CN116790749A - Nucleic acid molecule composition for typing detection of cervical cancer and cervical intraepithelial neoplasia, kit and application thereof - Google Patents

Abstract

The application discloses a nucleic acid molecule composition for typing detection of cervical cancer and cervical intraepithelial neoplasia, a kit and application thereof, wherein the nucleic acid molecule composition comprises at least one of nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.20, at least one of nucleotide sequences shown as SEQ ID No.21 to SEQ ID No.40, at least one of nucleotide sequences shown as SEQ ID No.41 to SEQ ID No.60 and at least one of nucleotide sequences shown as SEQ ID No.61 to SEQ ID No.80, and the nucleic acid molecule composition can be used as a molecular marker for screening, monitoring and diagnosing cervical cancer and cervical intraepithelial neoplasia, and has the advantages of high sensitivity, strong specificity, high accuracy and ideal stability.

Description

Nucleic acid molecule composition for typing detection of cervical cancer and cervical intraepithelial neoplasia, kit and application thereof

Technical Field

The application relates to the technical field of biomedicine, in particular to a nucleic acid molecule composition for typing detection of cervical cancer and cervical intraepithelial neoplasia, a kit and application thereof.

Background

Cervical cancer is one of malignant tumors which seriously threatens the life health of women, about 20 tens of thousands of women die from cervical cancer annually in the world, and the cervical cancer is listed in two-cancer (cervical cancer and breast cancer) important prevention and treatment plans in China. Cervical cancer includes cervical squamous cell carcinoma and cervical adenocarcinoma, wherein cervical adenocarcinoma is less common than cervical squamous cell carcinoma, and wherein the proportion of cervical cancer patients suffering from cervical squamous cell carcinoma is about 80% to 85% and the proportion of cervical adenocarcinoma patients suffering from cervical adenocarcinoma is about 10% to 15%. Cervical pre-lesions are also called cervical intraepithelial neoplasias, and cervical intraepithelial neoplasias are classified into CIN1, CIN2, and CIN3 according to the CIN (Cervical Intraepithelial Neoplasia) classification system, wherein CIN1 is a mild cervical intraepithelial neoplasias, CIN2 is a moderate cervical intraepithelial neoplasias, CIN3 is a high cervical intraepithelial neoplasias, and CIN3 is also called carcinoma in situ (Carcinoma In Situ, CIS) since the next stage of CIN3 is cervical cancer.

At present, three methods widely used for screening cervical cancer and cervical intraepithelial neoplasia are mainly adopted, wherein the first method is cytological screening, the second method is acetic acid/iodine staining macroscopic observation, and the third method is human papillomavirus (Human Papilloma Virus, HPV) detection, wherein the cytological screening and the acetic acid/iodine staining macroscopic observation have the defects of inconvenient sampling, low accuracy and incapability of distinguishing cervical lesion types, and the HPV detection has the defects of high price and incapability of distinguishing cervical lesion types, so that the type of cervical lesion can not be obtained through one-time detection by the existing screening method.

Disclosure of Invention

Aiming at the defects of the prior art, the application provides a nucleic acid molecule composition for typing detection of cervical cancer and cervical intraepithelial neoplasia, a kit and application thereof.

The technical scheme of the application is as follows:

in a first aspect, the present application provides a nucleic acid molecule composition for typing detection of cervical cancer and cervical intraepithelial neoplasia, the nucleic acid molecule composition comprising:

a first nucleic acid molecule comprising a nucleotide sequence as shown in any one or more of SEQ ID No.1 to SEQ ID No. 20;

a second nucleic acid molecule comprising a nucleotide sequence as shown in any one or more of SEQ ID No.21 to SEQ ID No. 40;

a third nucleic acid molecule comprising a nucleotide sequence as shown in any one or more of SEQ ID No.41 to SEQ ID No. 60; and

a fourth nucleic acid molecule comprising a nucleotide sequence as shown in any one or more of SEQ ID No.61 to SEQ ID No. 80.

Further, the first nucleic acid molecule comprises at least two of the nucleotide sequences as set forth in SEQ ID No.1 through SEQ ID No. 20;

and/or the second nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.21 to SEQ ID No. 40;

And/or, the third nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.41 to SEQ ID No. 60;

and/or, the fourth nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.61 to SEQ ID No. 80.

Alternatively, the first nucleic acid molecule comprises a collection of all nucleotide sequences as set forth in SEQ ID No.1 to SEQ ID No. 20;

and/or, the second nucleic acid molecule comprises a collection of all nucleotide sequences as set forth in SEQ ID No.21 to SEQ ID No. 40;

and/or, the third nucleic acid molecule comprises a collection of all nucleotide sequences as set forth in SEQ ID No.41 through SEQ ID No. 60;

and/or, the fourth nucleic acid molecule comprises a collection of all nucleotide sequences as set forth in SEQ ID No.61 through SEQ ID No. 80.

In a second aspect, the present application provides a kit for typing detection of cervical cancer and cervical intraepithelial neoplasia, the kit comprising: a reagent capable of specifically detecting the methylation level of any one or more of the nucleotide sequences shown in SEQ ID Nos. 1 to 20, 21 to 40, 41 to 60, and 61 to 80 in the DNA of a sample, wherein the methylation level is the ratio of the number of methylated CpG sites in each nucleotide sequence to the total number of CpG sites in the nucleotide sequence.

Further, the kit also comprises a modifying reagent and a detecting reagent;

wherein the modifying reagent is used for differentially modifying methylated CpG sites and unmethylated CpG sites in the DNA of the test sample;

the detection reagent is used to determine or assist in determining whether each CpG site of the nucleotide sequence of any one or more of SEQ ID Nos. 1 to 20 in the DNA of the test sample is methylated or unmethylated, and whether each CpG site of the nucleotide sequence of any one or more of SEQ ID Nos. 21 to 40 in the DNA of the test sample is methylated or unmethylated, and whether each CpG site of the nucleotide sequence of any one or more of SEQ ID Nos. 41 to 60 in the DNA of the test sample is methylated or unmethylated, and determining or assist in determining whether each CpG site of the nucleotide sequence of any one or more of SEQ ID No.61 to 80 in the DNA of the test sample is methylated or unmethylated.

Further, the modifying agent includes bisulfite and derivatives thereof;

And/or the detection reagent comprises a PCR reagent, wherein the PCR reagent comprises a primer pair and/or a specific probe for specifically amplifying any one or a plurality of nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.20, a primer pair and/or a specific probe for specifically amplifying any one or a plurality of nucleotide sequences shown in SEQ ID No.21 to SEQ ID No.40, a primer pair and/or a specific probe for specifically amplifying any one or a plurality of nucleotide sequences shown in SEQ ID No.41 to SEQ ID No.60, and a primer pair and/or a specific probe for specifically amplifying any one or a plurality of nucleotide sequences shown in SEQ ID No.61 to SEQ ID No. 80.

Further, the kit further comprises a DNA extraction reagent for extracting and/or purifying DNA from the test sample.

Further, the test sample is blood, urine, cervical scraping, vaginal secretion, vaginal douche, cervical epithelial cells, post-operative ex vivo tissue, or a purified DNA sample.

In a third aspect, the present application provides a method for using the kit for typing detection of cervical cancer and cervical intraepithelial neoplasia according to any one of the second aspect, the method comprising the steps of:

Providing a sample to be tested, and treating the sample to be tested or the DNA of the sample to be tested by adopting the modifying reagent to obtain a modified DNA sample;

selecting a detection sequence group of the DNA sample, wherein the detection sequence group comprises a first detection sequence subgroup, a second detection sequence subgroup, a third detection sequence subgroup and a fourth detection sequence subgroup; and

reacting said detection reagent with said DNA sample and determining the CpG methylation level of each nucleotide sequence in said set of detection sequences;

wherein the first detection sequence subgroup comprises any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.10, and/or any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No.11 to SEQ ID No. 20;

the second detection sequence subgroup comprises any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No. 21-SEQ ID No.30, and/or any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No. 31-SEQ ID No. 40;

the third detection sequence subgroup comprises any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No.41 to SEQ ID No.50, and/or any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No.51 to SEQ ID No. 60;

The fourth detection sequence subgroup comprises any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No. 61-SEQ ID No.70, and/or any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No. 71-SEQ ID No. 80.

Further, comparing the methylation level of each nucleotide sequence in the detection sequence set to a corresponding predetermined threshold;

wherein the preset threshold is the average value of CpG methylation levels of corresponding nucleotide sequences in DNA of a normal individual set, and the normal individual set comprises three or more normal individuals.

Further, the using method further comprises the steps of:

determining that the subject of the test sample is suffering from cervical adenocarcinoma if the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 1 to 10 is above 0.6, respectively, and/or the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 11 to 20 is below 0.3, respectively;

determining that the subject of the test sample is suffering from cervical squamous carcinoma if the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 21 to 30 is above 0.7, respectively, and/or the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 31 to 40 is below 0.3, respectively;

Determining that the subject of the test sample has a low grade cervical intraepithelial neoplasia if the CpG methylation level of at least eight of the nucleotide sequences set forth in SEQ ID nos. 41 to 50 is above 0.72, respectively, and/or the CpG methylation level of at least five of the nucleotide sequences set forth in SEQ ID nos. 51 to 60 is below 0.46, respectively; and

and if the CpG methylation level of at least eight nucleotide sequences in the nucleotide sequences shown in SEQ ID No.61 to SEQ ID No.70 is above 0.65 and/or the CpG methylation level of at least five nucleotide sequences in the nucleotide sequences shown in SEQ ID No.71 to SEQ ID No.80 is below 0.39, respectively, determining that the subject of the test sample suffers from high-grade cervical intraepithelial neoplasia.

The beneficial effects are that: the application provides a nucleic acid molecule composition for typing detection of cervical cancer and cervical intraepithelial neoplasia, a kit and application thereof, wherein the nucleic acid molecule composition comprises at least one of nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.20, at least one of nucleotide sequences shown as SEQ ID No.21 to SEQ ID No.40, at least one of nucleotide sequences shown as SEQ ID No.41 to SEQ ID No.60 and at least one of nucleotide sequences shown as SEQ ID No.61 to SEQ ID No.80, and the nucleic acid molecule composition can be used as a molecular marker for screening, monitoring and diagnosing cervical cancer and cervical intraepithelial neoplasia, has the advantages of high sensitivity, strong specificity, high accuracy and ideal stability, can be applied to clinical practice to guide cervical cancer patients and cervical cancer premalignant treatment schemes and prevention schemes of high-risk crowds, and has important clinical, scientific and social values.

The kit screens, monitors and performs parting diagnosis on the grade of cervical lesions by detecting the CpG methylation level of the detection sequence group in the DNA of the tested sample, can acquire the type of cervical lesions only by one-time detection, can realize noninvasive detection, and has wide application prospect.

Detailed Description

The application researches a nucleic acid molecule composition, a kit and application thereof for typing detection of cervical cancer and cervical intraepithelial neoplasia, wherein the nucleic acid molecule composition comprises a first nucleic acid molecule, a second nucleic acid molecule, a third nucleic acid molecule and a fourth nucleic acid molecule, wherein the first nucleic acid molecule comprises any one or a plurality of nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.20, the second nucleic acid molecule comprises any one or a plurality of nucleotide sequences shown as SEQ ID No.21 to SEQ ID No.40, the third nucleic acid molecule comprises any one or a plurality of nucleotide sequences shown as SEQ ID No.41 to SEQ ID No.60, and the fourth nucleic acid molecule comprises any one or a plurality of nucleotide sequences shown as SEQ ID No.61 to SEQ ID No. 80.

Wherein, the specific information of the nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 is shown in the following Table 1:

Table 1 specific information on nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10

Specific information on the nucleotide sequences shown in SEQ ID No.11 to SEQ ID No.20 is given in Table 2 below:

table 2 specific information on nucleotide sequences shown in SEQ ID No.11 to SEQ ID No.20

Specific information on the nucleotide sequences shown in SEQ ID No.21 to SEQ ID No.30 is given in Table 3 below:

specific information on nucleotide sequences shown in Table 3 EQ ID No.21 to SEQ ID No.30

Specific information on the nucleotide sequences shown in SEQ ID No.31 to SEQ ID No.40 is given in Table 4 below:

table 4 specific information on nucleotide sequences shown in SEQ ID No.31 to SEQ ID No.40

SEQ ID No.	Promoter region	Name of adjacent gene	Gene interval
				31	chr1:72340480-72340801	NEGR1	Promoter
32	chr15:18471329-18471349	CHEK2P2	Promoter
				33	chr15:44714201-44714818	B2M	Intron
34	chr2:20576796-20576861	HS1BP3-IT1	Exon
				35	chr20:26714703-26714968	MIR663AHG	Promoter
36	chr3:148298566-148298764	LINC02046	Intron
				37	chr5:37111851-37112131	CPLANE1	Intron
38	chr6:31853884-31853903	NEU1	Exon
				39	chr7:103155522-103155914	NAPEPLD	Promoter
40	chr9:3477305-3477524	RFX3	Intron

Specific information on the nucleotide sequences shown in SEQ ID No.41 to SEQ ID No.50 is given in Table 5 below:

table 5 specific information on nucleotide sequences shown in SEQ ID No.41 to SEQ ID No.50

Specific information on the nucleotide sequences shown in SEQ ID No.51 to SEQ ID No.60 is given in Table 6 below:

table 6 specific information on nucleotide sequences shown as SEQ ID No.51 to SEQ ID No.60

Specific information on the nucleotide sequences shown in SEQ ID No.61 to SEQ ID No.70 is given in Table 7 below:

TABLE 7 specific information on nucleotide sequences shown in SEQ ID No.61 to SEQ ID No.70

Specific information on the nucleotide sequences shown in SEQ ID No.71 to SEQ ID No.80 is given in Table 8 below:

Table 8 specific information on nucleotide sequences shown as SEQ ID No.71 to SEQ ID No.80

Specifically, the first nucleic acid molecule may comprise one or more of the nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.10, and the methylation level of the nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.10 in cervical adenocarcinoma patients is significantly higher than that of normal individuals.

In some embodiments of the application, the first nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.10, e.g., the first nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.1 to SEQ ID No.3, the first nucleic acid molecule further comprises the nucleotide sequence shown as SEQ ID No.1 to SEQ ID No.5, and the first nucleic acid molecule further comprises the nucleotide sequence shown as SEQ ID No.1 to SEQ ID No. 10.

The first nucleic acid molecule may further comprise one or more of the nucleotide sequences shown as SEQ ID No.11 to SEQ ID No.20, wherein the methylation level of the nucleotide sequences shown as SEQ ID No.11 to SEQ ID No.20 is significantly lower in patients suffering from cervical adenocarcinoma than in normal individuals.

In some embodiments of the application, the first nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.11 to SEQ ID No.20, e.g., the first nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.11 to SEQ ID No.13, the first nucleic acid molecule further comprises the nucleotide sequence shown as SEQ ID No.11 to SEQ ID No.15, and the first nucleic acid molecule further comprises the nucleotide sequence shown as SEQ ID No.11 to SEQ ID No. 20.

In some embodiments of the application, the first nucleic acid molecule may comprise the nucleotide sequence of one or more of the nucleotide sequences set forth in SEQ ID No.1 to SEQ ID No.10, and the nucleotide sequence of one or more of the nucleotide sequences set forth in SEQ ID No.11 to SEQ ID No. 20.

Specifically, the second nucleic acid molecule may comprise one or more of the nucleotide sequences shown as SEQ ID No.21 to SEQ ID No.30, wherein the methylation level of the nucleotide sequences shown as SEQ ID No.21 to SEQ ID No.30 in cervical squamous carcinoma patients is significantly higher than that of normal individuals.

In some embodiments of the application, the second nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.21 to SEQ ID No.30, e.g., the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.21 to SEQ ID No.23, the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.21 to SEQ ID No.25, and the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.21 to SEQ ID No. 30.

The second nucleic acid molecule may further comprise one or more of the nucleotide sequences shown as SEQ ID No.31 to SEQ ID No.40, wherein the methylation level of the nucleotide sequences shown as SEQ ID No.31 to SEQ ID No.40 is significantly lower in cervical squamous carcinoma patients than in normal individuals.

In some embodiments of the application, the second nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.31 to SEQ ID No.40, e.g., the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.31 to SEQ ID No.33, the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.31 to SEQ ID No.35, and the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.31 to SEQ ID No. 40.

In some embodiments of the application, the second nucleic acid molecule may comprise the nucleotide sequence of one or more of the nucleotide sequences set forth in SEQ ID No.21 to SEQ ID No.30, and the nucleotide sequence of one or more of the nucleotide sequences set forth in SEQ ID No.31 to SEQ ID No. 40.

Specifically, the third nucleic acid molecule may comprise one or more of the nucleotide sequences shown as SEQ ID No.41 to SEQ ID No.50, and the methylation level of the nucleotide sequences shown as SEQ ID No.41 to SEQ ID No.50 in patients with low grade cervical intraepithelial neoplasia is significantly higher than that of normal individuals.

In some embodiments of the application, the third nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.41 to SEQ ID No.50, e.g., the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.41 to SEQ ID No.43, and the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.41 to SEQ ID No.45, and the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.41 to SEQ ID No. 50.

The third nucleic acid molecule may further comprise one or more of the nucleotide sequences shown as SEQ ID No.51 to SEQ ID No.60, wherein the methylation level of the nucleotide sequences shown as SEQ ID No.51 to SEQ ID No.60 is significantly lower in patients with low grade cervical intraepithelial neoplasia than in normal individuals.

In some embodiments of the application, the third nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.51 to SEQ ID No.60, e.g., the third nucleic acid molecule comprises the nucleotide sequences shown as SEQ ID No.51 to SEQ ID No.53, the third nucleic acid molecule comprises the nucleotide sequences shown as SEQ ID No.51 to SEQ ID No.55, and the third nucleic acid molecule comprises the nucleotide sequences shown as SEQ ID No.51 to SEQ ID No. 60.

In some embodiments of the application, the third nucleic acid molecule may comprise the nucleotide sequence of one or more of the nucleotide sequences set forth in SEQ ID No.41 to SEQ ID No.50, and the nucleotide sequence of one or more of the nucleotide sequences set forth in SEQ ID No.51 to SEQ ID No. 60.

In particular, the fourth nucleic acid molecule may comprise one or more of the nucleotide sequences shown as SEQ ID No.61 to SEQ ID No.70, wherein the methylation level of the nucleotide sequences shown as SEQ ID No.61 to SEQ ID No.70 is significantly higher in patients with high grade cervical intraepithelial neoplasia than in normal individuals.

In some embodiments of the application, the fourth nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.61 to SEQ ID No.70, e.g., the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.61 to SEQ ID No.63, and the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.61 to SEQ ID No.65, and the second nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.61 to SEQ ID No. 70.

The fourth nucleic acid molecule may further comprise one or more of the nucleotide sequences shown as SEQ ID No.71 to SEQ ID No.80, wherein the methylation level of the nucleotide sequences shown as SEQ ID No.71 to SEQ ID No.80 is significantly lower in patients with high grade cervical intraepithelial neoplasia than in normal individuals.

In some embodiments of the application, the fourth nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.71 to SEQ ID No.80, e.g., the fourth nucleic acid molecule comprises the nucleotide sequence shown as SEQ ID No.71 to SEQ ID No.73, further comprises the nucleotide sequence shown as SEQ ID No.71 to SEQ ID No.75, and further comprises the nucleotide sequence shown as SEQ ID No.71 to SEQ ID No. 80.

In some embodiments of the application, the fourth nucleic acid molecule may comprise the nucleotide sequence of one or more of the nucleotide sequences set forth in SEQ ID No.61 through SEQ ID No.70, and the nucleotide sequence of one or more of the nucleotide sequences set forth in SEQ ID No.71 through SEQ ID No. 80.

The embodiment of the application also provides a kit for parting detection of cervical cancer and cervical intraepithelial neoplasia, which comprises: a reagent capable of specifically detecting the methylation level of any one or more of the nucleotide sequences shown in SEQ ID Nos. 1 to 20, 21 to 40, 41 to 60, and 61 to 80 in the DNA of a sample, wherein the methylation level is the ratio of the number of methylated CpG sites in each nucleotide sequence to the total number of CpG sites in the nucleotide sequence.

As used herein, "CpG sites" refers to cytosine-phosphate-guanine (methyl CpG) sites.

As used herein, "methylation level" refers to the methylation level of CpG sites, specifically the ratio of the number of methylated CpG sites in each nucleotide sequence to the total number of CpG sites in the nucleotide sequence. Thus, the number of CpG sites of different nucleotide sequences may not be the same, but the methylation level of CpG sites may be the same; furthermore, the same level of methylation of CpG sites of different nucleotide sequences does not mean that the same CpG site of different nucleotide sequences will be methylated equally or not.

In some embodiments of the application, the kit further comprises a modifying reagent and a detection reagent.

The modifying reagent is used for differentially modifying methylated CpG sites and unmethylated CpG sites in the DNA of the test sample.

The detection reagent is used to determine or assist in determining whether each CpG site of the nucleotide sequence of any one or more of SEQ ID No.1 to SEQ ID No.20 in the DNA of the test sample is methylated or unmethylated, and determining or assist in determining whether each CpG site of the nucleotide sequence of any one or more of SEQ ID No.21 to SEQ ID No.40 in the DNA of the test sample is methylated or unmethylated, and determining or assist in determining whether each CpG site of the nucleotide sequence of any one or more of SEQ ID No.41 to SEQ ID No.60 in the DNA of the test sample is methylated or unmethylated, and determining or assist in determining whether each CpG site of the nucleotide sequence of any one or more of SEQ ID No.61 to SEQ ID No.80 in the DNA of the test sample is methylated or unmethylated.

In some embodiments of the application, the modifying agent comprises bisulfite and derivatives thereof.

In some embodiments of the application, the detection reagent comprises a PCR reagent comprising a primer pair and/or a specific probe that specifically amplifies the nucleotide sequence shown in any one or more of SEQ ID Nos. 1 to 20, and a primer pair and/or a specific probe that specifically amplifies the nucleotide sequence shown in any one or more of SEQ ID Nos. 21 to 40, and a primer pair and/or a specific probe that specifically amplifies the nucleotide sequence shown in any one or more of SEQ ID Nos. 41 to 60, and a primer pair and/or a specific probe that specifically amplifies the nucleotide sequence shown in any one or more of SEQ ID Nos. 61 to 80.

As used herein, "primer" refers to an oligonucleotide sequence that serves as an initiator of a DNA replication reaction in a PCR reaction, and a person skilled in the art knows the method of designing a primer pair for PCR amplification thereof based on the nucleotide sequence of known sequence information.

As used herein, a "probe" refers to a nucleic acid sequence (DNA or RNA) that carries a detectable label and is of known sequence and is complementary to a gene of interest.

In some embodiments of the application, the kit further comprises a positive control and/or a negative control.

The positive control comprises any one nucleotide sequence or a combination of nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.20, any one nucleotide sequence or a combination of nucleotide sequences shown as SEQ ID No.21 to SEQ ID No.40, any one nucleotide sequence or a combination of nucleotide sequences shown as SEQ ID No.41 to SEQ ID No.60, and any one nucleotide sequence or a combination of nucleotide sequences shown as SEQ ID No.61 to SEQ ID No.80, wherein each nucleotide sequence of any one nucleotide sequence or each nucleotide sequence contains a methylation CpG site.

The negative control comprises any one nucleotide sequence or a combination of nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.20, any one nucleotide sequence or a combination of nucleotide sequences shown as SEQ ID No.21 to SEQ ID No.40, any one nucleotide sequence or a combination of nucleotide sequences shown as SEQ ID No.41 to SEQ ID No.60, and any one nucleotide sequence or a combination of nucleotide sequences shown as SEQ ID No.61 to SEQ ID No.80, wherein each nucleotide sequence of any one nucleotide sequence or combination does not contain a methylation CpG site.

In some embodiments of the application, the kit further comprises DNA extraction reagents for extracting and/or purifying DNA from the test sample.

In some embodiments of the application, the test sample is blood, urine, cervical scraping, vaginal secretions, vaginal douche, cervical epithelial cells, post-operative ex vivo tissue, or a purified DNA sample, e.g., tumor material, tissue, or body fluid derived from a surgical resection or biopsy (e.g., cells from a patient biopsy), e.g., blood or living tissue. Tissue may be removed by any suitable method, such as needle biopsy, aspiration, scraping, resection using surgical resection. Suitable samples comprise total tumor material, i.e. Tumor Infiltrating Leukocytes (TILs), stroma and tumor cells. Optionally, the sample may be resected tumor fragments. Samples may be obtained at one or more time points. Optionally, the sample may be processed (e.g., fixed, stored, frozen, lysed, homogenized, DNA or RNA extracted, cDNA converted, ultrafiltered, diluted (e.g., diluted with saline, buffer, or physiologically acceptable diluent, etc.), concentrated, evaporated, centrifuged, separated, filtered, etc.) using one or more post-collection preparation or storage techniques prior to analysis of the material by the methods of the present application.

Correspondingly, the embodiment of the application also provides a use method of the kit, which comprises the following steps:

s1, treating a sample to be tested, and treating the sample to be tested or DNA of the sample to be tested by using a modifying reagent to obtain a modified DNA sample;

s2, selecting a detection sequence group of the DNA sample, wherein the detection sequence group comprises a first detection sequence subgroup, a second detection sequence subgroup, a third detection sequence subgroup and a fourth detection sequence subgroup; and

s3, reacting the DNA sample with a detection reagent, and determining the CpG methylation level of each nucleotide sequence in the detection sequence group.

Wherein the first detection sequence subgroup comprises any one nucleotide sequence or a combination of at least two nucleotide sequences as shown in SEQ ID No.1 to SEQ ID No.10, and/or any one nucleotide sequence or a combination of at least two nucleotide sequences as shown in SEQ ID No.11 to SEQ ID No. 20; the second detection sequence subgroup comprises any one nucleotide sequence or a combination of at least two nucleotide sequences as shown in SEQ ID No.21 to SEQ ID No.30, and/or any one nucleotide sequence or a combination of at least two nucleotide sequences as shown in SEQ ID No.31 to SEQ ID No. 40; the third detection sequence subgroup comprises any one nucleotide sequence or a combination of at least two nucleotide sequences as shown in SEQ ID No.41 to SEQ ID No.50, and/or any one nucleotide sequence or a combination of at least two nucleotide sequences as shown in SEQ ID No.51 to SEQ ID No. 60; the fourth subset of detection sequences comprises any one nucleotide sequence or a combination of at least two nucleotide sequences as shown in SEQ ID No.61 to SEQ ID No.70 and/or any one nucleotide sequence or a combination of at least two nucleotide sequences as shown in SEQ ID No.71 to SEQ ID No. 80.

It will be appreciated that the methylation level of each nucleotide sequence can be detected using DNA methylation level detection methods known in the art, such as methylation specific PCR methods, bisulfite treatment and sequencing methods, restriction enzyme analysis methods in combination with sodium bisulfite, fluorescent quantitative PCR methods, methylation sensitive high resolution melting curve analysis methods, or pyrosequencing methods, and that the process of converting the results obtained by the methods of detecting DNA methylation to methylation levels is known to those skilled in the art.

In some embodiments of the application, the method of use further comprises the steps of: comparing the methylation level of each nucleotide sequence in the detection sequence group with a corresponding preset threshold, wherein the preset threshold can be a mean value of the methylation levels of the corresponding nucleotide sequences in a normal individual set, and the normal individual set comprises three or more normal individuals.

Further, the using method further comprises the steps of:

determining that the subject of the test sample is suffering from cervical adenocarcinoma if the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 1 to 10 is above 0.6, respectively, and/or the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 11 to 20 is below 0.3, respectively;

Determining that the subject of the test sample is suffering from cervical squamous carcinoma if the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 21 to 30 is above 0.7, respectively, and/or the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 31 to 40 is below 0.3, respectively;

determining that the subject of the test sample has a low grade cervical intraepithelial neoplasia if the CpG methylation level of at least eight of the nucleotide sequences set forth in SEQ ID nos. 41 to 50 is above 0.72, respectively, and/or the CpG methylation level of at least five of the nucleotide sequences set forth in SEQ ID nos. 51 to 60 is below 0.46, respectively; and

and if the CpG methylation level of at least eight nucleotide sequences in the nucleotide sequences shown in SEQ ID No.61 to SEQ ID No.70 is above 0.65 and/or the CpG methylation level of at least five nucleotide sequences in the nucleotide sequences shown in SEQ ID No.71 to SEQ ID No.80 is below 0.39, respectively, determining that the subject of the test sample suffers from high-grade cervical intraepithelial neoplasia.

The following description of the technical solutions in the embodiments of the present application will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to fall within the scope of the application. As used herein, "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defining "first" and "second" may explicitly or implicitly include one or more of the described features. As used herein, "patient" refers to humans and other mammals that are first diagnosed with cervical adenocarcinoma, and the patient may be an individual undergoing treatment for cervical adenocarcinoma, or any individual desiring to be analyzed or treated using the methods of the application. Optionally, the patient may be cervical adenocarcinoma that is in stage I, II, III or IV, for example, may be a stage I or II patient, or may also be a stage II or III patient, or may also be a stage III or IV patient. As used herein, "subject" refers to any individual that is detected, analyzed, or treated using the present application, including humans and other mammals that are first diagnosed with cervical adenocarcinoma, as well as humans and other mammals that are not afflicted with cervical adenocarcinoma.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are illustrative only and should not be construed as limiting the application.

Unless otherwise indicated, the starting materials and reagents used in the following examples are commercially available or may be prepared by methods known in the art.

The whole genome extraction, construction of WGBS library, WGBS sequencing and data processing involved in the embodiment of the application are all completed by Beijing NOX and Zhigen technologies Co.

The kit Tiangen DP304 related in the embodiment of the application is purchased from Tiangen Biochemical technology (Beijing) limited company; EZ DNA Methylation-GoldTM kit is available from ZYMO RESEARCH, inc. of USA; DNA concentration determination kits were purchased from Life technologies, california, USA;

agilent Bioanalyzer 2100 instruments involved in embodiments of the present application are purchased from Agilent; qubit 2.0 fluorometer was purchased from Life Technologies company, usa; spectrophotometers were purchased from IMPLEN company, usa.

All operations in the embodiments of the present application conform to the manufacturer's instructions.

The samples to be tested related in the embodiment of the application are derived from a biological library of a third hospital affiliated to Guangzhou medical university, and relate to in vitro samples of 18 patients, wherein 6 patients are diagnosed with low-grade cervical intraepithelial neoplasia (Cervical Intraepithelial Neoplasias, CIN 1) for the first time in histology, and the corresponding samples to be tested are cervical lesion tissues subjected to surgical excision, namely, sample 1 to sample 6; the first histologically diagnosed high grade cervical intraepithelial neoplasia (Cervical Intraepithelial Neoplasias, cin 3) in 5 patients, corresponding test samples were surgically excised cervical lesions, samples 7 through 11, respectively; 2 patients were histologically diagnosed with cervical adenocarcinoma for the first time (Adenocarcinoma of Cervix, CCA), and the corresponding test samples were surgically excised cervical carcinoma variegated tissue (stored at-80 ℃), sample 12 and sample 13, respectively; the first histologically diagnosed cervical squamous cell carcinoma (Squamous Cell Carcinoma, CCS) in 5 patients, the corresponding test samples were surgically excised cervical cancer metatissue (stored at-80 ℃), samples 14 to 18, respectively. The study protocol did not include cervical cancer recurrent patients and had been approved by each central ethics committee and written informed consent was obtained for each patient prior to surgery.

The control group related to the embodiment of the application is derived from in-vitro samples of 5 healthy females (each index of the body is normal and the cervix is not diseased), and the in-vitro samples are cervical scraping plates which correspond to the controls 1 to 5 respectively.

Wherein, the whole genome DNA extraction, whole genome DNA methylation sequencing (Whole Genome Bisulfite Sequencing, WGBS) and sequencing result analysis operations of sample 1 to sample 18 and control 1 to control 5 specifically comprise the following steps:

s10, respectively carrying out whole genome extraction operation on each tested sample/control sample by using a TiangenDP304 kit, and monitoring degradation and pollution of DNA by agarose gel electrophoresis to obtain whole genome DNA;

s20, measuring the purity of the whole genome DNA of the step S10 by using a spectrophotometer (IMPLEN, CA, USA), and using the same2.0 detection of the concentration of Whole genomic DNA by a DNA concentration determination kit used in combination with a fluorometer, then sonicating the whole genomic DNA with an ultrasonic DNA disrupter (Covaris S220) to a plurality of DNA fragments of 200bp to 300bp, and then adding end repair and adenylation to fragment the whole genomic DNA (total amount of 5.2. Mu.g) doped with 26ng lambda. DNA;

s30, connecting the cytosine methylated bar code to the DNA fragment obtained after ultrasonic treatment in the step S20 according to the specification conditions of the manufacturer to obtain a connected DNA fragment;

S40, adopting a EZ DNA Methylation-GoldTM kit to process the connected DNA fragment in the step S30 twice to obtain a single-stranded DNA fragment subjected to bisulphite treatment;

s50, carrying out PCR amplification on the single-stranded DNA fragments subjected to bisulphite treatment in the step S40 by adopting a KAPA HiFi HotStart Uracil and Readymix (2X) kit to obtain a WGBS library, determining the concentration of the WGBS library by using a Qubit 2.0 fluorometer and fluorescent quantitative PCR, and determining the size of the insert on a Agilent Bioanalyzer 2100 system;

s60, sequencing the WGBS library in the step S50, wherein the sequencing depth is at least 30×, and analyzing sequencing data by using MOABS series software to obtain the CpG dinucleotide methylation level of each DNA fragment in the WGBS library, the depth and coverage of the CpG dinucleotides in the whole genome DNA and the average methylation level of the whole genome DNA, wherein the average methylation level of the whole genome DNA refers to the average value of the CpG dinucleotide methylation levels of each DNA fragment in the WGBS library.

Example 1

This example compares the specificity of the nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.20 as biomarkers in cervical adenocarcinoma detection diagnosis for normal individuals and diseased individuals, and compares the DNA sequencing results of normal individuals (control 1 to control 5) and subjects clinically diagnosed for cervical adenocarcinoma for the first time, as shown in Table 9 below:

TABLE 9 CpG site methylation levels of the nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.20 in the whole genome DNA of sample 12, sample 13 and controls 1 to 5

As can be seen from the experimental data in Table 9, for ten nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 in the DNA of cervical adenocarcinoma subjects, the CpG site methylation level of each nucleotide sequence was higher than the average value of the CpG site methylation levels of the corresponding nucleotide sequences in the control group (control 1 to control 5), respectively, and the CpG site methylation level of at least five nucleotide sequences (of ten nucleotide sequences shown in SEQ ID No.1 to SEQ ID No. 10) in the DNA of cervical adenocarcinoma subjects was 0.6 or more (. Gtoreq.60%) and the CpG site methylation level of at least five nucleotide sequences (of ten nucleotide sequences shown in SEQ ID No.1 to SEQ ID No. 10) in the DNA of normal individuals (.ltoreq.20%).

In addition, for ten nucleotide sequences shown in SEQ ID No.11 to SEQ ID No.20 in the DNA of a cervical adenocarcinoma subject, the CpG site methylation level of each nucleotide sequence was lower than the average value of the CpG site methylation levels of the corresponding nucleotide sequences in the control group (control 1 to control 5), respectively, and the CpG site methylation level of at least five nucleotide sequences (of ten nucleotide sequences shown in SEQ ID No.11 to SEQ ID No. 20) in the DNA of a cervical adenocarcinoma subject was 0.3 or less (30%) and the CpG site methylation level of ten nucleotide sequences shown in SEQ ID No.11 to SEQ ID No.20 in the DNA of a normal individual (control 1 to control 5) was 0.7 or more (70%).

Thus, the level of methylation of CpG sites of the ten nucleotide sequences shown in SEQ ID No.1 through SEQ ID No.10 has a good differentiation between cervical adenocarcinoma subjects and normal individuals, demonstrating that the ten nucleotide sequences shown in SEQ ID No.1 through SEQ ID No.10 can be used as molecular markers for cervical adenocarcinoma screening, diagnosis and monitoring.

Example 2

In this example, ten nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 were used as molecular markers for cervical adenocarcinoma diagnosis, and DNA of samples from CIN1 subjects (samples 1 to 6), CIN3 subjects (samples 7 to 11), CCA subjects (samples 12 and 13) and CCS subjects were analyzed by sequencing and comparing, and the results are shown in Table 10:

TABLE 10 methylation levels of CpG sites of ten nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.20 in the whole genome DNA of samples 1 to 18 and controls 1 to 5

Note that: NULL indicates a NULL value, 0.00 indicates no methylation occurs, and other values represent methylation levels.

As can be seen from Table 10, controls 1 to 5 constitute a normal individual set having an average value of 0.118 ((0.089+0.09+0.105+0.201+0.103)/5) of methylation levels of nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10, the average value of methylation levels of CpG sites of nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 in the test sample DNA (sample 1 to sample 6) of CIN1 patient being 1.4 to 3.0 times the average value of normal individual sets, the average value of methylation levels of CpG sites of nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 in the test sample DNA (sample 7 to sample 11) of CIN3 patient being 1.3 to 3.7 times the average value of normal individual sets, the average value of the methylation level of CpG sites of the nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.10 in the sample DNA (sample 12 and sample 13) of the CCA patient is 4.5 to 6.7 times the average value of the normal individual groups, and the average value of the methylation level of CpG sites of the nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.10 in the sample DNA (sample 14 to sample 18) of the CCS patient is 1.8 to 3.5 times the average value of the normal individual groups.

It can be seen that the level of methylation of CpG sites of the nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 in the DNA of the test sample of a patient suffering from CCA is significantly higher than that of a patient suffering from cervical intraepithelial neoplasia (CIN 1 to CIN 3), CCS and normal individuals.

Example 3

In this example, ten nucleotide sequences shown in SEQ ID No.11 to SEQ ID No.20 were used as molecular markers for diagnosis of cervical adenocarcinoma, and DNA sequencing comparison analyses were performed on samples of CIN1 subjects (samples 1 to 6), CIN3 subjects (samples 7 to 11), CCA subjects (samples 12 and 13), and CCS subjects, and the results are shown in Table 11:

TABLE 11 CpG site methylation levels of ten nucleotide sequences shown in SEQ ID No.11 to SEQ ID No.20 in the whole genome DNA of samples 1 to 18 and controls 1 to 5

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As can be seen from Table 11, controls 1 to 5 constitute a normal individual set having an average value of 0.884 ((0.922+0.877+0.839+0.889)/5) of methylation levels of nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10, the average value of CpG site methylation levels of nucleotide sequences shown in SEQ ID No.11 to SEQ ID No.20 in the sample DNA (sample 1 to sample 6) of the patient with CIN1 being 36% to 75% of the average value of normal individual sets, the average value of CpG site methylation levels of nucleotide sequences shown in SEQ ID No.11 to SEQ ID No.20 in the sample DNA (sample 7 to sample 11) of the patient with CIN3 being 47% to 76% of the average value of CpG site methylation levels of nucleotide sequences shown in SEQ ID No.11 to SEQ ID No.20, the average value of CpG sites shown in the sample DNA (sample 12 and sample 13) of the patient with CCID No.11 to SEQ ID No.20, and the average value of CpG site methylation levels of nucleotide sequences shown in the sample 14% to 20 of the sample 14% to sample 14% of the patient with CIN3 being the average value of CpG sites being 15% to 18% of the average value of CpG sites shown in the sample DNA of the sample 14 to sample 9 of the patient with CIN.

It can be seen that the level of methylation of CpG sites in the DNA of the test samples of patients suffering from CCA, as shown by the nucleotide sequences shown by SEQ ID No.11 to SEQ ID No.20, is significantly lower than that of patients suffering from cervical intraepithelial neoplasias (CIN 1 to CIN 3), CCS and normal individuals.

From examples 2 and 3, it is understood that when the average value of the methylation levels of CpG sites of the nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 in the sample DNA is 4.5 to 6.7 times the average value of the normal individuals, and the average value of the methylation levels of CpG sites of the nucleotide sequences shown in SEQ ID No.11 to SEQ ID No.20 in the sample DNA is 15% to 22% of the average value of the normal individuals, the subject corresponding to the sample DNA suffers from CCA.

Example 4

To verify the specificity of the nucleotide sequences shown in SEQ ID Nos. 21 to 40 provided by the present application as biomarkers in cervical squamous carcinoma detection diagnosis for diseased individuals and normal individuals, the DNA sequencing results of normal individuals and subjects clinically diagnosed for the first time as cervical squamous carcinoma were compared, and the results are shown in Table 12, wherein NULL indicates no result.

Table 12 CpG site methylation levels of the nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.20 in the whole genome DNA of SCC samples and 5 control samples

As can be seen from Table 12, the methylation level of CpG sites in the patients with SCC is significantly higher than that of the control samples, the methylation level of SEQ ID No.1-10 is about 0-0.3 in normal individuals, the methylation level of SEQ ID No.1-10 is about 0.4-1.0 (about 40% -100%), about 4-10 times in normal individuals, and the methylation level of at least 5 sequences in SEQ ID No.1-10 is above 0.7 (. Gtoreq.70%); the methylation level of CpG sites of SEQ ID No.11-20 is obviously lower than that of each control sample, the methylation level of SEQ ID No.11-20 of a normal individual is 0.65-1, the methylation level of the patient with SCC is only about 0.07-0.5 (about 7% -50%), and the methylation level of at least 5 sequences in SEQ ID No.11-20 is below 0.3 (less than or equal to 30%). As can be seen, SEQ ID Nos. 1-20 have good discrimination between SCC patients and normal individuals.

Example 5

To further verify the specificity of SEQ ID nos. 1-20 provided herein as biomarkers in cervical squamous carcinoma detection diagnosis for cervical squamous carcinoma afflicted individuals and other cervical afflicted individuals, the DNA sequencing results of CIN1 subjects, CIN3 subjects, CCA subjects, and CCS subjects diagnosed for the first time clinically were compared and the results are shown in table 13 and table 14, wherein NULL indicates no result.

TABLE 13 CpG site methylation level of the nucleotide sequences shown in SEQ ID No.1 to SEQ ID.10 in SCC samples and other cervical lesion samples

TABLE 14 CpG site methylation level of the nucleotide sequences shown in SEQ ID No.31 to SEQ ID No. 40 in SCC samples and other cervical lesions samples

As can be seen from tables 13 and 14, the methylation levels in SEQ ID Nos. 1-10 are significantly higher in SCC patients than in CIN1, CIN3 and CCA patients, with at least 5 sequences having methylation levels above 0.7 (. Gtoreq.70%); methylation levels in SEQ ID Nos. 11-20 are significantly lower in SCC patients than CIN1, CIN3 and CCA subjects, with methylation levels of at least 5 sequences below 0.3 (.ltoreq.30%). It follows that the methylation levels of SEQ ID Nos. 1-20 can significantly distinguish SCC patients from other cervical lesions.

Example 6

This example compares the specificity of the nucleotide sequences shown in SEQ ID Nos. 41 to 60 as biomarkers in cervical lesion typing tests for CIN1 subjects and other cervical lesion subjects (CIN 3 subjects, CCA subjects, CCS subjects), and the results are shown in Table 15 and Table 16, wherein NULL indicates no results.

TABLE 15 CpG site methylation levels of ten nucleotide sequences shown in SEQ ID No.41 to SEQ ID No.50 in the genomic DNAs of samples 1 to 18 and controls 1 to 5

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As shown in the experimental data in Table 15, the methylation levels of SEQ ID No.41 to SEQ ID No.50 were significantly higher than those of the respective controls, the methylation levels of SEQ ID No.41 to SEQ ID No.50 were about 0.13 to 0.63 in the normal subjects, and about 0.73 to 1 (about 73% to 100%) in the CIN1 subjects, and the methylation levels of at least 8 sequences in the CIN1 subjects were 0.72 or higher (. Gtoreq.72%). From this, it can be seen that there is an abnormal increase in methylation level in at least 10 nucleotide sequence intervals including SEQ ID No.41 to SEQ ID No.50 in a CIN1 subject, in which the methylation-elevated level of SEQ ID No.41 is changed most and the methylation-elevated level of SEQ ID No.42 to 50 is changed less in sequence, as compared with a normal subject, and therefore, ten nucleotide sequences shown in SEQ ID No.41 to SEQ ID No.50 can be used as molecular markers of CIN1 and the methylation level or the average value of the methylation levels of any one or more of the ten nucleotide sequences can be used as an evaluation index of CIN 1.

TABLE 16 methylation levels of CpG sites of ten nucleotide sequences shown in SEQ ID No.51 to SEQ ID No.60 in the whole genome DNA of samples 1 to 18 and controls 1 to 5

As shown in the experimental data in Table 15, the methylation levels of SEQ ID No.51 to SEQ ID No.60 were significantly lower than those of the respective controls, the methylation levels of SEQ ID No.51 to SEQ ID No.60 were about 0.71 to 0.95 in the normal subjects, and about 0.11 to 0.34 (about 11% to 34%) in the CIN1 subjects, and the methylation levels of at least 8 sequences were 0.46% or less (46%). It can be seen that there is an abnormally reduced methylation level of at least 10 nucleotide sequences (the nucleotide sequences shown in SEQ ID No.51 to SEQ ID No. 60) in CIN1 subjects as compared with normal individuals, and therefore, ten nucleotide sequences shown in SEQ ID No.51 to SEQ ID No.60 can be used as molecular markers of CIN1, and the methylation level or the average value of the methylation level of any one or more of the ten nucleotide sequences can be used as an evaluation index of CIN 1.

Example 7

This example compares the specificity of the nucleotide sequences shown in SEQ ID Nos. 61 to 80 as biomarkers in cervical lesion typing tests for CIN3 subjects and other cervical lesion subjects (CIN 1 subjects, CCA subjects, CCS subjects), and the results are shown in Table 17 and Table 18, wherein NULL indicates no results.

TABLE 17 methylation levels of CpG sites of ten nucleotide sequences shown in SEQ ID No.61 to SEQ ID No.70 in the whole genome DNA of samples 1 to 18 and controls 1 to 5

As shown in the experimental data in Table 17, the methylation levels of SEQ ID No.61 to SEQ ID No.70 were significantly higher for CIN3 subjects than for the respective controls, wherein the methylation levels of SEQ ID No.61 to SEQ ID No.70 were about 0.46 for normal subjects, about 0.61 to about 0.93 (about 61% to 93%) for CIN3 subjects, and at least 8 sequences were at a methylation level of 0.65 or higher (. Gtoreq.65%). It can be seen that there is an abnormally elevated methylation level of at least 10 nucleotide sequences in CIN3 subjects compared to normal individuals, and that these 10 intervals include the nucleotide sequences shown in SEQ ID No.61 to SEQ ID No.70, and therefore, ten nucleotide sequences shown in SEQ ID No.61 to SEQ ID No.70 can be used as molecular markers for CIN3, and the methylation level or the average value of the methylation level of any one or more of these ten nucleotide sequences can be used as an evaluation index for CIN 3.

TABLE 18 methylation levels of CpG sites of ten nucleotide sequences shown in SEQ ID No.71 to SEQ ID No.80 in the whole genome DNA of samples 1 to 18 and controls 1 to 5

As shown in the experimental data in Table 18, the methylation levels of SEQ ID No.71 to SEQ ID No.80 were significantly lower than those of the respective controls, the methylation levels of SEQ ID No.71 to SEQ ID No.80 were about 0.65 to 1 in the normal subjects, and 0 to 0.48 (about 0% to 48%) in the CIN3 subjects, and the methylation levels of at least 8 sequences were 0.39 or less (. Ltoreq.39%). It can be seen that there is an abnormal decrease in methylation level in at least 10 nucleotide sequence intervals including the nucleotide sequences shown in SEQ ID No.71 to SEQ ID No.80 in CIN3 subjects as compared with normal individuals, and therefore, ten nucleotide sequences shown in SEQ ID No.71 to SEQ ID No.80 can be used as molecular markers of CIN3, and the methylation level or the average value of the methylation level of any one or more of the ten nucleotide sequences can be used as an evaluation index of CIN 3.

In the foregoing embodiments, the descriptions of the embodiments are emphasized, and for parts of one embodiment that are not described in detail, reference may be made to related descriptions of other embodiments.

The nucleic acid molecule composition, the kit and the application thereof for typing detection of cervical cancer and cervical intraepithelial neoplasia provided by the embodiment of the application are described in detail. The principles and embodiments of the present application have been described herein with reference to specific examples, the description of the above examples is only for aiding in understanding the technical solution of the present application and its core ideas; those of ordinary skill in the art will appreciate that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims (11)

1. A nucleic acid molecule composition for the genotyping detection of cervical cancer and cervical intraepithelial neoplasia, the nucleic acid molecule composition comprising:

a first nucleic acid molecule comprising a nucleotide sequence as shown in any one or more of SEQ ID No.1 to SEQ ID No. 20;

a second nucleic acid molecule comprising a nucleotide sequence as shown in any one or more of SEQ ID No.21 to SEQ ID No. 40;

a third nucleic acid molecule comprising a nucleotide sequence as shown in any one or more of SEQ ID No.41 to SEQ ID No. 60; and

a fourth nucleic acid molecule comprising a nucleotide sequence as shown in any one or more of SEQ ID No.61 to SEQ ID No. 80.

2. The nucleic acid molecule composition of claim 1, wherein the first nucleic acid molecule comprises at least three of the nucleotide sequences set forth in SEQ ID nos. 1 to 20;

and/or the second nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.21 to SEQ ID No. 40;

and/or, the third nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.41 to SEQ ID No. 60;

and/or, the fourth nucleic acid molecule comprises at least three of the nucleotide sequences shown as SEQ ID No.61 to SEQ ID No. 80.

3. The nucleic acid molecule composition of claim 1, wherein the first nucleic acid molecule comprises a collection of all nucleotide sequences as set forth in SEQ ID nos. 1 to 20;

and/or, the second nucleic acid molecule comprises a collection of all nucleotide sequences as set forth in SEQ ID No.21 to SEQ ID No. 40;

and/or, the third nucleic acid molecule comprises a collection of all nucleotide sequences as set forth in SEQ ID No.41 through SEQ ID No. 60;

and/or, the fourth nucleic acid molecule comprises a collection of all nucleotide sequences as set forth in SEQ ID No.61 through SEQ ID No. 80.

4. A kit for typing detection of cervical cancer and cervical intraepithelial neoplasia, the kit comprising: a reagent capable of specifically detecting the methylation level of any one or more of the nucleotide sequences shown in SEQ ID Nos. 1 to 20, 21 to 40, 41 to 60, and 61 to 80 in the DNA of a sample, wherein the methylation level is the ratio of the number of methylated CpG sites in each nucleotide sequence to the total number of CpG sites in the nucleotide sequence.

5. The kit of claim 4, further comprising a modification reagent and a detection reagent;

wherein the modifying reagent is used for differentially modifying methylated CpG sites and unmethylated CpG sites in the DNA of the test sample;

the detection reagent is used to determine or assist in determining whether each CpG site of the nucleotide sequence of any one or more of SEQ ID Nos. 1 to 20 in the DNA of the test sample is methylated or unmethylated, and whether each CpG site of the nucleotide sequence of any one or more of SEQ ID Nos. 21 to 40 in the DNA of the test sample is methylated or unmethylated, and whether each CpG site of the nucleotide sequence of any one or more of SEQ ID Nos. 41 to 60 in the DNA of the test sample is methylated or unmethylated, and determining or assist in determining whether each CpG site of the nucleotide sequence of any one or more of SEQ ID No.61 to 80 in the DNA of the test sample is methylated or unmethylated.

6. The kit of claim 5, wherein the modifying reagent comprises bisulfite and derivatives thereof;

And/or the detection reagent comprises a PCR reagent, wherein the PCR reagent comprises a primer pair and/or a specific probe for specifically amplifying any one or a plurality of nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.20, a primer pair and/or a specific probe for specifically amplifying any one or a plurality of nucleotide sequences shown in SEQ ID No.21 to SEQ ID No.40, a primer pair and/or a specific probe for specifically amplifying any one or a plurality of nucleotide sequences shown in SEQ ID No.41 to SEQ ID No.60, and a primer pair and/or a specific probe for specifically amplifying any one or a plurality of nucleotide sequences shown in SEQ ID No.61 to SEQ ID No. 80.

7. The kit of claim 4, further comprising a DNA extraction reagent for extracting and/or purifying DNA from the test sample.

8. The kit of claim 4, wherein the test sample is blood, urine, cervical scraping, vaginal secretions, vaginal douches, cervical epithelial cells, post-operative ex vivo tissue, or purified DNA sample.

9. The method of use of a kit for typing detection of cervical cancer and cervical intraepithelial neoplasia according to any one of claims 4 to 8, wherein the method of use comprises the steps of:

Providing a sample to be tested, and treating the sample to be tested or the DNA of the sample to be tested by adopting the modifying reagent to obtain a modified DNA sample;

selecting a detection sequence group of the DNA sample, wherein the detection sequence group comprises a first detection sequence subgroup, a second detection sequence subgroup, a third detection sequence subgroup and a fourth detection sequence subgroup; and

reacting said detection reagent with said DNA sample and determining the CpG methylation level of each nucleotide sequence in said set of detection sequences;

wherein the first detection sequence subgroup comprises any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No.1 to SEQ ID No.10, and/or any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No.11 to SEQ ID No. 20;

the second detection sequence subgroup comprises any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No. 21-SEQ ID No.30, and/or any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No. 31-SEQ ID No. 40;

the third detection sequence subgroup comprises any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No.41 to SEQ ID No.50, and/or any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No.51 to SEQ ID No. 60;

The fourth detection sequence subgroup comprises any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No. 61-SEQ ID No.70, and/or any one nucleotide sequence or a combination of at least two nucleotide sequences shown as SEQ ID No. 71-SEQ ID No. 80.

10. The method of use of claim 9, further comprising the steps of:

comparing the methylation level of each nucleotide sequence in the detection sequence group with a corresponding preset threshold value;

wherein the preset threshold is the average value of CpG methylation levels of corresponding nucleotide sequences in DNA of a normal individual set, and the normal individual set comprises three or more normal individuals.

11. The method of use of claim 9, further comprising the steps of:

determining that the subject of the test sample is suffering from cervical adenocarcinoma if the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 1 to 10 is above 0.6, respectively, and/or the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 11 to 20 is below 0.3, respectively;

Determining that the subject of the test sample is suffering from cervical squamous carcinoma if the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 21 to 30 is above 0.7, respectively, and/or the CpG methylation level of at least five of the nucleotide sequences shown in SEQ ID nos. 31 to 40 is below 0.3, respectively;

determining that the subject of the test sample has a low grade cervical intraepithelial neoplasia if the CpG methylation level of at least eight of the nucleotide sequences set forth in SEQ ID nos. 41 to 50 is above 0.72, respectively, and/or the CpG methylation level of at least five of the nucleotide sequences set forth in SEQ ID nos. 51 to 60 is below 0.46, respectively; and

and if the CpG methylation level of at least eight nucleotide sequences in the nucleotide sequences shown in SEQ ID No.61 to SEQ ID No.70 is above 0.65 and/or the CpG methylation level of at least five nucleotide sequences in the nucleotide sequences shown in SEQ ID No.71 to SEQ ID No.80 is below 0.39, respectively, determining that the subject of the test sample suffers from high-grade cervical intraepithelial neoplasia.