WO2023088335A1 - Reagent and kit for detecting endometrial cancer, and method for using kit - Google Patents

Abstract

The present application discloses a reagent and kit for detecting endometrial cancer, and a method for using the kit. The reagent and the kit diagnose the endometrial cancer or precancerous lesions by specifically detecting the methylation level of a CpG dinucleotide site in a CAPDPS gene CpG island in a DNA sample, and have ideal sensitivity and specificity. Moreover, the reagent and the kit have a good detection effect in tissue samples, cervical exfoliated cell samples and plasma samples, and can achieve non-invasive or minimally invasive detection.

Description

A reagent, kit and method for using the kit for detecting endometrial cancer

This application claims priority to a Chinese patent application with application number 202111367602.7 and titled "Reagents and kits for detection of endometrial cancer" filed at the China Patent Office on November 18, 2021, the entire contents of which are incorporated by reference incorporated in this application.

technical field

The present application relates to the technical field of biomedicine, in particular to a reagent, a kit and a method for using the kit for detecting endometrial cancer.

Background technique

Endometrial cancer (endometrial cancer) is a kind of epithelial tumor on the endometrium derived from the abnormal proliferation of endometrial cells. serious threat.

Endometrial cancer mostly occurs in postmenopausal women. The possible reason is that the secretion level of estrogen is unstable after menopause. In addition, smoking, high blood pressure, overweight and genetic diseases may all lead to the occurrence of cancer. Endometrial cancer is mainly composed of two types, type I: it mostly occurs in women who are in menopause or premenopause, and mostly has a history of exposure to unopposed endogenous or exogenous estrogen, and female estrogen There is a clear association of excesses such as obesity, anovulatory uterine bleeding, infertility, polycystic ovary syndrome, postmenopause, and endometrial hyperplasia, the most common type of endometrial cancer, reaching 80%-85%, mostly endometrioid carcinoma, the degree of tumor differentiation is good, the degree of malignancy is generally low, sensitive to progesterone drugs, the prognosis is good, and the 5-year survival rate is about 74%. Type II: non-estrogen-dependent, mostly occurs in atrophic endometrium, morphologically manifested as poorly differentiated carcinoma, mostly serous papillary carcinoma, clear cell carcinoma and other rare tumors, this type of tumor is a high-grade malignant tumor, Deep myometrial invasion has occurred, and the prognosis is relatively poor, with a 5-year survival rate of 27%-42%. Endometrial cancer sometimes goes through a latent period of 5-8 years from precancerous lesions to cancer. Without treatment, nearly 50% of precancerous lesions will eventually develop into endometrial cancer. In China, the five-year relative survival rate of patients with endometrial cancer is generally around 55%. The survival rate is related to the cancer stage. The five-year survival rate of patients with FIGO stage I-II is between 74%-91%, stage III is between 57%-66%, and stage IV is between 20%-26%. , Therefore, early diagnosis and treatment is the key to improving the survival rate.

technical problem

At present, the diagnosis of endometrial cancer is mainly based on the subject's medical history, clinical examination, pathological examination and various auxiliary examination results. The main detection methods include: B-ultrasound examination, diagnostic curettage, hysteroscopy and lymphography. These methods have disadvantages such as low detection rate and easy to cause severe physiological pain to patients. Therefore, how to provide a minimally invasive/non-invasive endometrial cancer detection reagent suitable for sampling is of great significance.

technical solution

In view of this, the present application provides a reagent, a kit and a method for using the kit for detecting endometrial cancer.

In a first aspect, the present application provides a reagent for detecting endometrial cancer, the reagent comprising: capable of specifically detecting at least one CpG dinucleotide site methyl group in a target nucleotide sequence in a DNA sample A detection reagent at the K level, the target nucleotide sequence is derived from the full length or partial region of the CpG island of the CADPS gene, and the partial region includes at least one CpG dinucleotide site.

Optionally, the nucleotide sequence of the CpG island of the CADPS gene includes one or both of the nucleotide sequences shown in SEQ ID NO.22 and SEQ ID NO.23.

Optionally, the nucleotide sequence of the partial region includes one or more of the nucleotide sequences shown in SEQ ID NO.24 to SEQ ID NO.29.

Optionally, the detection reagents include PCR reagents, and the PCR reagents include one or more of specific primer pairs and specific fluorescent probes capable of detecting the methylation level of the target nucleotide sequence.

Optionally, the primer pair includes any one or more combinations of the following primer pairs:

(a) the combination of SEQ ID NO.1 and SEQ ID NO.2;

(b) the combination of SEQ ID NO.4 and SEQ ID NO.5;

(c) the combination of SEQ ID NO.7 and SEQ ID NO.8;

(d) the combination of SEQ ID NO.10 and SEQ ID NO.11;

(e) the combination of SEQ ID NO.13 and SEQ ID NO.14;

(f) the combination of SEQ ID NO.16 and SEQ ID NO.17.

Optionally, the specific fluorescent probe is selected from SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15 and SEQ ID NO.18 one or more.

Optionally, the reagents also include reaction reagents that can differentially modify methylated sites and unmethylated sites in the DNA sample; after treating the DNA sample with the reaction reagent , the detection reagent can determine the methylation level of cytosine in a specific CpG dinucleotide site in the target nucleotide sequence through a methylation detection method.

Optionally, the reactant includes bisulfite or a derivative of bisulfite.

Optionally, the methylation detection method includes: methylation-specific PCR method, bisulfite sequencing method, genome-wide methylation sequencing method, pyrosequencing method, methylation-specific high performance liquid chromatography One or more of DNA analysis method, digital PCR method, methylation-specific high-resolution melting curve method, and methylation-sensitive restriction endonuclease method.

In the second aspect, the present application also provides a kit for detecting endometrial cancer, the kit including any one of the reagents in the first aspect.

Optionally, the kit further includes one or more of sampling devices, nucleic acid extraction reagents and nucleic acid purification reagents.

In a third aspect, the present application also provides a method for using a kit for detecting endometrial cancer, the method for using includes the step of: using a detection reagent to detect the target nucleotide sequence of a DNA sample derived from a test subject Whether a methylation reaction occurs at the CpG dinucleotide site in the middle; compare the methylation level of the target nucleotide sequence in the DNA sample from the test subject and the healthy person, and judge the target DNA sample from the test subject. Whether the methylation level of the nucleotide sequence is higher than the methylation level of the target nucleotide sequence in a DNA sample from a healthy person;

Wherein, the target nucleotide sequence is derived from the full-length or partial region of the CpG island of the CADPS gene, and the partial region includes at least one CpG dinucleotide site.

Optionally, the nucleotide sequence of the CpG island of the CADPS gene includes one or both of the nucleotide sequences shown in SEQ ID NO.22 and SEQ ID NO.23.

Optionally, the nucleotide sequence of the partial region includes one or more of the nucleotide sequences shown in SEQ ID NO.24 to SEQ ID NO.29.

Optionally, the DNA sample is from an in vitro biological sample of a mammal, and the in vitro biological sample of a mammal is from one or more of blood, endometrial tissue, and endometrial cell samples.

Optionally, the detection reagents include PCR reagents, and the PCR reagents include one or more of specific primer pairs and specific fluorescent probes capable of detecting the methylation level of the target nucleotide sequence.

Optionally, the primer pair includes any one or more combinations of the following primer pairs:

(a) the combination of SEQ ID NO.1 and SEQ ID NO.2;

(b) the combination of SEQ ID NO.4 and SEQ ID NO.5;

(c) the combination of SEQ ID NO.7 and SEQ ID NO.8;

(d) the combination of SEQ ID NO.10 and SEQ ID NO.11;

(e) the combination of SEQ ID NO.13 and SEQ ID NO.14;

(f) the combination of SEQ ID NO.16 and SEQ ID NO.17.

Optionally, the specific fluorescent probe is selected from SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15 and SEQ ID NO.18 one or more.

Optionally, before the step of using a detection reagent to detect whether a methylation reaction occurs at the CpG dinucleotide site in the target nucleotide sequence of the DNA sample derived from the subject, the method further includes the step : processing the DNA sample with a reaction reagent, the reaction reagent is used to differentially modify the methylation site and the non-methylation site in the DNA sample.

Optionally, using the DNA sample treated with the reaction reagent as a template, carry out methylation-specific PCR on the target nucleotide sequence, and determine the DNA sample by a methylation detection method. Whether the cytosine at the CpG dinucleotide site in the target nucleotide sequence is methylated or unmethylated.

Beneficial effect

The application provides a reagent, a kit and a method for using the kit for detecting endometrial cancer, by specifically detecting the methylation level of the CpG dinucleotide site in the CpG island of the CADPS gene in a DNA sample, For the diagnosis of endometrial cancer, it has high sensitivity and specificity for type I or type II endometrial cancer, and has good detection results in tissue samples, cervical exfoliated cell samples and plasma samples , for example: in tissue samples, the sensitivity can reach 100%, and the specificity is over 90%; in cervical exfoliated cell samples, the specificity can reach over 85%, and the sensitivity is 90%-100%, which can be used for non-invasive diagnosis of endometrial cancer. Or minimally invasive detection, it also has a certain detection rate for precancerous lesions of endometrial cancer, which has clinical practical significance.

Embodiments of the present invention

The following will clearly and completely describe the technical solutions in the embodiments of the application with reference to the drawings in the embodiments of the application. Apparently, the described embodiments are only some of the embodiments of the application, not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making creative efforts belong to the scope of protection of this application. It should be noted that the description sequence of the following embodiments is not intended to limit the preferred sequence of the embodiments.

Various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be construed as a rigid limitation on the scope of the application; therefore, the described range should be regarded as The description has specifically disclosed all possible subranges as well as individual values within that range. For example, a description of a range from 1 to 6 should be considered to have specifically disclosed subranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and Single numbers within the stated ranges, eg 1, 2, 3, 4, 5 and 6, apply regardless of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.

In this application, the term "and/or" is used to describe the relationship between associated objects, indicating that there may be three relationships, for example, "A and/or B" may indicate three situations: the first situation is that A exists alone ; The second case is the presence of A and B at the same time; the third case is the case of B alone, wherein A and B can be singular or plural respectively.

In this application, the term "at least one" means one or more, and "multiple" means two or more. The terms "at least one (individual)" and "any of the following circumstances" refer to any combination of these species (individuals), including any combination of a single species (individuals) or a plurality of species (individuals).

The term "DNA methylation level" is the same as the general understanding, referring to whether the cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/ratio/percentage of methylation , representing both qualitative and quantitative concepts. For example, if a cytosine (C) residue in a nucleic acid sequence is methylated, it can be called "hypermethylated" or has "increased methylation". In some cases, different detection indicators are used to compare the DNA methylation level. For example, in some cases, the comparison can be made according to the Ct value detected by the sample. In some cases, the methylation ratio of the marker in the sample can be calculated, that is, the methylation Number of methylated molecules/(number of methylated molecules + number of unmethylated molecules)×100, and then compare them. In some cases, statistical analysis and integration of each indicator is required to obtain the final judgment indicator .

The term "CpG island" refers to a region on DNA, which is rich in a large number of cytosine and guanine linked by phosphate bonds, which are mainly located in the promoter and exon regions of genes, and are rich in CpG dinuclear The region of nucleotides, the length is between 200bp～3000bp, and the total content of G and C exceeds 50%. For this embodiment, the CpG island of CADPS includes the following sequence (5'-3'): SEQ ID NO.22, located at Human chromosome 3, for example, with the GRCh38/hg38 genome as a reference, the sequence is located in the Chr3:62873943-62875515 region (the positions of the sites or regions mentioned in this article are all referenced to GRCh38/hg38) . Correspondingly, the nucleotide sequence of the CpG island of the CADPS gene also includes the reverse complementary nucleotide sequence SEQ ID NO.23 of the above-mentioned SEQ ID NO.22. Therefore, for the embodiment of the present application, the target nucleotide sequence as the detection target of methylation level may include SEQ ID NO.22 and/or SEQ ID NO.23.

In the embodiments of the present application, the "complementary" nucleotide sequence refers to one-to-one complementary bases. For example, the DNA sequence in the human genome includes a sense strand and a corresponding complementary antisense strand. The sense strand and the antisense strand have meanings known in the art. Generally speaking, the antisense strand (negative strand, nonsense strand) is mRNA transcription The template strand combined with time; the non-template strand is used to store the coding information of mRNA, and the non-template strand is the sense strand (positive strand, sense strand). It is understandable that in a DNA double-strand, only a certain strand is a sense strand in a certain part of the region, and part of it is an antisense strand, and it may be completely opposite in another region.

Those skilled in the art can understand that due to individual differences, there may be slight differences in the base sequence of genes in the same region of the same chromosome. Therefore in the embodiment of the present application, the target nucleotide sequence can also be selected from at least 70%, at least 80%, at least 90%, at least 95% of SEQ ID NO.22, and/or SEQ ID NO.23 Or sequences of at least 99% similarity.

"Similarity" between two nucleic acid sequences, the percentage expressing the statistically significant percentage of identical nucleotides between the two sequences to be compared obtained after the best alignment, the two sequences The difference between is randomly distributed over its entire length. Typically, such sequence comparisons can be performed manually or by means of computer programs including, but not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S. , F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Handbook, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine similarity.

In some embodiments, the nucleotide sequence of the CpG island region is at least one of SEQ ID NO.24 to SEQ ID NO.29, or is selected from a Sequences of at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% similarity.

Specifically, SEQ ID NO.24 shows the nucleotide sequence on the sense strand of the Chr3:62874785-62874899 region. SEQ ID NO.25 shows the nucleotide sequence on the sense strand of the Chr3:62875096-62875225 region. SEQ ID NO.26 shows the nucleotide sequence on the sense strand of the Chr3:62875250-62875421 region. SEQ ID NO.27 shows the nucleotide sequence on the negative sense strand of the Chr3:62875225-62875108 region. SEQ ID NO.28 shows the nucleotide sequence on the negative sense strand of the Chr3:62875068-62874902 region. SEQ ID NO.29 shows the nucleotide sequence on the negative sense strand of the Chr3:62874798-62874689 region.

In some embodiments, the detection reagents include: PCR reagents, the PCR reagents include one or more of specific primer pairs and specific fluorescent probes capable of detecting the methylation level of the target nucleotide sequence .

The term "primer" refers to an oligonucleotide, naturally occurring or synthetically produced in a purified restriction digest, when subjected to conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced (e.g., between nucleotides and In the presence of an inducing agent such as DNA polymerase and at the appropriate temperature and pH), it can serve as the starting point for synthesis. Primers are preferably single-stranded for maximum efficiency of amplification, but may also be double-stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primers are oligodeoxyribonucleotides. Primers must be long enough to prime the synthesis of extension products in the presence of an inducing agent. The exact length of primers will depend on many factors including temperature, source of primers, and method used.

The term "probe" refers to an oligonucleotide (e.g., a nucleotide sequence) naturally occurring in a purified restriction digest or produced synthetically, recombinantly, or by PCR amplification that is capable of interacting with another target oligonucleotide. Nucleotide hybridization. Probes can be single-stranded or double-stranded. Probes can be used for the detection, identification and isolation of specific gene sequences.

The inventors found in the course of their research that the methylation level of CpG islands in the CADPS gene is associated with endometrial cancer, and a large number of and long-term experiments have proved that: detecting changes in the methylation level of the CpG island region in the CADPS gene (such as Increased methylation level), can be used for diagnosis or auxiliary diagnosis of endometrial cancer, with high sensitivity and specificity.

In view of this, the present application provides a reagent for detecting endometrial cancer. The reagents include: a detection reagent capable of specifically detecting the methylation level of at least one CpG (cytosine-phosphate-guanine) dinucleotide site in a target nucleotide sequence in a DNA sample, and the target nucleoside The acid sequence is derived from the full length or partial region of the CpG island of the CADPS gene, wherein the partial region includes at least one CpG dinucleotide site.

Optionally, the nucleotide sequence of the CpG island of the CADPS gene includes one or both of the nucleotide sequences shown in SEQ ID NO.22 and SEQ ID NO.23.

Optionally, the nucleotide sequence of the partial region includes one or more of the nucleotide sequences shown in SEQ ID NO.24 to SEQ ID NO.29.

Optionally, the detection reagents include PCR reagents, and the PCR reagents include one or more of specific primer pairs and specific fluorescent probes capable of detecting the methylation level of the target nucleotide sequence.

As an exemplary scheme, the primer pair includes any one of the following primer pairs:

(a) the combination of SEQ ID NO.1 and SEQ ID NO.2;

(b) the combination of SEQ ID NO.4 and SEQ ID NO.5;

(c) the combination of SEQ ID NO.7 and SEQ ID NO.8;

(d) the combination of SEQ ID NO.10 and SEQ ID NO.11;

(e) the combination of SEQ ID NO.13 and SEQ ID NO.14;

(f) the combination of SEQ ID NO.16 and SEQ ID NO.17.

It can be understood that the primer pair can also be selected from a sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the sequence shown in any group of (a) to (f) above Similarity primers.

As an exemplary solution, the probe is selected from at least one of: SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15 and SEQ ID NO.18 , or selected from probes having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% sequence identity to the aforementioned sequences.

In the embodiments of the present application, the probe is a Taqman probe, which is labeled with a fluorescent reporter group and a fluorescent quencher group. In some embodiments, the 5' end of the probe is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a fluorescent quencher group MGB.

In some embodiments, the reagents also include reaction reagents, which can differentially modify methylated sites and unmethylated sites in the DNA sample; after the DNA sample is treated with the reaction reagent, the detection reagent can The detection method determines the methylation level of cytosines at specific CpG dinucleotide sites in the target nucleotide sequence.

As an exemplary embodiment, the reaction reagent includes bisulfite or a derivative thereof.

As an exemplary solution, the methylation detection method includes: methylation-specific PCR method, sequencing method (for example: bisulfite sequencing method, genome-wide methylation sequencing method and pyrosequencing method), methylation Methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve method and methylation-sensitive restriction enzyme method. Correspondingly, the detection reagent is a necessary reagent capable of realizing the above methylation detection method, such as a PCR reagent including the aforementioned primer pair and Taqman probe.

The present application also provides a kit for detecting endometrial cancer, which includes any one of the reagents described above.

In some embodiments, the kit further includes one or more of sampling devices, nucleic acid extraction reagents, and nucleic acid purification reagents.

The present application also provides a chip for the detection and diagnosis of endometrial cancer, the chip can specifically detect at least one CpG (cytosine-phosphate-guanine) dinucleoside in the target nucleotide sequence in the DNA sample A reagent for detecting the methylation level of an acid site, the target nucleotide sequence is derived from the full length or partial region of the CpG island of the CADPS gene.

The present application also provides a method for using the kit described in the above examples. It can be understood that the method is essentially a method for detecting the methylation of the CADPS gene. The method includes the steps of: using a detection reagent to detect Whether the methylation reaction occurs at the CpG dinucleotide site in the target nucleotide sequence of the DNA sample; compare the methylation level of the target nucleotide sequence in the DNA sample from the subject and the healthy person, and judge the source It depends on whether the methylation level of the target nucleotide sequence in the DNA sample of the test subject is higher than the methylation level of the target nucleotide sequence in the DNA sample from a healthy person. Target nucleotide sequences and detection reagents are as described above.

As used in this application, "subject" refers to any individual who is detected, analyzed or treated by the technical scheme of this application, for example, it may be an individual who is first diagnosed with endometrial cancer and/or endometrial precancerous lesions, or It can be an individual who is receiving treatment for endometrial cancer and/or endometrial precancerous lesions, or any individual who wishes to use the method of this application for analysis or treatment, or who is not suffering from endometrial cancer and/or uterine Individuals with precancerous endometrial cancer who are at risk.

In some embodiments, the DNA sample is from an ex vivo biological sample of a mammal, including a human, non-human primate. The ex vivo biological sample of the mammal may be from at least one of blood, endometrial tissue, and endometrial cell samples. Specifically, the blood samples include: whole blood samples, serum samples, plasma samples and blood cell samples; the endometrial cell samples can be exfoliated cell samples derived from the uterus, including: cervical exfoliated cell samples and endometrial exfoliated cells samples cell sample. When the sample comes from exfoliated cell samples derived from the uterus, the reagent can be applied to non-invasive detection, which can reduce the sampling pain of patients and improve the popularity of detection reagents.

In some embodiments, before the step of using the detection reagent to detect whether the methylation reaction occurs at the CpG dinucleotide site in the target nucleotide sequence of the DNA sample derived from the subject, the method of using the kit further includes Step: using a reaction reagent to process the DNA sample, and the reaction reagent is used to differentially modify the methylation site and the non-methylation site in the DNA sample. Reagents refer to the previous description.

In some embodiments, the use of a detection reagent to detect whether a methylation reaction occurs at the CpG dinucleotide site in the target nucleotide sequence of the DNA sample includes the steps of: using the DNA sample treated with the reaction reagent as a template, Perform methylation-specific PCR on the target nucleotide sequence to determine whether the cytosine at the CpG dinucleotide site in the target nucleotide sequence of the DNA sample is methylated or unmethylated by a methylation detection method of.

As an exemplary solution, the method for using the kit includes the following steps:

(1) DNA samples are extracted.

In (1), the DNA sample may be a cervical exfoliated cell sample, an endometrial tissue sample or a blood sample.

(2) adding a reaction reagent, and performing bisulfite conversion treatment on the DNA sample.

In (2), the reaction reagent is used to differentially modify methylated sites and unmethylated sites in the DNA sample. Specifically, it can be selected from bisulfite or its derivatives.

(3) adding detection reagents, performing PCR amplification reaction, and detecting whether methylation reaction occurs at the CpG dinucleotide site in the target nucleotide sequence in the DNA sample. The target nucleotide sequence is derived from the full length or partial region of the CpG island of the CADPS gene. Specifically, the detection reagent can determine whether the cytosine in the specific CpG of the target nucleotide is methylated or unmethylated by a methylation detection method, or further calculate or evaluate the target nucleotide sequence The proportion of methylated CpG dinucleotide sites in .

The present application also provides an application, including using the detection reagent for the detection, diagnosis or auxiliary diagnosis of type I endometrial cancer or type II endometrial cancer; or, using the detection reagent for type I endometrial cancer Detection of precancerous lesions of endometrial cancer or type II endometrial cancer. Specifically, the reagent can be applied to detect at least one of papillary serous adenocarcinoma, clear cell carcinoma, squamous cell carcinoma and endometrioid carcinoma.

In some embodiments, the detection reagent can also be used for the detection of stage I, stage II, stage III and stage IV endometrial cancer.

It should be noted that the stage I, stage II, stage III and stage IV endometrial cancer referred to in this application can refer to the FIGO staging system defined by the International Federation of Obstetrics and Gynecology. For the embodiment of the present application, even if the patient is in stage I, stage II, stage III and stage IV or earlier lesions, the detection kit of the present application can detect it, which is of great significance for improving the survival rate of patients.

The technical solutions in the embodiments of the present application are clearly and completely described below. Apparently, the described embodiments are only some of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making creative efforts belong to the scope of protection of this application. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as are familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the content described can be applied to this application. The preferred implementation methods and materials described in this article are only for demonstration purposes, but cannot limit the content of this application.

Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by methods known in the art.

Example

The embodiment is used to provide the reagents for endometrial cancer detection, a total of 6 groups of reagents, respectively embodiment 1 to embodiment 6, the reagents of each embodiment include a pair of methylation-specific primers of the target gene and a specific taqman Probes, wherein the sequence information of the target gene, primer pairs and probes are shown in Table 1.

Table 1. Methylated primer pairs and probes of six regions in the CpG island of CADPS gene

The reagents provided in Examples 1 to 6 can be used to diagnose endometrial cancer through the following methods, and the specific steps include:

(1) Sample DNA extraction

If the sample is a formalin-fixed, paraffin-embedded tissue sample (FFPE sample): use the QIAamp DNA FFPE Tissue Kit to extract tissue DNA. For details, refer to the kit instruction manual.

If the sample is cervical exfoliated cell sample: use Tiangen Biochemical Technology (Beijing) Co., Ltd. Blood/Cell/Tissue Genomic DNA Extraction Kit (DP304) to extract cervical exfoliated cell DNA. For specific operations, refer to the kit instruction manual.

If the sample is a plasma sample: use the plasma cfDNA extraction reagent (Nucleic Acid Extraction Reagent Ehan Jibei No. 20210740) of Wuhan Amison Life Science and Technology Co., Ltd., and refer to the kit instruction manual for specific operations.

(2) Bisulfite conversion

The nucleic acid conversion kit used is the EZ DNA Methylation-Gold TM Kit of ZYMO RESEARCH. For specific experimental operations, please refer to the kit manual.

(3) Methylation-specific PCR

Using the reagents provided in Examples 1 to 6 of the present application, methylation detection was performed on 6 regions in the CpG island. Specifically, the DNA converted by bisulfite is subjected to methylation-specific PCR reaction to detect the methylation status of CADPS gene region 1 to region 6, and each region is detected separately, that is, each time in a PCR tube Only the detection primer and probe of one embodiment are added, and the detection probe of the internal reference gene is added at the same time. ACTB is used as an internal reference gene, wherein the upstream primer of ACTB is: AAGGTGGTTGGGTGGTTGTTTTG (SEQ ID NO.19); the downstream primer of ACTB is: AATAACACCCCCACCCCTGC (SEQ ID NO.20); the probe of ACTB is: GGAGTGGTTTTTGGGTTTG (SEQ ID NO.21). The luminescent group of the region 1-6 probe is FAM, the quenching group is MGB, the luminescent group of ACTB is VIC, and the quenching group is BHQ1.

Adopt Platinum II Taq hot-start DNA polymerase to carry out PCR amplification, PCR reaction solution configuration system is shown in Table 2:

Table 2

components Specification Volume (μL) PCR buffer 5× 4 dNTPs 2.5mM each 2 Regional upstream primers 10μM 0.4 Region Downstream Primers 10μM 0.4 area probe 10μM 0.4 ACTB upstream primer 10μM 0.4 ACTB downstream primer 10μM 0.4 ACTB probe 10μM 0.4 Taq enzyme / 0.3 Sample DNA to be tested / 5

purified water / make up to 25

As shown in Table 3, when detecting the methylation status of any region of the CADPS gene region 1 to region 6 in the sample, only the primer probe corresponding to a certain region, ACTB primer probe, buffer, dNTP, DNase and sample DNA were added to the reaction system according to the volume in Table 2. Three replicate wells were set up for each sample.

The PCR reaction conditions are shown in Table 3 below.

table 3

(4) Quality control

Negative and positive controls were tested simultaneously at each assay.

Negative control was purified water.

The positive control is Hec-1A cell line genome, the concentration is 10 ³ copies/μL.

The negative control should have no amplification, the positive control should have a significant exponential growth period, and the Ct value of the internal reference gene of the sample to be tested should be ≤ 35. After the negative control, positive control and internal reference genes all meet the above requirements, it indicates that the experiment is valid. The judgment of the next sample result can be carried out. Otherwise, the test is invalid and must be tested again.

(5) PCR data analysis

Ct value reading: After the PCR is completed, adjust the baseline of the target region and the internal reference gene respectively, set the fluorescence value before the minimum Ct value of the sample in one PCR 1-2 cycles earlier as the baseline value, and set the threshold value at S-type amplification At the inflection point of the curve, the Ct value of the target gene to be detected and the Ct value of the internal reference gene ACTB of each sample were obtained.

Result analysis and interpretation method: If the Ct value of the region to be detected in a well is ≤38, it is considered that the region is detected to be methylated in the well, and when methylation is detected in at least two wells in the three multiple wells When it is methylated, it is determined that the region is methylation-positive in the sample, otherwise it is methylation-negative. Calculate the sensitivity of regions 1 to 6 in precancerous endometrial lesion samples and endometrial cancer samples, and detect the specificity of each region in paracancerous samples or normal (ie healthy people) samples.

Sensitivity% = number of positive methylation / (total number of endometrial precancerous lesions or endometrial cancer samples) × 100%

Specificity % = number of negative methylation/(total number of paracancerous samples or normal samples) × 100%

Experimental example 1

This experimental example is used to detect clinical tissue samples using the detection reagents and methods provided in the previous embodiments.

20 samples of endometrial cancer and 20 samples of paracancerous tissues were collected from a hospital in Wuhan. All samples were FFPE samples prepared by professional medical personnel. Of the 20 samples of endometrial cancer, 13 were intrauterine Membranous carcinoma, 2 cases were papillary serous adenocarcinoma, 3 cases were clear cell carcinoma, 2 cases were squamous cell carcinoma. For 40 samples, the method described in the examples was used to extract DNA from tissue samples, and the extracted DNA was subjected to bisulfite conversion, and the converted DNA was used as a template, and the reagents provided in Examples 1 to 6 were used to Regions 1 to 6 were detected by methylation-specific PCR, and the methylation status of each sample in Examples 1 to 6 was counted, and the sensitivity and specificity were calculated. The results are shown in Table 4.

Table 4 Methylation detection results in clinical tissue samples

It can be seen from the results in Table 4 that among the 20 endometrial cancer samples, the sensitivity of the methylation detection in Examples 1 to 6 was not lower than 95%, among which Examples 1, 3 and The sensitivity of Example 6 was 100%. In 20 cases of endometrial cancer samples, endometrioid carcinoma belongs to type I endometrial cancer, papillary serous adenocarcinoma, clear cell carcinoma and squamous cell carcinoma belong to type II endometrial cancer, the above results show that the embodiment Examples 1 to 6 have good detection results for both type I and type II endometrial cancer; in 20 cases of paracancerous samples, at most 2 of examples 1 to 6 were detected to be positive for methylation, and the specific None less than 90%.

Experimental example 2

This experimental example is mainly for the detection of cervical exfoliated cell samples.

10 samples of endometrial cancer, 15 samples of precancerous endometrial lesions and 20 samples of normal endometrium were taken from a hospital in Wuhan. All samples were cervical exfoliated cell samples. The samples were collected by professional medical personnel. The sample information As shown in Table 5. For 45 samples, the method described in the examples was used to extract the DNA of the cell samples, and the extracted DNA was subjected to bisulfite conversion, and the converted DNA was used as a template, and the reagents provided in Examples 1 to 6 were used to Regions 1 to 6 were detected by methylation-specific PCR, and the methylation status of each sample in Examples 1 to 6 was counted, and the sensitivity and specificity were calculated. The results are shown in Table 5.

Table 5 Clinical sample information and methylation detection results of exfoliated cells

As can be seen from the results in Table 5, in the 20 cases of cervical exfoliated cell samples, the detection sensitivity of methylation in Examples 1 to 6 is not less than 90%, which is consistent with the results of Experimental Example 1. Example 1 1～Example 6 all have good detection effect to I type and II type endometrial cancer, in addition, the detection sensitivity of embodiment 1～Example 6 is 73.3% or 80% for the precancerous lesion, shows that embodiment 1 ~ The reagents provided in Example 6 also have a good detection effect on endometrial precancerous lesions. Among the 20 normal samples, 3 cases were positive for methylation in Example 1, and the specificity was 85%. The number of positive cases for methylation in Examples 2 to 5 was not more than 2, and the specificity was not low. at 90%.

Experimental example 3

This experimental example is mainly for the detection of plasma samples.

30 endometrial cancer plasma samples and 45 healthy human plasma samples were taken from a hospital in Wuhan. The samples were collected by professional medical personnel. The sample information is shown in Table 6. For 75 samples, the method described in the examples was used to extract DNA from cell samples, and the extracted DNA was converted to bisulfite, and the converted DNA was used as a template, and the reagents provided in Examples 1 to 6 were used to Regions 1 to 6 were detected by methylation-specific PCR, and the methylation status of each sample in Examples 1 to 6 was counted, and the sensitivity and specificity were calculated. The results are shown in Table 6.

Table 6. Detection results in plasma samples

From the results in Table 6, it can be seen that in plasma samples, the overall sensitivity of Examples 1 to 6 to I-IV stage endometrial cancer samples is between 60% and 70%, among which, Example 3, Example 6 The sensitivity of Example 4 and Example 6 is the highest, which can reach 70%. The six groups of examples can all detect endometrial cancers of stage I, stage II, stage III and stage IV. Among the 45 healthy human plasma samples, the detection specificities of the six groups of embodiments are all above 90%.

In summary, according to the above experimental results, it can be concluded that for cervical exfoliated cell samples, tissue samples and plasma samples, the detection reagents provided by this application have high sensitivity and specificity for the detection of endometrial cancer. When the reagent is applied to a plasma sample, the sampling process can be simplified and the trauma of sampling can be reduced. When the reagent is applied to a cervical exfoliated cell sample, the reagent can be sampled through a vaginal swab, thereby reducing pain for patients and improving the popularity of the detection reagent. On the other hand, for various types of endometrial cancer, such as type I endometrioid carcinoma, type II papillary serous adenocarcinoma, clear cell carcinoma and squamous cell carcinoma, the detection reagents provided by the application all have Better detection effect. Moreover, the detection reagents provided by the present application can detect all stages of endometrial cancer, including stage I, stage II, stage III and stage IV endometrial cancer. In addition, it is also worth noting that the reagents provided by this application can still detect precancerous lesions, so it is of great significance for early screening, early treatment intervention and improvement of patient prognosis.

In the foregoing embodiments, the descriptions of each embodiment have their own emphases, and for parts not described in detail in a certain embodiment, reference may be made to relevant descriptions of other embodiments.

A reagent, a kit and a method for using the kit for detecting endometrial cancer provided in the embodiments of the present application have been described in detail above. In this paper, specific examples are used to illustrate the principles and implementation methods of the present application. , the description of the above embodiments is only used to help understand the method of the present application and its core idea; at the same time, for those skilled in the art, according to the idea of the present application, there will be changes in the specific implementation and application scope, To sum up, the contents of this specification should not be understood as limiting the application.

Claims (20)

A reagent for detecting endometrial cancer, wherein the reagent includes: a detection reagent capable of specifically detecting the methylation level of at least one CpG dinucleotide site in a target nucleotide sequence in a DNA sample, The target nucleotide sequence is derived from the full length or partial region of the CpG island of the CADPS gene, and the partial region includes at least one CpG dinucleotide site. The reagent according to claim 1, wherein the nucleotide sequence of the CADPS gene CpG island comprises one or both of the nucleotide sequences shown in SEQ ID NO.22 and SEQ ID NO.23. The reagent according to claim 1, wherein the nucleotide sequence of the partial region comprises one or more of the nucleotide sequences shown in SEQ ID NO.24 to SEQ ID NO.29. The reagent according to claim 1, wherein the detection reagent comprises a PCR reagent, and the PCR reagent comprises a pair of specific primers capable of detecting the methylation level of the target nucleotide sequence and a specific fluorescent probe. one or more of . The reagent according to claim 4, wherein the primer pair comprises any one or more combinations of the following primer pairs: (a) the combination of SEQ ID NO.1 and SEQ ID NO.2; (b) the combination of SEQ ID NO.4 and SEQ ID NO.5; (c) the combination of SEQ ID NO.7 and SEQ ID NO.8; (d) the combination of SEQ ID NO.10 and SEQ ID NO.11; (e) the combination of SEQ ID NO.13 and SEQ ID NO.14; (f) the combination of SEQ ID NO.16 and SEQ ID NO.17. The reagent according to claim 4 or 5, wherein the specific fluorescent probe is selected from SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO. 15 and one or more of SEQ ID NO.18. The reagent according to claim 1, wherein the reagent also includes a reaction reagent that can differentially modify the methylation site and the non-methylation site in the DNA sample; After the reagent treats the DNA sample, the detection reagent can determine the methylation level of cytosine in a specific CpG dinucleotide site in the target nucleotide sequence by a methylation detection method. The reagent according to claim 7, wherein the reaction reagent comprises bisulfite or a derivative of bisulfite. The reagent according to claim 7, wherein the methylation detection method comprises: methylation-specific PCR method, bisulfite sequencing method, genome-wide methylation sequencing method, pyrosequencing method, methylation One or more of methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve method, and methylation-sensitive restriction endonuclease method. A kit for detecting endometrial cancer, wherein the kit includes the reagent according to any one of claims 1-9. The kit according to claim 10, wherein the kit further comprises one or more of a sampling device, a nucleic acid extraction reagent and a nucleic acid purification reagent. A method for using a kit for detecting endometrial cancer, wherein the method includes the step of: using a detection reagent to detect the CpG dinucleotide position in the target nucleotide sequence of a DNA sample derived from a test subject Whether a methylation reaction occurs at the point; compare the methylation level of the target nucleotide sequence in the DNA sample from the subject and the healthy person, and judge the methylation of the target nucleotide sequence in the DNA sample from the subject Whether the level is higher than the methylation level of the target nucleotide sequence in a DNA sample from a healthy person; Wherein, the target nucleotide sequence is derived from the full-length or partial region of the CpG island of the CADPS gene, and the partial region includes at least one CpG dinucleotide site. The method according to claim 12, wherein the nucleotide sequence of the CADPS gene CpG island comprises one or both of the nucleotide sequences shown in SEQ ID NO.22 and SEQ ID NO.23. The method according to claim 12, wherein the nucleotide sequence of the partial region comprises one or more of the nucleotide sequences shown in SEQ ID NO.24 to SEQ ID NO.29. The method according to claim 12, wherein the DNA sample is from a mammalian biological sample in vitro, and the mammalian biological sample in vitro is from blood, endometrial tissue, and endometrial cell samples one or more of. The use method according to claim 12, wherein the detection reagent comprises a PCR reagent, and the PCR reagent comprises a pair of specific primers capable of detecting the methylation level of the target nucleotide sequence and a specific fluorescent probe one or more of. The method according to claim 16, wherein the primer pair comprises any one or more combinations of the following primer pairs: (a) the combination of SEQ ID NO.1 and SEQ ID NO.2; (b) the combination of SEQ ID NO.4 and SEQ ID NO.5; (c) the combination of SEQ ID NO.7 and SEQ ID NO.8; (d) the combination of SEQ ID NO.10 and SEQ ID NO.11; (e) the combination of SEQ ID NO.13 and SEQ ID NO.14; (f) the combination of SEQ ID NO.16 and SEQ ID NO.17. The use method according to claim 16 or 17, wherein the specific fluorescent probe is selected from SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO .15 and one or more of SEQ ID NO.18. The use method according to claim 12, wherein, before the step of using a detection reagent to detect whether a methylation reaction occurs at the CpG dinucleotide site in the target nucleotide sequence of the DNA sample derived from the subject , the use method further includes the step of: using a reaction reagent to process the DNA sample, and the reaction reagent is used to differentially modify the methylation site and the non-methylation site in the DNA sample. The use method according to claim 19, wherein said detection of whether a methylation reaction occurs at the CpG dinucleotide site in the target nucleotide sequence of the DNA sample using a detection reagent comprises the step of: The processed DNA sample is used as a template, and methylation-specific PCR is performed on the target nucleotide sequence, and the CpG dinucleoside in the target nucleotide sequence of the DNA sample is determined by a methylation detection method Whether the cytosine at the acid site is methylated or unmethylated.