Nucleic acid sequence combination for detecting cervical cell gene methylation, kit and application

Abstract

The invention relates to the technical field of biological detection, in particular to a nucleic acid sequence combination for detecting cervical cell gene methylation, a kit and application. The nucleic acid sequence combination for detecting the methylation of the cervical cell gene, provided by the invention, can specifically detect the methylation of ABCC9 and LINC00605 genes of an endometrial cancer cervical exfoliated cell sample, which are different from those of a healthy human cervical exfoliated cell sample, and the kit for detecting the methylation of the cervical cell gene, which is prepared by utilizing the nucleic acid sequence combination, can be used for detecting the methylation of the cervical cell gene. The kit is applied to diagnosis / screening of endometrial cancer, has relatively high detection sensitivity and specificity on stage I and stage II endometrial cancer, is simple to operate and beneficial to popularization and application, and noninvasive material drawing is realized.

Description

Technical Field

[0001] The present application relates to the field of biological testing technology, and in particular to a nucleic acid sequence combination and a kit and application for detecting cervical cell gene methylation. Background Art

[0002] Endometrial cancer (EC), also known as uterine body cancer, is a group of epithelial malignant tumors that occur in the endometrium. It is common in perimenopausal and postmenopausal women. Studies have shown that its incidence rate is increasing year by year, and it is one of the three most common gynecological malignancies in Chinese women. The incidence rate of endometrial cancer in urban residents is higher than that in rural residents. It ranks second among malignant tumors of the female reproductive system in my country. In some developed cities, its incidence rate ranks first among gynecological malignancies.

[0003] There are many methods for detecting endometrial cancer, including blood biochemical tests, tumor marker tests (CA125, CA19-9, CEA, CP2 or HE4), imaging tests (transabdominal or transvaginal ultrasound, MRI, CT, PET-CT), cytology, segmented diagnostic curettage, etc. However, the above detection methods have problems such as low sensitivity in early detection, invasive damage, and inconvenient sampling. Summary of the invention

[0004] In order to solve the above problems, the present application provides a nucleic acid sequence combination, a kit and an application for detecting cervical cell gene methylation. The present application obtains a detection combination with high sensitivity and specificity by screening DNA methylation genes of endometrial cancer and optimizing primers and probes of specific methylation gene sequences, thereby realizing early non-invasive screening and diagnosis of endometrial cancer.

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

[0006] In a first aspect, the present application provides a nucleic acid sequence combination for detecting gene methylation in cervical cells, the nucleic acid sequence combination comprising a detection primer pair and a detection probe, the detection primer pair comprising: a first primer pair for detecting methylation of the ABCC9 gene, the base sequence of the first primer pair being as shown in SEQ ID NOs.7 to 8 (SEQ ID NO.7: 5'-GCGTTTTGAGGACGGTATGC-3', SEQ ID NO.8: 5'-CCGCGA AACACAAACCG-3'); and / or, a second primer pair for detecting methylation of the LINC00605 gene, the base sequence of the second primer pair being as shown in SEQ ID NOs.11 to 12 (SEQ ID NO.11: 5'-GCGTTTT GTGGGTGGAAGTC-3', SEQ ID NO.12: 5'-CCGGCCGACTATACCTAACG-3'), the detection probe comprising: a first probe for detecting methylation of the ABCC9 gene, the base sequence of the first probe being as shown in SEQ ID NO.18 (SEQ ID NO.19: 5'-GCGTTTT GTGGGTGGAAGTC-3', SEQ ID NO.20: 5'-CCGGCCGACTATACCTAACG-3'). NO.18:5'-AACTTCTACCCCTAAAACT-3'); and / or, a second probe for methylation detection of the LINC00605 gene, the base sequence of the second probe is shown in SE Q ID NO.19 (SEQ ID NO.19:5'-TCGGGAGCGAGGTTA-3'), 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.

[0007] The ABCC9 gene belongs to the ATP-binding cassette C subfamily and is closely related to the body's stress response, vascular regulation, neuronal excitability, and neurotransmitter regulation. The LINC00605 gene belongs to the LINC family, which is involved in the activation of mitotic genes. The inventors of the present application have found that the sensitivity and specificity of diagnosing endometrial cancer by detecting the methylation of the ABCC9 gene and the LINC00605 gene are better than those of existing traditional technologies.

[0008] Furthermore, the first nucleic acid sequence combination and / or the second nucleic acid sequence combination further include: 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. Among them, SEQ ID NO.15: 5'-AAGGTGGTTGGGTGGTTGTTTTG-3', SEQ ID NO.16: 5'-AATAACACCCCCACCCTGC-3', SEQ ID NO.17: 5'-GGAGT GGTTTTTGGGTTTG-3'.

[0009] Furthermore, 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.

[0010] In a second aspect, the present application provides the use of the above-mentioned nucleic acid sequence combination for detecting cervical cell gene methylation in the preparation of a kit for diagnosing / screening endometrial cancer.

[0011] In a third aspect, the present application provides a kit for diagnosing / screening endometrial cancer, which diagnoses / screens endometrial cancer by detecting or measuring the methylation status of the ABCC9 gene and / or LINC00605 gene in the DNA of the test sample; the kit includes the above-mentioned combination of nucleic acid sequences for detecting cervical cell gene methylation.

[0012] Furthermore, the above-mentioned kit also includes at least one of a PCR reaction buffer, a DNA polymerase, a DNA extraction reagent or a DNA methylation conversion reagent.

[0013] Furthermore, the above-mentioned DNA methylation conversion reagent includes bisulfite.

[0014] Furthermore, the test sample DNA is extracted DNA from human exfoliated cervical cells;

[0015] Taking hg38 as the reference genome, the physical location of the target region of the ABCC9 gene is the negative chain Chr12: 21941832-21942230; the physical location of the target region of the LINC00605 gene is the negative chain Chr14: 103188889-103189287. When detecting the methylation level of the target region of the above gene, the detection can be performed on the full-length region of any of the above genes, and the detection can also be performed on a partial region of any of the above genes.

[0016] Furthermore, the method for using the above kit comprises the following steps:

[0017] (1) isolating the nucleic acid of the target gene in the biological sample to be tested;

[0018] (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;

[0019] (3) using the above-mentioned nucleic acid sequence combination combined with qPCR amplification technology to detect the methylation status of each group obtained in step (2);

[0020] (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;

[0021] (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.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This application optimizes the primers and probes of specific methylated gene sequences to obtain a nucleic acid sequence combination that can detect the methylation of the marker genes ABCC9 and LINC00605, with high sensitivity and specificity. At the same time, since the nucleic acid sequence combination only needs to detect the methylation level of the marker gene of the target biological sample, a trace amount of biological sample can be used to extract, amplify and detect the marker gene, so that a non-invasive and convenient sampling method can be selected in practical applications.

[0024] 2. The kit for diagnosing / screening endometrial cancer prepared by using the combination of nucleic acid sequences provided in this application has high detection sensitivity for both stage I and stage II endometrial cancer by detecting the methylation level of a single target region or a combination of target regions, and has excellent detection specificity for healthy human cervical exfoliated cell samples. It is applied to the screening / early diagnosis of endometrial cancer, with simple operation and non-invasive sampling, which is conducive to promotion and application. DETAILED DESCRIPTION

[0025] The technical scheme of the present invention 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 invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial sources.

[0026] Example 1 A nucleic acid sequence combination for detecting gene methylation in cervical cells

[0027] This example provides a nucleic acid sequence combination for detecting cervical cell gene methylation, and the base sequence specifically includes:

[0028] The first primer pair for detecting methylation of the ABCC9 gene, the base sequence is shown in SEQ ID NOs.7-8. The second primer pair for detecting methylation of the LINC00605 gene, the base sequence is shown in SEQ ID NOs.11-12. The first probe for detecting methylation of the ABCC9 gene, the base sequence is shown in SEQ ID NO.18. The second probe for detecting methylation of the LINC00605 gene, the base sequence is shown in SEQ ID NO.19. The third primer pair for detecting the internal reference gene ACTB, the base sequence is shown in SEQ ID NOs.15-16. The third probe for detecting the internal reference gene ACTB, the base sequence is shown in SEQ ID NO.17.

[0029] The first primer pair and the first probe are combined to obtain a first nucleic acid sequence combination for pairing, and the second primer pair and the second probe are combined to obtain a second nucleic acid sequence combination for pairing. The third primer pair and the third probe are combined to obtain a third nucleic acid sequence combination for pairing. The first, second, and third nucleic acid sequence combinations constitute the nucleic acid sequence combination for detecting cervical cell gene methylation in this example. The 5'-end fluorescent groups of the first, second, and third probes are VIC, ROX, and FAM, respectively, and the 3'-ends of the first, second, and third probes are labeled with MGB as a quenching group. In other preferred examples, the fluorescent reporter group can also be one of HEX, NED, TET, JOE, CY3, or CY5; the fluorescent quenching group can also be one of TAMRA, MGB, BHQ, BHQ1, BHQ2, or BHQ3.

[0030] Taking hg38 as the reference genome, the target region of the ABCC9 gene in this example is located at the negative chain Chr12: 21941832-21942230, and the original DNA sequence of its target region is shown in SEQ ID NO.1; the target region of the LINC00605 gene is located at the negative chain Chr14: 103188889-103189287, and the original DNA sequence of its target region is shown in SEQ ID NO.4. In this example, the first and second nucleic acid sequence combinations obtained by optimizing the primers and probes of the specific methylated gene sequence can accurately and specifically detect the methylation of the ABCC9 and LINC00605 genes.

[0031] Example 2 A kit for diagnosing / screening endometrial cancer

[0032] This example provides a kit for diagnosing endometrial cancer, which includes: a nucleic acid sequence combination for detecting cervical cell gene methylation provided in Example 1, a PCR reaction buffer, a DNA polymerase, a DNA extraction reagent, and a DNA methylation conversion reagent. The DNA methylation conversion reagent in this example is bisulfite, and the test sample DNA is human exfoliated cervical cell DNA.

[0033] The LINC00605 gene and ABCC9 gene used for the diagnosis of endometrial cancer in the kit provided in this example are highly methylated in endometrial cancer tissue samples, but are low methylated or unmethylated in adjacent normal tissue samples. Therefore, the kit provided in this example is used for the early screening and diagnosis of endometrial cancer and has high detection sensitivity and specificity.

[0034] Example 3: Method for using a kit for diagnosing / screening endometrial cancer

[0035] This example provides a method for using the kit for diagnosing / screening endometrial cancer as provided in Example 2. The specific method of use is as follows:

[0036] (1) Isolation of nucleic acid of target gene in human exfoliated cervical cell sample: Extract genomic DNA of tissue sample from paraffin-embedded sample using QIAamp DNA FFPE Tissue Kit (56404), and the specific operation is carried out according to the instruction manual of the kit.

[0037] (2) The DNA extracted in step (1) is subjected to bisulfite conversion treatment using a nucleic acid conversion reagent to obtain bisulfite-converted DNA, which is then purified for subsequent experiments, i.e., the detection group; 3 copies / μl of ACTB amplicon sequence, 10 3 copies / μl of fully methylated ABCC9 amplicon sequence and 10 3 The mixed plasmid containing fully methylated LINC00605 amplicon sequences at 100 copies / μl was used as the positive control group; TE buffer was used as the negative control group;

[0038] (3) using a nucleic acid sequence combination, PCR reaction buffer, DNA polymerase, DNA extraction reagent and DNA methylation conversion reagent provided in the kit in combination with qPCR amplification technology to detect the methylation status of each group obtained in step (2);

[0039] (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;

[0040] (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.

[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments.

[0042] Example 1 Determination of target regions of endometrial cancer biomarkers based on DNA methylation

[0043] 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 ABCC9 and LINC00605 showed higher methylation levels (β-value) 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 ABCC9 is negative chain Chr12: 21941832-21942230, and the physical location of the target region of gene LINC00605 is negative chain Chr14: 103188889-103189287. The above two target regions have significant differences in methylation levels in cancer tissue and adjacent normal tissue, so genes ABCC9 and LINC00605 can be used as methylation molecular markers for diagnosing endometrial cancer.

[0044] In this example, a total of 68 cancer tissue samples clinically diagnosed as endometrial cancer and 68 adjacent normal tissue samples were collected as training sets, GRCh38.p14 was used as the reference genome, and the target gene ABCC9 and the target gene LINC00605 were tried as methylation molecular markers for diagnosing endometrial cancer. The Sanger sequencing method was used 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 the areas with tissue sensitivity ≥80% and tissue specificity ≥80% were selected for the next step of verification. The specific steps are as follows:

[0045] 1. Sample collection

[0046] 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.

[0047] 2. Extract DNA from samples

[0048] For paraffin-embedded samples, QIAamp DNA FFPE Tissue Kit (56404) was used to extract genomic DNA from tissue samples. The specific operations were carried out according to the instructions of the kit.

[0049] 3. Bisulfite conversion The extracted DNA was bisulfite converted using the nucleic acid conversion reagent (Ehan Equipment No. 20200843) of Wuhan Aimisen Life Science Technology Co., Ltd., and the converted DNA was then purified for subsequent experiments. For specific operation steps, please refer to the kit instructions.

[0050] 4. PCR amplification and Sanger sequencing

[0051] The methylation molecular markers used for the diagnosis of endometrial cancer need to be highly methylated in endometrial cancer tissue samples, and low or unmethylated in adjacent normal tissue samples. Therefore, in this example, the amplification primer pairs shown above were used for PCR reaction to amplify the target region in the tissue sample. The target genes ABCC9 and LINC00605 were used as methylation molecular markers for diagnosing endometrial cancer (see Table 1), and the methylation molecular markers shown in Table 2 were used for 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.

[0052] Table 1 DNA sequence information of target region

[0053]

[0054]

[0055] Table 2 Primer pairs for amplification of target regions

[0056]

[0057] Table 3SYBR Green PCR reaction system

[0058] 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 ACTB upstream primer (10 μM) 0.5 ACTB downstream primer (10 μM) 0.5 Template DNA 4 Ultrapure water Up to 25

[0059] Table 4SYBR Green PCR reaction program

[0060]

[0061] 5. Results Analysis

[0062] 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.

[0063] 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.

[0064] Table 5 Sensitivity and specificity of CpG sites in the target region on the training set samples

[0065]

[0066] As shown in Table 5, the sensitivity of diagnosing endometrial cancer tissue by sequencing analysis of target region 1 and target region 2 was 88.2% and 91.2%, respectively, and the specificity for adjacent tissue samples was 83.6% 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.

[0067] Example 2 Performance of Sanger sequencing in detecting cervical exfoliated cell samples in the target region of the test set samples

[0068] In this example, the Sanger sequencing method was used to evaluate the differentially methylated sites screened in 48 exfoliated samples of endometrial cancer and 48 exfoliated cell samples of healthy subjects (sequencing set samples). The specific process is as follows:

[0069] 1. Extraction of DNA Samples

[0070] 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.

[0071] 2. Bisulfite conversion and purification

[0072] 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.

[0073] 3. PCR amplification and Sanger sequencing are the same as in Example 1.

[0074] 4. Analysis of results The same as in Example 1, the test results are shown in Table 6.

[0075] Table 6 Sensitivity and specificity of different CpG sites in exfoliated cell samples

[0076]

[0077] 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 89.6% and 93.8%, respectively, and the sensitivity and specificity of target region 2 for detecting exfoliated cell samples were 91.7% and 95.8%, respectively.

[0078] Example 3 Detection of exfoliated cell samples of early clinical endometrial cancer based on qMSP method

[0079] 1. Sample collection

[0080] 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.

[0081] This implementation diagnoses endometrial cancer patients by detecting the methylation level of the target area in the exfoliated cell 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.

[0082] 2. Extraction, transformation and purification of sample DNA

[0083] 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, please refer to the kit instructions. After the extraction is completed, elute with 50μL of purified water. Use the Wuhan Aimisen Life Science Technology Co., Ltd. Nucleic Acid Purification Reagent (Ehan Equipment No. 20200843) to perform bisulfite conversion on the extracted DNA of the exfoliated cell samples. For specific operations, please refer to the kit instructions. After the conversion is completed, elute with 30μL of purified water.

[0084] 3.qMSP reaction

[0085] 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: AACTTCTACCCCTAAAACT), 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: TCGGGAGCGAGGTTA), 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.

[0086] Table 7 Methylation primer pairs and detection probes for target region 1 and target region 2

[0087]

[0088] 4. Determination of test effectiveness

[0089] Using the exfoliated cell sample DNA converted and purified by bisulfite as a template, the methylation 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 the 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.

[0090] Table 8 TaqMan PCR reaction system

[0091] 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

[0092] Table 9 TaqMan PCR program

[0093]

[0094]

[0095] 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.

[0096] 5. Interpretation of results

[0097] 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 the target region amplified 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 one of the target regions 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.

[0098] Table 10 Performance of qMSP method in detecting exfoliated cell samples of subjects in different target areas

[0099]

[0100] 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 ABCC9 gene and the LINC00605 gene in cervical exfoliated cell samples. The sensitivity of the target region 1 of the ABCC9 gene in detecting cervical exfoliated cell samples of stage I endometrial cancer is 92.5%, and the sensitivity of the target region 2 of the LINC00605 gene in detecting cervical exfoliated cell samples of stage I endometrial cancer is 86.4%; the sensitivity of the target region 2 of the LINC00605 gene in detecting cervical exfoliated cell samples of stage I endometrial cancer is 92.5%, and the sensitivity of the target region 2 of the LINC00605 gene in detecting cervical exfoliated cell samples of stage II endometrial cancer is 91.0%. The specificity of detecting cervical exfoliated cell samples of healthy people is greater than or equal to 94%.

[0101] The target region of the ABCC9 gene and the target region of the LINC00605 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 93.5%; 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 relatively high level, with a detection specificity of 92.9%.

[0102] In summary, the kit provided by the present invention has excellent performance in diagnosing cervical exfoliated cell samples of patients with early endometrial cancer by detecting the methylation level of a single target region or a combination of target regions. It has high detection sensitivity for both stage I and stage II endometrial cancer, and has excellent detection specificity and high detection accuracy for cervical exfoliated cell samples of healthy subjects.

[0103] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment according to the technical essence of the present invention shall fall within the protection scope of the present invention. The present invention is further described above with the help of specific embodiments, but it should be understood that the specific description here should not be understood as limiting the essence and scope of the present invention. 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 invention.

Claims

1. A nucleic acid sequence combination for detecting gene methylation in cervical cells, 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 ABCC9 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 LINC00605 gene, the base sequence of the second primer pair is shown in SEQ ID NOs. 11 to 12; The detection probe includes: a first probe for detecting methylation of the ABCC9 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 LINC00605 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.

2. The nucleic acid sequence combination for detecting cervical cell gene methylation according to claim 1, 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.

3. The nucleic acid sequence combination for detecting cervical cell gene methylation according to claim 2, 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.

4. Use of the nucleic acid sequence combination for detecting cervical cell gene methylation according to any one of claims 1 to 3 in the preparation of a kit for diagnosing / screening endometrial cancer.

5. A kit for diagnosing / screening endometrial cancer, characterized in that: The kit diagnoses / screens endometrial cancer by detecting or measuring the methylation status of the ABCC9 gene and / or the LINC00605 gene in the DNA of the test sample; The kit comprises the nucleic acid sequence combination for detecting cervical cell gene methylation according to any one of claims 1 to 3.

6. The kit for diagnosing / screening endometrial cancer according to claim 5, 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.

7. The kit for diagnosing / screening endometrial cancer according to claim 6, characterized in that: The DNA methylation conversion reagent includes bisulfite.

8. The kit for diagnosing / screening endometrial cancer according to claim 5, characterized in that: The test sample DNA is extracted from human cervical exfoliated cells; The physical position of the target region of the ABCC9 gene is negative chain Chr12: 21941832-21942230, and the physical position of the target region of the LINC00605 gene is negative chain Chr14: 103188889-103189287.

9. The kit for diagnosing / screening endometrial cancer according to claim 5, 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 to be tested; (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; (3) using the nucleic acid sequence combination as described in claim 1 in combination with qPCR amplification technology and MSP detection technology to detect the methylation status 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 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.

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