Kit for diagnosing / screening endometrial cancer and application thereof

Abstract

The invention relates to the technical field of biomarkers, in particular to a kit for diagnosing / screening endometrial cancer. The kit provided by the invention is applied to early diagnosis / screening of endometrial cancer, can specifically detect methylation of the LINC01551 gene and the C5orf38 gene in an endometrial cancer cervical exfoliated cell sample, has relatively high detection sensitivity and specificity, and provides a new way for early diagnosis / screening of clinical endometrial cancer.

Description

Technical Field

[0001] The present application relates to the field of biomarker technology, and in particular to a kit for diagnosing / screening endometrial cancer and its application. Background Art

[0002] Endometrial cancer is one of the three most common malignant tumors of the female reproductive system, accounting for 20%-30% of malignant tumors of the female reproductive system, and is more common in postmenopausal women. In recent years, the incidence of endometrial cancer has shown a clear upward trend worldwide, and is gradually becoming younger.

[0003] At present, there is no effective means to screen endometrial cancer in the early asymptomatic stage. Clinical diagnosis of endometrial cancer is only performed through imaging examinations, endometrial biopsy or laboratory diagnosis when clinical symptoms such as vaginal bleeding, vaginal discharge and lower abdominal pain are shown: imaging examinations are mainly used for preliminary judgment; endometrial biopsy is prone to false positives and is an invasive examination. The uterus will be pulled during the sampling process, and there is a certain risk of infection; commonly used clinical laboratory test markers such as CA125 and HE4 have low detection sensitivity, making it difficult to effectively screen patients. Summary of the invention

[0004] In order to solve the above problems, the present application provides a kit for diagnosing / screening endometrial cancer and its application. The kit described in the present application has high detection sensitivity and good specificity, and can achieve non-invasive early screening and diagnosis of endometrial cancer.

[0005] The present application solves the technical problem by adopting the following technical solutions:

[0006] In a first aspect, the present application provides a kit for diagnosing / screening endometrial cancer, the kit comprising a nucleic acid sequence combination for detecting / measuring marker gene methylation. The above-mentioned marker gene is LINC01551 gene and / or C5orf38 gene. In an optional embodiment, the method for detecting / measuring the methylation of the marker gene includes: methylation-specific PCR method, bisulfite sequencing method, whole genome methylation sequencing method, pyrophosphate sequencing method, methylation-specific high-performance liquid chromatography method, digital PCR method, methylation-specific high-resolution melting curve method and methylation-sensitive restriction endonuclease method.

[0007] In an optional embodiment, taking hg38 as the reference genome, the target region of the LINC01551 gene is selected from the full length or partial region with a physical position of negative chain Chr14: 28785124-28785522; the target region of the C5orf38 gene is selected from the full length or partial region with a physical position of positive chain Chr5: 2755751-2756149.

[0008] In an optional embodiment, the nucleic acid sequence combination includes a detection primer pair and a detection probe. Specifically, the detection primer pair includes: a first primer pair for detecting methylation of the LINC01551 gene, the base sequence of the first primer pair is shown in SEQ ID NO.7-8, and / or, a second primer pair for detecting methylation of the C5orf38 gene, the base sequence of the second primer pair is shown in SEQ ID NO.11-12. The detection probe includes: a first probe for detecting methylation of the LINC01551 gene, the base sequence of the first probe is shown in SEQ ID NO.18; and / or, a second probe for detecting methylation of the C5orf38 gene, the base sequence of the second probe is shown in SEQ ID NO.19. The first primer pair and the first probe are combined to obtain a first nucleic acid sequence combination, and the second primer pair and the second probe are combined to obtain a second nucleic acid sequence combination.

[0009] In an optional embodiment, the first nucleic acid sequence combination and / or the second nucleic acid sequence combination further comprises a third primer pair for detecting the internal reference gene ACTB and a third probe for detecting the internal reference gene ACTB, the base sequence of the third primer pair is shown in SEQ ID NO.15-16, and the base sequence of the third probe is shown in SEQ ID NO.17. In an optional embodiment, the 5' end of the detection probe comprises a fluorescent reporter group, which is any one of FAM, VIC, HEX, NED, ROX, TET, JOE, CY3, and CY5; the 3' end comprises a fluorescent quencher group, which is any one of TAMRA, MGB, BHQ, BHQ1, BHQ2, and BHQ3.

[0010] In an optional embodiment, the above-mentioned kit further includes at least one of a PCR reaction buffer, a DNA polymerase, a DNA extraction reagent or a DNA methylation conversion reagent.

[0011] In an optional embodiment, the method for using the above kit comprises the following steps:

[0012] (1) isolating the nucleic acid of the target gene in the human cervical exfoliated cell sample to be tested;

[0013] (2) treating the nucleic acid obtained in step (1) with bisulfite to obtain bisulfite-converted DNA to obtain a test group; using a mixed plasmid containing an ACTB sequence and a fully methylated target gene sequence as a positive control group; and using TE buffer as a negative control group;

[0014] (3) using the above-mentioned nucleic acid sequence combination combined with qPCR technology to detect the methylation level of each group obtained in step (2);

[0015] (4) The results were interpreted when all of the following requirements were met: ① there was no amplification in the negative control group; ② there was an obvious exponential growth period in the positive control group, and the Ct value of the target gene in the positive control group was between 26 and 30; ③ the Ct value of the internal reference gene ACTB in the positive control group was ≤34;

[0016] (5) Combined detection of the target regions of the detection group: if the Ct value is ≤38, it is judged as positive; if the Ct value is >38, it is judged as negative; if any target region is methylation positive, the sample is judged as a positive sample for endometrial cancer; if both target regions are methylation negative, the sample is judged as a negative sample for endometrial cancer.

[0017] In a second aspect, the present application provides the use of the above-mentioned kit in diagnosing / screening endometrial cancer.

[0018] Compared with the prior art, the beneficial effects of this application are:

[0019] The kit provided in the present application contains a methylation marker, which can specifically detect the methylation of the LINC01551 gene and the C5orf38 gene in cell samples of endometrial cancer patients and distinguish them from cell samples of healthy people, and the detection has high sensitivity and specificity.

[0020] The nucleic acid sequence combination designed for the methylation marker gene sequence in the present application is used to prepare a kit for diagnosing / screening endometrial cancer, which can detect the methylation levels of the endometrial cancer-related LINC01551 gene and C5orf38 gene with high sensitivity and specificity. It is applied to the screening / early diagnosis of endometrial cancer, has the advantages of simple operation, non-invasive sampling, and is conducive to popularization and application. DETAILED DESCRIPTION

[0021] The technical scheme of the present application will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the protection scope of the present application. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0023] The term "methylation" is a form of chemical modification of DNA that can change genetic expression without changing the DNA sequence. DNA methylation refers to the covalent binding of a methyl group to the 5th carbon position of cytosine in genomic CpG dinucleotides under the action of DNA methyltransferase. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby regulating gene expression.

[0024] The term "methylation level" refers to whether cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency / ratio / percentage of methylation, which represents both qualitative and quantitative concepts. In practical applications, different detection indicators can be used to compare DNA methylation levels according to actual conditions. For example, in some cases, the comparison can be made based on the Ct value of the sample test; in some cases, the proportion of gene methylation in the sample can be calculated, that is, the number of methylated molecules / (the number of methylated molecules + the number of unmethylated molecules) × 100, and then compared; in some cases, it is also necessary to statistically analyze and integrate the various indicators to obtain the final judgment indicator.

[0025] The term "diagnosis" refers to determining the health status of a subject, including the presence or absence of a disease, response to treatment, recurrence risk assessment, assessment of the risk and extent of canceration, and prognosis. In some cases, the term "diagnosis" refers to a single factor used to determine, verify, or confirm the clinical status of a patient. In some embodiments, "detecting" endometrial cancer refers to detecting the presence or absence of the disease, that is, determining whether a subject has endometrial cancer.

[0026] The term "patient" or "subject" refers to an object that is observed, tested, or experimented on. In some embodiments, the subject can be a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates) and rodents (e.g., mice and rats). In some embodiments, the mammal can be a human.

[0027] The term "sample" or "sample" refers to a biological material obtained from a subject and comprises genomic DNA from all chromosomes, preferably genomic DNA covering the entire genome. If the subject suffers from cancer, the sample comprises cancer cells or free genomic DNA (including target DNA) from cancer cells, preferably circulating genomic DNA from cancer cells. The sample can be from any suitable tissue or biological fluid, such as blood, saliva, plasma, serum, urine, cerebrospinal fluid (CSF), feces, buccal or buccal-pharyngeal swabs, surgical specimens, samples obtained from biopsies, or tissue samples embedded in paraffin. Methods for obtaining samples from subjects are well known to those skilled in the art. Preferably, the sample is a tumor biopsy or a liquid sample, and the liquid sample is preferably blood, serum, plasma. In the case where the cancer is endometrial cancer, it is also considered that the sample comprises exfoliated cells from the cervix. Generally speaking, in samples comprising target DNA from endometrial cancer cells, especially extracellular target DNA, there is also a target DNA from non-cancerous cells that is not highly methylated in contrast to the target DNA from cancer cells.

[0028] Example 1

[0029] A kit for diagnosing / screening endometrial cancer

[0030] This example provides a kit for diagnosing endometrial cancer, which includes: a nucleic acid sequence combination for detecting methylation of cervical cell marker genes, a PCR reaction buffer, a DNA polymerase, a DNA extraction reagent, and a DNA methylation conversion reagent.

[0031] The nucleotide sequence of the marker LINC01551 gene in this example is shown in SEQ ID NO.1 above, and the nucleotide sequence of the marker C5orf38 gene is shown in SEQ ID NO.2.

[0032] The nucleic acid sequence combination for detecting cervical cell gene methylation in this example specifically includes:

[0033] The first primer pair for detecting methylation of the LINC01551 gene has a base sequence as shown in SEQ ID NOs.7-8; the second primer pair for detecting methylation of the C5orf38 gene has a base sequence as shown in SEQ ID NOs.11-12; the first probe for detecting methylation of the LINC01551 gene has a base sequence as shown in SEQ ID NO.18; the second probe for detecting methylation of the C5orf38 gene has a base sequence as shown in SEQ ID NO.19; the third primer pair for detecting the internal reference gene ACTB has a base sequence as shown in SEQ ID NOs.15-16; the third probe for detecting the internal reference gene ACTB has a base sequence as shown in SEQ ID NO.17.

[0034] The probe is a single-stranded or double-stranded DNA with a length of tens to thousands of base pairs. It can utilize the molecular denaturation, renaturation and high accuracy of base complementary pairing to hybridize with complementary non-labeled single-stranded DNA or RNA in the sample to be tested by hydrogen bonding to form a double-stranded hybrid. After washing away the unpaired probe, the hybridization reaction results can be detected by using a detection system such as radioautography or enzyme-linked reaction. In this application, the region complementary to or hybridized with the probe is the specific target region.

[0035] The kit provided in this example is used to diagnose the methylation of LINC01551 gene and C5orf38 gene in cervical tissue samples of endometrial cancer, and can be applied to the early screening and diagnosis of endometrial cancer with high detection sensitivity and specificity.

[0036] Example 2

[0037] How to use the kit

[0038] This example provides a method for using the kit prepared in Example 1, and the specific steps are as follows:

[0039] Step (1) isolating the nucleic acid of the target gene in the biological sample of human cervical exfoliated cells to be tested;

[0040] Step (2) treating the nucleic acid obtained in step (1) with bisulfite to obtain a detection group; 3 copies / μl of ACTB amplicon sequence, 10 3 copies / μl of fully methylated LINC01551 amplicon sequence and 10 3 The mixed plasmid containing 100 copies / μl of the fully methylated C5orf38 amplicon sequence was used as the positive control group; the TE buffer was used as the negative control group;

[0041] Step (3) using the nucleic acid sequence combination described in Example 1, combined with qPCR technology to detect the methylation level of each group obtained in step (2);

[0042] Step (4) When all the following requirements are met, the results are interpreted: ① there is no amplification in the negative control group; ② there is an obvious exponential growth period in the positive control group, and the Ct value of the target gene in the positive control group is between 26 and 30; ③ the Ct value of the internal reference gene ACTB in the positive control group is ≤34;

[0043] Step (5) performs combined detection on the target regions of the detection group. If the Ct value is ≤38, it is judged as methylation positive; if the Ct value is >38, it is judged as methylation negative; if any one of the target regions is methylation positive, the sample is judged as an endometrial cancer positive sample; when both target regions are methylation negative, the sample is judged as an endometrial cancer negative sample.

[0044] The above technical solution is described in detail below in conjunction with specific embodiments.

[0045] Example 1 Determination of target regions of endometrial cancer biomarkers based on DNA methylation

[0046] The inventors analyzed the methylation levels of each gene in endometrial cancer tissue and adjacent normal tissue in the TCGA database and found that genes LINC01551 and C5orf38 showed higher methylation levels in cancer tissue, and showed extremely low methylation levels in adjacent normal tissue. Taking hg38 as the reference genome, the physical location of the target region of gene LINC01551 is negative chain Chr14: 28785124-28785522, and the physical location of the target region of gene C5orf38 is positive chain Chr5: 2755751-2756149. The above two target regions have significant differences in methylation levels in cancer tissue and adjacent normal tissue, so genes LINC01551 and C5orf38 can be used as methylation molecular markers for diagnosing endometrial cancer.

[0047] The inventors collected 68 cancer tissue samples clinically diagnosed as endometrial cancer and 68 adjacent normal tissue samples as training sets, and used GRCh38.p14 as the reference genome. They tried to use the target gene LINC01551 (Chr14: 28785124-28785522 negative chain) and the target gene C5orf38 (Chr5: 2755751-2756149 positive chain) as methylation molecular markers for diagnosing endometrial cancer, and used Sanger sequencing to verify their methylation levels in endometrial cancer tissue samples and adjacent normal tissue samples to clarify whether they can be used as methylation molecular markers for diagnosing endometrial cancer, and selected areas with tissue sensitivity ≥ 80% and tissue specificity ≥ 80% for further verification. The specific steps are as follows:

[0048] 1. Sample collection

[0049] All tissue samples in the above training set were formalin-fixed and paraffin-embedded samples. The collection process of all samples has been approved by the Ethics Committee, and all volunteers signed informed consent forms before sample collection.

[0050] 2. Extraction of sample DNA

[0051] For paraffin-embedded tissue samples, QIAamp DNA FFPE Tissue Kit (56404) was used to extract genomic DNA of tissue samples. The specific operations were carried out according to the instructions of the kit.

[0052] 3. Bisulfite Conversion

[0053] The extracted DNA was bisulfite converted using the nucleic acid conversion reagent (Ehan Equipment No. 20200843) of Wuhan Aimeisen Life Science Technology Co., Ltd., and then the converted DNA was purified for subsequent experiments. For specific operation steps, please refer to the kit manual.

[0054] 4. PCR amplification and Sanger sequencing

[0055] The methylation molecular markers used for the diagnosis of endometrial cancer need to be highly methylated in endometrial cancer tissue samples, and low methylated or unmethylated in adjacent normal tissue samples. Therefore, in this example, the above-mentioned target genes LINC01551 and C5orf38 are used as methylation molecular markers for diagnosing endometrial cancer (see Table 1), and the amplification primer pairs shown in Table 2 are used to perform PCR reaction, amplify the target region in the tissue sample, and perform Sanger sequencing. Specifically, first prepare the PCR reaction system in Table 3, use the sample DNA converted by bisulfite as the template, and add SYBR Green PCRmix, methylated primer pairs and non-methylated primer pairs of the target region (see Table 2) and upstream and downstream primers of the internal reference gene ACTB: 5'-AAGGTGGTTGGGTGGTTGTTTTG-3' (SEQ ID NO.15), 5'-AATAACACCCCCACCCTGC-3' (SEQID NO.16), then perform PCR amplification according to the reaction procedure shown in Table 4, and finally send the amplified product to the company for Sanger sequencing.

[0056] Table 1 DNA sequence information of target region

[0057]

[0058]

[0059]

[0060] Table 2 Primer pairs for amplification of target regions

[0061]

[0062] Table 3SYBR Green PCR reaction system

[0063] Components Dosage (μL) SYBR Green PCR Mix 17.5 Methylated and unmethylated upstream primers (10 μM) 0.5 each Methylated and unmethylated downstream primers (10 μM) 0.5 each ACTB upstream primer (10 μM) 0.5 ACTB downstream primer (10 μM) 0.5 Template DNA 4 Ultrapure water Up to 25

[0064] Table 4SYBR Green PCR reaction program

[0065]

[0066] 5. Results Analysis

[0067] Analyze the sequencing peak graph and take the methylation status of the key CpG sites of Sanger sequencing as the standard. Specifically, the methylation of cytosine in a CpG nucleotide is divided into two types: methylated and unmethylated, among which methylation is divided into complete methylation and partial methylation. If the sequencing results of cytosine at a CpG dinucleotide site show that there are both C and T at the position of cytosine, the site is considered to be partially methylated. If more than 95% of the Cs in the CpG dinucleotide sites in a certain amplicon are methylated, the sample is considered to be methylated in this area.

[0068] According to the above standards, the sequencing results of the amplified products were compared with the pathological results, and the sensitivity and specificity of the target region were calculated according to the methylation status of the CpG sites of the amplified products. Sensitivity = the proportion of samples with positive pathological results that detected positive methylation; specificity = the proportion of samples with negative pathological results that detected negative methylation. The test results are shown in Table 5.

[0069] Table 5 Sensitivity and specificity of CpG sites in the target region on the training set samples

[0070]

[0071] As shown in Table 5, the sensitivity of diagnosing endometrial cancer tissue by sequencing analysis of target region 1 and target region 2 was 86.8% and 89.7%, respectively, and the specificity for adjacent tissue samples was 85.3% and 82.4%, respectively. Both target region 1 and target region 2 met the sensitivity ≥ 80% and the specificity ≥ 80%. Therefore, using target region 1 and target region 2 as target regions, the methylation levels of the above regions can effectively distinguish endometrial cancer tissue samples from adjacent normal tissue samples, so target region 1 and target region 2 can be verified in the next step.

[0072] Example 2 Performance of Sanger sequencing in detecting cervical exfoliated cell samples in the target region of the test set samples

[0073] In this example, the differential methylation sites screened were evaluated in 48 exfoliated samples of endometrial cancer and 48 exfoliated cell samples of healthy subjects using the Sanger sequencing method. The specific process is as follows:

[0074] 1. Extraction of DNA Samples

[0075] The collected exfoliated cell samples were used to extract DNA from the exfoliated cell samples using the nucleic acid extraction kit of Wuhan Aimison Life Science Technology Co., Ltd. (Ehan Equipment No. 20210740). For specific operations, please refer to the kit instructions.

[0076] 2. Bisulfite conversion and purification

[0077] The extracted genomic DNA of each sample was subjected to bisulfite conversion. The nucleic acid conversion kit used was the nucleic acid purification reagent of Wuhan Aimison Life Science Technology Co., Ltd. (Ehan Xiebei 20200843). For specific experimental operations, please refer to the instructions of the kit. After the conversion is completed, 30 μL of purified water is used for elution.

[0078] 3. PCR amplification and Sanger sequencing are the same as in Example 1.

[0079] 4. Analysis of results The same as in Example 1, the test results are shown in Table 6.

[0080] Table 6 Sensitivity and specificity of different CpG sites in exfoliated cell samples

[0081] Target area Amplicon location Sensitivity Specificity Target area 1 Chr14:28785234-28785394 negative chain 85.4％(41 / 48) 95.8％(46 / 48) Target area 2 Chr5:2755874-2756004 positive chain 87.5％(42 / 48) 93.8％(45 / 48)

[0082] As shown in Table 6, non-invasive diagnosis can be achieved by detecting the methylation level of the target region in the cervical exfoliated cell samples to distinguish between endometrial cancer patients and healthy people. The sensitivity and specificity of target region 1 for detecting exfoliated cell samples were 85.4% and 95.8%, respectively, and the sensitivity and specificity of target region 2 for detecting exfoliated cell samples were 87.5% and 93.8%, respectively.

[0083] Example 3 Detection of exfoliated cell samples of early clinical endometrial cancer based on qMSP method

[0084] 1. Sample collection

[0085] In this example, a total of 168 samples of cervical exfoliated cells from healthy people were collected, and 62 samples of cervical exfoliated cells with pathological diagnosis of endometrial cancer were included. Among the 62 patients with endometrial cancer, 40 were in stage I and 22 were in stage II. All patients did not receive radiotherapy or chemotherapy before surgery. In this example, stage I / II endometrial cancer is defined as early endometrial cancer based on clinical experience. The collection process of all samples was approved by the Ethics Committee, all volunteers signed informed consent forms, and all samples were anonymized.

[0086] This implementation diagnoses endometrial cancer patients by detecting the methylation level of the target area in the exfoliated cervical samples of the subjects. During the detection process, the collected samples are blinded and then tested by the test operator. The result reader compares the test results with the pathological results (gold standard) according to the interpretation criteria to examine the clinical effectiveness of the target area of ​​endometrial cancer.

[0087] 2. Extraction, transformation and purification of sample DNA

[0088] The collected exfoliated cell samples were used to extract the DNA of the exfoliated cell samples using the Wuhan Aimisen Life Science Technology Co., Ltd. Nucleic Acid Extraction Kit (Ehan Equipment No. 20210836). For specific operations, refer to the kit instructions. After the extraction is completed, elute with 50 μL of purified water. Use the Wuhan Aimisen Life Science Technology Co., Ltd. Nucleic Acid Purification Reagent (Ehan Equipment No. 20200843) to convert the extracted exfoliated cell sample DNA into bisulfite. For specific operations, refer to the kit instructions. After the conversion is completed, elute with 30 μL of purified water.

[0089] 3.qMSP reaction

[0090] The DNA sequences of target region 1 and target region 2 after bisulfite conversion are used as templates to design primer pairs and TaqMan detection probes for methylation-specific fluorescence quantitative PCR (qMSP). The primer pairs are required to have good amplification specificity, that is, the primer pairs only amplify methylated DNA templates, but not unmethylated DNA templates, and will not cause other non-specific amplifications; in addition, the primer pairs are required to have good amplification efficiency, that is, the amplification efficiency of each target region is between 90% and 110%. The nucleotide sequences of the primer pairs and detection probes that meet the above requirements are shown in Table 7. The probes used in Table 7 are all TaqMan probes, the 5' end of the probe is a fluorescent group, such as FAM, VIC, HEX, NED, ROX, TET, JOE, CY3, CY5, etc., and the 3' end is a fluorescent quenching group, such as TAMRA, MGB, BHQ, BHQ1, BHQ2, BHQ3, etc. The qPCR reaction based on TaqMan probe can detect multiple target genes simultaneously in one reaction system. At this time, it is only necessary that the fluorescent groups carried by the 5' end of each target gene-specific detection probe are different. In the present embodiment, each qPCR reaction system needs to detect two target genes, namely the target region (endometrial cancer methylation molecular marker) and the internal reference gene ACTB. The detection probe sequence of ACTB (SEQ ID NO.17.GGAGTGGTTTTTGGGTTTG), the 5' end fluorescent group of the probe is FAM, and the 3' end is the non-fluorescent quenching group MGB. The detection probe sequence of target region 1 (SEQ ID NO.18.CGCCAAAACTCCGATAC), the 5' end fluorescent group of the probe is ROX, and the 3' end is the non-fluorescent quenching group MGB. The detection probe sequence of target region 2 (SEQ ID NO.19.CTACCAAAACTAAAACTACG), the 5' end fluorescent group of the probe is VIC, and the 3' end is the non-fluorescent quenching group MGB. The above primers and probes are artificially synthesized for standby use.

[0091] Table 7 Methylation primer pairs and detection probes for target region 1 and target region 2

[0092]

[0093] 4. Determination of test effectiveness

[0094] Using human cervical exfoliated sample DNA converted and purified by bisulfite as a template, the methylated primer pairs and detection probes provided in Table 7 were used for qMSP reaction. The amplification system of qMSP is shown in Table 8, and the amplification procedure is shown in Table 9. When performing qPCR reaction on each sample, the amount of the internal reference gene ACTB needs to be detected to monitor the sample quality and result interpretation. In each PCR reaction plate, in addition to setting up experimental wells for detecting the samples to be tested, positive control wells and negative control wells also need to be set up at the same time. The template of the positive control well is 10 3 copies / μL of plasmid containing the transformed ACTB sequence and 10 3 The plasmid mixture containing the target region DNA sequence after complete methylation and bisulfite conversion (equal volume mixture) was 1000 copies / μL, and the other components were the same as those in the experimental tube; the template of the negative control well was TE buffer, and the other components were also the same as those in the experimental tube.

[0095] Table 8 TaqMan PCR reaction system

[0096] Components Specification Volume (μL) Platinum II PCR Buffer 5× 5 dNTPs 2.5mM each 3 Methylated upstream primers for each target region 10μM 0.5 Methylated downstream primers for each target region 10μM 0.5 Detection probes for each target region 10μM 0.5 ACTB gene upstream primers 10μM 0.5 ACTB gene downstream primers 10μM 0.5 ACTB gene detection probe 10μM 0.5 DNA polymerase / 0.5 DNA sample to be tested / 5 Purified water / Up to 25

[0097] Table 9 TaqMan PCR program

[0098]

[0099] After the qPCR reaction is completed, adjust the baseline (generally, set the signal value corresponding to the 3rd to 15th cycle as the baseline level) and set the threshold. The threshold must be within the exponential amplification period. The straight line that crosses the threshold and is parallel to the X-axis is called the threshold line. The number of cycles corresponding to the intersection of the threshold line and the amplification curve is the Ct value. To analyze the results of the qPCR reaction, it is required that ① the negative control tube has no amplification; ② the positive control PCR tube has an obvious exponential growth period, and the Ct value of the target gene in the positive control PCR tube is between 26 and 30; ③ the Ct value of the internal reference gene ACTB of the sample to be tested is less than or equal to 34. If the positive control, negative control and internal reference gene meet the above requirements, the test results of the sample to be tested can be analyzed and the results can be interpreted. Otherwise, the experiment is invalid and must be retested.

[0100] 5. Interpretation of results

[0101] For exfoliated cell samples, the positive judgment value is 38. If the Ct value of the target region amplified using a pair of methylation primers and detection probes is ≤38, the sample is judged to be methylation-positive in this amplified region; if the Ct value of amplification using a pair of methylation primers and detection probes is >38, the sample is judged to be methylation-negative in this amplified region. In the target region combination detection, if any target region in the region combination in the sample to be tested is methylation-positive, the sample is a positive sample for endometrial cancer. Only when both target regions in the region combination in the sample to be tested are methylation-negative, the sample is a negative sample for endometrial cancer. The test results are shown in Table 10.

[0102] Table 10 Performance of qMSP method in detecting exfoliated cell samples of subjects in different target areas

[0103]

[0104]

[0105] As shown in Table 10, the qMSP method has good performance in diagnosing early endometrial cancer by detecting the methylation levels of the target regions of the LINC01551 gene and the C5orf38 gene in cervical exfoliated cell samples. The sensitivity of the target region 1 of the LINC01551 gene in detecting cervical exfoliated cell samples of stage I endometrial cancer is 87.5%, and the sensitivity of the target region 2 of the C5orf38 gene in detecting cervical exfoliated cell samples of stage I endometrial cancer is 86.4%; the sensitivity of the target region 2 of the C5orf38 gene in detecting cervical exfoliated cell samples of stage I endometrial cancer is 90.0%, and the sensitivity of the target region 2 of the C5orf38 gene in detecting cervical exfoliated cell samples of stage II endometrial cancer is 86.4%. The specificity of detecting cervical exfoliated cell samples of healthy people is greater than or equal to 93%.

[0106] The target region of the LINC01551 gene and the target region of the C5orf38 gene were combined, that is, target region 1 and target region 2 were combined for detection. It was found that the overall sensitivity of the target region combination detection of endometrial cancer cervical exfoliated cell samples was higher than that of a single target region detection, and the total sensitivity of the detection could reach up to 95.2%; the specificity of the target region combination detection of healthy human cervical exfoliated cell samples was slightly lower than that of a single target region detection, but still at a high level, with a combined detection specificity of 92.3%.

[0107] In summary, the methylation markers provided in the present application can specifically detect the methylation of the LINC01551 gene and the C5orf38 gene in cervical exfoliated cell samples of endometrial cancer and cervical exfoliated cell samples of healthy people, have high sensitivity and specificity, and are used for the screening / early diagnosis of endometrial cancer. They have the advantages of simple operation, non-invasive sampling, and are conducive to popularization and application.

[0108] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present application shall fall within the protection scope of the present application. The present application is further described above with the help of specific embodiments, but it should be understood that the specific description here should not be construed as limiting the essence and scope of the present application. Various modifications made to the above embodiment by ordinary technicians in this field after reading this specification shall fall within the scope of protection of the present application.

Claims

1. A kit for diagnosing / screening endometrial cancer, characterized in that: The kit includes a combination of nucleic acid sequences for detecting / measuring marker gene methylation levels; The marker genes include LINC01551 gene and / or C5orf38 gene; the nucleotide sequence of the LINC01551 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the C5orf38 gene is shown in SEQ ID NO.

2.

2. The kit according to claim 1, characterized in that Methods for detecting / measuring marker gene methylation levels include: methylation-specific PCR, bisulfite sequencing, whole genome methylation sequencing, pyrophosphate sequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high resolution melting curve method or methylation-sensitive restriction endonuclease method.

3. The kit according to claim 1, characterized in that Taking hg38 as the reference genome, the target region of the LINC01551 gene is selected from the full length or partial region whose physical position is the negative chain Chr14: 28785124-28785522; the target region of the C5orf38 gene is selected from the full length or partial region whose physical position is the positive chain Chr5: 2755751-2756149.

4. The kit according to claim 1, characterized in that The nucleic acid sequence combination includes a detection primer pair and a detection probe; The detection primer pair includes: a first primer pair for detecting methylation of the LINC01551 gene, the base sequence of the first primer pair is shown in SEQ ID NOs. 7 to 8; and / or, a second primer pair for detecting methylation of the C5orf38 gene, the base sequence of the second primer pair is shown in SEQ ID NOs. 11 to 12; The detection probes include: a first probe for detecting methylation of the LINC01551 gene, the base sequence of which is shown in SEQ ID NO.18; and / or a second probe for detecting methylation of the C5orf38 gene, the base sequence of which is shown in SEQ ID NO.19; The first primer pair and the first probe are combined to obtain a first nucleic acid sequence combination, and the second primer pair and the second probe are combined to obtain a second nucleic acid sequence combination.

5. The kit according to claim 4, characterized in that The first nucleic acid sequence combination and / or the second nucleic acid sequence combination also includes a third primer pair for detecting the internal reference gene ACTB and a third probe for detecting the internal reference gene ACTB, the base sequence of the third primer pair is shown in SEQ ID NO.15-16, and the base sequence of the third probe is shown in SEQ ID NO.

17.

6. The kit according to claim 5, characterized in that The 5' end of the detection probe contains a fluorescent reporter group, which is any one of FAM, VIC, HEX, NED, ROX, TET, JOE, CY3 or CY5; the 3' end of the detection probe contains a fluorescent quencher group, which is any one of TAMRA, MGB, BHQ, BHQ1, BHQ2 or BHQ3.

7. The kit according to claim 1, characterized in that The kit also includes at least one of a PCR reaction buffer, a DNA polymerase, a DNA extraction reagent or a DNA methylation conversion reagent.

8. The kit according to claim 1, characterized in that The method for using the kit comprises the following steps: (1) isolating the nucleic acid of the target gene in the biological sample of human cervical exfoliated cells to be tested; (2) treating the nucleic acid obtained in step (1) with bisulfite to obtain a test group; using a mixed plasmid containing the ACTB sequence and the fully methylated target gene sequence as a positive control group; and using TE buffer as a negative control group; (3) using the nucleic acid sequence combination as described in claim 4 in combination with qPCR technology to detect the methylation level of each group obtained in step (2); (4) The results were interpreted when all of the following requirements were met: ① there was no amplification in the negative control group; ② there was an obvious exponential growth period in the positive control group, and the Ct value of the target gene in the positive control group was between 26 and 30; ③ the Ct value of the internal reference gene ACTB in the positive control group was ≤34; (5) Combined detection of the target regions of the detection group: if the Ct value is ≤38, it is judged as methylation positive; if the Ct value is >38, it is judged as methylation negative; if any target region is methylation positive, the sample is judged as an endometrial cancer positive sample; if both target regions are methylation negative, the sample is judged as an endometrial cancer negative sample.

9. Use of the kit according to any one of claims 1 to 8 in diagnosing / screening endometrial cancer.

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