Primer probe combination for detecting gene methylation of cervical cancer and endometrial cancer and its application

Abstract

The present application embodiment proposes a cervical cancer and endometrial cancer gene methylation detection primer probe combination and its use. The cervical cancer and endometrial cancer gene methylation detection primer probe combination provided in the present application embodiment designs specific gene methylation detection primers and probes for multiple methylation detection sites on cervical cancer and endometrial cancer high methylation candidate genes, covering multiple methylated CpG sites. The present application embodiment combines the methylation conditions of different genes for joint detection, improves the sensitivity and specificity of the kit through multiple ways, and can achieve early screening and accurate detection of cervical cancer and endometrial cancer.

Description

Technical Field

[0001] The embodiments of the present application relate to the field of nucleic acid detection technology, and specifically to a primer probe combination for detecting methylation of cervical cancer and endometrial cancer genes and uses thereof. Background Art

[0002] Cervical cancer and endometrial cancer are common malignant tumors in the female reproductive system. The treatment effect of cervical cancer and endometrial cancer is closely related to the clinical stage. At present, patients who are discovered early and receive standardized treatment can often be cured well, that is, early diagnosis and early treatment are the key to improving the cure rate; at the same time, cervical cancer and endometrial cancer have the possibility of recurrence. Therefore, how to detect cervical cancer and endometrial cancer as early as possible and how to detect the recurrence of cervical cancer and endometrial cancer in time after surgery are of great clinical significance.

[0003] At present, cervical cancer screening includes cervical cytology testing and human papillomavirus (HPV) testing; among them, cytology testing includes traditional Pap smear and liquid-based cytology. The traditional Pap smear may lead to a certain degree of missed diagnosis or misdiagnosis due to its preparation method, and its accuracy and sensitivity are low; liquid-based cytology is affected by factors such as the doctor's subjective experience, sampling, and preparation, and its sensitivity and specificity are not high; HPV testing has high sensitivity, but there is a certain degree of false positives, and repeated examinations will increase the risk of reinfection, which can easily cause psychological panic in patients.

[0004] The existing screening methods for endometrial cancer mainly include: (1) cervical Pap test and liquid-based cytology smear, but the sensitivity is low; (2) transvaginal ultrasound examination, which has the problem of high sensitivity and poor specificity; (3) endometrial exfoliated cytology examination, the sample processing and preparation process is cumbersome, lacks a unified interpretation standard, and has low sensitivity and specificity.

[0005] Therefore, finding new non-invasive and accurate screening methods for cervical cancer and endometrial cancer is an urgent clinical problem that needs to be solved. Summary of the invention

[0006] The purpose of the embodiments of the present application is to provide a primer probe combination for detecting gene methylation of cervical cancer and endometrial cancer and its use.

[0007] To achieve the above objectives, the present application proposes the following technical solutions:

[0008] In a first aspect, the present invention provides a primer-probe combination for detecting gene methylation in cervical cancer and endometrial cancer, the primer-probe combination comprising:

[0009] A PCR primer-probe combination for detecting PAX1 methylation, comprising an upstream primer as shown in any one of SEQ ID NO.1 to SEQ ID NO.3, a downstream primer as shown in any one of SEQ ID NO.4 to SEQ ID NO.6, and a fluorescent probe as shown in any one of SEQ ID NO.7 to SEQ ID NO.9;

[0010] A PCR primer-probe combination for detecting PCDH10 methylation, comprising an upstream primer as shown in any one of SEQ ID NO.10 to SEQ ID NO.12, a downstream primer as shown in any one of SEQ ID NO.13 to SEQ ID NO.15, and a fluorescent probe as shown in any one of SEQ ID NO.16 to SEQ ID NO.18;

[0011] The PCR primer-probe combination for SOX1 methylation detection includes an upstream primer as shown in any one of SEQ ID NO.19 to SEQ ID NO.21, a downstream primer as shown in any one of SEQ ID NO.22 to SEQ ID NO.24, and a fluorescent probe as shown in any one of SEQ ID NO.25 to SEQ ID NO.27.

[0012] As an embodiment, the primer-probe combination further includes an internal standard primer-probe combination for detecting an internal reference gene GAPDH;

[0013] The internal standard primer-probe combination includes an upstream primer as shown in any one of SEQ ID NO.28 to SEQ ID NO.30, a downstream primer as shown in any one of SEQ ID NO.31 to SEQ ID NO.33, and a fluorescent probe as shown in any one of SEQ ID NO.34 to SEQ ID NO.36.

[0014] As an embodiment, the 5' end of the fluorescent probe contains a fluorescent group;

[0015] The fluorescent group is selected from FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3 or CY5.

[0016] As an embodiment, the 3' end of the fluorescent probe comprises a quenching group;

[0017] The quenching group is selected from BHQ, TAMRA or DABCYL.

[0018] In a second aspect, the embodiments of the present application propose the use of the primer-probe combination described in the first aspect in the preparation of a kit for detecting cervical cancer and endometrial cancer.

[0019] In a third aspect, an embodiment of the present application provides a cervical cancer and endometrial cancer gene methylation detection kit, wherein the detection kit comprises the primer-probe combination described in the first aspect.

[0020] As an embodiment, the detection kit further comprises an amplification premix;

[0021] The amplification premix comprises DNA polymerase, buffer and dNTPs, wherein the content of the DNA polymerase is 3% to 6%, the content of the buffer is 30% to 40%, and the content of the dNTPs is 15% to 30%, based on the total volume of the amplification premix.

[0022] As an embodiment, the detection kit further includes a negative control product, and the negative control product is a healthy human leukocyte DNA.

[0023] As an embodiment, the detection kit also includes a strong positive quality control product, and the strong positive quality control product is Hela cell line DNA.

[0024] In a fourth aspect, the present application embodiment proposes a non-disease diagnostic detection method for detecting gene methylation of cervical cancer and endometrial cancer, using the detection kit as described in the third aspect, the detection method comprising:

[0025] Obtaining DNA of the sample to be tested;

[0026] The DNA of the sample to be tested is subjected to bisulfite conversion treatment to obtain converted DNA;

[0027] The methylation status of the converted DNA was detected using a detection kit.

[0028] The embodiments of the present application have at least the following beneficial effects:

[0029] The cervical cancer and endometrial cancer gene methylation detection primer and probe combination provided in the embodiments of the present application designs specific gene methylation detection primers and probes for multiple methylation detection sites on high-methylation candidate genes of cervical cancer and endometrial cancer, covering multiple methylated CpG sites. The embodiments of the present application combine the methylation conditions of different genes for joint detection, improve the sensitivity and specificity of the kit through multiple approaches, and can achieve early screening and accurate detection of cervical cancer and endometrial cancer.

[0030] Additional aspects and advantages of the present application will be partially given in the following description, which will become apparent from the following description, or will be understood through the practice of the present application. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the embodiments described below 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 creative work are within the scope of protection of the present application.

[0032] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0033] It should also be understood that the terms used in this specification of the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present application. As used in the specification of the embodiments of the present application and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0034] The technical solution of the embodiment of the present application is described in detail below.

[0035] First, the primer and probe combination for detecting methylation of cervical cancer and endometrial cancer genes according to the first aspect of this embodiment is described.

[0036] Primer probe combination for detecting gene methylation in cervical cancer and endometrial cancer

[0037] This embodiment provides a primer-probe combination for detecting gene methylation in cervical cancer and endometrial cancer, and the primer-probe combination includes:

[0038] A PCR primer-probe combination for detecting PAX1 methylation, comprising an upstream primer as shown in any one of SEQ ID NO.1 to SEQ ID NO.3, a downstream primer as shown in any one of SEQ ID NO.4 to SEQ ID NO.6, and a fluorescent probe (or probe 3) as shown in any one of SEQ ID NO.7 to SEQ ID NO.9;

[0039] A PCR primer-probe combination for PCDH10 methylation detection, comprising an upstream primer as shown in any one of SEQ ID NO.10 to SEQ ID NO.12, a downstream primer as shown in any one of SEQ ID NO.13 to SEQ ID NO.15, and a fluorescent probe (or probe 2) as shown in any one of SEQ ID NO.16 to SEQ ID NO.18;

[0040] The PCR primer-probe combination for SOX1 methylation detection includes an upstream primer as shown in any one of SEQ ID NO.19 to SEQ ID NO.21, a downstream primer as shown in any one of SEQ ID NO.22 to SEQ ID NO.24, and a fluorescent probe (or probe 1) as shown in any one of SEQ ID NO.25 to SEQ ID NO.27.

[0041] It can be understood that the gene methylation detection sites of this embodiment include SOX1, PCDH10, and PAX1. For multiple specific methylation detection sites such as SOX1, PCDH10, and PAX1, this embodiment designs specific gene methylation detection primers and probes to cover multiple methylated CpG sites; therefore, this embodiment combines the methylation conditions of different genes for joint detection, and improves the sensitivity and specificity of the kit through multiple pathways, which can achieve early screening and accurate detection of cervical cancer and endometrial cancer.

[0042] Among them, the primers or probes used in this embodiment can adopt any one of the corresponding sequences; for example, the upstream primer for PAX1 methylation detection (or PAX1-F1) can adopt the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; the upstream primer for PCDH10 methylation detection (or PCDH10-F1) can adopt the nucleotide sequence shown in SEQID NO.10, SEQ ID NO.11 or SEQ ID NO.12; the upstream primer for SOX1 methylation detection (or SOX1-PrimerF1) can adopt the nucleotide sequence shown in SEQ ID NO.19, SEQ ID NO.20 or SEQ ID NO.21.

[0043] As an implementation mode, the primer-probe combination of this embodiment also includes an internal standard primer-probe combination for detecting an internal reference gene GAPDH;

[0044] The internal standard primer probe combination includes an upstream primer as shown in any one of SEQ ID NO.28 to SEQ ID NO.30, a downstream primer as shown in any one of SEQ ID NO.31 to SEQ ID NO.33, and a fluorescent probe (or probe 4) as shown in any one of SEQ ID NO.34 to SEQ ID NO.36.

[0045] In this embodiment, upper and lower primers and fluorescent probes of the above-mentioned nucleotide sequence are designed for the internal reference gene GAPDH; wherein, the upper and lower primers and fluorescent probes provided in this embodiment can monitor the extraction of samples and the amplification process of PCR reaction, timely discover problems that may occur in different links of the whole process and monitor possible inhibitory factors.

[0046] Similarly, the above-mentioned internal standard primer probe can also adopt any one of the corresponding sequences; for example, the upstream primer can adopt the nucleotide sequence shown in SEQ ID NO.28, SEQ ID NO.29 or SEQ ID NO.30; the downstream primer can adopt the nucleotide sequence shown in SEQ ID NO.31, SEQ ID NO.32 or SEQ ID NO.33. The fluorescent probe can adopt the nucleotide sequence shown in SEQ ID NO.34, SEQ ID NO.35 or SEQ ID NO.36.

[0047] Specifically, the detection genes and corresponding detection channels of this embodiment are shown in Table 1 below.

[0048] Table 1:

[0049]

[0050] As a preferred embodiment, the 5' end of the above fluorescent probes contains a fluorescent group, and the 3' end of the fluorescent probes contains a quenching group; wherein the fluorescent group is selected from FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3 or CY5, and the quenching group is selected from BHQ, TAMRA or DABCYL.

[0051] Among them, when the fluorescent probe is intact, the fluorescent group at the 5' end is restricted by the quenching group at the 3' end and fluorescence cannot be detected. When the fluorescent probe binds to the target DNA template, the fluorescent group at the 5' end will be freed and emit fluorescence. Therefore, this embodiment can detect the methylation status of genes such as SOX1, PCDH10, and PAX1 by detecting the fluorescence intensity in the reaction system.

[0052] The primer-probe combination provided in this embodiment is used in combination with bisulfite modification and TaqMan probe in the actual detection process; first, this embodiment uses bisulfite to convert unmethylated C bases into U bases, and then converts them into T bases during PCR amplification, while methylated C bases remain unchanged, so methylated and unmethylated C bases can be distinguished. At the same time, TaqMan probes are used as a platform for fluorescent quantitative PCR instrument. During PCR reaction, the probe can specifically anneal and bind to the complementary sequence between the forward primer and the reverse primer. When the probe exists in a complete form, due to energy resonance transfer, the fluorescent group only emits weak fluorescence. After the specific probe is combined with the corresponding sequence, the DNA polymerase exerts 5' to 3' exonuclease activity, cuts off the reporter fluorescent group, and detaches from the quenching effect of the quenching fluorescent group at the 3' end to emit fluorescence. According to the detected fluorescence, the methylation status of genes such as SOX1, PCDH10, and PAX1 is detected.

[0053] Next, the use of the primer-probe combination according to the second aspect of this embodiment will be described.

[0054] use

[0055] This embodiment proposes the use of the primer-probe combination described in the first aspect in preparing a kit for detecting cervical cancer and endometrial cancer.

[0056] As mentioned above, in view of the fact that this embodiment targets multiple specific methylation detection sites such as SOX1, PCDH10, PAX1, etc., specific gene methylation detection primers and probes are designed to cover multiple methylated CpG sites; thus, by combining the methylation conditions of different genes for joint detection, the sensitivity and specificity of the kit are improved through multiple ways, which can achieve early screening and accurate detection of cervical cancer and endometrial cancer.

[0057] Therefore, this embodiment can prepare a methylation detection kit for detecting cervical cancer and endometrial cancer based on the primer-probe combination described in the first aspect; the methylation detection kit has the advantages of high sensitivity and high specificity.

[0058] Next, the detection kit according to the third aspect of this embodiment will be described.

[0059] Detection Kits

[0060] The cervical cancer and endometrial cancer gene methylation detection kit of this embodiment comprises the primer-probe combination described in the first aspect.

[0061] It should be noted that in the following examples, the mixture of the PCR primer probe combination for SOX1 methylation detection, the PCR primer probe combination for PCDH10 methylation detection, the PCR primer probe combination for PAX1 methylation detection and the GAPDH internal standard primer probe combination is recorded as the primer probe mixture.

[0062] The kit of this embodiment is applicable to cervical cancer or endometrial cancer tissue and peripheral blood samples, and the kit is used to detect cervical cancer and endometrial cancer.

[0063] As an embodiment, the detection kit further includes an amplification premix (or PCR Mix);

[0064] The amplification premix comprises DNA polymerase, buffer and dNTPs, wherein the content of the DNA polymerase is 3% to 6%, the content of the buffer is 30% to 40%, and the content of the dNTPs is 15% to 30%, based on the total volume of the amplification premix.

[0065] Usually, in addition to DNA polymerase, buffer and dNTPs, the amplification premix is ​​also dissolved and mixed with pure water. The amplification premix can be selected from existing conventional amplification premixes, such as the amplification premix purchased from Takara Biotechnology (Dalian) Co., Ltd., item number R007A.

[0066] Exemplarily, in each 10.5uL amplification premix, the content of DNA polymerase is 0.5uL / 10.5uL, the content of buffer is 4uL / 10.5uL, the content of dNTPs is 3uL / 10.5uL, and the content of pure water is 3uL / 10.5uL.

[0067] The buffer of this embodiment is a buffer (PCR buffer) conventionally used in the art; for example, the buffer includes: Tris-HCl and bovine serum albumin.

[0068] As an embodiment, the detection kit further comprises a negative control product and a strong positive control product, wherein the negative control product is a healthy human leukocyte DNA, and the strong positive control product is a Hela cell line DNA.

[0069] It is understandable that the negative control product test result is negative and the strong positive control product test result is positive. Negative control can exclude false positive results caused by reagent or environmental contamination; positive control can monitor whether the reagent performance or personnel operation is normal to exclude false negative results.

[0070] Illustratively, in the kit of this embodiment, the final concentration and dosage of each component are shown in Tables 2 and 3 below.

[0071] Table 2: Primer-probe mixture

[0072]

[0073]

[0074] Table 3: PCR MIX

[0075]

[0076] In summary, the kit of this embodiment combines the methylation conditions of different genes for joint detection, improves the sensitivity and specificity of the kit through multiple approaches, and can achieve early screening and accurate detection of cervical cancer and endometrial cancer.

[0077] The following describes the detection method of the fourth aspect of this embodiment.

[0078] Detection Methods

[0079] This embodiment is based on the detection kit of the third aspect, and further describes the detection method of this embodiment. The detection method includes:

[0080] (1) Obtaining DNA from a sample to be tested;

[0081] (2) treating the DNA of the sample to be tested with bisulfite to obtain converted DNA;

[0082] (3) Use a detection kit to detect the methylation status of the converted DNA.

[0083] It is understandable that the detection method of this embodiment does not involve specific disease diagnosis and treatment.

[0084] The following is a further description of the relevant steps in the detection method of this embodiment.

[0085] In step (1), the biological sample (DNA sample) can be cervical cancer or endometrial cancer tissue, normal tissue adjacent to the cancer and peripheral blood; taking cervical exfoliated cell samples as an example, it can adopt the sampling operation currently known in the art.

[0086] For example: the medical staff first exposes the cervix with a speculum, and wipes away the excess secretions from the cervical opening with a cotton swab. The cervical brush is placed at the cervical opening, and rotated 5 times in one direction to obtain a sufficient amount of epithelial cell samples. The cervical brush head is then placed in a sample tube containing cell preservation fluid, and the cervical brush handle is broken along the crease of the brush handle, leaving the brush head in the sample tube, and the tube cap is tightened to mark the sample.

[0087] Among them, after sampling, the DNA of the sample can be extracted using an existing DNA extraction kit or nucleic acid extraction reagent to obtain the DNA of the sample to be tested.

[0088] For example, the nucleic acid extraction or purification reagent of Anhui Medical Technology Co., Ltd. (medical device registration number: Anhui Wuxixibei 20220023 or Anhui Wuxixibei 20190002) can be used to extract the DNA of each sample. For specific operations, please refer to the kit instructions.

[0089] Exemplarily, DNA is extracted by a nucleic acid extraction or purification reagent produced by Anhui Dajian Medical Technology Co., Ltd. (medical device registration number: Anhui Wuxi Xiebei 20220023), and the specific steps are as follows:

[0090] (a) Take a 1.5 mL centrifuge tube, add 200 μL of the sample (plasma) and 20 μL of pancreatic lipase, vortex and shake, mix thoroughly and leave at room temperature for 5 min;

[0091] (b) Add 20 μL of proteinase K and 360 μL of lysis buffer to the centrifuge tube, vortex and shake to mix thoroughly, centrifuge briefly, and place in a 70°C water bath for 10 min;

[0092] (c) Briefly centrifuge, add 200 μL of pre-cooled isopropanol to the centrifuge tube, vortex to mix thoroughly, briefly centrifuge to remove droplets on the inner wall of the tube cap, and stand at -20°C for 5 min;

[0093] (d) adding the solution and flocculent precipitate from step c to the adsorption column (the adsorption column is placed in a collection tube), centrifuging at 13000 rpm for 1 min, discarding the waste liquid, and reusing the collection tube;

[0094] (e) Add 600 μL of rinse solution I (precooled) to the adsorption column, centrifuge at 13000 rpm for 1 min, and discard the waste solution;

[0095] (f) Add 600 μL of rinse solution II (precooled) to the adsorption column, centrifuge at 13000 rpm for 1 min, and discard the waste solution;

[0096] (g) Place the adsorption column in a clean 1.5 mL centrifuge tube and centrifuge at 13,000 rpm for 3 min. Discard the centrifuge tube and waste liquid.

[0097] (h) Place the adsorption column in a clean 1.5 mL centrifuge tube, open the tube cap, and let it dry for 3 min;

[0098] (i) Add 100 μL of elution buffer to the middle of the adsorption column, let it stand at room temperature for 3 min, centrifuge at 13,000 rpm for 2 min, collect the DNA into a centrifuge tube, and store it at -20°C.

[0099] Next, the DNA extracted in the above step is subjected to bisulfite conversion and purification.

[0100] Step (2), subjecting the DNA sample to be tested to bisulfite conversion treatment to obtain converted DNA, namely Bis-DNA.

[0101] Wherein, the bisulfite conversion treatment can be performed by using a nucleic acid extraction or purification reagent (centrifugal column type) produced by Anhui Dajian Medical Technology Co., Ltd.; illustratively, the specific steps are as follows:

[0102] (a) Take 45 μL of the DNA sample to be tested and place it in a new 1.5 mL centrifuge tube, add 5 μL of conversion buffer, and place it in a metal bath and incubate at 37°C for 15 min;

[0103] (b) After incubation, add 100 μL of the pre-prepared transformation solution to each sample, mix well and centrifuge briefly, and incubate in a metal bath at 50°C in the dark for 12-16 hours;

[0104] (c) The samples were incubated on ice (0-4°C) for 10 min;

[0105] (d) Place the adsorption column in the collection tube and add 400 μL of binding solution to the adsorption column;

[0106] (e) Add the sample from step c to the adsorption column (containing the binding solution), cap the tube tightly, invert it several times to mix, centrifuge at full speed (14000 rpm) for 30 seconds, and discard the waste liquid;

[0107] (f) Add 100 μL of rinse solution to the adsorption column, centrifuge at full speed for 30 s, and discard the waste solution;

[0108] (g) Add 200 μL of desulfurization solution to the adsorption column, incubate at room temperature (25°C) for 20 min, then centrifuge at full speed for 30 s and discard the waste liquid;

[0109] (h) Add 200 μL of rinse solution to the adsorption column and centrifuge at full speed for 30 s. Repeat the process of adding 200 μL of rinse solution and centrifuging at full speed for 30 s. Discard the waste solution and the collection tube.

[0110] (i) Place the adsorption column in a 1.5 mL sterile centrifuge tube, add 30 μL of elution solution to the middle of the adsorption membrane, elute the transformed DNA, centrifuge at full speed for 1 min, collect the Bis-DNA, and store at -20°C.

[0111] Next, after obtaining Bis-DNA, a detection kit is used to perform detection through PCR reaction.

[0112] The PCR reaction conditions are shown in Table 4.

[0113] Table 4: Reaction conditions

[0114]

[0115] The following is a further explanation of the result analysis method and quality control standards of the detection method.

[0116] Results Analysis

[0117] Baseline setting: The baseline can be automatically output by the instrument, or it can be manually adjusted according to the instrument's instructions.

[0118] Threshold setting: It should be set at the exponential amplification stage of the fluorescence signal. The fluorescence curve above the threshold should have a typical "S" curve.

[0119] It should be noted that the following methods can be used to effectively interpret the test results: if the internal standard gene test (VIC channel) has typical amplification, the analysis can continue; if the internal standard gene test (VIC channel) has no amplification, the test needs to be repeated.

[0120] Positive judgment value:

[0121] A total of 181 samples (25 cases of cervical cancer, 53 cases of endometrial cancer, and 103 cases of control group) were used to study the positive judgment value.

[0122] Among them, when the SOX1 gene △Ct≤5, the Youden index is the largest, and the sensitivity of the kit diagnosis is 88.46% and the specificity is 85.44%. Therefore, the SOX1 gene △Ct of 5 is selected as the optimal cut-off value of the kit, that is, the SOX1 gene positive judgment value of the kit is set as the sample △Ct≤5, which is methylation positive, and when △Ct>5, it is judged as methylation negative.

[0123] When the PCDH10 gene △Ct≤3, the Youden index is the largest, and the diagnostic sensitivity of the kit is 83.33% and the specificity is 91.26%. Therefore, the PCDH10 gene △Ct of 3 is selected as the optimal cut-off value of the kit, that is, the PCDH10 gene positive judgment value of the kit is set as the sample △Ct≤3, which is methylation positive, and when △Ct>3, it is judged as methylation negative.

[0124] When the PAX1 gene △Ct≤10, the Youden index is the largest, and the diagnostic sensitivity of the kit is 70.51% and the specificity is 95.15%. Therefore, the PAX1 gene △Ct of 10 is selected as the optimal cut-off value of the kit, that is, the PAX1 gene positive judgment value of the kit is set as the sample △Ct≤10, which is methylation positive, and when △Ct>10, it is judged as methylation negative.

[0125] It should be noted that:

[0126] 1. If the gene is not amplified, the maximum cycle number of 35 is used as the gene Ct value;

[0127] 2. △Ct = Ct value of target gene (Ct value is an integer) - Ct value of internal standard gene (Ct value is an integer).

[0128] Kit Validity:

[0129] (1) Negative control: When the target gene is detected through the FAM channel, there is no amplification curve or there is an amplification curve, but the Ct value is greater than 25; when the internal standard gene is detected through the VIC channel, there is an amplification curve and the Ct value is ≤25.

[0130] (2) Positive control: FAM alone shows amplification, or both FAM and VIC channel detection show amplification (showing a typical S-shaped curve) and the Ct value of the FAM channel is ≤25.

[0131] The present application is further described below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application.

[0132] In the following examples, the materials, reagents and instruments used can be purchased from commercial sources unless otherwise specified.

[0133] The present application will be further described below in conjunction with specific embodiments.

[0134] Example 1

[0135] Screening and optimization of candidate genes with hypermethylation associated with cervical cancer and endometrial cancer.

[0136] This example screened out cervical cancer genes and endometrial cancer-related hypermethylated genes by collecting literature, designed primers and probes in the hypermethylated regions of the related genes, and set specific primers and probes for the internal reference gene GAPDH. Among them, the PCR primer and probe combination for the methylation of the related genes is shown in Table 5 below.

[0137] Table 5: Primer probe combinations

[0138]

[0139]

[0140] Based on the primers designed above, the methylation status of genes in cancer tissue samples, adjacent normal tissue samples, and peripheral blood samples was detected, and the screened differential methylation sites were evaluated.

[0141] The specific steps include:

[0142] Extraction of sample DNA

[0143] Take DNA samples to be tested (cervical cancer or endometrial cancer tissue, normal tissue adjacent to the cancer, and peripheral blood), and use the nucleic acid extraction or purification reagent produced by Anhui Dajian Medical Technology Co., Ltd. (product registration number: Wanwu Xiebei 20220023) to extract high-purity DNA.

[0144] The specific steps are as follows:

[0145] (a) Take a 1.5 mL centrifuge tube, add 200 μL of the sample to be tested and 20 μL of pancreatic lipase, vortex shake, mix thoroughly, and place at room temperature for 5 min;

[0146] (b) Add 20 μL of proteinase K and 360 μL of lysis buffer to the centrifuge tube, vortex and shake to mix thoroughly, centrifuge briefly, and place in a 70°C water bath for 10 min;

[0147] (c) Briefly centrifuge, add 200 μL of pre-cooled isopropanol to the centrifuge tube, vortex to mix thoroughly, briefly centrifuge to remove droplets on the inner wall of the tube cap, and stand at -20°C for 5 min;

[0148] (d) adding the solution and flocculent precipitate from step c to the adsorption column (the adsorption column is placed in a collection tube), centrifuging at 13000 rpm for 1 min, discarding the waste liquid, and reusing the collection tube;

[0149] (e) Add 600 μL of rinse solution I (precooled) to the adsorption column, centrifuge at 13000 rpm for 1 min, and discard the waste solution;

[0150] (f) Add 600 μL of rinse solution II (precooled) to the adsorption column, centrifuge at 13000 rpm for 1 min, and discard the waste solution;

[0151] (g) Place the adsorption column in a clean 1.5 mL centrifuge tube and centrifuge at 13,000 rpm for 3 min. Discard the centrifuge tube and waste liquid.

[0152] (h) Place the adsorption column in a clean 1.5 mL centrifuge tube, open the tube cap, and let it dry for 3 min;

[0153] (i) Add 100 μL of elution buffer to the middle of the adsorption column, let it stand at room temperature for 3 min, centrifuge at 13,000 rpm for 2 min, collect the DNA sample into a centrifuge tube, and store it at -20°C.

[0154] Bisulfite conversion and purification

[0155] Next, the DNA extracted in the above step is subjected to bisulfite conversion and purification to obtain Bis-DNA.

[0156] In this embodiment, the bisulfite conversion treatment is performed using a nucleic acid extraction or purification reagent (centrifugal column type) produced by Anhui Dajian Medical Technology Co., Ltd. (Anhui Wuxi Medical Equipment No. 20200003); the specific steps are as follows:

[0157] (a) Take 45 μL of the DNA sample to be tested and place it in a new 1.5 mL centrifuge tube, add 5 μL of conversion buffer, and place it in a metal bath and incubate at 37°C for 15 min;

[0158] (b) After incubation, add 100 μL of the pre-prepared transformation solution to each sample, mix well and centrifuge briefly, and incubate in a metal bath at 50°C in the dark for 12-16 hours;

[0159] (c) The samples were incubated on ice (0-4°C) for 10 min;

[0160] (d) Place the adsorption column in the collection tube and add 400 μL of binding solution to the adsorption column;

[0161] (e) Add the sample from step c to the adsorption column (containing the binding solution), cap the tube tightly, invert it several times to mix, centrifuge at full speed (14000 rpm) for 30 seconds, and discard the waste liquid;

[0162] (f) Add 100 μL of rinse solution to the adsorption column, centrifuge at full speed for 30 s, and discard the waste solution;

[0163] (g) Add 200 μL of desulfurization solution to the adsorption column, incubate at room temperature (25°C) for 20 min, then centrifuge at full speed for 30 s and discard the waste liquid;

[0164] (h) Add 200 μL of rinse solution to the adsorption column and centrifuge at full speed for 30 s. Repeat the process of adding 200 μL of rinse solution and centrifuging at full speed for 30 s. Discard the waste solution and the collection tube.

[0165] (i) Place the adsorption column in a 1.5 mL sterile centrifuge tube, add 30 μL of elution solution to the middle of the adsorption membrane, elute the transformed DNA, centrifuge at full speed for 1 min, collect the Bis-DNA, and store at -20°C.

[0166] Next, after obtaining Bis-DNA, the Bis-DNA converted by bisulfite was used as a template, and PCR Mix and a methylation primer probe combination of methylation biomarkers of cervical cancer and endometrial cancer (target regions of SOX1, PCDH10, and PAX1) were added for PCR amplification. The PCR reaction system and reaction conditions are shown in Tables 6 and 7.

[0167] Table 6: PCR reaction system

[0168]

[0169] Table 7: Reaction conditions

[0170]

[0171] Pyrosequencing

[0172] The PCR product is subjected to pyrophosphate sequencing, and the sequencing peak graph is analyzed, and the methylation status of the key CpG sites of pyrophosphate sequencing is used as the standard. Specifically, the methylation of cytosine in a CpG nucleotide is divided into two types: methylated and unmethylated, among which methylation is further divided into complete methylation and partial methylation; if the sequencing result of cytosine at a certain CpG dinucleotide site shows that there are both C and T at the position of cytosine, then 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, then the sample is considered to be methylated in this area.

[0173] Results Analysis

[0174] After the PCR reaction, the sequencing results of the amplified products were compared with the pathological results, and the methylation status of the CpG sites of the amplified products was calculated to calculate the sensitivity and specificity of the target region. The sensitivity (trimethylation positive sample / pathological result) was positive for the sample; the specificity (trimethylation negative sample / pathological result) was negative for the sample. The test results are shown in Table 8.

[0175] Table 8: Sensitivity and specificity of biomarkers on the training set

[0176]

[0177] According to the test results in Table 9, it can be seen that the methylation levels of SOX1 gene, PCDH10 gene and PAX1 gene as biomarkers for cervical cancer and endometrial cancer in cancer tissue samples are significantly higher than their methylation levels in adjacent normal tissue samples. According to the above preliminary screening criteria, by detecting the methylation levels of the respective regions of SOX1 gene, PCDH10 gene and PAX1 gene, the specificity and sensitivity meet the requirements.

[0178] Example 2

[0179] A test kit based on the qMSP method for detecting cervical cancer and endometrial cancer samples.

[0180] The DNA sample extraction, bisulfite conversion and purification treatment are the same as those in Example 1. The sample is a cervical exfoliated cell sample.

[0181] The reaction system and reaction conditions of qPCR reaction are shown in Tables 9 and 10.

[0182] Table 9: Reaction system

[0183]

[0184] Table 10: Reaction conditions

[0185]

[0186] Among them, the composition of the primer-probe mixture and PCR MIX is shown in Table 11-12.

[0187] Table 11: Primer-probe mixture

[0188]

[0189]

[0190] Table 12: PCR MIX

[0191]

[0192]

[0193] Among them, PCR MIX is an amplification premix purchased from Takara Biotechnology (Dalian) Co., Ltd., with product number R007A.

[0194] The reaction system in Table 9 was thoroughly mixed, centrifuged instantaneously, and dispensed into 0.2 mL PCR reaction tubes with 20 μL per tube; the PCR reaction tubes were placed in the corresponding fluorescent PCR detector (ABI 7500 fluorescent PCR amplifier), the order of sample placement was recorded, and then methylation fluorescent quantitative PCR amplification detection was performed.

[0195] Test Results

[0196] In this example, when the negative quality control product and the positive quality control product meet the validity judgment of the kit, 181 exfoliated cell samples were tested, including 103 samples of benign lesions, 25 samples of cervical cancer patients, and 53 samples of endometrial cancer; the sensitivity and specificity of the single gene methylation detection results were analyzed; the comparison of the test results with the clinical results is shown in Table 13.

[0197] Table 13:

[0198]

[0199]

[0200] According to the test results in Table 13, the cervical cancer and endometrial cancer gene methylation detection primer probe combination provided in this embodiment has high sensitivity and significant specificity for multiple specific methylation detection sites such as SOX1, PCDH10, PAX1, etc.

[0201] In summary, the cervical cancer and endometrial cancer gene methylation detection primer and probe combination provided in this embodiment designs specific gene methylation detection primers and probes for multiple methylation detection sites on high-methylation candidate genes of cervical cancer and endometrial cancer, covering multiple methylated CpG sites. This embodiment combines the methylation conditions of different genes for joint detection, improves the sensitivity and specificity of the kit through multiple ways, and can achieve early screening and accurate detection of cervical cancer and endometrial cancer.

[0202] The technical solutions provided by the embodiments of the present application are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present application. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present application. At the same time, for those skilled in the art, according to the embodiments of the present application, there will be changes in the specific implementation methods and application scopes.

[0203] In summary, the contents of this specification should not be understood as limiting the present application.

Claims

1. A primer probe combination for detecting gene methylation in cervical cancer and endometrial cancer, characterized in that: The primer-probe combination comprises: A PCR primer-probe combination for detecting PAX1 methylation, comprising an upstream primer as shown in any one of SEQ ID NO.1 to SEQ ID NO.3, a downstream primer as shown in any one of SEQ ID NO.4 to SEQ ID NO.6, and a fluorescent probe as shown in any one of SEQ ID NO.7 to SEQ ID NO.9; The PCR primer probe combination for PCDH10 methylation detection includes SEQ ID An upstream primer as shown in any one of SEQ ID NO.10 to SEQ ID NO.12, a downstream primer as shown in any one of SEQ ID NO.13 to SEQ ID NO.15, and a fluorescent probe as shown in any one of SEQ ID NO.16 to SEQ ID NO.18; The PCR primer-probe combination for SOX1 methylation detection includes an upstream primer as shown in any one of SEQ ID NO.19 to SEQ ID NO.21, a downstream primer as shown in any one of SEQ ID NO.22 to SEQ ID NO.24, and a fluorescent probe as shown in any one of SEQ ID NO.25 to SEQID NO.

27.

2. The primer probe combination for detecting gene methylation of cervical cancer and endometrial cancer according to claim 1, characterized in that: The primer-probe combination also includes an internal standard primer-probe combination for detecting an internal reference gene GAPDH; The internal standard primer-probe combination includes an upstream primer as shown in any one of SEQ ID NO.28 to SEQ ID NO.30, a downstream primer as shown in any one of SEQ ID NO.31 to SEQ ID NO.33, and a fluorescent probe as shown in any one of SEQ ID NO.34 to SEQ ID NO.

36.

3. The primer probe combination for detecting gene methylation of cervical cancer and endometrial cancer according to claim 1, characterized in that: The 5' end of the fluorescent probe contains a fluorescent group; The fluorescent group is selected from FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3 or CY5.

4. The primer probe combination for detecting gene methylation of cervical cancer and endometrial cancer according to claim 1, characterized in that: The 3' end of the fluorescent probe contains a quenching group; The quenching group is selected from BHQ, TAMRA or DABCYL.

5. Use of the primer-probe combination according to any one of claims 1 to 4 in preparing a kit for detecting cervical cancer and endometrial cancer.

6. A cervical cancer and endometrial cancer gene methylation detection kit, characterized in that: The detection kit comprises the primer-probe combination according to any one of claims 1 to 4.

7. The cervical cancer and endometrial cancer gene methylation detection kit according to claim 6, characterized in that: The detection kit also includes an amplification premix; The amplification premix comprises DNA polymerase, buffer and dNTPs, wherein the content of the DNA polymerase is 3% to 6%, the content of the buffer is 30% to 40%, and the content of the dNTPs is 15% to 30%, based on the total volume of the amplification premix.

8. The cervical cancer and endometrial cancer gene methylation detection kit according to claim 6, characterized in that: The detection kit also includes a negative quality control product, which is the DNA of healthy human leukocytes.

9. The cervical cancer and endometrial cancer gene methylation detection kit according to claim 6, characterized in that: The detection kit also includes a strong positive quality control product, which is Hela cell line DNA.

10. A non-disease diagnostic method for detecting gene methylation of cervical cancer and endometrial cancer, using the detection kit according to any one of claims 6 to 9, characterized in that: The detection method comprises: Obtaining DNA of the sample to be tested; The DNA of the sample to be tested is subjected to bisulfite conversion treatment to obtain converted DNA; The methylation status of the converted DNA was detected using a detection kit.

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