CN116200497A - Methylation detection reagent for human cervical cells and cell type determination method - Google Patents

Abstract

The invention provides a reagent for detecting methylation of human cervical cells and a method for determining cell types. In the present invention, 20 nucleotide sequences specific to abnormal methylation of human cervical cancer cells are used as positive reference reagents and negative reference reagents respectively, thereby improving the sensitivity and accuracy of methylation detection. This application uses the 10 specific sequences related to the abnormal increase of the methylation level of cervical cancer cells and/or multiple sequences among the 10 specific sequences related to the abnormal decrease of the methylation level of cervical cancer cells The average value of the methylation level is used to judge whether the cervical cells to be tested have abnormal methylation levels, so as to judge whether the cervical cells to be tested have differentiated into cervical cancer cells, which has the advantages of high sensitivity, good specificity, and is not easy to cause misjudgment Effect.

Description

Methylation detection reagent for human cervical cells and method for determining cell types

Technical Field

The invention belongs to the technical field of biomedicine and relates to a methylation detection reagent for human cervical cells and a method for determining cell types.

Background Art

At present, cervical cancer is a common and frequent type of cancer among women, so it has received increasing attention. In the early stages of cervical cancer, it usually first develops into low-grade squamous intraepithelial lesions (LGSIL) and high-grade squamous intraepithelial lesions (HGSIL). Among them, low-grade squamous intraepithelial lesions are also called cervical intraepithelial neoplasias-phase 1 (CIN1), and high-grade squamous intraepithelial lesions include cervical intraepithelial neoplasias-phase 2 (CIN2) and cervical intraepithelial neoplasias-phase 3 (CIN3). However, if it is not treated actively, it may often develop into advanced cancer, such as squamous cell carcinoma (SCC) or adenomatous carcinoma (ACC).

Current studies have shown that in the progression and development of cervical cancer, certain specific sites of specific genes (such as some tumor suppressor genes) will undergo site-specific hypermethylation, that is, the methylation level will increase. Therefore, kits that can detect the methylation level of specific sites of different genes are often designed to detect whether the cervical cells to be tested are cervical cancer cells. However, each of the above-mentioned kits often only detects the methylation level of each specific gene. Since there are many genes that are highly methylated in the whole genome of cervical cancer cells, and the methylation levels of different genes vary with the different types of cancer cells, the methylation levels of the same gene at different stages are also different. If only one kit is used to detect the methylation level of a gene, its test results are often difficult to reflect the specific stage of cervical cancer. In order to obtain reliable test results, multiple kits need to be used to simultaneously detect multiple genes corresponding to them, which will increase the number of test kits used and the number of tests, and may cause over-testing. In addition, if a certain kit is used and the gene does not undergo abnormal methylation, it will cause inaccurate test results.

Summary of the invention

The purpose of the present application is to provide a methylation detection reagent for human cervical cells, which detects the methylation level of the cervical cells to be tested, so as to determine whether the cervical cells to be tested are cervical cancer cells based on the methylation level of specific polynucleotide sequences of the cervical cells to be tested.

Another object of the present application is to provide a method for determining the cell type of human cervical cells, which compares the average methylation level of a specific sequence of the cervical cells to be tested with the average methylation level of the same sequence of normal cervical cells, thereby determining whether the cell type of the cervical cells to be tested is cervical cancer cells.

In order to achieve the above purpose, the technical solution of this application is as follows:

A positive reference reagent is selected from any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20, wherein the sequence contains methylated cytosine-phosphate-guanine.

In some embodiments, the methylation level of each sequence in the positive reference reagent is selected from any value between 60% and 100%, and the methylation levels of different sequences are the same or different. The methylation level is defined as the percentage of the number of methylated cytosine-phosphate-guanine in each sequence to the total number of cytosine-phosphate-guanine in the sequence.

The above-mentioned positive reference reagent is used as a positive control having the same nucleotide sequence as each sequence when detecting the methylation level of any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20 in the genome of ex vivo cervical cells.

A negative reference reagent is selected from any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20, and the sequence does not contain methylated cytosine-phosphate-guanine.

The above-mentioned negative reference reagent is used as a negative control having the same nucleotide sequence as each sequence when detecting the methylation level of any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20 in the genome of ex vivo cervical cells.

A detection reagent combination that can specifically detect the methylation level of one or a combination of several sequences shown in SEQ ID No:1 to SEQ ID No:20 in a DNA sample, wherein the methylation level is defined as the percentage of the number of methylated cytosine-phosphate-guanine in each DNA sequence to the total number of cytosine-phosphate-guanine in the DNA sequence.

In some embodiments, the detection reagent combination further comprises: a reaction reagent and a detection reagent. The reaction reagent can differentially modify methylation sites and non-methylation sites in a DNA sample. After treating the DNA sample with the reaction reagent, the detection reagent can determine whether each cytosine-phosphate-guanine in the sequence shown in SEQ ID No: 1 to SEQ ID No: 20 is methylated or non-methylated.

In some embodiments, the above-mentioned detection reagent combination further includes: a positive reference reagent and/or a negative reference reagent.

The positive reference reagent may be selected from any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20, wherein the sequences shown in SEQ ID No: 1 to SEQ ID No: 20 contain methylated cytosine-phosphate-guanine, and the methylation level of each sequence segment in the positive reference reagent is selected from any value between 60% and 100%, and the methylation levels of different sequence segments are the same or different, and the methylation level is defined as the percentage of the number of methylated cytosine-phosphate-guanine in each sequence segment to the total number of cytosine-phosphate-guanine in the sequence segment.

The negative reference reagent can be selected from any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20, and the sequence does not contain methylated cytosine-phosphate-guanine.

In some embodiments, in the above-mentioned detection reagent combination, the DNA sample is from human ex vivo cervical cells, and the ex vivo cervical cells are selected from human cervical epithelial cells or cervical tissue cells obtained by puncture.

A DNA methylation detection kit, comprising: any one of the above detection reagent combinations.

A PCR reagent combination includes: a PCR buffer, and PCR primer pairs that can specifically and one-to-one amplify sequences shown as SEQ ID No: 1 to SEQ ID No: 20, each PCR primer pair including a forward primer and a reverse primer.

A PCR kit comprises: the above-mentioned PCR reagent combination and bisulfite.

A method for determining the type of cervical cells in vitro, comprising the following steps:

Select any three or more sequences from the sequences shown in SEQ ID No: 1 to SEQ ID No: 10 in the genome of the ex vivo cervical cells of the individual to be tested as the target sequence group;

Determine the methylation level of the whole genome of ex vivo cervical cells, select the methylation level of each sequence in the target sequence group, and obtain the average methylation level of the target sequence group; the methylation level is defined as the percentage of the number of methylated cytosine-phosphate-guanine in each sequence to the total number of cytosine-phosphate-guanine in the sequence;

Compare the average methylation level with the reference methylation level;

When the average methylation level is greater than or equal to a specific multiple of the reference methylation level, the cell type of the isolated cervical cells is determined to be cervical cancer cells; otherwise, the cell type of the isolated cervical cells is determined to be non-cervical cancer cells.

In some embodiments, the cervical cancer cells are squamous cell carcinoma cells or adenocarcinoma cells.

In some embodiments, the reference methylation level is an average of the methylation levels of sequences having the same nucleotide sequence as the target sequence group in ex vivo cervical cells of a set of normal individuals, and the set of normal individuals includes three or more normal individuals.

In some embodiments, the specific multiple is any value between 2 and 7 times.

A method for determining the type of cervical cells in vitro, comprising the following steps:

Select any three or more sequences from the sequences shown in SEQ ID No: 11 to SEQ ID No: 20 in the genome of the ex vivo cervical cells of the individual to be tested as the target sequence group;

Determine the methylation level of the whole genome of ex vivo cervical cells, select the methylation level of each sequence in the target sequence group, and obtain the average methylation level of the target sequence group; the methylation level is defined as the percentage of the number of methylated cytosine-phosphate-guanine in each sequence to the total number of cytosine-phosphate-guanine in the sequence;

Compare the average methylation level with the reference methylation level;

When the average methylation level is less than or equal to a specific percentage of the reference methylation level, the cell type of the isolated cervical cells is determined to be cervical cancer cells; otherwise, the cell type of the isolated cervical cells is determined to be non-cervical cancer cells.

In some embodiments, the cervical cancer cells are squamous cell carcinoma cells or adenocarcinoma cells.

In some embodiments, the reference methylation level is an average of the methylation levels of sequences having the same nucleotide sequence as the target sequence group in ex vivo cervical cells of a set of normal individuals, and the set of normal individuals includes three or more normal individuals.

In some embodiments, the specific percentage is any value between 20% and 65%.

A method for determining the type of cervical cells in vitro, comprising the following steps:

Select any two or more sequences from the sequences shown in SEQ ID No: 1 to SEQ ID No: 10 in the genome of the ex vivo cervical cells of the individual to be tested as a high methylation sequence group, and select any two or more sequences from the sequences shown in SEQ ID No: 11 to SEQ ID No: 20 in the genome as a low methylation sequence group;

Determine the methylation level of the whole genome of ex vivo cervical cells, select the methylation level of each sequence in the high methylation sequence group, and obtain the average methylation level of the high methylation sequence group as the high methylation level; select the methylation level of each sequence in the low methylation sequence group, and obtain the average methylation level of the low methylation sequence group as the low methylation level; the methylation level is defined as the percentage of the number of methylated cytosine-phosphate-guanine in each sequence to the total number of cytosine-phosphate-guanine in the sequence;

comparing the magnitude of the hypermethylation level to a reference hypermethylation level, and comparing the magnitude of the hypomethylation level to a reference hypomethylation level;

When the high methylation level is greater than or equal to a specific multiple of the reference high methylation level, and when the low methylation level is less than or equal to a specific percentage of the reference low methylation level, the cell type of the ex vivo cervical cells is determined to be cervical cancer cells; otherwise, the cell type of the ex vivo cervical cells is determined to be non-cervical cancer cells.

In some embodiments, the cervical cancer cells are squamous cell carcinoma cells or adenocarcinoma cells.

In some embodiments, the reference high methylation level is the average methylation level of sequences having the same nucleotide sequence as the high methylation sequence group in ex vivo cervical cells of a set of normal individuals; the reference low methylation level is the average methylation level of sequences having the same nucleotide sequence as the low methylation sequence group in ex vivo cervical cells of a set of normal individuals, and the set of normal individuals includes three or more normal individuals.

In some embodiments, the specific multiple is any value between 2 and 7 times.

In some embodiments, the specific percentage is any value between 20% and 65%.

Due to the adoption of the above technical solutions, some embodiments of the present application achieve the following technical effects:

First, some embodiments of the present application use positive reference reagents and/or negative reference reagents having the same nucleotide sequence as the specific sequence indicating the methylation level when detecting the methylation level of the cervical cells to be tested, so as to correct the detection result of the methylation level, thereby improving the accuracy of the methylation level detection.

Second, some embodiments of the present application screened out 10 specific sequences (SEQ ID No: 1 to SEQ ID No: 10) related to abnormally increased methylation levels of cervical cancer cells, and used the average methylation level of any combination of three or more sequences in these 10 sequences to determine whether the cervical cells to be tested have abnormally increased methylation levels. These 10 specific sequences are located at different positions in the whole genome of cervical cells, and have specificity and sensitivity to increased methylation of cervical cancer cells. Experiments have shown that any combination of three sequences can effectively detect whether the cervical cells to be tested have abnormally increased methylation levels, and can thus be used to determine whether the cervical cells to be tested have differentiated into cervical cancer cells.

Third, some embodiments of the present application screened out 10 specific sequences (SEQ ID No: 11 to SEQ ID No: 20) related to the abnormal reduction of methylation levels in cervical cancer cells, and used the average methylation level of any combination of three or more sequences in these 10 sequences to determine whether the cervical cells to be tested have an abnormal reduction in methylation levels. These 10 specific sequences are located at different positions in the whole genome of cervical cells, and have specificity and sensitivity to the reduction of methylation in cervical cancer cells. Experiments have shown that any combination of three sequences can effectively detect whether the cervical cells to be tested have an abnormal reduction in methylation levels, and can thus be used to determine whether the cervical cells to be tested have differentiated into cervical cancer cells.

Fourth, some embodiments of the present application also use a combination of any two or more sequences in the sequences shown in SEQ ID No: 1 to SEQ ID No: 10 and any two or more sequences in the sequences shown in SEQ ID No: 11 to SEQ ID No: 20 to determine whether the methylation level of the cervical cells to be tested is abnormal. Any two or more sequences in the sequences shown in SEQ ID No: 1 to SEQ ID No: 10 can be used as indicators to indicate whether the methylation level of the cervical cells to be tested is abnormally increased, and any two or more sequences in the sequences shown in SEQ ID No: 11 to SEQ ID No: 20 can be used as indicators to indicate whether the methylation level of the cervical cells to be tested is abnormally decreased. The present application combines the two indicators of abnormally increased and abnormally decreased methylation levels for judgment, which can more accurately determine whether the cervical cells to be tested have abnormal methylation levels, thereby more accurately determining whether the cervical cells to be tested have differentiated into cervical cancer cells.

Fifth, in the determination method of cervical cell type in some embodiments of the present application, the methylation level of a specific sequence can be obtained by using whole genome methylation level detection, or by using methylation-specific PCR method and supplemented by positive reference reagents and/or negative reference reagents to detect methylation levels. Therefore, the determination method of the present application has strong adaptability and better sensitivity. In addition, if whole genome methylation level detection is used, the determination method of the present application only needs one test to obtain the methylation level of all specific fragments, avoiding the use of multiple detection kits for multiple tests. Although the number of tests and the workload are reduced, it still has high detection efficiency and detection sensitivity.

Sixth, some embodiments of the present application use the average value of the methylation levels of multiple specific sequences as an indicator for judging whether the methylation of the cervical cells to be tested is abnormal, rather than comparing the methylation level of each specific sequence of the cervical cells to be tested with the methylation level of the same sequence of normal cervical cells. This can prevent the phenomenon of large differences in the methylation levels of a single specific sequence due to individual differences, thereby avoiding misjudgment. In addition, the method of the present application does not compare whether each site is methylated, which can prevent misjudgment caused by differences in site methylation. All of the above are helpful to improve the accuracy of methylation level judgment.

DETAILED DESCRIPTION

The technology of the present application is described in detail below in conjunction with specific implementations. It should be noted that the following specific implementations are only used to help those skilled in the art understand the present application, and are not intended to limit the present application.

The present invention is further described below with reference to the embodiments.

Embodiment 1

This embodiment provides a positive reference reagent for detecting the methylation level of ex vivo cervical cells, which is selected from any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20.

Optionally, the positive reference reagent can be selected from any one of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20. For example, it can be selected from the sequence shown in SEQ ID No: 1; it can be selected from the sequence shown in SEQ ID No: 2; it can be selected from the sequence shown in SEQ ID No: 3; and so on. If only the methylation level of a specific gene in a DNA sample of an ex vivo cervical cell to be tested needs to be detected, the positive reference reagent can be selected from a sequence with the same nucleotide sequence as the specific gene. Optionally, the positive reference reagent can be selected from any combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20. For example, it can be selected from any combination of two or more of the sequences shown in SEQ ID No: 1 to SEQ ID No: 10; it can be selected from any combination of two or more of the sequences shown in SEQ ID No: 11 to SEQ ID No: 20; it can be selected from any combination of one or more of the sequences shown in SEQ ID No: 1 to SEQ ID No: 10, and selected from any combination of one or more of the sequences shown in SEQ ID No: 11 to SEQ ID No: 20. If it is necessary to detect the methylation levels of multiple specific genes in the DNA sample of the ex vivo cervical cells to be tested, the positive reference reagent may contain a sequence identical to the nucleotide sequence of each specific gene.

As a positive reference reagent, each of the above-mentioned sequences should contain methylated cytosine-phosphate-guanine (Methylated CpG). Methylation in this article refers to the methylation of CpG sites and does not represent the methylation of other sites. The average methylation levels in different sequences can be the same or different. The average methylation level (or the average value of the methylation level) is defined as the percentage of the number of methylated CpGs in a sequence to the total number of CpGs. Therefore, the number of CpGs in different sequences may be different, but the average methylation level may be the same. The same average methylation level does not mean that the same CpG site in different samples will be equally methylated or equally unmethylated. It is also possible that the methylation of certain specific CpG sites in different samples is different, but the average methylation level of these different samples is the same.

In order to achieve a better positive reference effect, the average methylation level of different sequences in the positive reference reagent is at least 50%, or 60%, or 70%, or 80%, or 90%, or 100%. When the average methylation level of a certain sequence is 100%, it means that all CpG sites in the sequence are methylated.

The positive reference reagent is generally carried out simultaneously with the methylation test of the DNA sample. When the average methylation level of a certain sequence in the DNA sample (for example, the whole genome sample of cervical cells to be tested) is tested, the average methylation level of the sequence with the same nucleotide sequence as that of the sequence in the positive reference reagent is also tested. Then, the result of the average methylation level of the positive reference reagent obtained is compared with the result of the reagent manual. If the result of the average methylation level of the positive reference reagent obtained is basically consistent with the result of the reagent manual, it means that this test is valid. If the results of the two are very different, it means that there is a problem with the result of this test and further testing is required.

For example, when detecting the methylation level of any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20 in the genome of ex vivo cervical cells, a sequence having the same nucleotide sequence as the above sequence is selected in the positive reference reagent, and the methylation level of the selected one or more sequences is simultaneously determined, and then the average methylation level of the positive reference reagent is obtained, and then the result of the average methylation level of the positive reference reagent is compared with the result in the reagent manual. When comparing the average methylation level, the principle of comparing the same sequences separately is adopted, that is, the sequences in the genome of ex vivo cervical cells having the same nucleotide sequence as the positive reference reagent are compared one by one.

Embodiment 2

This embodiment provides a negative reference reagent for detecting the methylation level of ex vivo cervical cells, which is selected from any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20.

Optionally, the negative reference reagent can be selected from any one of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20. For example, it can be selected from the sequence shown in SEQ ID No: 1; it can be selected from the sequence shown in SEQ ID No: 2; it can be selected from the sequence shown in SEQ ID No: 3; and so on. If only the methylation level of a specific gene in a DNA sample of an ex vivo cervical cell to be tested needs to be detected, the negative reference reagent can be selected from a sequence having the same nucleotide sequence as the specific gene.

Optionally, the negative reference reagent can be selected from any combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20. For example, it can be selected from any combination of two or more of the sequences shown in SEQ ID No: 1 to SEQ ID No: 10; it can be selected from any combination of two or more of the sequences shown in SEQ ID No: 11 to SEQ ID No: 20; it can be selected from any combination of one or more of the sequences shown in SEQ ID No: 1 to SEQ ID No: 10, and selected from any combination of one or more of the sequences shown in SEQ ID No: 11 to SEQ ID No: 20. If it is necessary to detect the methylation levels of multiple specific genes in the DNA sample to be tested of the in vitro cervical cells to be tested, the negative reference reagent can contain a sequence identical to the nucleotide sequence of each specific gene.

As a negative reference reagent, each of the above sequences should not contain methylated cytosine-phosphate-guanine (methylated CpG). That is, the CpG sites in each of the above sequences are not methylated. At this time, the average methylation level of different sequences in the negative reference reagent is 0%.

The negative reference reagent is generally carried out simultaneously with the methylation test of the DNA sample. When the average methylation level of a certain sequence in the DNA sample (for example, the whole genome sample of cervical cells to be tested) is tested, the average methylation level of the sequence with the same nucleotide sequence as that of the sequence in the negative reference reagent is also tested. Then, the result of the average methylation level of the negative reference reagent obtained is compared with the result of the reagent manual. If the result of the average methylation level of the negative reference reagent obtained is basically consistent with the result of the reagent manual (such as 0% excluding systematic errors and/or interference factors), it means that this test is valid. If the results of the two are very different, it means that there is a problem with the result of this test and further testing is required.

For example, when detecting the methylation level of any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20 in the genome of ex vivo cervical cells, a sequence having the same nucleotide sequence as the above sequence is selected in the negative reference reagent, and the methylation level of the selected one or more sequences is simultaneously determined, and then the average methylation level of the negative reference reagent is obtained, and then the average methylation level of the negative reference reagent is compared with the result in the reagent manual. When comparing the average methylation level, the principle of comparing the same sequences separately is adopted, that is, the sequences in the genome of ex vivo cervical cells having the same nucleotide sequence as the negative reference reagent are compared one by one.

When detecting the methylation of a DNA sample, the negative reference reagent of this embodiment and the positive reference reagent of embodiment 1 can be used simultaneously to improve the reliability of the detection. Of course, they can also be used separately.

Embodiment 3

This embodiment provides a method for screening the sequences SEQ ID No: 1 to SEQ ID No: 20 in the whole genome of cervical cancer cells. The method measures the methylation level of the whole genome of human cervical cells (including normal cervical cells and cervical cancer cells), and then screens based on the abnormally increased and abnormally decreased values of the methylation levels of normal cervical cells and cervical cancer cells, thereby screening out 20 sequences (SEQ ID No: 1 to SEQ ID No: 20) that are closely related to the occurrence of cervical cancer. These 20 sequences can be used as reference reagents in Examples 1 and 2.

Specifically, the method of this embodiment includes the following steps:

(1) Obtain a certain number of normal cervical cells from normal individuals and a certain number of cervical cancer cells (including squamous cell carcinoma cells or adenocarcinoma cells) from diseased individuals, and extract genomic DNA from the above two types of cells respectively.

(2) Fragmenting the above genomic DNA to obtain DNA fragments.

(3) The DNA fragments are subjected to end repair, adaptation ligation, bisulfite treatment, PCR amplification, library test, sequencing and bioinformatics analysis in sequence to finally obtain the methylation level index of the genomic DNA.

(4) Screen out 10 sequences whose average methylation levels are abnormally increased compared with the average methylation level of normal cervical cells (i.e., SEQ ID No: 1 to SEQ ID No: 10), and screen out 10 sequences whose average methylation levels are abnormally decreased compared with the average methylation level of normal cervical cells (i.e., SEQ ID No: 11 to SEQ ID No: 20).

Experiments have shown that the 20 sequences from SEQ ID No: 1 to SEQ ID No: 20 screened out through the above steps are only related to cervical cancer cells, and therefore can be used as specific biomarkers and specific reference reagents for detecting cervical cancer cells.

The basic information of the above 20 sequences is shown in Table 1 below:

Table 1 is a basic information table of SEQ ID No: 1 to SEQ ID No: 20

As shown in Table 1, in cervical cancer cells, the average methylation levels of SEQ ID No: 1 to SEQ ID No: 10 are abnormally elevated relative to normal cervical cells, referred to as hyper differentially methylated regions (Hyper differentially methylated regions, referred to as Hyper DMRs); while the average methylation levels of SEQ ID No: 11 to SEQ ID No: 20 are abnormally reduced relative to normal cervical cells, referred to as hypo differentially methylated regions (Hypodifferentially methylated regions, referred to as Hypo DMRs). The table also shows the accession number, adjacent gene name, gene interval, and distance to the adjacent gene interval of these 20 sequences (a negative sign indicates that the sequence is located upstream of the adjacent gene, a positive sign indicates that the sequence is located downstream of the adjacent gene, and 0 indicates that the sequence is located in the adjacent gene content in the table).

Exemplarily, SEQ ID No: 1 is located in the intron of the ARHGAP42 gene, and its distance from the ARHGAP42 gene is 0. SEQ ID No: 2 is located in the promoter of the NDN gene, and it is located 2281 upstream of the NDN gene. A negative distance means that the gene is upstream of the transcriptional start site (TSS). A positive distance means that it is downstream of the transcriptional start site. The distances expressed above are the distances of the relevant genes from the TSS.

The results of determining the methylation levels of the above SEQ ID No: 1 to SEQ ID No: 20 in normal human cervical cells are shown in Table 2.

Table 2 is a table showing the methylation levels of SEQ ID No: 1 to SEQ ID No: 20 in normal human cervical cells

From the test results of normal cervical cells of a certain number of normal individuals in step (1), 5 test results are randomly selected and presented as the test results of the normal group (Control) in Table 2. Among them, the serial numbers Ctr_1 to Ctr_5 represent the number of test samples of normal cervical cells. After calculation, it can be known that the average methylation level of SEQ ID No: 1 to SEQ ID No: 10 of normal cervical cells is low, which is below 34% (i.e., 0.34). The methylation level of SEQ ID No: 11 to SEQ ID No: 20 of normal cervical cells is high, which is above 71% (i.e., 0.71). Therefore, if it is detected that the methylation level of SEQ ID No: 1 to SEQ ID No: 10 in the cervical cell sample of the individual to be tested increases to a certain value, it means that the cervical cell sample of the individual to be tested is a cervical cancer cell (possibly a squamous cell carcinoma cell or adenocarcinoma cell). In addition, if the methylation levels of SEQ ID No: 1 to SEQ ID No: 10 in the cervical cell sample of the tested individual are detected to be reduced to a certain value, it means that the cervical cell sample of the tested individual is a cervical cancer cell (possibly a squamous cell carcinoma cell or an adenocarcinoma cell).

The results of determining the methylation levels of the above SEQ ID No: 1 to SEQ ID No: 20 in abnormal human cervical cells (such as adenocarcinoma cells) are shown in Table 3.

Table 3 is a table showing the methylation levels of SEQ ID No: 1 to SEQ ID No: 20 in human abnormal cervical cells

Two test results are randomly selected from the test results of cervical cancer cells of a certain number of diseased individuals in step (1) and are presented in Table 3 as the test results of the diseased group (such as the CCA group).

As can be seen from Table 2 and Table 3, the methylation levels of SEQ ID No: 1 to SEQ ID No: 10 in normal cervical cells are low, while the methylation levels in cervical cancer cells are high. For the same sequence, the increased methylation level is at least 1.5 times, or at least 2 times, or at least 3 times, or at least 4 times, or at least 5 times, or at least 6 times, but not more than 7 times, of the normal methylation level. From the above experimental test results, for the same sequence, the increased methylation level reached up to 6.57 times the normal methylation level. Because the methylation levels of SEQ ID No: 1 to SEQ ID No: 10 in cervical cancer cells have increased by a large multiple, it is possible to judge whether the in vitro cervical cells to be tested are cervical cancer cells based on the multiples of the increased methylation levels of the above sequences in the cervical cell samples to be tested.

As shown in Table 2 and Table 3, the methylation levels of SEQ ID No:11 to SEQ ID No:20 in normal cervical cells are relatively high, while the methylation levels in cervical cancer cells are relatively low, and the variation trend is just opposite to that of SEQ ID No:1 to SEQ ID No:10. For the same sequence, the reduced methylation level is at most 65% or less, or at most 55% or less, or at most 40% or less of the normal methylation level, and the lowest reaches 20%. From the above experimental test results, for the same sequence, the reduced methylation level reaches up to 64% of the normal methylation level. Because the methylation levels of SEQ ID No:11 to SEQ ID No:20 in cervical cancer cells have been greatly reduced, it is possible to judge whether the in vitro cervical cells to be tested are cervical cancer cells based on the percentage of the reduced methylation levels of the above sequences in the cervical cell samples to be tested.

The present application uses the average value of the methylation levels of multiple specific sequences as an indicator for judging whether the methylation of the cervical cells to be tested is abnormal, rather than comparing the methylation level of each specific sequence of the cervical cells to be tested with the methylation level of the same sequence of normal cervical cells. This can prevent the phenomenon of large differences in the methylation level of a single specific sequence due to individual differences, and help improve the accuracy of methylation level judgment. This is because the average methylation levels of different specific fragments of the same individual are similar, with no difference in order of magnitude.

The results of determining the methylation levels of the above SEQ ID No: 1 to SEQ ID No: 20 in abnormal human cervical cells (such as squamous cell carcinoma cells) are shown in Table 4.

Table 4 is a table showing the methylation levels of SEQ ID No: 1 to SEQ ID No: 20 in human abnormal cervical cells

Five test results are randomly selected from the test results of cervical cancer cells of a certain number of diseased individuals in step (1) and are presented in Table 4 as the test results of the diseased group (such as the CCS group).

It can be seen from Tables 2, 3 and 4 that the increased methylation levels of SEQ ID No: 1 to SEQ ID No: 10 in the CCS group are similar to the increased methylation levels of the CCA group, and the decreased methylation levels of SEQ ID No: 11 to SEQ ID No: 20 in the CCS group are similar to the decreased methylation levels of the CCA group. Therefore, SEQ ID No: 1 to SEQ ID No: 10 can be used as biomarkers and reference reagents for detecting cervical cancer cells (including squamous cell carcinoma cells and adenocarcinoma cells).

When detecting and judging the methylation levels of SEQ ID No:1 to SEQ ID No:20 of cervical cancer cells, because the error of judging whether the cervical cell sample to be tested is cervical cancer cells based on the methylation level of only one of the sequences is relatively large, the average methylation level of the combination of two or more sequences is generally used to judge whether the cervical cell sample to be tested is cervical cancer cells.

For example, if the whole genome methylation detection method is used to detect the methylation level of the whole genome of the cervical cells to be tested, three sequences can be randomly selected from the 10 sequences of SEQ ID No: 1 to SEQ ID No: 10, and the methylation levels of the three sequences are averaged to obtain the average methylation level, which is then compared with the average methylation level of the three sequences with the same nucleotide sequence as the sequences of normal cervical cells, so as to determine whether the cervical cells to be tested are cervical cancer cells based on the degree of increased methylation. In other embodiments, more sequences can also be selected.

For example, if the whole genome methylation detection method is used to detect the methylation level of the whole genome of the cervical cells to be tested, 3 sequences can be randomly selected from the 20 sequences of SEQ ID No: 11 to SEQ ID No: 10, and the methylation levels of the 3 sequences are averaged to obtain the average methylation level, which is then compared with the average methylation level of the same 3 sequences of normal cervical cells, so as to determine whether the cervical cells to be tested are cervical cancer cells according to the extent of methylation reduction. In other embodiments, more sequences can also be selected.

Exemplarily, if the whole genome methylation detection method is used to detect the methylation level of the whole genome of the cervical cells to be tested, two sequences can be selected from the 10 sequences of SEQ ID No: 1 to SEQ ID No: 10, and the methylation levels of the two sequences are averaged to obtain the average methylation level of the two sequences, and then compared with the average methylation level of the two sequences with the same sequence of normal cervical cells (which can be temporarily measured on site or measured in advance). At the same time, two sequences are selected from the 10 sequences of SEQ ID No: 11 to SEQ ID No: 20, and the methylation levels of the two sequences are averaged to obtain the average methylation level of the two sequences, and then compared with the average methylation level of the two sequences with the same sequence of normal cervical cells. Then, according to the degree of methylation reduction from SEQ ID No: 1 to SEQ ID No: 10, and at the same time combined with the degree of methylation increase from SEQ ID No: 11 to SEQ ID No: 20, it is judged whether the cervical cells to be tested are cervical cancer cells. In the above situations, if it is necessary to measure the methylation level of the corresponding sequence of normal cervical cells on site, positive reference reagents and/or negative reference reagents can be used during the measurement.

For example, if the methylation-specific PCR (MSP) method is used to detect the methylation level, it is necessary to specifically detect the methylation level of the selected sequence in the genome of the cervical cells to be tested. During the detection, the positive reference reagent and/or the negative reference reagent can be used to correct the detection result of the methylation level, and then make a corresponding judgment. When detecting the methylation level of the selected sequence in the genome of the cervical cells to be tested, the methylation level of the same selected sequence of normal cervical cells can also be detected at the same time. The selection method of the selected sequence is as shown in the above cases.

Embodiment 4

This embodiment provides a detection reagent combination, which can specifically detect the DNA methylation level of one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20 in a DNA sample. The detection reagent combination includes: a reaction reagent and a detection reagent.

Among them, the reaction reagent can differentially modify the methylated sites and unmethylated sites in the DNA sample, so that the methylated sites will not be demethylated and the unmethylated sites will not be remethylated in the subsequent reaction process, which is conducive to ensuring the accuracy of the methylation level determination. Specifically, taking the methylation-specific PCR detection method as an example, the DNA sample is converted by bisulfite. During the bisulfite conversion process, the cytosine in the DNA (single-stranded DNA) is converted into a cytosine-bisulfite derivative, and this reaction is reversible; then the cytosine-bisulfite derivative undergoes an irreversible hydrolysis and deamination process to form a uracil-bisulfite derivative. Finally, under high pH conditions, the uracil-bisulfite derivative is desulfonated to form uracil. Only unmethylated cytosine undergoes base changes under bisulfite treatment, and 5-mC and 5-hmC remain unchanged. The converted DNA is amplified by PCR, and uracil (U) is converted to thymine (T). Thus, the methylation level can be determined.

The detection reagent is used to determine whether each cytosine-phosphate-guanine in the sequence shown in SEQ ID No: 1 to SEQ ID No: 20 is methylated or unmethylated after the DNA sample is treated with the reaction reagent.

Whether the whole genome methylation detection method is used to determine the methylation level of the whole genome, or the methylation-specific PCR method is used to determine the methylation level of certain sequences in the sequences shown in SEQ ID No: 1 to SEQ ID No: 20, the above-mentioned reaction reagents and detection reagents can be used.

In addition, when determining the methylation level of normal cervical cells as a control, or when using a methylation-specific PCR method to determine the methylation level of the cervical cells to be tested, a positive reference reagent and/or a negative reference reagent can also be used. Among them, the positive reference reagent is selected from any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20, and the sequences shown in SEQ ID No: 1 to SEQ ID No: 20 contain methylated cytosine-phosphate-guanine. The negative reference reagent is selected from any one or a combination of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20, and the sequences do not contain methylated cytosine-phosphate-guanine. The positive reference reagent and the negative reference reagent are beneficial to reducing the systematic error of methylation detection.

The DNA samples come from human ex vivo cervical cells, which are selected from human cervical epithelial cells or cervical tissue cells obtained by puncture.

The various reagents in the detection reagent combination of this embodiment are placed in a container at a certain concentration, thereby becoming a component of the DNA methylation detection kit. The DNA methylation detection kit can be used to determine whether the ex vivo cervical cells are cervical cancer cells.

Embodiment 5

This embodiment provides a PCR reagent combination, which can specifically amplify the sequences shown in SEQ ID No: 1 to SEQ ID No: 20 and has PCR primer pairs corresponding to the sequences one by one, and each PCR primer pair includes a forward primer and a reverse primer. The various reagents in the PCR reagent combination are placed in a container at a certain concentration, thereby becoming a component of a PCR kit. The PCR kit also includes an amplification reagent that can specifically amplify the selected sequence in the genome of the cervical cell to be tested, including but not limited to a PCR buffer, etc.

Embodiment 6

This embodiment provides a method for determining the type of cervical cells in vitro, which comprises the following steps:

(1) selecting any three or more sequences from the sequences shown in SEQ ID No: 1 to SEQ ID No: 10 in the genome of the ex vivo cervical cells of the individual to be tested as the target sequence group;

(2) Determine the methylation level of the whole genome of ex vivo cervical cells, select the methylation level of each sequence in the target sequence group, and obtain the average methylation level of the target sequence group; the methylation level is defined as the percentage of the number of methylated cytosine-phosphate-guanine in each sequence to the total number of cytosine-phosphate-guanine in the sequence;

(3) Compare the average methylation level with the reference methylation level;

(4) When the average methylation level is greater than or equal to a specific multiple of the reference methylation level, the cell type of the isolated cervical cells is determined to be cervical cancer cells; otherwise, the cell type of the isolated cervical cells is determined to be non-cervical cancer cells.

Among them, cervical cancer cells are squamous cell carcinoma cells or adenocarcinoma cells.

The reference methylation level is an average value of the methylation levels of sequences having the same nucleotide sequence as the target sequence group in ex vivo cervical cells of a set of normal individuals, and the set of normal individuals includes three or more normal individuals.

The specific multiple is any value between 2 and 7. For example, illustratively, values such as 3, 4, 5, and 6 can be selected.

Embodiment 7

This embodiment provides a method for determining the type of cervical cells in vitro, comprising the following steps:

Select any three or more sequences from the sequences shown in SEQ ID No: 11 to SEQ ID No: 20 in the genome of the ex vivo cervical cells of the individual to be tested as the target sequence group;

Determine the methylation level of the whole genome of ex vivo cervical cells, select the methylation level of each sequence in the target sequence group, and obtain the average methylation level of the target sequence group; the methylation level is defined as the percentage of the number of methylated cytosine-phosphate-guanine in each sequence to the total number of cytosine-phosphate-guanine in the sequence;

Compare the average methylation level with the reference methylation level;

When the average methylation level is less than or equal to a specific percentage of the reference methylation level, the cell type of the isolated cervical cells is determined to be cervical cancer cells; otherwise, the cell type of the isolated cervical cells is determined to be non-cervical cancer cells.

The cervical cancer cells are squamous cell cancer cells or adenocarcinoma cells. The reference methylation level is the average value of the methylation levels of sequences having the same nucleotide sequence as the target sequence group in the in vitro cervical cells of a normal individual set, and the normal individual set includes three or more normal individuals. The specific percentage is any value between 20% and 65%.

Embodiment 8

This embodiment provides a method for determining the type of cervical cells in vitro, which comprises the following steps:

Select any two or more sequences from the sequences shown in SEQ ID No: 1 to SEQ ID No: 10 in the genome of the ex vivo cervical cells of the individual to be tested as a high methylation sequence group, and select any two or more sequences from the sequences shown in SEQ ID No: 11 to SEQ ID No: 20 in the genome as a low methylation sequence group;

Determine the methylation level of the whole genome of ex vivo cervical cells, select the methylation level of each sequence in the high methylation sequence group, and obtain the average methylation level of the high methylation sequence group as the high methylation level; select the methylation level of each sequence in the low methylation sequence group, and obtain the average methylation level of the low methylation sequence group as the low methylation level; the methylation level is defined as the percentage of the number of methylated cytosine-phosphate-guanine in each sequence to the total number of cytosine-phosphate-guanine in the sequence;

comparing the magnitude of the hypermethylation level to a reference hypermethylation level, and comparing the magnitude of the hypomethylation level to a reference hypomethylation level;

When the high methylation level is greater than or equal to a specific multiple of the reference high methylation level, and when the low methylation level is less than or equal to a specific percentage of the reference low methylation level, the cell type of the ex vivo cervical cells is determined to be cervical cancer cells; otherwise, the cell type of the ex vivo cervical cells is determined to be non-cervical cancer cells.

Wherein, the cervical cancer cells are squamous cell cancer cells or adenocarcinoma cells. The reference high methylation level is the average value of the methylation level of the sequence with the same nucleotide sequence as the high methylation sequence group in the in vitro cervical cells of the normal individual set; the reference low methylation level is the average value of the methylation level of the sequence with the same nucleotide sequence as the low methylation sequence group in the in vitro cervical cells of the normal individual set, and the normal individual set includes three or more normal individuals. The specific multiple is any value between 2 and 7 times. The specific percentage is any value between 20% and 65%.

The present application has been described by the above-mentioned relevant embodiments, but the above-mentioned embodiments are only examples for implementing the present application. It must be pointed out that the disclosed embodiments do not limit the scope of the present application. On the contrary, modifications and equivalent settings contained in the spirit and scope of the claims are included in the scope of the present application.

Claims (18)

1. A positive reference reagent, characterized in that it is selected from any one or combination of several of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20, containing methylated cells in the sequence Pyrimidine-phosphate-guanine. 2. The positive reference reagent according to claim 1, wherein the methylation level of each sequence in the positive reference reagent is selected from any value between 60% and 100%, and the methylation level of different sequences is The methylation level is the same or different, and the methylation level is defined as the percentage of the amount of methylated cytosine-phosphate-guanine in each sequence to the total amount of cytosine-phosphate-guanine in the sequence. 3. positive reference reagent as claimed in claim 1 detects the methyl group of any one or several combination in the sequence shown in SEQ ID No:1 to SEQ ID No:20 in the genome of isolated cervical cell Use as a positive control having the same nucleotide sequence as each of the described sequences at the Kl level. 4. A negative reference reagent, characterized in that it is selected from any one or combination of several of the sequences shown in SEQ ID No:1 to SEQ ID No:20, which does not contain methylated Cytosine-phosphate-guanine. 5. negative reference reagent as claimed in claim 4 detects the methyl group of any one or several combinations in the sequence shown in SEQ ID No:1 to SEQ ID No:20 in the genome of detection isolated cervical cell Use as a negative control having the same nucleotide sequence as each of the described sequences at the Kl level. 6. A combination of detection reagents, characterized in that it can specifically detect the methylation level of one or more combinations of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20 in a DNA sample, said Methylation level is defined as the percentage of methylated cytosine-phosphate-guanine in each DNA sequence to the total number of cytosine-phosphate-guanine in the DNA sequence. 7. detection reagent combination as claimed in claim 6, is characterized in that, comprises: Reagents capable of differentially modifying methylated sites and unmethylated sites in said DNA sample; and A detection reagent, after treating the DNA sample with the reaction reagent, it can be determined whether each cytosine-phosphate-guanine in the sequences shown in SEQ ID No: 1 to SEQ ID No: 20 is methylated or non-methylated. Methylation. 8. The detection reagent combination according to claim 7, further comprising: a positive reference reagent and/or a negative reference reagent; Wherein, the positive reference reagent is selected from any one or combination of several of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20, which are shown in SEQ ID No: 1 to SEQ ID No: 20 The sequence contains methylated cytosine-phosphate-guanine, the methylation level of each sequence in the positive reference reagent is selected from any value between 60% and 100%, and the methylation of different sequences The levels are the same or different, and the methylation level is defined as the percentage of the amount of methylated cytosine-phosphate-guanine in each sequence to the total amount of cytosine-phosphate-guanine in the sequence; The negative reference reagent is selected from any one or combination of several of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20, which do not contain methylated cytosine-phosphate-guanine . 9. detection reagent combination as claimed in claim 6 is characterized in that, described DNA sample is from the isolated cervical cell of human, and described isolated cervical cell is selected from human cervical epidermal cell or the cervical tissue cell that obtains by puncture method . 10. A DNA methylation detection kit, characterized in that it comprises: the detection reagent combination according to any one of claims 6-9. 11. A combination of PCR reagents, characterized in that it comprises: a PCR buffer, and a pair of PCR primers that can specifically amplify the sequence shown in SEQ ID No: 1 to SEQ ID No: 20, each The PCR primer pair includes a forward primer and a reverse primer. 12. A PCR kit, characterized in that it comprises: the PCR reagent combination as claimed in claim 11, and bisulfite. 13. A method for determining the type of isolated cervical cells, comprising the steps of: Select any three or more sequences in the genome of the isolated cervical cells of the individual to be tested as the target sequence group as shown in SEQ ID No: 1 to SEQ ID No: 10; Measuring the methylation level of the entire genome of the isolated cervical cells, selecting the methylation level of each sequence in the target sequence group, and obtaining the average methylation level of the target sequence group; the methylation Level is defined as the amount of methylated C-P-G in each sequence as a percentage of the total amount of C-P-G in that sequence; comparing the average methylation level with a reference methylation level; When the average methylation level is greater than or equal to a specific multiple of the reference methylation level, it is determined that the cell type of the isolated cervical cell is cervical cancer cell, otherwise, the cell type of the isolated cervical cell is determined for non-cervical cancer cells. 14. The method according to claim 13, wherein the cervical cancer cells are squamous cancer cells or adenocarcinoma cells; and/or, The reference methylation level is the average value of the methylation levels of sequences having the same nucleotide sequence as the target sequence group in the isolated cervical cells of a collection of normal individuals, and the collection of normal individuals includes three or three more than normal individuals; and/or, The specific multiple is any value from 2 to 7 times. 15. A method for determining the type of isolated cervical cells, comprising the steps of: Select any three or more sequences in the genome of the isolated cervical cells of the individual to be tested as the target sequence group as shown in SEQ ID No: 11 to SEQ ID No: 20; Measuring the methylation level of the entire genome of the isolated cervical cells, selecting the methylation level of each sequence in the target sequence group, and obtaining the average methylation level of the target sequence group; the methylation Level is defined as the amount of methylated C-P-G in each sequence as a percentage of the total amount of C-P-G in that sequence; comparing the average methylation level with a reference methylation level; When the average methylation level is less than or equal to a specific percentage of the reference methylation level, it is determined that the cell type of the isolated cervical cell is cervical cancer cell, otherwise, the cell type of the isolated cervical cell is determined for non-cervical cancer cells. 16. The method according to claim 15, wherein the cervical cancer cells are squamous cancer cells or adenocarcinoma cells; and/or, The reference methylation level is the average value of the methylation levels of sequences having the same nucleotide sequence as the target sequence group in the isolated cervical cells of a collection of normal individuals, and the collection of normal individuals includes three or three more than normal individuals; and/or, The specific percentage is any value from 20% to 65%. 17. A method for determining the type of isolated cervical cells, comprising the following steps: Select any two or more sequences in the sequence shown in SEQ ID No:1 to SEQ ID No:10 in the genome of the isolated cervical cell of the individual to be tested as the hypermethylation sequence group, and select the sequence group in the genome Any two or more sequences in the sequences shown in SEQ ID No:11 to SEQ ID No:20 are used as the hypomethylation sequence group; Measuring the methylation level of the whole genome of the isolated cervical cells, selecting the methylation level of each sequence in the hypermethylation sequence group, and obtaining the average methylation level of the hypermethylation sequence group as the hypermethylation level ; select the methylation level of each sequence in the low methylation sequence group, and obtain the average methylation level of the low methylation sequence group as the low methylation level; the methylation level is defined is the percentage of the amount of methylated cytosine-phosphate-guanine in each sequence to the total amount of cytosine-phosphate-guanine in the sequence; comparing the magnitude of the hypermethylation level with a reference hypermethylation level, and comparing the magnitude of the hypomethylation level with a reference hypomethylation level; When the high methylation level is greater than or equal to a specific multiple of the reference high methylation level, and when the low methylation level is less than or equal to a specific percentage of the reference low methylation level, it is determined that the in vitro The cell type of the cervical cells is cervical cancer cells, otherwise, the cell type of the isolated cervical cells is determined to be non-cervical cancer cells. 18. The method according to claim 17, wherein the cervical cancer cells are squamous cancer cells or adenocarcinoma cells; and/or, The reference hypermethylation level is the average value of the methylation levels of the sequences having the same nucleotide sequence as the hypermethylation sequence group in the isolated cervical cells of normal individuals; the reference hypomethylation level is normal The average value of methylation levels of sequences having the same nucleotide sequence as the hypomethylated sequence group in the isolated cervical cells of a collection of individuals, the collection of normal individuals includes three or more normal individuals; and /or, The specific multiple is any value from 2 to 7 times; and/or, The specific percentage is any value from 20% to 65%.