CN113549694A - Novel cervical cancer methylation gene detection method - Google Patents

Novel cervical cancer methylation gene detection method Download PDF

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CN113549694A
CN113549694A CN202110994929.0A CN202110994929A CN113549694A CN 113549694 A CN113549694 A CN 113549694A CN 202110994929 A CN202110994929 A CN 202110994929A CN 113549694 A CN113549694 A CN 113549694A
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actb
fam19a4
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喻德华
贾树芹
毛玉
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Shenzhen You Shengkang Bioscience Co ltd
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Abstract

The invention relates to the technical field of cervical cancer detection, and discloses a novel cervical cancer methylation gene detection method, which comprises the following steps: performing probe fluorescent quantitative PCR amplification on the bis-DNA converted by the bisulfite by adopting a PCR amplification solution, which specifically comprises the following steps: designing forward and reverse primers, a detection probe and a specific blocking probe aiming at a bis-DNA positive strand and a bis-DNA negative strand respectively, specifically binding a methylation sequence target gene PAX1, FAM19A4, hsa-mir124-2, ATP10A, HAS1 and an internal reference ACTB (beta-actin) gene by using the detection probe, and binding an unmethylated sequence by using the specific blocking probe; and determining the methylation value of the sample to be detected according to the relative fluorescence CT values of PCR amplification results of PAX1, FAM19A4, hsa-mir124-2, ATP10A, HAS1 and ACTB genes, and respectively calculating the methylation values of PAX1, FAM19A4, hsa-mir124-2, ATP10A and HAS1 relative to the ACTB genes. The invention obtains the technical effect of improving the detection sensitivity and specificity of cervical cancer methylation genes, and discovers a unique new methylation target: HAS1, ATP 10A.

Description

Novel cervical cancer methylation gene detection method
Technical Field
The invention relates to the technical field of cervical cancer detection, in particular to a novel cervical cancer methylation gene detection method.
Background
As can be seen by analyzing the existing cervical cancer screening technical data, the prior art has many defects, which are specifically as follows:
firstly, HPV screening: high sensitivity, but low specificity, and most HPV infections can be automatically cleared by the body.
II, a liquid-based cytology detection method (TCT): obviously improves the detection rate of the canceration cells, and correspondingly reduces the times of repeated papanicolaou tests, thereby reducing unnecessary worry of patients caused by repeated tests. But the level of the obtained material, the slice preparation and the reading of the slice is different. The sample contains many normal cervical cells, relatively few abnormal cells which are possibly not detected easily cause false negative, and the omission ratio is high. Errors remain unavoidable and the test results remain inaccurate.
Thirdly, colposcopy examination: cervical lesions can be found in time, but physical injury of iatrogenic genital tracts can be caused, postmenopausal women have low estrogen level, genital tract epithelium is thinner, lesions are more hidden and are difficult to identify.
Fourthly, the traditional methylation detection method comprises the following steps: methylation specific PCR and bisulfite sequencing. However, the methods have the defects of complicated operation, low accuracy and low sensitivity, and limit the wide application of the methods in clinical laboratories; meanwhile, results with large influence mostly come from European and American laboratories, research objects are mostly white women abroad, and research targets are not necessarily suitable for Chinese women; as can be seen by analyzing a large amount of technical data, most methylated Real-time PCR research methods only detect one strand of DNA, and the detection sensitivity is low. The single PCR detection technology is used, only one target is detected in one reaction system, the detection efficiency is low, and the detection cost is high.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the following technical purposes are achieved:
screening out a unique new methylation target and a target combination which are really suitable for Chinese females;
the detection efficiency is high, results can be obtained within about 2 hours, the results are objective, the dependence on the experience of doctors is reduced, and the popularization is convenient;
thirdly, the detection sensitivity and specificity are improved; the invention respectively designs a primer and a specific probe aiming at the positive strand and the negative strand of DNA converted by bisulfite, and simultaneously combines the use of a closed probe to prevent the probe from being combined to an unmethylated strand;
fourthly, the detection cost is reduced, and the detection efficiency is improved; detecting a plurality of target genes simultaneously in a reaction system by using a multiplex PCR detection technology;
the invention provides a novel cervical cancer methylation gene detection method.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme:
a novel cervical cancer methylation gene detection method comprises the following steps:
extracting genome DNA of cervical exfoliated cells;
step two, performing bisulfite conversion on the extracted genome DNA to obtain bis-DNA;
step three, performing probe fluorescent quantitative PCR amplification on bis-DNA by adopting a PCR amplification solution, which comprises the following specific steps:
designing forward and reverse primers, a detection probe and a specific blocking probe aiming at a bis-DNA positive strand and a bis-DNA negative strand respectively, specifically binding a methylation sequence target gene PAX1, FAM19A4, hsa-mir124-2, ATP10A, HAS1 and an internal reference ACTB (beta-actin) gene by using the detection probe, and binding an unmethylated sequence by using the specific blocking probe to avoid the binding with the primers;
and step four, determining the methylation value of the sample to be detected according to the relative fluorescence CT values of the PCR amplification results of the PAX1, FAM19A4, hsa-mir124-2, ATP10A, HAS1 and ACTB genes, and respectively calculating the methylation values of the PAX1, FAM19A4, hsa-mir124-2, ATP10A and HAS1 relative to the ACTB genes.
Furthermore, the cervical cells PAX1, FAM19A4, hsa-mir124-2, ATP10A and HAS1 have an average value of more than 12 relative to the methylation value of the ACTB gene, and can be determined as a cervical cancer patient.
Furthermore, the cervical cells PAX1, FAM19A4, hsa-mir124-2, ATP10A and HAS1 were judged to be non-cervical cancer when the average value of the methylation value of the ACTB gene was 0.9 or less.
Further, the reaction conditions of the amplification are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15S, annealing at 62 ℃ for 5S, and annealing at 58 ℃ for 40S for 50 cycles.
Further, the PCR reaction solution consists of 3 parts, including a detection solution, a negative control and a positive control;
the detection solution comprises 9 systems:
the detection solution comprises 9 systems, wherein the system 1-5 is a 4-fold system, the system 6-9 is a 5-fold system, and the systems comprise the following components:
the system 1 is a 4-fold system and is used for detecting genes PAX1, FAM19A4, hsa-mir-124-2 and ACTB;
the system 2 is a 4-fold system and is used for detecting genes PAX1, FAM19A4, HAS1 and ACTB;
the system 3 is a 4-fold system and is used for detecting genes PAX1, FAM19A4, ATP10A and ACTB;
the system 4 is a 4-fold system and is used for detecting genes PAX1, hsa-mir-124-2, HAS1 and ACTB;
the system 5 is a 4-fold system and is used for detecting genes PAX1, hsa-mir-124-2, ATP10A and ACTB;
the system 6 is a 5-fold system and is used for detecting genes PAX1, FAM19A4, hsa-mir124-2, ATP10A and ACTB;
the system 7 is a 5-fold system and is used for detecting genes PAX1, FAM19A4, hsa-mir124-2, HAS1 and ACTB;
the system 8 is a 5-fold system and is used for detecting genes PAX1, FAM19A4, ATP10A, HAS1 and ACTB;
system 9 is a 5-fold system and is used to detect genes PAX1, hsa-mir124-2, ATP10A, HAS1 and ACTB.
Further, the detection solution further comprises: 0.3. mu.M deoxyribonucleoside triphosphate, MgCl2The concentration was 5mM, the DNA polymerase concentration was 0.1U/. mu.L, and the glycerol ratio was 1%.
Furthermore, the solvent of the PCR amplification solution is nuclease-free water.
(III) advantageous technical effects
Compared with the prior art, the invention has the following beneficial technical effects:
1. double-strand methylation detection technology: according to the invention, primers and specific probes are respectively designed for the positive strand and the negative strand of the DNA after bisulfite conversion, so that the detection sensitivity is greatly improved;
2. use of blocking probes: the invention carries out PCR amplification on DNA which is extracted from human cervical exfoliated cells and is converted by bisulfite, and simultaneously detects a target gene and an internal reference ACTB (beta-actin) gene; using specific blocking probes and detection probes to distinguish between methylated and unmethylated gene sequences; blocking the probe from binding to unmethylated sequences, preventing binding to the primer; the detection probe can specifically bind to the methylated sequence; the internal reference ACTB gene is used for evaluating the quality of DNA in the detection;
3. unique new methylation targets were discovered: HAS1, ATP 10A; 9 target combinations were found;
1、PAX1、FAM19A4、hsa-mir-124-2；2、PAX1、FAM19A4、HAS1；3、 PAX1、FAM19A4、ATP10A；4、PAX1、hsa-mir-124-2、HAS1；5、PAX1、 hsa-mir-124-2、ATP10A；6、PAX1、FAM19A4、hsa-mir124-2、ATP10A；7、 PAX1、FAM19A4、hsa-mir124-2、HAS1；8、PAX1、FAM19A4、ATP10A、 HAS1；9、PAX1、hsa-mir124-2、ATP10A、HAS1。
drawings
FIG. 1 is a flow chart of the detection steps of the novel cervical cancer methylation gene detection method of the invention.
Detailed Description
Selecting an outpatient of cervical abnormality who is treated in a hospital as a research object, excluding pregnancy and cervical resection history, patients with malignant tumor history, and cervical precancerous lesion groups (cervical intraepithelial neoplasia CIN I, CIN II and CIN III), sampling in a non-menstrual period, prohibiting sexual life within three days before examination, prohibiting repeated cleaning and medication of vagina, collecting a cervical orifice and a cervical tube shed cell sample by using a cervical tube brush, and storing the sample at-20 ℃ for later use;
the DNA extraction kit is an FFPE nucleic acid extraction or purification kit produced by Yoshikon biotechnology limited;
primers were purchased from England Weiji (Shanghai) trade Co., Ltd;
the probes were purchased from Nanjing Kinsrui Biotechnology, Inc.;
MgCl2purchased from Sigma company;
DNA polymerase was purchased from Takara bioengineering (Dalian) Inc.;
deoxyribonucleoside triphosphates were purchased from philippine bio-ltd;
nuclease-free water was purchased from semer feishel ltd;
the working concentration of proteinase K (protease K) is 20-40mg/mL, and the proteinase K is purchased from Saint biotechnologies (Shanghai) Co., Ltd;
example (b):
a novel cervical cancer methylation gene detection method is shown in figure 1, and comprises the following steps:
the method comprises the following steps: extracting DNA, namely selecting an FFPE nucleic acid extraction or purification kit produced by Yongkang Biotechnology limited, wherein the extraction steps are strictly operated according to a specification;
the kit comprises lysis solution, binding solution, washing solution I, washing solution II and eluent;
the lysis solution: the cracking binding solution contains 200-450g/L guanidinium isothiocyanate, 5-15g/L citric acid, 5-30g/L trisodium citrate, 1-10g/L sodium chloride, 10-50% Triton X-100 by volume and 10-50% isopropanol by volume;
the binding liquid includes: 70-80% isopropanol and 12-234M NaCl;
the washing solution I comprises: 60-90% ethanol and 15-60mM Tris-HCl;
the washing solution II comprises: 60-90% ethanol and 5-15% isopropanol;
the eluent comprises: Tris-HCl 0-5g/L and EDTA-2Na 0.2-5g/L, wherein the pH value of the eluent is 7.0-8.5;
(1) extraction of sample DNA: adding 1.5mL of cervical exfoliated cell sample into a clean EP tube, centrifuging at 12000rpm for 2min, and removing supernatant;
sequentially adding 20 mu L of proteinase K and 180 mu L of lysis solution into the obtained precipitate, vortexing and shaking for 10s, incubating at 56 ℃ for 30min, and lysing cells;
(2) adding 200 mu L of binding solution and 200 mu L of absolute ethyl alcohol into a centrifuge tube, and immediately carrying out vortex oscillation and uniform mixing; part of white insoluble precipitate can be generated in the process, but the later DNA extraction is not influenced;
(3) adding the mixed solution obtained in the last step into an adsorption column, centrifuging at 8000rpm (or 6000 Xg) for 2min at room temperature, pouring out waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again;
(4) adding 500 μ L of washing solution I into adsorption column, centrifuging at 8000rpm (or 6000 × g) for 1min, removing waste liquid in the collection tube, and placing the adsorption column back into the collection tube;
(5) adding 500 μ L of washing solution II into adsorption column, centrifuging at 8000rpm (or 6000 Xg) for 1min, removing waste liquid in the collection tube, and placing the adsorption column back into the collection tube;
(6) repeating the previous step;
(7) placing the adsorption column back into the collecting tube, centrifuging at 12000rpm (or 13400 Xg) for 2min, uncovering the adsorption column, and standing at room temperature for 5min to completely remove residual ethanol in the adsorption column;
(8) transferring the adsorption column into a new centrifuge tube, adding 30-100 μ L of eluent dropwise into the middle of the membrane, standing at room temperature for 2-5min, centrifuging at 12000rpm (or 13400 × g) for 2min, and storing the centrifuge tube with collected DNA at-20 deg.C;
step two: DNA transformation, namely transforming the extracted DNA by using a DNA transformation kit of the Yongkang Biotechnology limited company, wherein the transformation step is strictly operated according to a specification;
(1) adding 50 mu L of sample DNA into a 200uL centrifuge tube (the total amount of DNA is less than or equal to 2 mu g), and when the sample DNA is less than 50 mu L, filling the volume to 50 mu L with sterilized deionized water (self-prepared);
(2) adding 30 μ L bisulfite protective agent (PA-1) and 70 μ L bisulfite conversion reagent (conversion agent A), vortex-shaking for 30sec, and performing instantaneous centrifugation;
reaction procedure: 1. at 95 ℃ for 3 min; 2. at 95 ℃ for 1 min; 3. 10min at 80 ℃; 3 cycles; keeping the temperature at 25 ℃;
(3) adding 500 μ L Binding Solution (BS) (it must be confirmed that absolute ethanol has been added before first use according to the label), adding 5 μ L Acryl Carrier stock solution when the total amount of bisulfite converted DNA is less than 100ng DNA, shaking and mixing for 10sec, and centrifuging instantaneously;
transferring the solution into an adsorption column (the adsorption column is placed in a collecting pipe), centrifuging for 1min at 10000 Xg (or 12000rpm), pouring off waste liquid in the collecting pipe, and placing the adsorption column back into the collecting pipe again;
(4) adding 200 μ L of Washing Solution (WS), centrifuging at 10000 × g (or 12000rpm) for 1min, pouring off waste liquid in the centrifuge tube, and replacing the adsorption column in the collection tube;
(5) adding 700 μ L of prepared desulfonation reagent (DB), standing at room temperature for 10min, centrifuging at 10000 Xg (or 12000rpm) for 1min, pouring off waste liquid in a centrifuge tube, and putting the adsorption column back into the collection tube;
(6) adding 400 μ L of Washing Solution (WS), centrifuging at 10000 × g (or 12000rpm) for 1min, pouring off waste liquid in the centrifuge tube, and replacing the adsorption column in the collection tube;
(7) adding 400 μ L of Washing Solution (WS), and centrifuging at 10000 × g (or 12000rpm) for 1 min; then centrifuging again at 10000 Xg (or 12000rpm) for 1min, and discarding the collection tube;
(8) placing the centrifugal column into a clean centrifugal tube (EP tube), opening the cover of the adsorption column, and placing in a constant temperature oscillator or metal bath at 60 deg.C for 5 min;
(9) adding 50. mu.L of Eluent (EB) or ddH2O, standing at room temperature for 1min, centrifuging at 10000 Xg (or 12000rpm) for 1min, and removing the adsorption column to obtain DNA;
diluting the transformed bis-DNA to 500pg/μ L;
step three: performing probe fluorescent quantitative PCR amplification on bis-DNA by using PCR amplification solution;
wherein, the PCR reaction solution comprises the following components:
the PCR reaction solution product described in this example consists of 3 parts, including a detection solution, a negative control, and a positive control;
the detection solution comprises 9 systems:
the system 1-5 is a 4-fold system, the system 6-9 is a 5-fold system, and the systems comprise the following components:
the system 1 is a 4-fold system and is used for detecting genes PAX1, FAM19A4, hsa-mir-124-2 and ACTB;
the system 2 is a 4-fold system and is used for detecting genes PAX1, FAM19A4, HAS1 and ACTB;
the system 3 is a 4-fold system and is used for detecting genes PAX1, FAM19A4, ATP10A and ACTB;
the system 4 is a 4-fold system and is used for detecting genes PAX1, hsa-mir-124-2, HAS1 and ACTB;
the system 5 is a 4-fold system and is used for detecting genes PAX1, hsa-mir-124-2, ATP10A and ACTB;
the system 6 is a 5-fold system and is used for detecting genes PAX1, FAM19A4, hsa-mir124-2, ATP10A and ACTB;
the system 7 is a 5-fold system and is used for detecting genes PAX1, FAM19A4, hsa-mir124-2, HAS1 and ACTB;
the system 8 is a 5-fold system and is used for detecting genes PAX1, FAM19A4, ATP10A, HAS1 and ACTB;
system 9 is a 5-fold system and is used to detect genes PAX1, hsa-mir124-2, ATP10A, HAS1 and ACTB.
The detection liquid is mainly used for detecting the expression quantity of related genes;
the negative control is used for detecting whether the environment is polluted or not;
the positive control is mainly used for verifying whether the quality of the detection liquid is qualified or not;
wherein the PCR amplification solution comprises the following components in terms of the concentration of substances:
system 1:
first, positive chain of PAX 1: 0.25 μ M PAX1 forward primer, 0.25 μ M PAX1 reverse primer, 0.2 μ M PAX1 detection probe (fluorophore-FAM), 0.35 μ M PAX1 blocking probe;
II, PAX1 negative chain: 0.25 μ M PAX1 forward primer, 0.25 μ M PAX1 reverse primer, 0.2 μ M PAX1 detection probe (fluorophore-FAM), 0.35 μ M PAX1 blocking probe;
third, FAM19a4 positive strand: 0.2 μ M FAM19A4 forward primer, 0.2 μ M FAM19A4 reverse primer, 0.2 μ M FAM19A4 detection probe (fluorophore-ROX), 0.4 μ M FAM19A4 blocking probe;
fourth, FAM19a4 negative strand: 0.2 μ M FAM19A4 forward primer, 0.2 μ M FAM19A4 reverse primer, 0.2 μ M FAM19A4 detection probe (fluorophore-ROX), 0.4 μ M FAM19A4 blocking probe;
fifthly, hsa-mir124-2 positive strand: 0.15 μ M hsa-mir124-2 forward primer, 0.15 μ M hsa-mir124-2 reverse primer, 0.15 μ M hsa-mir124-2 detection probe (fluorophore-CY 5), 0.25 μ M hsa-mir124-2 blocking probe;
sixthly, hsa-mir124-2 negative strand: 0.15 μ M hsa-mir124-2 forward primer, 0.15 μ M hsa-mir124-2 reverse primer, 0.15 μ M hsa-mir124-2 detection probe (fluorophore-CY 5), 0.25 μ M hsa-mir124-2 blocking probe;
seventhly, ACTB plus chain: 0.2 μ M ACTB forward primer, 0.2 μ M ACTB reverse primer, 0.2 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe;
eight, ACTB minus strand: 0.2 μ M ACTB forward primer, 0.2 μ M ACTB reverse primer, 0.2 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe;
system 2:
first, positive chain of PAX 1: 0.30 μ M PAX1 forward primer, 0.30 μ M PAX1 reverse primer, 0.25 μ M PAX1 detection probe (fluorophore-FAM), 0.30 μ M PAX1 blocking probe;
II, PAX1 negative chain: 0.30 μ M PAX1 forward primer, 0.30 μ M PAX1 reverse primer, 0.25 μ M PAX1 detection probe (fluorophore-FAM), 0.30 μ M PAX1 blocking probe;
third, FAM19a4 positive strand: 0.25 μ M FAM19A4 forward primer, 0.25 μ M FAM19A4 reverse primer, 0.25 μ M FAM19A4 detection probe (fluorophore-ROX), 0.35 μ M FAM19A4 blocking probe;
fourth, FAM19a4 negative strand: 0.25 μ M FAM19A4 forward primer, 0.25 μ M FAM19A4 reverse primer, 0.25 μ M FAM19A4 detection probe (fluorophore-ROX), 0.35 μ M FAM19A4 blocking probe;
five, HAS1 positive strand: 0.35 μ M HAS1 forward primer, 0.35 μ M HAS1 reverse primer, 0.35 μ M HAS1 detection probe (fluorophore-FAM), 0.4 μ M HAS1 blocking probe;
six, HAS1 negative strand: 0.35 μ M HAS1 forward primer, 0.35 μ M HAS1 reverse primer, 0.35 μ M HAS1 detection probe (fluorophore-FAM), 0.4 μ M HAS1 blocking probe;
seventhly, ACTB plus chain: 0.2 μ M ACTB forward primer, 0.2 μ M ACTB reverse primer, 0.2 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe;
eight, ACTB minus strand: 0.2. mu.M ACTB forward primer, 0.2. mu.M ACTB reverse primer, 0.2. mu.M ACTB detection probe (fluorophore-JOE), 0.3. mu.M ACTB blocking probe.
System 3:
first, positive chain of PAX 1: 0.30 μ M PAX1 forward primer, 0.30 μ M PAX1 reverse primer, 0.25 μ M PAX1 detection probe (fluorophore-FAM), 0.30 μ M PAX1 blocking probe;
II, PAX1 negative chain: 0.30 μ M PAX1 forward primer, 0.30 μ M PAX1 reverse primer, 0.25 μ M PAX1 detection probe (fluorophore-FAM), 0.30 μ M PAX1 blocking probe;
third, FAM19a4 positive strand: 0.25 μ M FAM19A4 forward primer, 0.25 μ M FAM19A4 reverse primer, 0.25 μ M FAM19A4 detection probe (fluorophore-ROX), 0.35 μ M FAM19A4 blocking probe;
fourth, FAM19a4 negative strand: 0.25 μ M FAM19A4 forward primer, 0.25 μ M FAM19A4 reverse primer, 0.25 μ M FAM19A4 detection probe (fluorophore-ROX), 0.35 μ M FAM19A4 blocking probe;
fifth, ATP10A positive strand: 0.35 μ M ATP10A forward primer, 0.35 μ M ATP10A reverse primer, 0.35 μ M ATP10A detection probe (fluorophore-FAM), 0.4 μ M ATP10A blocking probe;
sixthly, ATP10A negative chain: 0.35 μ M ATP10A forward primer, 0.35 μ M ATP10A reverse primer, 0.35 μ M ATP10A detection probe (fluorophore-FAM), 0.4 μ M ATP10A blocking probe;
seventhly, ACTB plus chain: 0.2 μ M ACTB forward primer, 0.2 μ M ACTB reverse primer, 0.2 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe;
eight, ACTB minus strand: 0.2. mu.M ACTB forward primer, 0.2. mu.M ACTB reverse primer, 0.2. mu.M ACTB detection probe (fluorophore-JOE), 0.3. mu.M ACTB blocking probe.
System 4:
first, positive chain of PAX 1: 0.22. mu.M PAX1 forward primer, 0.22. mu.M PAX1 reverse primer, 0.22. mu.M PAX1 detection probe (fluorophore-FAM), 0.35. mu.M PAX1 blocking probe;
II, PAX1 negative chain: 0.22. mu.M PAX1 forward primer, 0.22. mu.M PAX1 reverse primer, 0.22. mu.M PAX1 detection probe (fluorophore-FAM), 0.35. mu.M PAX1 blocking probe;
thirdly, hsa-mir124-2 positive strand: 0.2 μ M hsa-mir124-2 forward primer, 0.2 μ M hsa-mir124-2 reverse primer, 0.15 μ M hsa-mir124-2 detection probe (fluorophore-CY 5), 0.25 μ M hsa-mir124-2 blocking probe;
fourthly, hsa-mir124-2 negative strand: 0.15 μ M hsa-mir124-2 forward primer, 0.15 μ M hsa-mir124-2 reverse primer, 0.15 μ M hsa-mir124-2 detection probe (fluorophore-CY 5), 0.25 μ M hsa-mir124-2 blocking probe;
five, HAS1 positive strand: 0.35 μ M HAS1 forward primer, 0.35 μ M HAS1 reverse primer, 0.35 μ M HAS1 detection probe (fluorophore-FAM), 0.4 μ M HAS1 blocking probe;
six, HAS1 negative strand: 0.35 μ M HAS1 forward primer, 0.35 μ M HAS1 reverse primer, 0.35 μ M HAS1 detection probe (fluorophore-FAM), 0.4 μ M HAS1 blocking probe;
seventhly, ACTB plus chain: 0.2 μ M ACTB forward primer, 0.2 μ M ACTB reverse primer, 0.2 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe;
eight, ACTB minus strand: 0.2. mu.M ACTB forward primer, 0.2. mu.M ACTB reverse primer, 0.2. mu.M ACTB detection probe (fluorophore-JOE), 0.3. mu.M ACTB blocking probe.
System 5:
first, positive chain of PAX 1: 0.22. mu.M PAX1 forward primer, 0.22. mu.M PAX1 reverse primer, 0.22. mu.M PAX1 detection probe (fluorophore-FAM), 0.35. mu.M PAX1 blocking probe;
II, PAX1 negative chain: 0.22. mu.M PAX1 forward primer, 0.22. mu.M PAX1 reverse primer, 0.22. mu.M PAX1 detection probe (fluorophore-FAM), 0.35. mu.M PAX1 blocking probe;
thirdly, hsa-mir124-2 positive strand: 0.2 μ M hsa-mir124-2 forward primer, 0.2 μ M hsa-mir124-2 reverse primer, 0.15 μ M hsa-mir124-2 detection probe (fluorophore-CY 5), 0.25 μ M hsa-mir124-2 blocking probe;
fourthly, hsa-mir124-2 negative strand: 0.15 μ M hsa-mir124-2 forward primer, 0.15 μ M hsa-mir124-2 reverse primer, 0.15 μ M hsa-mir124-2 detection probe (fluorophore-CY 5), 0.25 μ M hsa-mir124-2 blocking probe;
fifth, ATP10A positive strand: 0.35 μ M HAS1 forward primer, 0.35 μ M HAS1 reverse primer, 0.35 μ M HAS1 detection probe (fluorophore-FAM), 0.4 μ M HAS1 blocking probe;
sixthly, ATP10A negative chain: 0.35 μ M HAS1 forward primer, 0.35 μ M HAS1 reverse primer, 0.35 μ M HAS1 detection probe (fluorophore-FAM), 0.4 μ M HAS1 blocking probe;
seventhly, ACTB plus chain: 0.25 μ M ACTB forward primer, 0.25 μ M ACTB reverse primer, 0.25 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe;
eight, ACTB minus strand: 0.25 μ M ACTB forward primer, 0.25 μ M ACTB reverse primer, 0.25 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe.
System 6:
first, positive chain of PAX 1: 0.22. mu.M PAX1 forward primer, 0.22. mu.M PAX1 reverse primer, 0.22. mu.M PAX1 detection probe (fluorophore-FAM), 0.35. mu.M PAX1 blocking probe;
II, PAX1 negative chain: 0.22. mu.M PAX1 forward primer, 0.22. mu.M PAX1 reverse primer, 0.22. mu.M PAX1 detection probe (fluorophore-FAM), 0.35. mu.M PAX1 blocking probe;
third, FAM19a4 positive strand: 0.25 μ M FAM19A4 forward primer, 0.25 μ M FAM19A4 reverse primer, 0.25 μ M FAM19A4 detection probe (fluorophore-ROX), 0.35 μ M FAM19A4 blocking probe;
fourth, FAM19a4 negative strand: 0.25 μ M FAM19A4 forward primer, 0.25 μ M FAM19A4 reverse primer, 0.25 μ M FAM19A4 detection probe (fluorophore-ROX), 0.35 μ M FAM19A4 blocking probe;
fifthly, hsa-mir124-2 positive strand: 0.2 μ M hsa-mir124-2 forward primer, 0.2 μ M hsa-mir124-2 reverse primer, 0.15 μ M hsa-mir124-2 detection probe (fluorophore-CY 5), 0.25 μ M hsa-mir124-2 blocking probe;
sixthly, hsa-mir124-2 negative strand: 0.15 μ M hsa-mir124-2 forward primer, 0.15 μ M hsa-mir124-2 reverse primer, 0.15 μ M hsa-mir124-2 detection probe (fluorophore-CY 5), 0.25 μ M hsa-mir124-2 blocking probe;
seventhly, ATP10A positive strand: 0.35 μ M HAS1 forward primer, 0.35 μ M HAS1 reverse primer, 0.35 μ M HAS1 detection probe (fluorophore-FAM), 0.4 μ M HAS1 blocking probe;
eight, ATP10A negative strand: 0.35 μ M HAS1 forward primer, 0.35 μ M HAS1 reverse primer, 0.35 μ M HAS1 detection probe (fluorophore-FAM), 0.4 μ M HAS1 blocking probe;
nine, ACTB plus strand: 0.25 μ M ACTB forward primer, 0.25 μ M ACTB reverse primer, 0.25 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe;
ten, ACTB minus strand: 0.25 μ M ACTB forward primer, 0.25 μ M ACTB reverse primer, 0.25 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe.
System 7:
first, positive chain of PAX 1: 0.30 μ M PAX1 forward primer, 0.30 μ M PAX1 reverse primer, 0.30 μ M PAX1 detection probe (fluorophore-FAM), 0.30 μ M PAX1 blocking probe;
II, PAX1 negative chain: 0.30 μ M PAX1 forward primer, 0.30 μ M PAX1 reverse primer, 0.30 μ M PAX1 detection probe (fluorophore-FAM), 0.30 μ M PAX1 blocking probe;
third, FAM19a4 positive strand: 0.25 μ M FAM19A4 forward primer, 0.25 μ M FAM19A4 reverse primer, 0.25 μ M FAM19A4 detection probe (fluorophore-ROX), 0.35 μ M FAM19A4 blocking probe;
fourth, FAM19a4 negative strand: 0.25 μ M FAM19A4 forward primer, 0.25 μ M FAM19A4 reverse primer, 0.25 μ M FAM19A4 detection probe (fluorophore-ROX), 0.35 μ M FAM19A4 blocking probe;
fifthly, hsa-mir124-2 positive strand: 0.2 μ M hsa-mir124-2 forward primer, 0.2 μ M hsa-mir124-2 reverse primer, 0.15 μ M hsa-mir124-2 detection probe (fluorophore-CY 5), 0.25 μ M hsa-mir124-2 blocking probe;
sixthly, hsa-mir124-2 negative strand: 0.15 μ M hsa-mir124-2 forward primer, 0.15 μ M hsa-mir124-2 reverse primer, 0.15 μ M hsa-mir124-2 detection probe (fluorophore-CY 5), 0.25 μ M hsa-mir124-2 blocking probe;
seventh, HAS1 positive strand: 0.35 μ M HAS1 forward primer, 0.35 μ M HAS1 reverse primer, 0.35 μ M HAS1 detection probe (fluorophore-FAM), 0.4 μ M HAS1 blocking probe;
eight, HAS1 negative strand: 0.35 μ M HAS1 forward primer, 0.35 μ M HAS1 reverse primer, 0.35 μ M HAS1 detection probe (fluorophore-FAM), 0.4 μ M HAS1 blocking probe;
nine, ACTB plus strand: 0.25 μ M ACTB forward primer, 0.25 μ M ACTB reverse primer, 0.25 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe;
ten, ACTB minus strand: 0.25 μ M ACTB forward primer, 0.25 μ M ACTB reverse primer, 0.25 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe.
System 8:
first, positive chain of PAX 1: 0.30 μ M PAX1 forward primer, 0.30 μ M PAX1 reverse primer, 0.30 μ M PAX1 detection probe (fluorophore-FAM), 0.30 μ M PAX1 blocking probe;
II, PAX1 negative chain: 0.30 μ M PAX1 forward primer, 0.30 μ M PAX1 reverse primer, 0.30 μ M PAX1 detection probe (fluorophore-FAM), 0.30 μ M PAX1 blocking probe;
third, FAM19a4 positive strand: 0.25 μ M FAM19A4 forward primer, 0.25 μ M FAM19A4 reverse primer, 0.25 μ M FAM19A4 detection probe (fluorophore-ROX), 0.35 μ M FAM19A4 blocking probe;
fourth, FAM19a4 negative strand: 0.25 μ M FAM19A4 forward primer, 0.25 μ M FAM19A4 reverse primer, 0.25 μ M FAM19A4 detection probe (fluorophore-ROX), 0.35 μ M FAM19A4 blocking probe;
fifth, ATP10A positive strand: 0.2 μ M ATP10A forward primer, 0.2 μ M ATP10A reverse primer, 0.15 μ M ATP10A detection probe (fluorophore-CY 5), 0.25 μ M ATP10A blocking probe;
sixthly, ATP10A negative chain: 0.15 μ M ATP10A forward primer, 0.15 μ M ATP10A reverse primer, 0.15 μ M ATP10A detection probe (fluorophore-CY 5), 0.25 μ M ATP10A blocking probe;
seventh, HAS1 positive strand: 0.33. mu.M HAS1 forward primer, 0.33. mu.M HAS1 reverse primer, 0.33. mu.M HAS1 detection probe (fluorophore-FAM), 0.50. mu.M HAS1 blocking probe;
eight, HAS1 negative strand: 0.33. mu.M HAS1 forward primer, 0.33. mu.M HAS1 reverse primer, 0.33. mu.M HAS1 detection probe (fluorophore-FAM), 0.50. mu.M HAS1 blocking probe;
nine, ACTB plus strand: 0.25 μ M ACTB forward primer, 0.25 μ M ACTB reverse primer, 0.25 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe;
ten, ACTB minus strand: 0.25 μ M ACTB forward primer, 0.25 μ M ACTB reverse primer, 0.25 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe.
System 9:
first, positive chain of PAX 1: 0.30 μ M PAX1 forward primer, 0.30 μ M PAX1 reverse primer, 0.30 μ M PAX1 detection probe (fluorophore-FAM), 0.30 μ M PAX1 blocking probe;
II, PAX1 negative chain: 0.30 μ M PAX1 forward primer, 0.30 μ M PAX1 reverse primer, 0.30 μ M PAX1 detection probe (fluorophore-FAM), 0.30 μ M PAX1 blocking probe;
thirdly, hsa-mir124-2 positive strand: 0.20 μ M hsa-mir124-2 forward primer, 0.20 μ M hsa-mir124-2 reverse primer, 0.20 μ M hsa-mir124-2 detection probe (fluorophore-ROX), 0.30 μ M hsa-mir124-2 blocking probe;
fourthly, hsa-mir124-2 negative strand: 0.20 μ M hsa-mir124-2 forward primer, 0.20 μ M hsa-mir124-2 reverse primer, 0.20 μ M hsa-mir124-2 detection probe (fluorophore-ROX), 0.30 μ M hsa-mir124-2 blocking probe;
fifth, ATP10A positive strand: 0.2 μ M ATP10A forward primer, 0.2 μ M ATP10A reverse primer, 0.15 μ M ATP10A detection probe (fluorophore-CY 5), 0.25 μ M ATP10A blocking probe;
sixthly, ATP10A negative chain: 0.15 μ M ATP10A forward primer, 0.15 μ M ATP10A reverse primer, 0.15 μ M ATP10A detection probe (fluorophore-CY 5), 0.25 μ M ATP10A blocking probe;
seventh, HAS1 positive strand: 0.33. mu.M HAS1 forward primer, 0.33. mu.M HAS1 reverse primer, 0.33. mu.M HAS1 detection probe (fluorophore-FAM), 0.50. mu.M HAS1 blocking probe;
eight, HAS1 negative strand: 0.33. mu.M HAS1 forward primer, 0.33. mu.M HAS1 reverse primer, 0.33. mu.M HAS1 detection probe (fluorophore-FAM), 0.50. mu.M HAS1 blocking probe;
nine, ACTB plus strand: 0.25 μ M ACTB forward primer, 0.25 μ M ACTB reverse primer, 0.25 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe;
ten, ACTB minus strand: 0.25 μ M ACTB forward primer, 0.25 μ M ACTB reverse primer, 0.25 μ M ACTB detection probe (fluorophore-JOE), 0.3 μ M ACTB blocking probe.
System 1 also contains the corresponding ion concentrations: 0.3-0.6. mu.M deoxyribonucleoside triphosphate, MgCl2The concentration is 5-15mM, the concentration of DNA polymerase is 0.1-0.3U/mu L, the proportion of glycerol is 1-5%, and the solvent of the PCR amplification solution is nuclease-free water;
system 2 also contains the corresponding ion concentrations: 0.3-0.7 μ M deoxyribonucleoside triphosphate, MgCl2The concentration is 5-10mM, the concentration of DNA polymerase is 0.1-0.4U/muL, the proportion of glycerol is 1-4%, and the solvent of the PCR amplification solution is nuclease-free water;
system 3 also contains the corresponding ion concentrations: 0.4-0.7 μ M deoxyribonucleoside triphosphate, MgCl2The concentration is 7-10mM, the concentration of DNA polymerase is 0.2-0.4U/mu L, the proportion of glycerol is 3-5%, and the solvent of the PCR amplification solution is nuclease-free water;
system 4 also contains the corresponding ion concentrations: 0.5-0.7 μ M deoxyribonucleoside triphosphate, MgCl2The concentration is 7-12mM, the concentration of DNA polymerase is 0.3-0.6U/mu L, the proportion of glycerol is 2-7%, and the solvent of the PCR amplification solution is nuclease-free water;
system 5 also contains the corresponding ion concentrations: 0.6-0.8 μ M deoxyribonucleoside triphosphate, MgCl2The concentration is 9-12mM, the concentration of DNA polymerase is 0.1-0.3U/mu L, the proportion of glycerol is 2-7%, and the solvent of the PCR amplification solution is nuclease-free water;
system 6 also contains the corresponding ion concentrations: 0.4-0.8 μ M deoxyribonucleoside triphosphate, MgCl2The concentration is 5-12mM, the concentration of DNA polymerase is 0.1-0.25U/muL, the proportion of glycerol is 3-7%, and the solvent of the PCR amplification solution is nuclease-free water;
system 7 also contains the corresponding ion concentrations: 0.4-0.6. mu.M deoxyribonucleoside triphosphate, MgCl2The concentration is 5-8mM, the concentration of DNA polymerase is 0.2-0.3U/mu L, the proportion of glycerol is 3-10%, and the solvent of the PCR amplification solution is nuclease-free water;
system 8 also contains the corresponding ion concentrations: 0.5-0.7 μ M deoxyribonucleoside triphosphate, MgCl2The concentration is 4-9mM, the concentration of DNA polymerase is 0.25-0.40U/muL, the proportion of glycerol is 5-8%, and the solvent of the PCR amplification solution is nuclease-free water;
system 9 also contains the corresponding ion concentrations: 0.45-0.65 μ M deoxyribonucleoside triphosphate, MgCl2The concentration is 5-10mM, the concentration of DNA polymerase is 0.30-0.45U/muL, the proportion of glycerol is 5-10%, and the solvent of the PCR amplification solution is nuclease-free water;
the positive control and the negative control are respectively a negative and positive cell line, the concentration is respectively 500 pg/mu L, and the sequences of the primer and the probe are shown in the following table 1;
TABLE 1
Figure BDA0003233508650000181
Figure BDA0003233508650000191
The reaction conditions for the amplification are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15S, annealing at 62 ℃ for 5S, and annealing and extension at 58 ℃ for 40S (fluorescence is collected during annealing and extension), for 50 cycles; storing at 4 deg.C;
determining the methylation value of a sample to be detected according to the result of the fluorescent quantitative PCR amplification of the probe; according to 1, PAX1, FAM19A4, hsa-mir-124-2; 2. PAX1, FAM19a4, HAS 1; 3. PAX1, FAM19a4, ATP 10A; 4. PAX1, hsa-mir-124-2, HAS 1; 5. PAX1, hsa-mir-124-2, ATP 10A; 6. PAX1, FAM19A4, hsa-mir124-2, ATP 10A; 7. PAX1, FAM19A4, hsa-mir124-2, HAS 1; 8. PAX1, FAM19a4, ATP10A, HAS 1; 9. PAX1, hsa-mir124-2, ATP10A and HAS 1. Determining the methylation value of the sample to be detected according to the relative fluorescence CT value of the PCR amplification result of the ACTB gene;
extracting genome DNA of cervical exfoliated cells of 10 cervical cancer patients and 10 non-cervical cancer control populations according to the implementation method, then performing bisulfite transformation on the extracted genome DNA, respectively performing PCR amplification (ABI 7500 fluorescence quantitative instrument of ThermoFisher company) on the target and ACTB genes contained in 9 systems by taking the DNA after sulfite transformation as a template according to the PCR reaction system and the reaction conditions of the embodiment, and respectively calculating methylation values of the target contained in the 9 systems relative to the ACTB gene, wherein specific results are shown in the following table 2;
TABLE 2-1 (System 1)
Figure BDA0003233508650000201
Figure BDA0003233508650000211
TABLE 2-2 (System 2)
Serial number Mean methylation of cervical cancer group Mean methylation of non-cervical cancer group
1 14.58 0.68
2 14.73 0.32
3 17.64 0.24
4 13.27 0.16
5 15.23 0.58
6 16.87 0.61
7 15.71 0.23
8 13.19 0.19
9 17.87 0.10
10 14.67 0.14
Tables 2-3 (System 3)
Figure BDA0003233508650000212
Figure BDA0003233508650000221
Tables 2-4 (System 4)
Serial number Mean methylation of cervical cancer group Mean methylation of non-cervical cancer group
1 16.18 0.13
2 14.20 0.13
3 13.55 0.29
4 15.15 0.56
5 17.95 0.75
6 18.06 0.76
7 15.60 0.17
8 13.14 0.59
9 14.56 0.22
10 14.18 0.30
Tables 2-5 (System 5)
Figure BDA0003233508650000222
Figure BDA0003233508650000231
Tables 2-6 (System 6)
Serial number Mean methylation of cervical cancer group Mean methylation of non-cervical cancer group
1 14.50 0.28
2 13.53 0.76
3 15.53 0.67
4 13.67 0.44
5 17.90 0.46
6 13.20 0.66
7 14.23 0.54
8 16.01 0.19
9 14.41 0.70
10 17.42 0.43
Tables 2-7 (System 7)
Figure BDA0003233508650000232
Figure BDA0003233508650000241
Tables 2-8 (System 8)
Serial number Mean methylation of cervical cancer group Mean methylation of non-cervical cancer group
1 12.89 0.41
2 15.24 0.66
3 18.19 0.33
4 18.30 0.09
5 15.04 0.66
6 17.79 0.72
7 13.83 0.52
8 16.95 0.77
9 12.76 0.64
10 14.87 0.44
Tables 2-9 (System 9)
Figure BDA0003233508650000242
Figure BDA0003233508650000251
According to the test data, cervical cells 1, PAX1, FAM19A4 and hsa-mir-124-2 of the cervical cancer patient; 2. PAX1, FAM19a4, HAS 1; 3. PAX1, FAM19a4, ATP 10A; 4. PAX1, hsa-mir-124-2, HAS 1; 5. PAX1, hsa-mir-124-2, ATP 10A; 6. PAX1, FAM19A4, hsa-mir124-2, ATP 10A; 7. PAX1, FAM19A4, hsa-mir124-2, HAS 1; 8. PAX1, FAM19a4, ATP10A, HAS 1; 9. PAX1, hsa-mir124-2, ATP10A and HAS 1. Compared with the average value of the methylation value of the ACTB gene, the average value of the methylation value of the cervical cell above system of non-cervical cancer is above 12, and the average value of the methylation value of the ACTB gene above system of non-cervical cancer is below 0.9, namely, the methylation value of the cervical cancer patient is remarkably higher than that of the cervical cell of a non-cervical cancer control 1, PAX1, FAM19A4 and hsa-mir-124-2; 2. PAX1, FAM19a4, HAS 1; 3. PAX1, FAM19a4, ATP 10A; 4. PAX1, hsa-mir-124-2, HAS 1; 5. PAX1, hsa-mir-124-2, ATP 10A; 6. PAX1, FAM19A4, hsa-mir124-2, ATP 10A; 7. PAX1, FAM19A4, hsa-mir124-2, HAS 1; 8. PAX1, FAM19a4, ATP10A, HAS 1; 9. PAX1, hsa-mir124-2, ATP10A and HAS 1. Compared with the ACTB gene average value (P <0.01), the detection method can accurately distinguish cervical cancer patients from non-cervical cancer controls, so that the PAX1, FAM19A4, hsa-mir124-2, ATP10A and HAS1 genes can be used as auxiliary methods for detecting cervical cancer biomarkers.

Claims (7)

1. A novel cervical cancer methylation gene detection method is characterized by comprising the following steps:
extracting genome DNA of cervical exfoliated cells;
step two, performing bisulfite conversion on the extracted genome DNA to obtain bis-DNA;
performing probe fluorescent quantitative PCR amplification on bis-DNA by using a PCR amplification solution, specifically:
designing forward and reverse primers, a detection probe and a specific blocking probe aiming at a bis-DNA positive strand and a bis-DNA negative strand respectively, specifically binding a methylation sequence target gene PAX1, FAM19A4, hsa-mir124-2, ATP10A, HAS1 and an internal reference ACTB (beta-actin) gene by using the detection probe, and binding an unmethylated sequence by using the specific blocking probe to avoid the binding with the primers;
and step four, determining the methylation value of the sample to be detected according to the relative fluorescence CT values of the PCR amplification results of the PAX1, FAM19A4, hsa-mir124-2, ATP10A, HAS1 and ACTB genes, and respectively calculating the methylation values of the PAX1, FAM19A4, hsa-mir124-2, ATP10A and HAS1 relative to the ACTB genes.
2. The method of claim 1, wherein the cervical cells PAX1, FAM19A4, hsa-mir124-2, ATP10A and HAS1 have an average value of 12 or more with respect to the methylation value of the ACTB gene, and thus can be determined as a suspected cervical cancer patient.
3. The method of claim 1, wherein the cervical cells PAX1, FAM19A4, hsa-mir124-2, ATP10A and HAS1 have an average value of 0.9 or less relative to the methylation value of the ACTB gene, and thus can be determined as non-cervical cancer.
4. The method for detecting the methylation gene of the cervical cancer according to claim 1, wherein the amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15S, annealing at 62 ℃ for 5S, and annealing at 58 ℃ for 40S for 50 cycles.
5. The method for detecting the methylation gene of the cervical cancer according to claim 4, wherein the PCR reaction solution consists of 3 parts, including a detection solution, a negative control, and a positive control;
the detection solution comprises 9 systems, wherein the systems 1-5 are 4-fold systems, the systems 6-9 are 5-fold systems, and the systems comprise the following components:
the system 1 is a 4-fold system and is used for detecting genes PAX1, FAM19A4, hsa-mir-124-2 and ACTB;
the system 2 is a 4-fold system and is used for detecting genes PAX1, FAM19A4, HAS1 and ACTB;
the system 3 is a 4-fold system and is used for detecting genes PAX1, FAM19A4, ATP10A and ACTB;
the system 4 is a 4-fold system and is used for detecting genes PAX1, hsa-mir-124-2, HAS1 and ACTB;
the system 5 is a 4-fold system and is used for detecting genes PAX1, hsa-mir-124-2, ATP10A and ACTB;
the system 6 is a 5-fold system and is used for detecting genes PAX1, FAM19A4, hsa-mir124-2, ATP10A and ACTB;
the system 7 is a 5-fold system and is used for detecting genes PAX1, FAM19A4, hsa-mir124-2, HAS1 and ACTB;
the system 8 is a 5-fold system and is used for detecting genes PAX1, FAM19A4, ATP10A, HAS1 and ACTB;
system 9 is a 5-fold system and is used to detect genes PAX1, hsa-mir124-2, ATP10A, HAS1 and ACTB.
6. The method for detecting a methylation gene of cervical cancer according to claim 5, wherein the detection solution further comprises: 0.3. mu.M deoxyribonucleoside triphosphate, MgCl2The concentration is 5mM, the DNA polymerase is concentratedThe content was 0.1U/. mu.L, and the ratio of glycerin was 1%.
7. The method for detecting a methylated gene of cervical cancer according to claim 6, wherein the solvent of the PCR amplification solution is nuclease-free water.
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CN114214415A (en) * 2021-12-29 2022-03-22 广州安必平医药科技股份有限公司 Primer probe combination for methylation detection of cervical cancer related genes and application thereof
CN114672568A (en) * 2022-04-27 2022-06-28 亚能生物技术（深圳）有限公司 Kit for detecting cervical cell gene methylation
CN114672568B (en) * 2022-04-27 2023-01-24 亚能生物技术（深圳）有限公司 Kit for detecting cervical cell gene methylation

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Application publication date: 20211026