Human cervical cell methylation detection reagent and cell type determination method

Abstract

The present application provides a methylation detection reagent for human cervical cells and a cell type determination method. The present application uses 20 nucleotide sequences specific to methylation abnormalities of human cervical cancer cells as positive and negative reference reagents, respectively, thereby improving the sensitivity and accuracy of methylation detection. The present application uses the average value of the methylation levels of multiple sequences in the 10 specific sequences related to the abnormal increase in the methylation level of cervical cancer cells and / or the 10 specific sequences related to the abnormal decrease in the methylation level of cervical cancer cells to determine whether the methylation level of the test cervical cells is abnormal, thereby determining whether the test cervical cells have differentiated into cervical cancer cells, which has the beneficial effects of high sensitivity, good specificity, and less false positives.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and relates to a methylation detection reagent for human cervical cells and a cell type determination method. BACKGROUND

[0002] At present, cervical cancer is a common and frequently-occurring cancer type among women, and thus has attracted increasing attention. In the early stage of cervical cancer, low-grade squamous intraepithelial lesion (LGSIL) and high-grade squamous intraepithelial lesion (HGSIL) generally occur. The low-grade squamous intraepithelial lesion is also known as cervical intraepithelial neoplasias-phase 1 (CIN1), and the high-grade squamous intraepithelial lesion includes cervical intraepithelial neoplasias-phase 2 (CIN2) and cervical intraepithelial neoplasias-phase 3 (CIN3). However, if no active treatment is performed, the cervical cancer may develop into a late-stage cancer, such as squamous cell carcinoma (SCC) or adenomatous carcinoma (ACC).

[0003] Current studies have shown that, in the occurrence and development of cervical cancer, site-specific hypermethylation, i.e., an increase in methylation level, occurs at certain specific sites of certain genes (such as some tumor suppressor genes). Therefore, a kit capable of detecting the methylation level of the specific sites of different genes is often designed to detect whether the cervical cells to be detected are cervical cancer cells. However, each of the above kits is often used to detect the methylation level of each specific gene. Since there are many genes with site-specific hypermethylation in the whole genome of cervical cancer cells, and the methylation levels of different genes vary with the type of cancer cells, and the methylation levels of the same gene also vary at different stages, the detection result of using only one kit to detect the methylation level of one gene is often difficult to reflect the specific stage of cervical cancer. In order to obtain a reliable detection result, multiple kits need to be used to detect multiple corresponding genes at the same time, which increases the number of kits used and the number of detections, and may cause over-detection. In addition, if a certain kit is used, and the gene does not have abnormal methylation, the detection result will be inaccurate. SUMMARY

[0004] The present application aims 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 according to the methylation level of specific polynucleotide sequences of the cervical cells to be tested.

[0005] 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 specific sequences of the cervical cells to be tested with the average methylation of the same sequences of normal cervical cells, so as to determine whether the cell type of the cervical cells to be tested is cervical cancer cells.

[0006] In order to achieve the above-mentioned objects, the technical solutions of the present application are as follows:

[0007] A positive reference reagent selected from any one or a combination of several of the sequences shown as SEQ ID No: 1 to SEQ ID No: 20, which contains methylated cytosine-phosphate-guanine in the sequence.

[0008] In some embodiments, the methylation level of each sequence in the positive reference reagent is selected from any one value in the range of 60% to 100%, and the methylation levels of different sequences 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 to the total number of cytosine-phosphate-guanine in the sequence.

[0009] The above-mentioned positive reference reagent is used as a positive control with the same nucleotide sequence as each sequence when detecting the methylation level of any one or a combination of several of the sequences shown as SEQ ID No: 1 to SEQ ID No: 20 in the genome of the cervical cells to be tested.

[0010] A negative reference reagent selected from any one or a combination of several of the sequences shown as SEQ ID No: 1 to SEQ ID No: 20, which does not contain methylated cytosine-phosphate-guanine in the sequence.

[0011] The above-mentioned negative reference reagent is used as a negative control with the same nucleotide sequence as each sequence when detecting the methylation level of any one or a combination of several of the sequences shown as SEQ ID No: 1 to SEQ ID No: 20 in the genome of the cervical cells to be tested.

[0012] A detection reagent combination capable of specifically detecting the methylation level of one or a combination of several sequences as shown in SEQ ID No: 1 to SEQ ID No: 20 in a DNA sample, the methylation level being 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.

[0013] In some embodiments, the detection reagent combination further comprises: a reaction reagent and a detection reagent. The reaction reagent is capable of differentially modifying the methylated sites and the non-methylated sites in the DNA sample. After treating the DNA sample with the reaction reagent, the detection reagent is capable of determining whether each cytosine-phosphate-guanine in the sequences as shown in SEQ ID No: 1 to SEQ ID No: 20 is methylated or non-methylated.

[0014] In some embodiments, the detection reagent combination described above further comprises: a positive reference reagent and / or a negative reference reagent.

[0015] The positive reference reagent can be selected from any one or a combination of several sequences as shown in SEQ ID No: 1 to SEQ ID No: 20, which contains methylated cytosine-phosphate-guanine, the methylation level of each sequence in the positive reference reagent being selected from any one value in the range of 60% to 100%, the methylation levels of different sequences being the same or different, the methylation level being 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.

[0016] The negative reference reagent can be selected from any one or a combination of several sequences as shown in SEQ ID No: 1 to SEQ ID No: 20, which does not contain methylated cytosine-phosphate-guanine.

[0017] In some embodiments, in the detection reagent combination described above, the DNA sample is from an ex vivo cervical cell of a human, the ex vivo cervical cell being selected from a human cervical epithelial cell or a cervical tissue cell obtained by puncture.

[0018] A DNA methylation detection kit comprising: the detection reagent combination of any one of the above.

[0019] A PCR reagent combination comprising: a PCR buffer, and a PCR primer pair capable of specifically and one-to-one amplifying the sequences as shown in SEQ ID No: 1 to SEQ ID No: 20, each PCR primer pair comprising a forward primer and a reverse primer.

[0020] A PCR kit comprising: the PCR reagent combination described above, and a bisulfite reagent.

[0021] A method for determining the type of an ex vivo cervical cell, comprising the steps of:

[0022] selecting any three or more sequences among the sequences shown as SEQ ID No: 1 to SEQ ID No: 10 as a target sequence group in the genome of the ex vivo cervical cell of the individual to be tested;

[0023] determining the methylation level of the whole genome of the ex vivo cervical cell, 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 percentage of the number of methylated cytosine-phosphate-guanine in each sequence to the total number of cytosine-phosphate-guanine in the sequence;

[0024] comparing the average methylation level with the reference methylation level;

[0025] when the average methylation level is greater than or equal to a specific multiple of the reference methylation level, determining that the cell type of the ex vivo cervical cell is a cervical cancer cell, otherwise, determining that the cell type of the ex vivo cervical cell is a non-cervical cancer cell.

[0026] In some embodiments, the cervical cancer cell is a squamous cell carcinoma cell or an adenocarcinoma cell.

[0027] In some embodiments, the reference methylation level is the average of the methylation levels of the sequences having the same nucleotide sequence as the target sequence group in the ex vivo cervical cells of a set of normal individuals, and the set of normal individuals includes three or more normal individuals.

[0028] In some embodiments, the specific multiple is any one value from 2 to 7.

[0029] A method for determining the type of an ex vivo cervical cell, comprising the steps of:

[0030] selecting any three or more sequences among the sequences shown as SEQ ID No: 11 to SEQ ID No: 20 as a target sequence group in the genome of the ex vivo cervical cell of the individual to be tested;

[0031] determining the methylation level of the whole genome of the ex vivo cervical cell, 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 percentage of the number of methylated cytosine-phosphate-guanine in each sequence to the total number of cytosine-phosphate-guanine in the sequence;

[0032] comparing the average methylation level with the reference methylation level;

[0033] When the average methylation level is less than or equal to a specific percentage of the reference methylation level, the cell type of the exfoliated cervical cell is determined to be a cervical cancer cell, otherwise, the cell type of the exfoliated cervical cell is determined to be a non-cervical cancer cell.

[0034] In some embodiments, the cervical cancer cell is a squamous cell carcinoma cell or an adenocarcinoma cell.

[0035] In some embodiments, the reference methylation level is an average of methylation levels of sequences having the same nucleotide sequence as the target sequence group in exfoliated cervical cells of a normal individual set, the normal individual set comprising three or more normal individuals.

[0036] In some embodiments, the specific percentage is any value in a range from 20% to 65%.

[0037] A method for determining the cell type of an exfoliated cervical cell, comprising the steps of:

[0038] selecting any two or more sequences among the sequences shown as SEQ ID No: 1 to SEQ ID No: 10 as a high-methylation sequence group, and selecting any two or more sequences among the sequences shown as SEQ ID No: 11 to SEQ ID No: 20 as a low-methylation sequence group in the genome of the exfoliated cervical cell of the subject to be tested;

[0039] determining the methylation levels of the whole genome of the exfoliated cervical cell, selecting the methylation level of each sequence in the high-methylation sequence group, and obtaining the average methylation level of the high-methylation sequence group as a high-methylation level; selecting the methylation level of each sequence in the low-methylation sequence group, and obtaining the average methylation level of the low-methylation sequence group as a 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;

[0040] comparing the high-methylation level with the reference high-methylation level, and comparing the low-methylation level with the reference low-methylation level;

[0041] 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 exfoliated cervical cell is determined to be a cervical cancer cell, otherwise, the cell type of the exfoliated cervical cell is determined to be a non-cervical cancer cell.

[0042] In some embodiments, the cervical cancer cell is a squamous cell carcinoma cell or an adenocarcinoma cell.

[0043] In some embodiments, the reference high methylation level is the average of the methylation levels of sequences having the same nucleotide sequence as the high methylation sequence group in the ex vivo cervical cells of a set of normal individuals; and the reference low methylation level is the average of the methylation levels of sequences having the same nucleotide sequence as the low methylation sequence group in the ex vivo cervical cells of a set of normal individuals, the set of normal individuals including three or more normal individuals.

[0044] In some embodiments, the specific fold is any one of 2 to 7.

[0045] In some embodiments, the specific percentage is any one of 20% to 65%.

[0046] As the above technical solutions are adopted, some embodiments of the present application achieve the following technical effects:

[0047] First, some embodiments of the present application use positive 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, which can correct the detection result of the methylation level, thereby improving the accuracy of the methylation level detection.

[0048] Second, some embodiments of the present application screen out 10 specific sequences (SEQ ID No: 1 to SEQ ID No: 10) related to the abnormal increase of the methylation level of cervical cancer cells, and use the average methylation level of any combination of three or more of the 10 sequences to determine whether the cervical cells to be tested have an abnormal increase of the methylation level. The 10 specific sequences are respectively located at different positions of the whole genome of cervical cells, and have specificity and sensitivity to the increase of the methylation level of cervical cancer cells. Experiments have proved that any combination of three sequences can effectively detect whether the cervical cells to be tested have an abnormal increase of the methylation level, thereby being able to be used to determine whether the cervical cells to be tested have differentiated into cervical cancer cells.

[0049] Third, some embodiments of the present application screen out 10 specific sequences (SEQ ID No: 11 to SEQ ID No: 20) related to the abnormal decrease of the methylation level of cervical cancer cells, and use the average methylation level of any combination of three or more of the 10 sequences to determine whether the cervical cells to be tested have an abnormal decrease of the methylation level. The 10 specific sequences are respectively located at different positions of the whole genome of cervical cells, and have specificity and sensitivity to the decrease of the methylation level of cervical cancer cells. Experiments have proved that any combination of three sequences can effectively detect whether the cervical cells to be tested have an abnormal decrease of the methylation level, thereby being able to be used to determine whether the cervical cells to be tested have differentiated into cervical cancer cells.

[0050] Fourthly, some embodiments of the present application also adopt a combination of any two or more sequences shown in SEQ ID No: 1 to SEQ ID No: 10 and any two or more sequences shown in SEQ ID No: 11 to SEQ ID No: 20 to determine whether the methylation level of the cervical cell to be tested is abnormal. Any two or more sequences shown in SEQ ID No: 1 to SEQ ID No: 10 can be used as an index indicating whether the methylation level of the cervical cell to be tested is abnormally high, and any two or more sequences shown in SEQ ID No: 11 to SEQ ID No: 20 can be used as an index indicating whether the methylation level of the cervical cell to be tested is abnormally low. The present application combines the two indexes of abnormal increase and abnormal decrease of methylation level to determine whether the methylation level of the cervical cell to be tested is abnormal, so as to more accurately determine whether the cervical cell to be tested has been differentiated into a cervical cancer cell.

[0051] Fifthly, in the cervical cell type determination method of some embodiments of the present application, the methylation level of the specific sequence can be obtained by using whole genome methylation level detection, or by using methylation-specific PCR method and supplementing with positive reference reagent and / or negative reference reagent for methylation level detection, so that 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 can obtain the methylation levels of all specific fragments by only one detection, avoiding the use of multiple detection kits for multiple detections, although the number of detections and the workload are reduced, but the detection efficiency and sensitivity are still high.

[0052] Sixthly, some embodiments of the present application use the average value of the methylation levels of the multiple specific sequences as an index for determining whether the methylation of the cervical cell to be tested is abnormal, rather than comparing the methylation level of each specific sequence of the cervical cell to be tested with the methylation level of the same sequence of the normal cervical cell, which can prevent the phenomenon that the methylation level of a single specific sequence differs greatly 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 the misjudgment phenomenon caused by site methylation differences. The above all help to improve the accuracy of methylation level determination. DETAILED DESCRIPTION

[0053] The technology of the present application is described in detail below in combination with the specific embodiments. It should be understood that the following specific embodiments are only used to help those skilled in the art to understand the present application, but not to limit the present application.

[0054] The present application is further illustrated below in combination with the examples.

[0055] Embodiment One

[0056] The present 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 several of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20.

[0057] Alternatively, 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 to be tested of an ex vivo cervical cell to be tested needs to be detected, the positive reference reagent can be selected from a sequence identical in nucleotide sequence to the specific gene. Alternatively, the positive reference reagent can be selected from a combination of any several of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20. For example, it can be selected from a combination of any two or more of the sequences shown in SEQ ID No: 1 to SEQ ID No: 10; it can be selected from a combination of any two or more of the sequences shown in SEQ ID No: 11 to SEQ ID No: 20; it can be selected from a combination of any one or more of the sequences shown in SEQ ID No: 1 to SEQ ID No: 10 and a combination of any one or more of the sequences shown in SEQ ID No: 11 to SEQ ID No: 20. If the methylation levels of multiple specific genes in a DNA sample to be tested of an ex vivo cervical cell to be tested need to be detected, the positive reference reagent can comprise a sequence identical in nucleotide sequence to each of the specific genes.

[0058] As the positive reference reagent, each of the above sequences should contain methylated cytosine-phosphate-guanine (Methylated CpG). The methylation herein refers to the methylation of CpG sites, and does not represent the methylation of other sites. The average methylation level 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 can be different, but the average methylation level can be the same. The same average methylation level does not mean that the same CpG site in different samples is 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.

[0059] 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 more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more. When the average methylation level of a sequence is 100%, it means that all CpG sites in the sequence are methylated.

[0060] The positive reference reagent is generally detected synchronously with the methylation of the DNA sample. When the average methylation level of a sequence in the DNA sample (for example, the whole genome sample of the cervical cells to be detected) is detected, the average methylation level of the sequence with the same nucleotide sequence in the positive reference reagent is also detected. Then, the obtained average methylation level of the positive reference reagent is compared with the result in the reagent instruction. If the obtained average methylation level of the positive reference reagent is basically consistent with the result in the reagent instruction, it means that the detection is effective. If the results are quite different, it means that the detection result has a problem and needs to be further detected.

[0061] For example, when the methylation level of any one or a combination of several sequences shown in SEQ ID No: 1 to SEQ ID No: 20 in the genome of the cervical cells ex vivo is detected, a sequence with the same nucleotide sequence as the above sequence is selected in the positive reference reagent, and the methylation level of the selected sequence or sequences is determined at the same time, and then the average methylation level of the positive reference reagent is obtained. Then, the obtained average methylation level of the positive reference reagent is compared with the result in the reagent instruction. When comparing the average methylation level, the same sequence is used for comparison, that is, the sequence with the same nucleotide sequence in the positive reference reagent is compared one by one.

[0062] Example Two

[0063] The embodiment provides a negative reference reagent for detecting the methylation level of cervical cells ex vivo, which is selected from any one or a combination of several sequences shown in SEQ ID No: 1 to SEQ ID No: 20.

[0064] 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 the DNA sample of the test ex vivo cervical cells needs to be detected, the negative reference reagent can be selected from a sequence having the same nucleotide sequence as the specific gene.

[0065] Optionally, the negative reference reagent can be selected from a combination of any several of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20. For example, it can be selected from a combination of any two or more of the sequences shown in SEQ ID No: 1 to SEQ ID No: 10; it can be selected from a combination of any two or more of the sequences shown in SEQ ID No: 11 to SEQ ID No: 20; it can be selected from a combination of any one or more of the sequences shown in SEQ ID No: 1 to SEQ ID No: 10 and a combination of any one or more of the sequences shown in SEQ ID No: 11 to SEQ ID No: 20. If the methylation levels of multiple specific genes in the DNA sample of the test ex vivo cervical cells need to be detected, the negative reference reagent can contain a sequence having the same nucleotide sequence as each specific gene.

[0066] As the negative reference reagent, the above sequences should not contain methylated cytosine-phosphate-guanine (methylated CpG). That is, the CpG sites in the above sequences are not methylated. At this time, the average methylation level of different sequences in the negative reference reagent is 0%.

[0067] The negative reference reagent is generally detected synchronously with the methylation detection of the DNA sample. When the average methylation level of a sequence in the DNA sample (e.g., the whole genome sample of the test cervical cells) is detected, the average methylation level of a sequence having the same nucleotide sequence as the sequence in the negative reference reagent is also detected at the same time. Then, the obtained average methylation level of the negative reference reagent is compared with the result in the reagent instruction. If the obtained average methylation level of the negative reference reagent is basically consistent with the result in the reagent instruction (e.g., both are 0% excluding systematic errors and / or interference factors), it indicates that the current detection is valid. If the results of the two are quite different, it indicates that there is a problem with the current detection result, which needs to be further detected.

[0068] For example, in detecting the methylation level of any one of the sequences shown as SEQ ID No: 1 to SEQ ID No: 20 or a combination of several thereof in the genome of the ex vivo cervical cells, a sequence having the same nucleotide sequence as the above sequences is selected in the negative reference reagent, and the methylation level of the selected sequence or sequences is measured at the same time, and then the average methylation level of the negative reference reagent is obtained, and then the obtained average methylation level of the negative reference reagent is compared with the result of the reagent manual. In comparing the average methylation level, the same sequence is compared respectively, i.e., the sequences having the same nucleotide sequence in the genome of the ex vivo cervical cells and the negative reference reagent are compared one by one.

[0069] In detecting the methylation of the DNA sample, the negative reference reagent of the present embodiment can be used together with the positive reference reagent of Embodiment 1 in order to improve the reliability of the detection. Of course, it can also be used alone.

[0070] Embodiment Three

[0071] The present embodiment provides a method of screening the sequences of SEQ ID No: 1 to SEQ ID No: 20 in the whole genome of the cervical cancer cells. The method measures the methylation level of the whole genome of the human cervical cells (including normal cervical cells and cervical cancer cells), and then screens based on the abnormal increase and abnormal decrease of the values of the methylation levels of the normal cervical cells and the cervical cancer cells, thereby screening out the 20 sequences (SEQ ID No: 1 to SEQ ID No: 20) closely related to the generation of cervical cancer. The 20 sequences can be used as the reference reagents of Embodiment 1 and Embodiment 2.

[0072] Specifically, the method of the present embodiment includes the following steps:

[0073] (1) A certain number of normal cervical cells of normal individuals and a certain number of cervical cancer cells (including squamous cell carcinoma cells or adenocarcinoma cells) of diseased individuals are obtained, and the genomic DNA (Genomics DNA) of the above two types of cells is extracted respectively.

[0074] (2) The above genomic DNA is fragmented to obtain DNA fragments.

[0075] (3) The DNA fragments are subjected to end repair, adaption ligation, bisulfite treatment, PCR amplification, library test, sequencing and bio-informatics analysis in sequence, and finally the methylation level index of genomic DNA is obtained.

[0076] (4) The 10 sequences (i.e. SEQ ID No: 1 to SEQ ID No: 10) with abnormal increase in average methylation level compared with the average methylation level of normal cervical cells are screened out, and the 10 sequences (i.e. SEQ ID No: 11 to SEQ ID No: 20) with abnormal decrease in average methylation level compared with the average methylation level of normal cervical cells are screened out.

[0077] Experiments show that the 20 sequences of SEQ ID No: 1 to SEQ ID No: 20 screened out by the above steps are only related to cervical cancer cells, and thus can be used as specific biomarkers and specific reference reagents for detecting cervical cancer cells.

[0078] The basic information of the above 20 sequences is shown in Table 1:

[0079] Table 1 is a basic information table of SEQ ID No: 1 to SEQ ID No: 20

[0080]

[0081]

[0082] As shown in Table 1, the average methylation levels of SEQ ID No: 1 to SEQ ID No: 10 are abnormally increased in cervical cancer cells relative to normal cervical cells, which are referred to as hyper differentially methylated regions (Hyper DMRs); while the average methylation levels of SEQ ID No: 11 to SEQ ID No: 20 are abnormally decreased in cervical cancer cells relative to normal cervical cells, which are referred to as hypo differentially methylated regions (Hypo DMRs). The table also shows the accession number, the name of the adjacent gene, the gene interval, and the distance from the adjacent gene interval (a negative number indicates that the sequence is located upstream of the adjacent gene, a positive number indicates that the sequence is located downstream of the adjacent gene, and 0 indicates that the sequence is located in the content of the adjacent gene in the table) of the 20 sequences.

[0083] For example, SEQ ID No: 1 is located in the intron of the ARHGAP42 gene, and the distance from the ARHGAP42 gene is 0. SEQ ID No: 2 is located in the promoter of the NDN gene, which is located 2281 upstream of the NDN gene. The distance is negative, which means that the gene is upstream of the transcriptional start site (TSS). The distance is positive, which means that it is downstream of the transcriptional start site. The distance indicated above is the distance of the relevant gene from the TSS.

[0084] The determination results of the methylation levels of SEQ ID No: 1 to SEQ ID No: 20 in human normal cervical cells are shown in Table 2.

[0085] Table 2 is a table of methylation levels of SEQ ID No: 1 to SEQ ID No: 20 in human normal cervical cells

[0086]

[0087]

[0088] Five detection results were randomly selected from any of the detection results of normal cervical cells of a certain number of normal individuals in step (1) as the detection results of the normal group (Control) and presented in Table 2. Among them, serial numbers Ctr_1 to Ctr_5 represent the number of detection samples of normal cervical cells. It is calculated that the average methylation levels of SEQ ID No: 1 to SEQ ID No: 10 of normal cervical cells are low, below 34% (i.e. 0.34). The methylation levels of SEQ ID No: 11 to SEQ ID No: 20 of normal cervical cells are high, above 71% (i.e. 0.71). Therefore, if the methylation levels of SEQ ID No: 1 to SEQ ID No: 10 in the cervical cell sample of the individual to be tested are detected to increase to a certain value, it means that the cervical cell sample of the individual to be tested is a cervical cancer cell (may be a squamous cell carcinoma cell or an 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 individual to be tested are detected to decrease to a certain value, it means that the cervical cell sample of the individual to be tested is a cervical cancer cell (may be a squamous cell carcinoma cell or an adenocarcinoma cell).

[0089] The determination results of the methylation levels of SEQ ID No: 1 to SEQ ID No: 20 in human abnormal cervical cells (such as adenocarcinoma cells) are shown in Table 3.

[0090] Table 3 is the methylation level table of SEQ ID No: 1 to SEQ ID No: 20 in human abnormal cervical cells

[0091]

[0092]

[0093]

[0094] Two detection results were randomly selected from the detection results of cervical cancer cells of a certain number of diseased individuals in step (1) as the detection results of the diseased group (such as the CCA group) and presented in Table 3.

[0095] As shown in 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 results, for the same sequence, the increased methylation level is up to 6.57 times of the normal methylation level. Because the methylation levels of SEQ ID No: 1 to SEQ ID No: 10 in cervical cancer cells have been greatly increased, the methylation level of the above sequences in the cervical cell sample to be tested can be used to determine whether the cervical cell to be tested in vitro is a cervical cancer cell.

[0096] As shown in 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 results, for the same sequence, the increased methylation level is up to 6.57 times of the normal methylation level. Because the methylation levels of SEQ ID No: 1 to SEQ ID No: 10 in cervical cancer cells have been greatly increased, the methylation level of the above sequences in the cervical cell sample to be tested can be used to determine whether the cervical cell to be tested in vitro is a cervical cancer cell.

[0097] The present application uses the average of the methylation levels of multiple specific sequences as an index to determine whether the methylation of the cervical cell to be tested is abnormal, rather than comparing the methylation level of each specific sequence of the cervical cell to be tested with the methylation level of the same sequence of the normal cervical cell, which can prevent the phenomenon that the methylation level of a single specific sequence differs greatly due to individual differences, and helps to improve the accuracy of methylation level determination. This is because the average methylation levels of different specific fragments of the same individual are similar and have no order of magnitude difference.

[0098] The determination results of the methylation levels of SEQ ID No: 1 to SEQ ID No: 20 in human abnormal cervical cells (such as squamous cell carcinoma cells) are shown in Table 4.

[0099] Table 4 is a table of methylation levels of SEQ ID No: 1 to SEQ ID No: 20 in human abnormal cervical cells

[0100]

[0101]

[0102] Five detection results were randomly selected from the detection results of a certain number of cervical cancer cells of diseased individuals in step (1) as the detection results of the diseased group (such as the CCS group) and are shown in Table 4.

[0103] As can be seen from Tables 2, 3 and 4, the elevated methylation levels of SEQ ID No: 1 to SEQ ID No: 10 in the CCS group are similar to the elevated methylation levels in the CCA group, and the reduced methylation levels of SEQ ID No: 11 to SEQ ID No: 20 in the CCS group are similar to the reduced methylation levels in the CCA group, and 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).

[0104] In detecting and determining the methylation levels of SEQ ID No: 1 to SEQ ID No: 20 in cervical cancer cells, because it is difficult to determine whether the cervical cell sample to be tested is a cervical cancer cell according to the methylation level of only one sequence, the average methylation level of the combination of two or more sequences is generally used to determine whether the cervical cell sample to be tested is a cervical cancer cell.

[0105] For example, if the whole genome methylation detection method is used to detect the methylation level of the whole genome of the cervical cell to be tested, three sequences can be selected from the ten sequences of SEQ ID No: 1 to SEQ ID No: 10, the average methylation level of the three sequences is obtained by averaging the methylation levels of the three sequences, and then compared with the average methylation level of the three sequences having the same nucleotide sequence as the sequence of the normal cervical cell, so as to determine whether the cervical cell to be tested is a cervical cancer cell according to the degree of methylation increase. In other embodiments, more sequences can also be selected.

[0106] Exemplarily, if the whole genome methylation detection method is used to detect the methylation level of the whole genome of the cervical cell to be tested, 3 sequences can be selected from the 20 sequences of SEQ ID No: 11 to SEQ ID No: 10, and the average methylation level of the 3 sequences is obtained by averaging the methylation levels of the 3 sequences, and then compared with the average methylation level of the same 3 sequences of the normal cervical cell, so as to determine whether the cervical cell to be tested is a cervical cancer cell according to the degree of methylation reduction. In other embodiments, more sequences can also be selected.

[0107] Exemplarily, if the whole genome methylation detection method is used to detect the methylation level of the whole genome of the cervical cell to be tested, 2 sequences can be selected from the 10 sequences of SEQ ID No: 1 to SEQ ID No: 10, and the average methylation level of the 2 sequences is obtained by averaging the methylation levels of the 2 sequences, and then compared with the average methylation level of the same 2 sequences of the normal cervical cell (which can be determined on site or in advance). At the same time, 2 sequences can be selected from the 10 sequences of SEQ ID No: 11 to SEQ ID No: 20, and the average methylation level of the 2 sequences is obtained by averaging the methylation levels of the 2 sequences, and then compared with the average methylation level of the same 2 sequences of the normal cervical cell. Then, according to the degree of methylation reduction of SEQ ID No: 1 to SEQ ID No: 10, and at the same time combined with the degree of methylation increase of SEQ ID No: 11 to SEQ ID No: 20, whether the cervical cell to be tested is a cervical cancer cell is determined. In the above cases, if it is necessary to determine the methylation level of the corresponding sequence of the normal cervical cell on site, positive reference reagents and / or negative reference reagents can be used during the determination.

[0108] Exemplarily, if the methylation-specific PCR (MSP) method is used to detect the methylation level, it is necessary to detect the methylation level of the selected sequence in the genome of the cervical cell to be tested, and the detection result of the methylation level can be corrected by using positive reference reagents and / or negative reference reagents, and then the corresponding judgment is made. When detecting the methylation level of the selected sequence in the genome of the cervical cell to be tested, the methylation level of the same selected sequence of the normal cervical cell can also be detected at the same time. The selection method of the selected sequence is as described above.

[0109] Example Four

[0110] The embodiment provides a detection reagent combination which can specifically detect the DNA methylation level of one or a combination of several sequences as shown in SEQ ID No: 1 to SEQ ID No: 20 in a DNA sample. The detection reagent combination comprises: a reaction reagent and a detection reagent.

[0111] The reaction reagent can differentially modify the methylation site and the non-methylation site in the DNA sample, so that the methylation site is not demethylated and the non-methylation site is not remethylated in the subsequent reaction process, which is beneficial to ensure the accuracy of the methylation level determination. Specifically, taking the methylation-specific PCR detection method as an example, the DNA sample is subjected to bisulfite conversion. In the bisulfite conversion process, the cytosine in the DNA (single-stranded DNA) is converted into cytosine-bisulfite derivative, and this reaction is reversible; then the cytosine-bisulfite derivative undergoes an irreversible hydrolysis deamination process to form uracil-bisulfite derivative. Finally, the uracil-bisulfite derivative is de-sulfonated to form uracil under high pH conditions. Only the unmethylated cytosine is changed in base under bisulfite treatment, and 5-mC and 5-hmC remain unchanged. The DNA after the conversion treatment is subjected to PCR amplification, and the uracil (U) is converted into thymine (T). Thus, the methylation level can be determined.

[0112] The detection reagent is used to determine whether each cytosine-phosphate-guanine in the sequence as 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.

[0113] 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 several sequences as shown in SEQ ID No: 1 to SEQ ID No: 20, the reaction reagent and the detection reagent described above can be used.

[0114] In addition, when the methylation level of normal cervical cells is determined as a control, or when the methylation level of the cervical cells to be tested is determined by using the methylation-specific PCR method, a positive reference reagent and / or a negative reference reagent can also be used. The positive reference reagent is selected from any one or a combination of several of the sequences shown in SEQ ID No: 1 to SEQ ID No: 20, which contain methylated cytosine-phosphate-guanine. The negative reference reagent is selected from any one or a 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. The positive reference reagent and the negative reference reagent are beneficial for reducing systematic errors in methylation detection.

[0115] The DNA sample is from an ex vivo cervical cell of a human, which is selected from a cervical epithelial cell of a human or a cervical tissue cell obtained by puncture.

[0116] The various reagents in the detection reagent combination of the present embodiment are placed in containers at certain concentrations, thereby becoming part of a DNA methylation detection kit. The DNA methylation detection kit can be used to determine whether the ex vivo cervical cell is a cervical cancer cell.

[0117] Example Five

[0118] The present embodiment provides a PCR reagent combination capable of specifically amplifying the sequences shown in SEQ ID No: 1 to SEQ ID No: 20 and a PCR primer pair corresponding to each sequence, each PCR primer pair comprising a forward primer and a reverse primer. The various reagents in the PCR reagent combination are placed in containers at certain concentrations, thereby becoming part of a PCR reagent kit. The PCR reagent kit further comprises amplification reagents capable of specifically amplifying the selected sequences in the genome of the cervical cells to be tested, including but not limited to PCR buffer, etc.

[0119] Example Six

[0120] The present embodiment provides a method for determining the type of an ex vivo cervical cell, which comprises the following steps:

[0121] (1) selecting any three or more of the sequences shown in SEQ ID No: 1 to SEQ ID No: 10 in the genome of the ex vivo cervical cell of the individual to be tested as a target sequence group;

[0122] (2) determining the methylation level of the whole genome of the exfoliated cervical cell, 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 percentage of the number of methylated cytosine-phosphate-guanine in each sequence to the total number of cytosine-phosphate-guanine in the sequence;

[0123] (3) comparing the average methylation level with the reference methylation level;

[0124] (4) when the average methylation level is greater than or equal to a certain multiple of the reference methylation level, the cell type of the exfoliated cervical cell is determined to be cervical cancer cell, otherwise, the cell type of the exfoliated cervical cell is determined to be non-cervical cancer cell.

[0125] Among them, the cervical cancer cell is squamous cell carcinoma or adenocarcinoma cell.

[0126] The reference methylation level is the average value of the methylation level of the sequence with the same nucleotide sequence as the target sequence group in the exfoliated cervical cell of the normal individual set, and the normal individual set includes three or more normal individuals.

[0127] The specific multiple is any one value in the range of 2 to 7. For example, 3, 4, 5, 6, etc. can be selected.

[0128] Example Seven

[0129] The present embodiment provides a method for determining the type of exfoliated cervical cell, comprising the following steps:

[0130] Selecting any three or more sequences in the sequences shown as SEQ ID No: 11 to SEQ ID No: 20 in the genome of the exfoliated cervical cell of the test individual as the target sequence group;

[0131] Determining the methylation level of the whole genome of the exfoliated cervical cell, 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 percentage of the number of methylated cytosine-phosphate-guanine in each sequence to the total number of cytosine-phosphate-guanine in the sequence;

[0132] Comparing the average methylation level with the reference methylation level;

[0133] When the average methylation level is less than or equal to a certain percentage of the reference methylation level, the cell type of the exfoliated cervical cell is determined to be cervical cancer cell, otherwise, the cell type of the exfoliated cervical cell is determined to be non-cervical cancer cell.

[0134] The cervical cancer cell is a squamous cell carcinoma or an adenocarcinoma cell. The reference high methylation level is an average of methylation levels of sequences having the same nucleotide sequence as the high methylation sequence group in exfoliated cervical cells of a normal individual set, and the reference low methylation level is an average of methylation levels of sequences having the same nucleotide sequence as the low methylation sequence group in exfoliated cervical cells of the normal individual set. The normal individual set includes three or more normal individuals. The specific percentage is any one value in a range of 20% to 65%.

[0135] Example Eight

[0136] The present example provides a method for determining the type of exfoliated cervical cells, which includes the following steps:

[0137] Any two or more of the sequences shown as SEQ ID No: 1 to SEQ ID No: 10 in the genome of the exfoliated cervical cells of the subject to be tested are selected as the high methylation sequence group, and any two or more of the sequences shown as SEQ ID No: 11 to SEQ ID No: 20 in the genome are selected as the low methylation sequence group;

[0138] The methylation levels of the whole genome of the exfoliated cervical cells are determined, the methylation level of each sequence in the high methylation sequence group is selected, and the average methylation level of the high methylation sequence group is obtained as the high methylation level; the methylation level of each sequence in the low methylation sequence group is selected, and the average methylation level of the low methylation sequence group is obtained 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;

[0139] The high methylation level is compared with the reference high methylation level, and the low methylation level is compared with the reference low methylation level;

[0140] 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 exfoliated cervical cells is determined to be a cervical cancer cell, otherwise, the cell type of the exfoliated cervical cells is determined to be a non-cervical cancer cell.

[0141] The cervical cancer cell is a squamous cell carcinoma or an adenocarcinoma cell. The reference high methylation level is an average of methylation levels of sequences having the same nucleotide sequence as the high methylation sequence group in exfoliated cervical cells of a normal individual set, and the reference low methylation level is an average of methylation levels of sequences having the same nucleotide sequence as the low methylation sequence group in exfoliated cervical cells of the normal individual set. The normal individual set includes three or more normal individuals. The specific percentage is any one value in a range of 20% to 65%.

[0142] The present application has been described in terms of the above embodiments, however, the embodiments are merely exemplary of the application. It must be noted that, in the above embodiments, specific details are set forth in order to provide a thorough understanding of the present application. However, it must be understood that the application can be practiced without these specific details. In other instances, well-known methods, procedures, components and circuits have not been described in detail as not to unnecessarily obscure the present application. Other embodiments can be apparent to those of ordinary skill in the art from this disclosure, which incorporates all such embodiments and alternative embodiments in the scope of the application.

Claims

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

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

3. A test reagent combination, characterized in that, Including positive control reagents and / or negative control reagents; The positive reference reagent is selected from any one or a combination of several 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 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 amount of methylated cytosine-phosphate-guanine in each sequence relative to the total amount of cytosine-phosphate-guanine in that sequence. The negative reference reagent is selected from any one or a combination of several sequences shown in SEQ ID No:1 to SEQ ID No:20, wherein the sequences do not contain methylated cytosine-phosphate-guanine; The detection reagent combination can specifically detect the methylation level of one or more combinations of 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 methylated cytosine-phosphate-guanine in each DNA sequence relative to the total amount of cytosine-phosphate-guanine in that DNA sequence.

4. The detection reagent combination as described in claim 3, characterized in that, Also includes: The reaction reagents can differentially modify methylated and unmethylated sites in the DNA sample; as well as The detection reagent, after treating the DNA sample with the reaction reagent, can determine whether each cytosine-phosphate-guanine in the sequences shown in SEQ ID No:1 to SEQ ID No:20 is methylated or unmethylated.

5. The detection reagent combination as described in claim 3, characterized in that, The DNA sample was derived from human cervical cells obtained in vitro, which were selected from human cervical epithelial cells or cervical tissue cells obtained by puncture.

6. A DNA methylation detection kit, characterized in that, include: The combination of detection reagents as described in any one of claims 3 to 5.

7. A PCR reagent combination, characterized in that, include: PCR buffer, and PCR primer pairs that specifically amplify the sequences shown in SEQ ID No:1 to SEQ ID No:20, each PCR primer pair including a forward primer and a reverse primer.

8. A PCR kit, characterized in that, include: The PCR reagent combination as described in claim 7, and bisulfite.

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