CN117721209B - Combined detection reagent and kit for cervical cancer detection - Google Patents
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Abstract
The invention relates to the technical field of gene diagnosis, in particular to a combined detection reagent and a kit for cervical cancer detection; the present disclosure relates to detection reagents designed to detect methylation of cervical cancer SOX1 gene, SEPTIN9 gene, TAC1 gene, ZIC1 gene; and screening and confirming four HPVs 16/18/52/58 with higher detection specificity and detection sensitivity, designing detection reagents for detecting HPV16/18/52/58 virus methylation, combining any combination of SOX1/SEPTIN9/TAC1 or SOX1/SEPTIN9/ZIC1 with HPV16/18/52/58 virus methylation detection, and realizing high-specificity and high-sensitivity detection of cervical cancer and precancerous lesions thereof, and is suitable for large-scale crowd screening and routine molecular diagnosis.
Description
Technical Field
The invention relates to the technical field of gene diagnosis, in particular to a combined detection reagent and a kit for cervical cancer detection.
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. In recent years, research has indicated that cervical cancer progression has a close correlation with epigenetic changes, and DNA methylation belongs to the common epigenetic phenomenon of cancer: the cancer suppressor gene promoter is highly methylated in cancer cells to cause abnormal expression of the cancer suppressor gene and even inactivation of the gene, so that malignant growth of the cells occurs, which is an important theoretical basis for the occurrence of cancer at present, cervical cancer methylation gene detection can discover cervical high-level lesions as early as possible, guide clinical intervention and become a new means for preventing and treating cervical cancer in the future.
Human Papillomavirus (HPV) persistent infection is a major factor in cervical lesions, so HPV detection is commonly used in the diagnosis of cytologically abnormal cervical lesions, and Human Papillomavirus (HPV) DNA methylation can be used as a novel biomarker for predicting cervical tumor progression, thereby avoiding oversubscription and psychological burden on patients due to HPV transient infection.
In the related art, cervical cancer methylation gene detection or human papillomavirus DNA methylation is adopted singly for detection, but the detection is limited by detection reagents or detection means of methylation tumor markers, so that the sensitivity and the specificity of the tumor markers cannot meet the requirements.
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 of the prior art, the invention aims to provide a combined detection reagent and a kit for cervical cancer detection.
The invention discovers that any combination of SOX1/SEPTIN9/TAC1 or SOX1/SEPTIN9/ZIC1 is combined with HPV16/18/52/58 virus methylation detection for the first time, and can realize high-specificity and high-sensitivity detection of cervical cancer and precancerous lesions thereof.
In a first aspect, the present application provides a primer, which adopts the following technical scheme:
a primer selected from the group consisting of the amino acid sequence shown as SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：21、SEQ ID NO：22、SEQ ID NO：25、SEQ ID NO：26、SEQ ID NO：29、SEQ ID NO：30, 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:17 and SEQ ID NO: 18. SEQ ID NO:21 and SEQ ID NO: 22. SEQ ID NO:25 and SEQ ID NO: 26. SEQ ID NO:29 and SEQ ID NO:30 or a complement thereof;
Preferably, the primer is selected from the group consisting of SEQ ID NO:17 and SEQ ID NO: 18. SEQ ID NO:21 and SEQ ID NO: 22. SEQ ID NO:25 and SEQ ID NO: 26. SEQ ID NO:29 and SEQ ID NO:30, at least one primer pair shown in seq id no;
More preferably, the primer is selected from the group consisting of SEQ ID NO:17 and SEQ ID NO: 18. SEQ ID NO:21 and SEQ ID NO: 22. SEQ ID NO:25 and SEQ ID NO: 26. SEQ ID NO:29 and SEQ ID NO: 30.
Preferably, the primer further comprises a sequence selected from the group consisting of SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 13. SEQ ID NO:14, 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 and SEQ ID NO: 2. SEQ ID NO:5 and SEQ ID NO: 6. SEQ ID NO:9 and SEQ ID NO: 10. SEQ ID NO:13 and SEQ ID NO:14 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:5 and SEQ ID NO: 6. SEQ ID NO:9 and SEQ ID NO: 10. SEQ ID NO:13 and SEQ ID NO:14, at least one primer pair shown in FIG. 14;
More preferably, the primer is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2. SEQ ID NO:5 and SEQ ID NO: 6. SEQ ID NO:9 and SEQ ID NO: 10. and/or SEQ ID NO:13 and SEQ ID NO: 14.
In a second aspect, the present application provides a probe, which adopts the following technical scheme:
a probe selected from the group consisting of SEQ ID NOs: 19. SEQ ID NO: 23. SEQ ID NO: 27. SEQ ID NO:31 or a complement thereof;
Wherein the probe is selected from the group consisting of SEQ ID NOs: 19. SEQ ID NO: 23. SEQ ID NO: 27. SEQ ID NO: 31;
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 or TAMRA, and the fluorescence reporting group is FAM, CY3, CY5, HEX or TET;
more preferably, the fluorescence quenching group is BHQ1 and the fluorescence reporting group is FAM.
Preferably, the probe further comprises a sequence selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO: 7. SEQ ID NO: 11. SEQ ID NO:15 or a complement thereof;
More preferably, the probe is selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO: 7. SEQ ID NO:11 and/or SEQ ID NO: 15.
In a third aspect, the present application provides the use of the above-described primers and/or the above-described probes in the preparation of a cervical cancer detection reagent or kit.
The primer and/or the probe are applied to the preparation of cervical cancer detection reagents or kits.
In a fourth aspect, the present application provides a cervical cancer detection reagent, which adopts the following technical scheme:
A cervical cancer detection reagent comprising the above primer and/or the above probe.
Preferably, the HPV types involved in pathogen HPV DNA methylation include four HPV types HPV16/18/52/58, and the corresponding genome or promoter region thereof is specifically as follows:
the detection region of the detection reagent for HPV16 is SEQ ID NO:20, a sequence shown in seq id no;
the detection region of the detection reagent for HPV18 is SEQ ID NO:24, a sequence shown in seq id no;
the detection region of the detection reagent against HPV52 is SEQ ID NO:28, a sequence shown in seq id no;
the detection region of the detection reagent against HPV58 is SEQ ID NO: 32.
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 TAC1 gene genome or a promoter region thereof, a ZIC1 gene genome or a promoter region thereof, specifically as follows:
the detection reagent is specific to SOX1 gene detection region as SEQ ID NO: 4;
The detection reagent is specific to SEPTIN9 gene detection region as SEQ ID NO: 8;
The detection reagent is used for detecting the TAC1 gene with the detection region of SEQ ID NO:12, a sequence shown in seq id no;
the detection reagent is specific to the ZIC1 gene detection region shown in SEQ ID NO: 16.
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; the primer pair is SEQ ID NO:33 and SEQ ID NO:34, the probe is SEQ ID NO:35.
In a specific embodiment, the application provides a cervical cancer detection reagent, which is used for detecting HPV16/18/52/58 virus methylation, and adopts the following technical scheme:
a cervical cancer detection reagent comprises a primer probe combination, wherein a primer pair and a probe are shown as follows;
The primer pair for HPV type 16 is selected from SEQ ID NO:17 and SEQ ID NO:18, a primer pair shown in FIG; the probe is selected from SEQ ID NO:19, a sequence shown in seq id no;
The primer pair for HPV type 18 is selected from SEQ ID NO:21 and SEQ ID NO:22, a primer pair shown in FIG. 22; the probe is selected from SEQ ID NO:23, a sequence shown in seq id no;
The primer pair for HPV type 52 is selected from SEQ ID NO:25 and SEQ ID NO:26, a primer pair shown in seq id no; the probe is selected from SEQ ID NO: 27;
the primer pair for HPV type 58 is selected from SEQ ID NO:29 and SEQ ID NO:30, a primer pair shown in FIG. 30; the probe is selected from SEQ ID NO: 31.
In a specific embodiment, the application provides a cervical cancer detection reagent, which is used for detecting SOX1/SEPTIN 9/TAC 1 methylation, and adopts the following technical scheme:
a cervical cancer detection reagent comprises a primer probe combination, wherein a primer pair and a probe are shown as follows;
The primer pair for SOX1 gene detection is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2; the probe is selected from SEQ ID NO:3, a sequence shown in 3;
The primer pair for SEPTIN9 gene detection is selected from SEQ ID NO:5 and SEQ ID NO: 6; the probe is selected from SEQ ID NO: 7;
The primer pair for TAC1 gene detection is selected from SEQ ID NO: 9. SEQ ID NO:10, a primer pair shown in FIG. 10; the probe is selected from SEQ ID NO: 11.
In a specific embodiment, the application provides a cervical cancer detection reagent for detecting SOX1/SEPTIN9/ZIC1 methylation, which adopts the following technical scheme:
a cervical cancer detection reagent comprises a primer probe combination, wherein a primer pair and a probe are shown as follows;
The primer pair for SOX1 gene detection is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2; the probe is selected from SEQ ID NO:3, a sequence shown in 3;
The primer pair for SEPTIN9 gene detection is selected from SEQ ID NO:5 and SEQ ID NO: 6; the probe is selected from SEQ ID NO: 7;
the primer pair for ZIC1 gene detection is selected from SEQ ID NO: 13. SEQ ID NO:14, a sequence shown in seq id no; the probe is selected from SEQ ID NO: 15.
In a specific embodiment, the application provides a cervical cancer detection reagent, which carries out joint detection on SOX1/SEPTIN9/TAC1 and HPV16/18/52/58 virus methylation, and adopts the following technical scheme:
a cervical cancer detection reagent comprises a primer probe combination, wherein a primer pair and a probe are shown as follows;
The primer pair for HPV type 16 is selected from SEQ ID NO:17 and SEQ ID NO:18, a primer pair shown in FIG; the probe is selected from SEQ ID NO:19, a sequence shown in seq id no;
The primer pair for HPV type 18 is selected from SEQ ID NO:21 and SEQ ID NO:22, a primer pair shown in FIG. 22; the probe is selected from SEQ ID NO:23, a sequence shown in seq id no;
The primer pair for HPV type 52 is selected from SEQ ID NO:25 and SEQ ID NO:26, a primer pair shown in seq id no; the probe is selected from SEQ ID NO: 27;
The primer pair for HPV type 58 is selected from SEQ ID NO:29 and SEQ ID NO:30, a primer pair shown in FIG. 30; the probe is selected from SEQ ID NO: 31;
The primer pair for SOX1 gene detection is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2; the probe is selected from SEQ ID NO:3, a sequence shown in 3;
The primer pair for SEPTIN9 gene detection is selected from SEQ ID NO:5 and SEQ ID NO: 6; the probe is selected from SEQ ID NO: 7;
The primer pair for TAC1 gene detection is selected from SEQ ID NO: 9. SEQ ID NO:10, a primer pair shown in FIG. 10; the probe is selected from SEQ ID NO: 11.
In a specific embodiment, the application provides a cervical cancer detection reagent, which carries out joint detection on SOX1/SEPTIN9/ZIC1 and HPV16/18/52/58 virus methylation, and adopts the following technical scheme:
a cervical cancer detection reagent comprises a primer probe combination, wherein a primer pair and a probe are shown as follows;
The primer pair for HPV type 16 is selected from SEQ ID NO:17 and SEQ ID NO:18, a primer pair shown in FIG; the probe is selected from SEQ ID NO:19, a sequence shown in seq id no;
The primer pair for HPV type 18 is selected from SEQ ID NO:21 and SEQ ID NO:22, a primer pair shown in FIG. 22; the probe is selected from SEQ ID NO:23, a sequence shown in seq id no;
The primer pair for HPV type 52 is selected from SEQ ID NO:25 and SEQ ID NO:26, a primer pair shown in seq id no; the probe is selected from SEQ ID NO: 27;
The primer pair for HPV type 58 is selected from SEQ ID NO:29 and SEQ ID NO:30, a primer pair shown in FIG. 30; the probe is selected from SEQ ID NO: 31;
The primer pair for SOX1 gene detection is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2; the probe is selected from SEQ ID NO:3, a sequence shown in 3;
The primer pair for SEPTIN9 gene detection is selected from SEQ ID NO:5 and SEQ ID NO: 6; the probe is selected from SEQ ID NO: 7;
the primer pair for ZIC1 gene detection is selected from SEQ ID NO: 13. SEQ ID NO:14, a sequence shown in seq id no; the probe is selected from SEQ ID NO: 15.
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 target gene for non-disease diagnosis, which adopts the following technical scheme:
A method for detecting methylation of a target gene for non-disease diagnosis, comprising 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 a target gene of the sample to be detected after modification of the conversion reagent by using the detection reagent or the kit;
Wherein; and (3) comparing and analyzing the detected delta Ct value obtained in the step (S2) with a critical value, and when the delta Ct value is smaller than the critical value, determining that the collected sample to be detected contains at least one methylated target gene.
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 target gene;
b. And a result judgment means: the detection component is used for quantitatively detecting the DNA methylation degree result of the target gene according to the detection result of the detection component and outputting the possibility or risk value of cervical cancer;
wherein the detection component for quantitatively detecting the DNA methylation degree of the target gene comprises 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 combined detection reagent and kit for cervical cancer detection, wherein genes involved in genome DNA methylation comprise SOX1 gene and promoter region thereof, SEPTIN9 gene and promoter region thereof, TAC1 gene and promoter region thereof, ZIC1 gene and promoter region thereof; the HPV types involved in the pathogen HPV DNA methylation comprise four HPV types HPV 16/18/52/58. The inventor firstly detects and obtains detection rates and combined detection results of SOX1, SEPTIN9, TAC1 and ZIC1 genes in tissue samples, and then screens and confirms four HPVs 16/18/52/58 in small batches of TCT samples (training sets) to have higher detection specificity and detection sensitivity. Finally, SOX1, SEPTIN9, TAC1, ZIC1 genes and HPV16/18/52/58 genes are verified simultaneously in a TCT large sample (verification group); the results show that the combination of any one of SOX1/SEPTIN9/TAC1 or SOX1/SEPTIN9/ZIC1 and HPV16/18/52/58 virus methylation detection can realize high-specificity and high-sensitivity detection of cervical cancer and precancerous lesions thereof.
Aiming at Genome methylation markers, the inventor searches and refers to a large number of documents and performs data analysis on THE CANCER Genome Atlas (TCGA) platform, and based on a 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 diagnostic significance for cervical cancer and precancerous lesions, and meanwhile, qPCR method is adopted to sequentially pass through the cervical paraffin samples and TCT cervical shedding cell samples for verification, and finally, SOX1/SEPTIN9/TAC1 or SOX1/SEPTIN9/ZIC1 combination is detected, so that cervical cancer and precancerous lesion patients are identified with high specificity and high sensitivity.
Aiming at pathogen HPV methylation markers, the inventor searches and refers to a large number of documents and comprehensively analyzes data of an Encyclopedia of DNA ELEMENTS (encoding) platform, and confirms that HPV16/18/52/58 type four HPV pathogen methylation has higher correlation with clinical pathology stage through sequencing analysis of 14 high-risk types of HPV and qPCR verification.
Finally, the inventor finds out that the result of research in a large sample verification set shows that any combination of SOX1/SEPTIN9/TAC1 or SOX1/SEPTIN9/ZIC1 is combined with HPV16/18/52/58 virus methylation detection, so that the cervical cancer and precancerous lesions thereof can be detected 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 combined detection reagent and a kit for cervical cancer detection, wherein SOX1, SEPTIN9, TAC1 and ZIC1 genes are detected in a tissue sample to obtain detection rates and combined detection results, and then HPV16/18/52/58 is screened and confirmed to have higher detection specificity and detection sensitivity; and simultaneously verifies SOX1, SEPTIN9, TAC1, ZIC1 genes and HPV16/18/52/58 genes; the result shows that the combination of any one of SOX1/SEPTIN9/TAC1 or SOX1/SEPTIN9/ZIC1 and HPV16/18/52/58 virus methylation detection can realize high-specificity and high-sensitivity detection of cervical cancer and precancerous lesions thereof; 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 determines SOX1, SEPT9, TAC1, ZIC1 preferred regions and HPV16/18/52/58 preferred regions after repeated research and comparison, specifically as follows:
For the SOX1 gene detection region SEQ ID NO: 4; the detection region for the SEPTIN9 gene is SEQ ID NO: 8; the detection region for the TAC1 gene is SEQ ID NO:12, a sequence shown in seq id no; the detection region for the ZIC1 gene is SEQ ID NO: 16.
The detection region for HPV16 is SEQ ID NO:20, a sequence shown in seq id no; the detection region for HPV18 is SEQ ID NO:24, a sequence shown in seq id no; the detection region for HPV52 is SEQ ID NO:28, a sequence shown in seq id no; the detection region for HPV58 is SEQ ID NO: 32.
In order to complete the invention, the inventor screens a large number of genes, finally confirms SOX1, SEPTIN9, TAC1 genes, ZIC1 genes, HPV16, HPV18, HPV52 and HPV58 as preferable genes to be tested, takes beta-actin genes as internal reference genes, researches the distribution situation of methylation sites of the genes, and designs detection primer probes for detection respectively; the detection primer probes of each gene are as follows:
TABLE 1 primer probe combinations
Example 2: detection of SOX1, SEPTIN9, TAC1, ZIC1 genes in Paraffin samples
Through screening genes, SOX1, SEPTIN9, TAC1 and ZIC1 genes are finally confirmed to be optimal to be detected genes, beta-actin genes are used as internal reference genes, the distribution situation of methylation sites of the genes is researched, and detection primer probes are designed and used for detection respectively. The detection primer probes of each gene 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:5, a sequence shown in seq id no;
SEPT9-MR1: SEQ ID NO: 6;
SEPT9-P1: SEQ ID NO: 7;
The detection primers and probes of TAC1 are as follows:
TAC1-MF2: SEQ ID NO: 9;
TAC1-MR2: SEQ ID NO:10, a sequence shown in seq id no;
TAC1-P2: SEQ ID NO:11, a sequence shown in seq id no;
The detection primers and probes of ZIC1 are as follows:
ZIC1-MF3: SEQ ID NO:13, a sequence shown in seq id no;
ZIC1-MR3: SEQ ID NO:14, a sequence shown in seq id no;
ZIC1-P3: SEQ ID NO:15, a sequence shown in seq id no;
the detection primers and probes of the beta-actin are as follows:
ACTB-MF SEQ ID NO:33, a sequence shown in seq id no;
ACTB-MR SEQ ID NO:34, a sequence shown in seq id no;
ACTB-P, SEQ ID NO: 35.
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
Cervical cancer and high grade squamous intraepithelial lesions (HSIL) and non-tumor patient paraffin tissue specimens were subjected to DNA extraction according to the protocol of Meiy Bio-company kit HiPureFFPE DNA Kit (D3126-03).
2. DNA modification
Bisulfite modification was performed with ZYMO RESEARCH Bio-company KIT EZ DNA Methylation TM KIT (D5002) instructions.
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 TAC1 is 7 and 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, judging that the marker is positive to the sample detection result, otherwise, judging that the marker is negative, wherein 50 cervical paraffin samples are detected as follows:
TABLE 4 detection results
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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 each gene has high sensitivity in the cervical paraffin samples, regardless of whether CIN2+ is analyzed as a whole or according to HSIL and cervical cancer group, respectively, at a specificity of 100%. Especially for HSIL group, single gene detection sensitivity is up to more than 80%, SOX1/SEPTIN9/TAC1 gene combined detection can be 100%, SOX1/SEPTIN9/ZIC1 combined detection can be 90%, and since cervical cancer development course is from squamous intraepithelial lesions (HSIL) to cervical cancer, high sensitivity detection of HSIL has important significance for early diagnosis of cervical cancer.
Example 3: detection of HPV16/18/52/58 Gene methylation in cervical exfoliated cell samples
Carrying out methylation research on the high-risk HPV in 14, finally confirming that HPV16/18/52/58 gene methylation has correlation with cervical high-level lesions and cervical cancer, simultaneously taking a beta-actin gene as an internal reference gene, researching the distribution situation of methylation sites of all genes, and designing detection primer probes for detection respectively; the detection primer probes of each gene are as follows:
the methylation detection primers and probes for HPV16 are:
HPV16-MF5 SEQ ID NO:17, a sequence shown in seq id no;
HPV16-MR5 SEQ ID NO:18, a sequence shown in seq id no;
HPV16-P5 SEQ ID NO:19, a sequence shown in seq id no;
the methylation detection primers and probes of HPV18 are:
HPV18-MF5: SEQ ID NO:21, a sequence shown in seq id no;
HPV18-MR5: SEQ ID NO:22, a sequence shown in seq id no;
HPV18-P5: SEQ ID NO:23, a sequence shown in seq id no;
the methylation detection primers and probes for HPV52 are:
HPV52-MF2: SEQ ID NO:25, a sequence shown in seq id no;
HPV52-MR2: SEQ ID NO:26, a sequence shown in seq id no;
HPV52-P2: SEQ ID NO: 27;
The methylation detection primers and probes for HPV58 are:
HPV58-MF3: SEQ ID NO:29, a sequence shown in seq id no;
HPV58-MR3: SEQ ID NO:30, a sequence shown in seq id no;
HPV58-P3: SEQ ID NO: 31;
the detection primers and probes of the beta-actin are as follows:
ACTB-MF SEQ ID NO:33, a sequence shown in seq id no;
ACTB-MR SEQ ID NO:34, a sequence shown in seq id no;
ACTB-P, SEQ ID NO: 35.
Sample detection:
Sample information: testing 100 HPV16/18/52/58 HPV positive TCT samples with single infection or comprehensive infection, wherein the TCT samples contain pathological information for defining different disease types; the sample comprises 28 HPV16 positive samples, 8 negative control samples, namely that the pathological result is less than or equal to CIN1 (the same applies below), and 20 positive control samples, namely that the pathological result is more than or equal to CIN2 (the same applies below); 25 HPV18 positive samples, 7 negative control samples, 18 positive control samples; 32 HPV52 positive samples, 10 negative control samples, 22 positive control samples; 34 HPV58 positive samples, 14 negative control samples and 20 positive control samples.
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
Bisulfite modification was performed with ZYMO RESEARCH Bio-company KIT EZ DNA Methylation TM KIT (D5002) instructions.
3. Amplification and detection
Preparing a liquid system:
TABLE 6 liquid distribution System
Amplification procedure:
TABLE 7 amplification procedure
4. Detection result
The positive judgment value of each HPV type is 39, when the Ct value of the detection result is smaller than 39, the methylation detection of the HPV type is positive to the detection result of the sample, otherwise, the detection result of 100 TCT samples is negative, and the detection result of 100 TCT samples is as follows:
TABLE 8 test results for 100 TCT samples
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Note that: when the amplification result is no amplification curve, uniformly assigning 45; 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 above results, it can be seen that methylation detection is performed on the corresponding types of single or compound infection of HPV16/18/52/58 in TCT samples, and the sensitivities of HPV16, HPV18 and HPV52 are as high as 85%, 88.9% and 77.3% respectively when the specificities are 100%; the sensitivity was 85% at a HPV58 specificity of 92.9%.
Therefore, 4 types have very high specificity and high sensitivity, particularly have the characteristic of being transient for HPV infection, and the time from HPV infection to high-level lesion recurrence to cervical cancer can last for several years or even 10 years, and HPV16/18/52/58 is the 4 types with the highest proportion among the types of HPV infection of China females; the split flow is carried out by carrying out further methylation detection on patients infected with the 4 types of patients, and has important significance.
Example 4: detection of SOX1, SEPTIN9, TAC1, ZIC1 genes and HPV16/18/52/58 methylation in cervical exfoliated cell samples
By methylation detection in cervical paraffin samples, SOX1, SEPTIN9, TAC1 and ZIC1 genes are confirmed to have high specificity and sensitivity no matter single detection or combined detection is carried out, meanwhile HPV16/18/52/58 is respectively subjected to HPV virus methylation detection in TCT samples, and the result shows that the detection has high specificity and high sensitivity. In order to verify the joint detection performance of the 4 genes and HPV16/18/52/58 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 a beta-actin gene as an internal reference gene, and researches the detection conditions of SOX1, SEPTIN9, TAC1, ZIC1 genes and HPV16/18/52/58 in the TCT samples; the detection primer probes of each gene 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 SEPT9 are as follows:
SEPT9-MF1: SEQ ID NO:5, a sequence shown in seq id no;
SEPT9-MR1: SEQ ID NO: 6;
SEPT9-P1: SEQ ID NO: 7;
The detection primers and probes of TAC1 are as follows:
TAC1-MF2: SEQ ID NO: 9;
TAC1-MR2: SEQ ID NO:10, a sequence shown in seq id no;
TAC1-P2: SEQ ID NO:11, a sequence shown in seq id no;
The detection primers and probes of ZIC1 are as follows:
ZIC1-MF3: SEQ ID NO:13, a sequence shown in seq id no;
ZIC1-MR3: SEQ ID NO:14, a sequence shown in seq id no;
ZIC1-P3: SEQ ID NO:15, a sequence shown in seq id no;
the methylation detection primers and probes for HPV16 are:
HPV16-MF5 SEQ ID NO:17, a sequence shown in seq id no;
HPV16-MR5 SEQ ID NO:18, a sequence shown in seq id no;
HPV16-P5 SEQ ID NO:19, a sequence shown in seq id no;
the methylation detection primers and probes of HPV18 are:
HPV18-MF5: SEQ ID NO:21, a sequence shown in seq id no;
HPV18-MR5: SEQ ID NO:22, a sequence shown in seq id no;
HPV18-P5: SEQ ID NO:23, a sequence shown in seq id no;
the methylation detection primers and probes for HPV52 are:
HPV52-MF2: SEQ ID NO:25, a sequence shown in seq id no;
HPV52-MR2: SEQ ID NO:26, a sequence shown in seq id no;
HPV52-P2: SEQ ID NO: 27;
The methylation detection primers and probes for HPV58 are:
HPV58-MF3: SEQ ID NO:29, a sequence shown in seq id no;
HPV58-MR3: SEQ ID NO:30, a sequence shown in seq id no;
HPV58-P3: SEQ ID NO: 31;
the detection primers and probes of the beta-actin are as follows:
ACTB-MF SEQ ID NO:33, a sequence shown in seq id no;
ACTB-MR SEQ ID NO:34, a sequence shown in seq id no;
ACTB-P, SEQ ID NO: 35.
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. In the other 99 negative control samples, 10 HPVs 16 positive, 10 HPVs 18 positive, 20 HPVs 52 positive and 22 HPVs 58 positive; of the 101 positive control samples, 30 were positive for HPV16, 25 were positive for HPV18, 27 were positive for HPV52, and 26 were positive for HPV 58.
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
Bisulfite modification was performed with ZYMO RESEARCH Bio-company KIT EZ DNA Methylation TM KIT (D5002) instructions.
3. Amplification and detection
Preparing a liquid system:
TABLE 10 liquid distribution System
Amplification procedure:
TABLE 11 amplification procedure
4. Detection result
The gene marker takes 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 TAC1 is 6, the positive judgment value delta Ct of ZIC1 is 5.5, the HPV methylation marker takes a Ct value 39 as a sample positive judgment standard, when the detection result delta Ct value is smaller than the corresponding positive judgment value, the marker is judged to be positive to a sample detection result, otherwise, the detection result of 200 TCT samples is negative, and the detection result of 200 TCT samples is as follows:
Table 12 200 TCT sample test results
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Note that: when no amplification curve is used, ct value is uniformly assigned to 45, and the space of HPV type indicates no HPV infection or other type (besides HPV 16/18/52/58) of HPV infection.
Analyzing the detection result, wherein the analysis result is as follows:
TABLE 13 HPV16/18/52/58 analysis results
TABLE 14 analysis results
From the results, in the TCT sample, under the condition that the high specificity of 97% is maintained, the combined detection of SOX1/SEPT9/TAC1 genes has the overall sensitivity of 88.1%, and for the HSIL group, the sensitivity is 79.7%, and the cervical cancer group can be detected by 100%; under the condition of maintaining 96% of high specificity, the combined detection of SOX1/SEPT9/ZIC1 genes has the overall sensitivity up to 88.1%, and for an HSIL group, the sensitivity also up to 79.7%, and the cervical cancer group can be detected by 100%; the genome combination and HPV16/18/52/58 virus methylation are combined, SOX1/SEPT9/TAC1 and HPV16/18/52/58 are combined, the specificity is kept at 97%, the overall sensitivity is improved to 92.1%, and the sensitivity is improved to 86.4% for the HSIL group. SOX1/SEPT9/ZIC1 and HPV16/18/52/58 are combined for detection, the specificity is kept at 94.9%, the overall sensitivity is improved to 93.1%, and the sensitivity is improved to 88.1% for the HSIL group. 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.
Claims (14)
1. A primer probe composition, characterized in that the composition consists of primer probe sequences for detection of HPV type 16, HPV type 18, HPV type 52, HPV type 58, SOX1 gene, SEPTIN9 gene and TAC1 gene, or of primer probe sequences for detection of HPV type 16, HPV type 18, HPV type 52, HPV type 58, SOX1 gene, SEPTIN9 gene and ZIC1 gene;
Wherein, the primer probe sequences for HPV16 type, HPV18 type, HPV52 type, HPV58 type, SOX1 gene, SEPTIN9 gene and TAC1 gene detection are as follows:
group one: the primer pair sequence for HPV type 16 detection is shown as SEQ ID NO:17 and SEQ ID NO: shown at 18; the probe sequence is shown in SEQ ID NO: 19;
group II: the primer pair sequence for HPV type 18 detection is shown as SEQ ID NO:21 and SEQ ID NO: shown at 22; the probe sequence is shown in SEQ ID NO: indicated at 23;
Group III: the primer pair sequence for HPV52 type detection is shown as SEQ ID NO:25 and SEQ ID NO: 26; the probe sequence is shown in SEQ ID NO: shown at 27;
group four: the primer pair sequence for HPV type 58 detection is shown in SEQ ID NO:29 and SEQ ID NO: shown at 30; the probe sequence is shown in SEQ ID NO: 31;
Group five: the primer pair sequence for SOX1 gene detection is shown as SEQ ID NO:1 and SEQ ID NO:2 is shown in the figure; the probe sequence is shown in SEQ ID NO:3 is shown in the figure;
Group six: the primer pair sequence for SEPTIN9 gene detection is shown in SEQ ID NO:5 and SEQ ID NO:6 is shown in the figure; the probe sequence is shown in SEQ ID NO: shown in figure 7;
Group seven: the primer pair sequence for TAC1 gene detection is shown as SEQ ID NO:9 and SEQ ID NO:10 is shown in the figure; the probe sequence is shown in SEQ ID NO: 11;
wherein, the primer probe sequences for HPV16 type, HPV18 type, HPV52 type, HPV58 type, SOX1 gene, SEPTIN9 gene and ZIC1 gene detection are as follows:
group one: the primer pair sequence for HPV type 16 detection is shown as SEQ ID NO:17 and SEQ ID NO: shown at 18; the probe sequence is shown in SEQ ID NO: 19;
group II: the primer pair sequence for HPV type 18 detection is shown as SEQ ID NO:21 and SEQ ID NO: shown at 22; the probe sequence is shown in SEQ ID NO: indicated at 23;
Group III: the primer pair sequence for HPV52 type detection is shown as SEQ ID NO:25 and SEQ ID NO: 26; the probe sequence is shown in SEQ ID NO: shown at 27;
group four: the primer pair sequence for HPV type 58 detection is shown in SEQ ID NO:29 and SEQ ID NO: shown at 30; the probe sequence is shown in SEQ ID NO: 31;
Group five: the primer pair sequence for SOX1 gene detection is shown as SEQ ID NO:1 and SEQ ID NO:2 is shown in the figure; the probe sequence is shown in SEQ ID NO:3 is shown in the figure;
Group six: the primer pair sequence for SEPTIN9 gene detection is shown in SEQ ID NO:5 and SEQ ID NO:6 is shown in the figure; the probe sequence is shown in SEQ ID NO: shown in figure 7;
Group eight: the primer pair sequence for ZIC1 gene detection is shown as SEQ ID NO:13 and SEQ ID NO: 14; the probe sequence is shown in SEQ ID NO: 15.
2. The primer probe composition of claim 1, wherein: the 3 'end of the probe is connected with a fluorescence quenching group, and the 5' end is connected with a fluorescence reporting group.
3. A primer probe composition according to claim 2, wherein: the fluorescence quenching group is BHQ, BHQ1, BHQ2 or TAMRA.
4. A primer probe composition according to claim 3, wherein: the fluorescence quenching group is BHQ1.
5. A primer probe composition according to claim 2, wherein: the fluorescent reporter group is FAM, CY3, CY5, HEX or TET.
6. The primer probe composition of claim 5, wherein: the fluorescent reporter group is FAM.
7. The application of the primer probe composition in preparing cervical cancer detection reagent or kit is characterized in that: the primer probe composition comprises the primer probe composition of claim 1.
8. A cervical cancer detection reagent, characterized in that: the detection reagent comprises the primer probe composition of claim 1.
9. The cervical cancer detection reagent according to claim 8, wherein: the detection reagent also comprises one or more of dNTPs, DNA polymerase, buffer solution, nuclease-free water and internal reference genes.
10. The cervical cancer detection reagent according to claim 9, wherein: the reference gene is beta-actin.
11. The cervical cancer detection reagent according to claim 10, wherein: the reference gene beta-actin detection reagent comprises a primer pair and a probe for detecting the reference gene; the primer pair has the sequence shown in SEQ ID NO:33 and SEQ ID NO:34, the sequence of the probe is shown as SEQ ID NO: shown at 35.
12. A cervical cancer detection kit, characterized in that: the detection kit comprises the cervical cancer detection reagent according to any one of claims 8 to 11.
13. A method for detecting methylation of a target gene for non-disease diagnosis, comprising 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 a target gene of a sample to be detected after modification of a conversion reagent by using the detection reagent according to any one of claims 8 to 11 or the kit according to claim 12;
Wherein; comparing and analyzing the detected delta Ct value obtained in the step S2 with a critical value, and when the delta Ct value is smaller than the critical value, determining that the collected sample to be detected contains at least one methylated target gene;
The conversion reagent is one or more of bisulfite, hydrazine salt or bisulphite.
14. 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 target gene;
b. And a result judgment means: the detection component is used for quantitatively detecting the DNA methylation degree result of the target gene according to the detection result of the detection component and outputting a risk value of cervical cancer;
Wherein the detection means for quantitatively detecting the degree of DNA methylation of a target gene comprises the detection reagent according to any one of claims 8 to 11 or the kit according to claim 12.
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