SEPT9 is 7, the positive judgment value delta Ct of ZIC1 is 7, when the detection result delta Ct value is smaller than the corresponding positive judgment value, the marker is judged to be positive to the sample detection result, otherwise, the marker is negative, and 50 cervical paraffin samples are detected as follows:
TABLE 4 detection results
Note that: and when the amplification curve is not generated, the Ct value is uniformly assigned to 45, and delta Ct value calculation is performed.
Analyzing the detection result, wherein the analysis result is as follows:
TABLE 5 analysis results
From the above results, it can be seen that the cervical paraffin samples have high sensitivity in the case of 100% specificity, regardless of whether CIN2+ is analyzed as a whole or according to HSIL and cervical cancer groups, respectively. Especially for HSIL group, the single gene detection sensitivity is up to more than 80%, and the combined detection of 3 genes can be detected by 90%, because the development course of cervical cancer is from squamous intraepithelial lesions (HSIL) to cervical cancer, the high sensitivity detection of HSIL has important significance for early diagnosis and early discovery of cervical cancer.
Example 3: detection of SOX1, SEPTIN9, ZIC1 genes in cervical exfoliated cell samples
By performing methylation detection in a cervical paraffin sample, the SOX1, SEPTIN9 and ZIC1 genes are confirmed to have high specificity and sensitivity no matter single detection or combined detection, and in order to verify the detection performance of the 3 genes in an actual clinical sample, namely a cervical exfoliated cell sample (hereinafter referred to as TCT sample), the inventor collects 200 TCT samples containing pathological information of different disease types, takes beta-actin genes as reference genes, and researches the detection conditions of the SOX1, SEPTIN9 and ZIC1 genes in the TCT samples.
The detection primer probes of each gene in this example are as follows:
the detection primers and probes of SOX1 are:
SOX1-MF2: SEQ ID NO:1, a sequence shown in seq id no;
SOX1-MR2: SEQ ID NO:2, a sequence shown in seq id no;
SOX1-P2: SEQ ID NO:3, a sequence shown in 3;
the detection primers and probes of SEPTIN9 are:
SEPT9-MF1: SEQ ID NO:22, a sequence shown in seq id no;
SEPT9-MR1: SEQ ID NO:23, a sequence shown in seq id no;
SEPT9-P1: SEQ ID NO:24, a sequence shown in seq id no;
the detection primers and probes of ZIC1 are as follows:
ZIC1-MF3: SEQ ID NO: 43.
ZIC1-MR3: SEQ ID NO:44, a sequence shown in seq id no;
ZIC1-P3: SEQ ID NO:45;
the detection primers and probes of the beta-actin are as follows:
ACTB-MF SEQ ID NO:58, and a sequence shown in seq id no;
ACTB-MR SEQ ID NO: 59;
ACTB-P, SEQ ID NO:60.
Sample detection:
sample information: 200 TCT samples containing pathological information of different disease types are tested, wherein the TCT samples comprise 99 negative control samples (less than or equal to CIN 1) and 101 positive control samples (less than or equal to CIN 2), and the negative control samples comprise 59 samples of cervical diseases other than cervical cancer and precancerous lesions thereof and 40 samples of low-grade squamous intraepithelial lesions (LSIL); the positive control samples included 59 high grade squamous intraepithelial lesions (HSIL), 42 cervical cancer samples, with the cervical cancer samples including 34 squamous cell carcinomas, 4 adenocarcinomas, 2 adenosquamous carcinomas, 1 small cell carcinoma, 1 cervical cancer sample of undefined type.
The test process comprises the following steps:
1. extraction of DNA
A quantity of TCT clinical samples were taken and the cells were centrifuged to obtain a pellet, which was subjected to DNA extraction according to the protocol of Meiyaku Bio-company kit HiPureFFPE DNA Kit (D3126-03).
2. DNA modification
EZ DNA Methylation with ZYMO RESEARCH Bio-Inc. kit TM KIT (D5002) instructions for bisulfite modification.
3. Amplification and detection
Preparing a liquid system:
TABLE 6 liquid distribution System
Amplification procedure:
TABLE 7 amplification procedure
/>
4. Detection result
Taking a delta Ct value (delta Ct=marker Ct value-ACTB Ct value) as a sample positive judgment standard, wherein the positive judgment value delta Ct of SOX1 is 4, the positive judgment value delta Ct of SEPT9 is 10, the positive judgment value delta Ct of ZIC1 is 5.5, and when the detection result delta Ct value is smaller than the corresponding positive judgment value, the marker is judged to be positive to the sample detection result, otherwise, the marker is negative, and 200 TCT sample detection results are as follows:
table 8 200 TCT sample test results
/>
/>
/>
/>
/>
/>
/>
Note that: when no amplification curve exists, the Ct value is uniformly assigned to 45, and delta Ct value calculation is performed; NA indicates that the corresponding type is not detected.
Analyzing the detection result, wherein the analysis result is as follows:
table 9 analysis results
From the results, in the TCT sample, under the condition that the specificity is more than or equal to 97%, the sensitivity of each gene in cervical cancer is more than 80%; however, in the HSIL group, the expression of each gene was greatly different from that of paraffin sample detection results (sensitivity was 54-73%), resulting in a decrease in the overall performance of each gene. Under the condition of maintaining 96% of high specificity, the combined detection of 3 genes has the overall sensitivity reaching 88.1%, and the sensitivity reaching 79.7% for the HSIL group, so that the cervical cancer group can be detected by 100%. Since the development course of cervical cancer is from squamous intraepithelial lesions (HSIL) to cervical cancer, the high-sensitivity detection of HSIL has important significance for early diagnosis and early discovery of cervical cancer.
Example 4: primer design and optimization for SOX1, SEPT9 and ZIC1
The primers and the probes have great influence on the detection effect of tumor markers, and in the research process, the inventor designs a plurality of pairs of primers and corresponding probes so as to find the probes and the primers which can improve the detection sensitivity and the specificity as much as possible, so that the detection reagent can be practically applied to clinical detection.
Table 10 primer probe combinations
Screening and detecting the above probes in 50 cervical paraffin samples, wherein the number of the samples in a control group is 20; 30 positive samples (pathology is larger than or equal to CIN2, and is abbreviated as CIN2+) and comprises 10 HSIL samples and 20 squamous carcinoma samples (14 cervical carcinoma samples in stage I/II and 6 cervical carcinoma samples in stage III/IV).
The procedure and amplification procedure were the same as in example 2.
The detection results are as follows:
TABLE 11 SOX1 different primer-probe pairs detection results
In the detection results of the SOX1 multiple pairs of primer probes, the results show that the detection performance (specificity and sensitivity) of the combination 2 is optimal, the combination 1 and the combination 6 times, and the performance of the combination 3, the combination 4, the combination 5 and the combination 7 is poor.
TABLE 12 detection results for SEPT9 different primer pairs
In the detection results of the SEPT9 multiple pairs of primer probes, the results show that the detection performance (specificity and sensitivity) of the combination 1 is optimal, the performance of the combination 2, the combination 3 and the combination 4 times is poorer, and the performance of the combination 5, the combination 6 and the combination 7 is poorer.
TABLE 13 detection results for ZIC1 different primer pairs
In the detection results of ZIC1 multiple pairs of primer probes, the results show that the detection performance (specificity and sensitivity) of the combination 3 is optimal, the combination 4 and the combination 5 times, and the performance of the combination 1 and the combination 2 is poor.
It is clear from Table 11, table 12 and Table 13 that the detection results are affected by the different primer pairs for the same region.
Claims (10)
1. A primer, characterized in that: the primer is selected from SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO: 32. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 49. SEQ ID NO:50, or an amino acid sequence having at least 80% or more homology thereto.
2. A primer according to claim 1, wherein: the primer is selected from SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO: 32. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 49. SEQ ID NO:50 or a complement thereof;
the primer is selected from SEQ ID NO:1 and SEQ ID NO: 2. SEQ ID NO:4 and SEQ ID NO: 5. SEQ ID NO:7 and SEQ ID NO: 8. SEQ ID NO:22 and SEQ ID NO: 23. SEQ ID NO:25 and SEQ ID NO: 26. SEQ ID NO:28 and SEQ ID NO: 29. SEQ ID NO:31 and SEQ ID NO: 32. SEQ ID NO:43 and SEQ ID NO: 44. SEQ ID NO:46 and SEQ ID NO: 47. SEQ ID NO:49 and SEQ ID NO:50, at least one primer pair shown in seq id no;
wherein the primer is selected from SEQ ID NO:1 and SEQ ID NO: 2. SEQ ID NO:22 and SEQ ID NO: 23. SEQ ID NO:43 and SEQ ID NO: 44.
3. A probe, characterized in that: the probe is selected from SEQ ID NO:3. SEQ ID NO: 6. SEQ ID NO:9. SEQ ID NO:24. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NO:33. SEQ ID NO:45. SEQ ID NO: 48. SEQ ID NO:51 or a complement thereof;
wherein the probe is selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO:24. SEQ ID NO:45;
the 3 'end of the probe is connected with a fluorescence quenching group, and the 5' end is connected with a fluorescence reporting group;
the fluorescence quenching group is BHQ, BHQ1, BHQ2, TAMRA or MGB, and the fluorescence reporting group is FAM, CY3, CY5, HEX, ROX or TET; wherein the fluorescence quenching group is BHQ1, and the fluorescence reporting group is FAM.
4. Use of the primer according to claim 1 or 2 and the probe according to claim 3 for preparing a cervical cancer detection reagent or kit.
5. A cervical cancer detection reagent, characterized in that: the detection reagent comprises the primer of claim 1 or 2 and the probe of claim 3.
6. The cervical cancer detection reagent according to claim 5, wherein: the detection reagent also comprises one or more of dNTPs, DNA polymerase, buffer solution and nuclease-free water.
7. The cervical cancer detection reagent according to claim 5, wherein: the detection reagent also comprises a detection reagent of an internal reference gene;
the reference gene is beta-actin or COL2A1;
wherein the reference gene is beta-actin;
the reference gene beta-actin detection reagent contains a primer pair and a probe for detecting the reference gene; the primer pair is SEQ ID NO:58 and SEQ ID NO:59, the probe is SEQ ID NO:60.
8. a cervical cancer detection kit, characterized in that: comprising the primer according to claim 1 or 2, or the probe according to claim 3, or the detection reagent according to any one of claims 5 to 7.
9. A method for detecting methylation of SOX1-SEPTIN9-ZIC1 gene for non-disease diagnosis, characterized in that the method comprises the steps of:
s1, extracting DNA from a sample to be detected, and then treating the sample with a conversion reagent to obtain a modified sample to be detected;
s2, detecting methylation of SOX1-SEPTIN9-ZIC1 of a sample to be detected modified by a conversion reagent by using the detection reagent according to any one of claims 5 to 7 or the kit according to claim 8;
comparing and analyzing the detected delta Ct value obtained in the step S2 with a critical value, and determining that the sample to be detected contains at least one of methylated SOX1 gene, SEPTIN9 gene and ZIC1 gene when the delta Ct value is smaller than the critical value and positive;
the conversion reagent is one or more of bisulfite, hydrazine salt or bisulphite.
10. A diagnostic system for cervical cancer, the diagnostic system comprising:
a. a detection member: a detection means for quantitatively detecting the degree of DNA methylation of the SOX1-SEPTIN9-ZIC1 gene;
b. and a result judgment means: the detection component is used for quantitatively detecting the DNA methylation degree result of the SOX1-SEPTIN9-ZIC1 gene and outputting the possibility or risk value of cervical cancer;
wherein the detection member for quantitatively detecting the DNA methylation degree of the SOX1-SEPTIN9-ZIC1 gene comprises the detection reagent according to any one of claims 5 to 7 or the kit according to claim 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410160371.XA CN117757945A (en) | 2024-02-05 | 2024-02-05 | Reagent and kit for detecting methylation of SOX1-SEPTIN9-ZIC1 gene of cervical cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410160371.XA CN117757945A (en) | 2024-02-05 | 2024-02-05 | Reagent and kit for detecting methylation of SOX1-SEPTIN9-ZIC1 gene of cervical cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117757945A true CN117757945A (en) | 2024-03-26 |
Family
ID=90322026
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410160371.XA Pending CN117757945A (en) | 2024-02-05 | 2024-02-05 | Reagent and kit for detecting methylation of SOX1-SEPTIN9-ZIC1 gene of cervical cancer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117757945A (en) |
-
2024
- 2024-02-05 CN CN202410160371.XA patent/CN117757945A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021128519A1 (en) | Combination of dna methylation biomarkers, and detection method therefor and kit thereof | |
| CN113355415A (en) | Detection reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer | |
| CN111826446A (en) | Primer, probe and kit for early screening and auxiliary diagnosis of bladder cancer | |
| CN113355414B (en) | Esophageal cancer detection kit and application thereof | |
| US11535897B2 (en) | Composite epigenetic biomarkers for accurate screening, diagnosis and prognosis of colorectal cancer | |
| CN117701720B (en) | Cervical cancer CLIP3 gene methylation detection reagent and kit | |
| CN117701721B (en) | Detection reagent and kit for methylation of SOX1-SEPTIN9-TAC1 gene of cervical cancer | |
| CN117757945A (en) | Reagent and kit for detecting methylation of SOX1-SEPTIN9-ZIC1 gene of cervical cancer | |
| CN117721209B (en) | Combined detection reagent and kit for cervical cancer detection | |
| CN116356027B (en) | Reagent and kit for detecting esophageal squamous carcinoma or precancerous lesions and application of reagent and kit | |
| CN115786502B (en) | Use of an agent for detecting the methylation level of a target region for the preparation of a diagnostic product for bladder cancer | |
| JP7539739B2 (en) | Fluorescent cross-linked RNase H mutant conjugates, miRNA combinations and uses thereof | |
| CN117327794A (en) | Reagent and kit for detecting benign and malignant lung nodules and application of reagent and kit | |
| CN117778582A (en) | Nucleic acid combination for detecting gastric cancer, kit and application | |
| CN118256621A (en) | Nucleic acid reagent for detecting gastric cancer, detection kit and application | |
| CN117106899A (en) | Primer pair, primer probe combination, kit and application for detecting benign and malignant lung nodule | |
| CN117265110A (en) | Kit for detecting lung squamous carcinoma and application thereof | |
| CN118064592A (en) | Nucleic acid combination for detecting prostate cancer, kit and application | |
| CN117904301A (en) | Primer probe composition and kit for detecting UTP3 and ZNF318 gene promoter methylation diagnosis of esophageal cancer | |
| CN118064593A (en) | Prostate cancer biomarker, prostate cancer detection kit and application | |
| CN117467763A (en) | Nucleic acid combination and kit for detecting methylation level of non-small cell lung cancer biomarker | |
| CN117248021A (en) | Nucleic acid combination, kit and application for detecting urothelial cancer or precancerous lesions | |
| CN116083586A (en) | Nucleic acid product, kit and application for diagnosing esophageal cancer | |
| CN117778573A (en) | Nucleic acid combination for detecting thyroid cancer biomarker, kit and application | |
| CN117867126A (en) | Primer probe composition for detecting esophageal cancer based on TaqMan probe melting curve method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |