WO2023088335A1 - Réactif et kit pour la détection du cancer endométrial, et procédé pour l'utilisation du kit - Google Patents

Réactif et kit pour la détection du cancer endométrial, et procédé pour l'utilisation du kit Download PDF

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WO2023088335A1
WO2023088335A1 PCT/CN2022/132415 CN2022132415W WO2023088335A1 WO 2023088335 A1 WO2023088335 A1 WO 2023088335A1 CN 2022132415 W CN2022132415 W CN 2022132415W WO 2023088335 A1 WO2023088335 A1 WO 2023088335A1
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seq
reagent
methylation
nucleotide sequence
dna sample
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PCT/CN2022/132415
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Chinese (zh)
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苏雅婷
郭洪
王方媛
郑义慧
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武汉艾米森生命科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • 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.
  • 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.
  • 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.
  • the present application provides a reagent, a kit and a method for using the kit for detecting endometrial cancer.
  • 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.
  • 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.
  • 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.
  • 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 primer pair includes any one or more combinations of the following primer pairs:
  • 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.
  • 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.
  • the reactant includes bisulfite or a derivative of bisulfite.
  • 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.
  • the present application also provides a kit for detecting endometrial cancer, the kit including any one of the reagents in the first aspect.
  • 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 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;
  • 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 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.
  • 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.
  • the DNA sample is from an in vitro biological sample of a mammal
  • the in vitro biological sample of a mammal is from one or more of blood, endometrial tissue, and endometrial cell samples.
  • 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 primer pair includes any one or more combinations of the following primer pairs:
  • 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.
  • 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.
  • 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.
  • 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,
  • endometrial cancer 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.
  • 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.
  • 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).
  • 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”.
  • 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.
  • 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 .
  • 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%.
  • 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) .
  • 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.
  • the "complementary" nucleotide sequence refers to one-to-one complementary bases.
  • 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.
  • the antisense strand (negative strand, nonsense strand) is mRNA transcription
  • the non-template strand is used to store the coding information of mRNA
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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 primer pair includes any one of the following primer pairs:
  • 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.
  • 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.
  • the probe is a Taqman probe, which is labeled with a fluorescent reporter group and a fluorescent quencher group.
  • 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.
  • 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.
  • the reaction reagent includes bisulfite or a derivative thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the reagent can be applied to non-invasive detection, which can reduce the sampling pain of patients and improve the popularity of detection reagents.
  • the method of using the kit 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.
  • 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.
  • the method for using the kit includes the following steps:
  • the DNA sample may be a cervical exfoliated cell sample, an endometrial tissue sample or a blood sample.
  • 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.
  • the target nucleotide sequence is derived from the full length or partial region of the CpG island of the CADPS gene.
  • 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.
  • the reagent can be applied to detect at least one of papillary serous adenocarcinoma, clear cell carcinoma, squamous cell carcinoma and endometrioid carcinoma.
  • the detection reagent can also be used for the detection of stage I, stage II, stage III and stage IV endometrial cancer.
  • 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.
  • the detection kit of the present application can detect it, which is of great significance for improving the survival rate of patients.
  • 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.
  • the reagents provided in Examples 1 to 6 can be used to diagnose endometrial cancer through the following methods, and the specific steps include:
  • FFPE sample 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.
  • 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.
  • 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.
  • plasma cfDNA extraction reagent Nucleic Acid Extraction Reagent Ehan Jibei No. 20210740
  • the nucleic acid conversion kit used is the EZ DNA Methylation-Gold TM Kit of ZYMO RESEARCH.
  • kit manual For specific experimental operations, please refer to the kit manual.
  • 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.
  • Negative control was purified water.
  • the positive control is Hec-1A cell line genome, the concentration is 10 3 copies/ ⁇ L.
  • 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.
  • 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%
  • This experimental example is used to detect clinical tissue samples using the detection reagents and methods provided in the previous embodiments.
  • 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.
  • Table 4 The results are shown in Table 4.
  • This experimental example is mainly for the detection of cervical exfoliated cell samples.
  • 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.
  • 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%.
  • This experimental example is mainly for the detection of plasma samples.
  • Example 6 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%.
  • the detection reagents provided by this application have high sensitivity and specificity for the detection of endometrial cancer.
  • the sampling process can be simplified and the trauma of sampling can be reduced.
  • the reagent can be sampled through a vaginal swab, thereby reducing pain for patients and improving the popularity of the detection reagent.
  • 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.

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Abstract

La présente invention concerne un réactif et un kit de détection du cancer endométrial, ainsi qu'un procédé d'utilisation du kit. Le réactif et le kit permettent de diagnostiquer le cancer endométrial ou les lésions précancéreuses en détectant particulièrement le niveau de méthylation d'un site de dinucléotide CpG dans un îlot CpG du gène CAPDPS dans un échantillon d'ADN, et possèdent une sensibilité et une spécificité idéales. En outre, le réactif et le kit possèdent un bonne capacité de détection dans les échantillons tissulaires, les échantillons de cellules exfoliées du col de l'utérus et les échantillons plasmatiques, et peuvent permettre une détection non invasive ou minimalement invasive.
PCT/CN2022/132415 2021-11-18 2022-11-17 Réactif et kit pour la détection du cancer endométrial, et procédé pour l'utilisation du kit WO2023088335A1 (fr)

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CN113913522B (zh) * 2021-11-18 2023-03-10 武汉艾米森生命科技有限公司 子宫内膜癌检测的试剂及试剂盒
CN116356029B (zh) * 2023-03-29 2023-12-12 北京优迅医疗器械有限公司 Txnrd1基因甲基化水平检测在子宫内膜癌诊断和/或筛查中的应用

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WO2014036314A2 (fr) * 2012-08-31 2014-03-06 Ignyta, Inc. Diagnostic d'arthrite rhumatoïde (ra) à l'aide de loci méthylés de façon différentielle identifiés dans des cellules mononucléées de sang périphériques, des lymphocytes t, des lymphocytes b et des monocytes
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CA2963831A1 (fr) * 2014-10-17 2016-04-21 The Hospital For Sick Children Marqueurs de methylation de l'adn pour les syndromes de croissance excessive
CN113913522A (zh) * 2021-11-18 2022-01-11 武汉艾米森生命科技有限公司 子宫内膜癌检测的试剂及试剂盒

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