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CN114561461B - Composition for detecting cervical cancer, kit and application thereof - Google Patents

Composition for detecting cervical cancer, kit and application thereof Download PDF

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CN114561461B
CN114561461B CN202011356693.XA CN202011356693A CN114561461B CN 114561461 B CN114561461 B CN 114561461B CN 202011356693 A CN202011356693 A CN 202011356693A CN 114561461 B CN114561461 B CN 114561461B
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CN114561461A (en
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邵琦
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Anhui Dajian Medical Technology Co ltd
GUANGZHOU TARGENE BIOTECH CO Ltd
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GUANGZHOU TARGENE BIOTECH CO Ltd
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Abstract

The invention provides a composition for detecting cervical cancer and a kit and application thereof, wherein a bisulphite modification method is adopted to carry out bisulphite conversion on a nucleic acid sample of the cervical cancer to be detected, a fluorescent quantitative PCR technology is combined, a literature research result, a TCGA methylation chip database and a transcriptome sequencing expression profile are comprehensively analyzed, cervical cancer hypermethylation candidate genes are screened through multiple data filtering analysis, specific gene methylation detection primers and probes are designed aiming at a plurality of methylation detection sites on the cervical cancer hypermethylation candidate genes, more than 10 methylation CpG sites are covered, a DNA sample to be detected which is modified by the bisulphite is amplified through a multiple PCR amplification technology, the methylation condition of a target gene in the sample to be detected is determined according to a PCR amplification result, and the sensitivity and the specificity of the kit are improved through multiple ways, so that early screening and diagnosis of the cervical cancer are realized.

Description

Composition for detecting cervical cancer, kit and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a composition for detecting cervical cancer, a kit and application thereof.
Background
Cervical cancer (cervical cancer) is one of the most common malignant tumors of the reproductive system of females, and is second among the global malignant tumors of females, and the morbidity and mortality rate are inferior to breast cancer, so that the physical and mental health of females is seriously threatened. Cervical cancer is mostly associated with high-risk human papillomavirus (HR-HPV) infection, and the onset of cervical cancer is most frequently aged 40 to 50 years, and recently, the onset of cervical cancer tends to be younger. Cervical cancer occurs in cervical regions, mostly squamous carcinoma, secondarily adenocarcinoma, adenosquamous carcinoma, rarely in the form of small cell carcinoma, clear cell carcinoma, etc. The cervical cancer is generally free from any symptoms in early stage, and the symptoms such as contact bleeding, abnormal vaginal bleeding and the like can only appear along with the progress of the disease, and the cervical cancer can be discovered only by active screening. The treatment effect of cervical cancer is closely related to clinical stage, early cancer cervical cancer usually has better prognosis, early detection of cervical cancer can increase the chance of retaining uterus in operation, the overall survival rate is improved, early diagnosis and early treatment are the keys for improving the cure rate, and meanwhile, the cervical cancer has the possibility of recurrence, so that the prognosis monitoring also needs to be considered, and the clinical significance is very important how to discover cervical cancer as early as possible and how to detect recurrence of cervical cancer after operation in time.
The current diagnostic techniques for cervical cancer are: 1) Cervical scraping cytology examination: is easily influenced by subjective factors, has certain false negative rate and is easy to leak diagnosis; 2) Imaging technology: MRI, B-ultrasonic and CT, but is not sensitive enough to smaller tumors and cannot be clearly diagnosed; 3) Colposcopy: whether the cancer is observed or not can not be observed by naked eyes, and the method is also not applicable to patients with vaginal stenosis, cervical surface necrosis and hemorrhage; 4) Cervical biopsy: the operation is complex, the requirement on the diagnosis level of a pathological diagnosis doctor is high, the grading standard is fuzzy, and the highly atypical cells (HSIL) and the low atypical cells (LSIL) are difficult to accurately distinguish. The invention adopts the latest methylation specific real-time fluorescence PCR (MSP-PCR) technology, can realize the extraction of genome from cervical exfoliated cells, and detects the methylation condition of cervical cancer related genes, so that the collection of samples is more convenient; the experimental result is stable, the repeatability is good, the method is very suitable for periodic inspection of high-risk groups and early diagnosis of related groups, and a new thought and method are provided for early diagnosis of cervical cancer.
Epigenetic is a hot field of recent tumor research, and methylation of DNA, histone modification, chromatin remodeling, non-coding RNA regulation and other epigenetic changes are considered to have close relation with tumor occurrence, wherein the methylation of DNA is the most common epigenetic change, can regulate cell proliferation, apoptosis and differentiation, and the level is closely related with the biological characteristics of the tumor. The research at present shows that cervical cancer is the same as other cancers and is the long-term result of various cancerogenic factors, the pathological change process is a complex process of multi-gene mutation accumulation, and abnormal methylation of various oncogenes and cancer suppressor genes is involved, wherein most abnormal methylation is hypermethylation of the cancer suppressor genes, and the hypermethylation often leads to transcriptional silencing of the cancer suppressor genes. DNA methylation abnormalities usually occur in early cancer and throughout the course of cancer development and progression, the methylation state of which changes once it is established that it requires prolonged continuous stimulation by the external environment, so that the detection of DNA methylation indicators can be used as important biological indicators for diagnosis, early screening and prognosis of cancer.
Methods for DNA methylation detection can be broadly divided into two categories: whole genome methylation analysis and specific site methylation detection. The whole genome methylation analysis has higher detection cost, and is often used as a means for screening and finding target genes with high flux; the specific site methylation detection method comprises a restriction enzyme analysis method (COBRA) combined with sodium bisulphite, a methylation specificity PCR Method (MSP), a methylation fluorescence quantification method (Methyllight), a methylation sensitivity high-resolution melting curve analysis method and the like, wherein the restriction enzyme analysis method can only obtain the methylation condition of a special enzyme cutting site, the methylation specificity PCR method is complicated in operation and easy to cause sample pollution based on common PCR and electrophoresis analysis, the methylation sensitivity high-resolution melting curve analysis method has high requirements on instruments, a fluorescent quantitative PCR instrument with a high-resolution melting (HRM) module is needed, and the methylation fluorescence quantification method is based on high flux and high sensitivity, does not need operations such as electrophoresis and hybridization after PCR, reduces pollution and operation errors, and is widely applied to DNA methylation detection. In the current methylation fluorescent quantitative method for detecting cervical cancer DNA methylation, the detection accuracy aiming at a single gene is not ideal, the diagnosis effect is limited, researchers often improve the detection sensitivity by combining the combined detection of a plurality of genes, but the combined detection of the plurality of genes can possibly lead to the reduction of the specificity, and meanwhile, if the detection is carried out by adopting a single-tube single-gene test mode, more reagent is required, the operation of experimenters is increased, and the experiment cost is higher.
Therefore, there is an urgent need in the market to develop a noninvasive cervical cancer detection method that is stable and reliable and has high sensitivity and specificity.
Disclosure of Invention
In view of the above, the invention provides a composition for detecting cervical cancer, a kit and application thereof, which are characterized in that a bisulphite modification method is adopted to carry out bisulphite conversion on a nucleic acid sample of cervical cancer to be detected, a fluorescent quantitative PCR technology is combined, a literature research result, a TCGA methylation chip database and a transcriptome sequencing expression profile are comprehensively analyzed, cervical cancer hypermethylation candidate genes are screened through multiple data filtering analysis, specific gene methylation detection primers and probes are designed aiming at a plurality of methylation detection sites on the cervical cancer hypermethylation candidate genes, more than 10 methylation CpG sites are covered, a DNA sample to be detected which is modified by the bisulphite is amplified through a multiple PCR amplification technology, the methylation condition of a target gene in the sample to be detected is determined according to a PCR amplification result, and the sensitivity and the specificity of the kit are improved through multiple ways, so that early screening and diagnosis of the cervical cancer are realized.
To achieve the above object, the first aspect of the present invention provides a cervical cancer gene methylation detection site, wherein the gene methylation detection site comprises one or more of PCDH10 and LMX1A, ZNF 582.
The second aspect of the invention provides a PCR primer probe combination for methylation detection of cervical cancer genes, which comprises one or more of the following nucleic acid sequence combinations shown in 1) -3):
1) The PCR primer and the probe for PCDH10 methylation detection comprise one or more of a primer probe combination 1, a primer probe combination 2 and a primer probe combination 3, wherein the primer probe combination 1 comprises an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2 and a fluorescent probe shown as SEQ ID NO.3, the primer probe combination 2 comprises an upstream primer shown as SEQ ID NO.4, a downstream primer shown as SEQ ID NO.5 and SEQ ID NO.6 and a fluorescent probe shown as SEQ ID NO.7, and the primer probe combination 3 comprises an upstream primer shown as SEQ ID NO.8, a downstream primer shown as SEQ ID NO.9 and SEQ ID NO.10 and a fluorescent probe shown as SEQ ID NO. 11;
2) The PCR primer and the probe for LMX1A methylation detection comprise one or more of a primer probe combination 4, a primer probe combination 5 and a primer probe combination 6, wherein the primer probe combination 4 comprises an upstream primer shown as SEQ ID NO.12, a downstream primer shown as SEQ ID NO.13 and a fluorescent probe shown as SEQ ID NO.14, the primer probe combination 5 comprises an upstream primer shown as SEQ ID NO.15, a downstream primer shown as SEQ ID NO.16 and a fluorescent probe shown as SEQ ID NO.17, and the primer probe combination 6 comprises an upstream primer shown as SEQ ID NO.18, a downstream primer shown as SEQ ID NO.19 and a fluorescent probe shown as SEQ ID NO. 20;
3) The PCR primer and the probe for the ZNF582 methylation detection comprise one or more of a primer probe combination 7, a primer probe combination 8 and a primer probe combination 9, wherein the primer probe combination 7 comprises an upstream primer shown as SEQ ID NO.21, a downstream primer shown as SEQ ID NO.22 and a fluorescent probe shown as SEQ ID NO.23, the primer probe combination 8 comprises an upstream primer shown as SEQ ID NO.24, a downstream primer shown as SEQ ID NO.25 and a fluorescent probe shown as SEQ ID NO.26, and the primer probe combination 9 comprises an upstream primer shown as SEQ ID NO.27, a downstream primer shown as SEQ ID NO.28 and a fluorescent probe shown as SEQ ID NO. 29.
In an embodiment of the invention, the PCR primer probe combination for methylation detection of cervical cancer genes further comprises a PCR primer for detecting an internal reference gene GAPDH and a probe, wherein the primer comprises an upstream primer shown as SEQ ID NO.30 and a downstream primer shown as SEQ ID NO.31, and the probe comprises a fluorescent probe shown as SEQ ID NO. 32.
In an embodiment of the present invention, the 5' end of the fluorescent probe includes a fluorescent reporter group, including any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY and CY 5.
In one embodiment of the invention, the 3' end of the fluorescent probe comprises a fluorescence quenching group, including any one of MGB, BHQ-1, BHQ-2 and BHQ-3.
In a preferred embodiment of the invention, the fluorescence quenching group is MGB.
The third aspect of the invention provides a cervical cancer gene methylation detection kit, which comprises the PCR primer probe combination according to the second aspect of the invention, and further comprises a positive quality control product and a negative quality control product.
In an embodiment of the invention, the positive quality control product is cervical cancer tissue DNA.
In an embodiment of the invention, the negative quality control is leukocyte DNA.
In one embodiment of the invention, the final concentration composition of the cervical cancer gene methylation detection kit reaction system comprises: 0.1-1. Mu.M PCR primer and 0.1-1. Mu.M probe.
In a preferred embodiment of the present invention, the final concentration composition of the cervical cancer gene methylation detection kit reaction system comprises: 0.1-0.5. Mu.MPCR primer, 0.1-0.5. Mu.M probe.
In one embodiment of the invention, the fluorescent quantitative PCR reaction conditions of the cervical cancer gene methylation detection kit are as follows:
in one embodiment of the invention, the fluorescent quantitative PCR reaction conditions of the cervical cancer gene methylation detection kit are as follows:
a fourth aspect of the present invention provides a method for detecting methylation of cervical cancer genes, comprising the steps of:
1) Isolating nucleic acid of a target gene in a biological sample to be tested;
2) Subjecting the nucleic acid obtained in step 1) to bisulfite conversion treatment to obtain bisulfite-converted DNA (Bis-DNA);
3) Detecting the methylation state of the Bis-DNA obtained in the step 2) by adopting a methylation fluorescent quantitative PCR technology.
In one embodiment of the present invention, the biological sample in step 1) includes one of a tissue and a cell.
In a preferred embodiment of the invention, the biological sample of step 1) comprises cervical exfoliated cells.
In a fifth aspect, the invention provides the application of the cervical cancer gene methylation detection site according to the first aspect of the invention, the PCR primer probe combination for cervical cancer gene methylation detection according to the second aspect of the invention, the cervical cancer gene methylation detection kit according to the third aspect of the invention or the detection method for cervical cancer gene methylation detection according to the fourth aspect of the invention in preparation of the cervical cancer detection kit.
The invention has the following beneficial effects:
1) Can be used as an important index for early screening, process monitoring and prognosis evaluation of cervical cancer: the cervical cancer gene methylation detection kit provided by the invention takes DNA methylation abnormality as a detection object, the DNA methylation abnormality usually occurs in early cancer and penetrates through the occurrence and development processes of the cancer, and the methylation state of the cervical cancer gene methylation detection kit can be changed once the methylation state is formed and needs to be continuously stimulated by the external environment for a long time, so that the detection of DNA methylation indexes can be used as important biological indexes for early screening, process monitoring and prognosis evaluation of cervical cancer;
2) Noninvasive detection: the cervical cancer gene methylation detection kit provided by the invention can assist in diagnosing cervical cancer by detecting the methylation state of cervical cancer related genes in cervical exfoliated cells, so that noninvasive detection is realized;
3) Single tube multiplex gene methylation detection: and joint inspection of single-tube multiple gene methylation detection sites is established, so that reagent consumption is reduced, consumable cost is lowered, and meanwhile, operation steps of experimenters are reduced, and labor cost is lowered.
4) The accuracy is high: the cervical cancer gene methylation detection kit provided by the invention screens cervical cancer hypermethylation candidate genes through multiple data analysis, designs specific gene methylation detection primers and probes aiming at a plurality of methylation detection sites on the cervical cancer hypermethylation candidate genes, covers more than 10 methylation CpG sites, adopts a multiple PCR amplification technology to amplify a DNA sample to be detected modified by bisulfite, carries out joint detection by combining methylation conditions of different genes or detects a plurality of different methylation detection regions of a single gene, and simultaneously, in order to further improve detection sensitivity, designs two upstream primers or two downstream primers for one methylation detection region of the single gene in a targeted manner, and improves the sensitivity and the specificity of the kit through multiple ways so as to realize accurate detection of early detection and diagnosis of cervical cancer.
Drawings
FIG. 1 is a positive diagram of typical detection results of methylation detection of cervical cancer genes, provided by the embodiment of the invention, wherein the CT value of the methylation detection sites of the genes is less than or equal to 25 and less than the CT value of the reference genes, and the detection results are judged to be positive;
FIG. 2 is a positive diagram of another typical detection result of methylation detection of cervical cancer genes, provided by the embodiment of the invention, wherein the CT value of the methylation detection sites of the genes is less than or equal to 25 and greater than that of the internal reference genes, the CT value is less than or equal to 5, and the result is judged to be positive;
FIG. 3 is a negative graph of typical detection results of methylation detection of cervical cancer genes, wherein the CT value of the methylation detection sites of the genes is larger than that of the reference genes and delta CT value is larger than 5, and the detection results are negative.
Detailed Description
The present invention is described in detail below by way of specific examples to enable those skilled in the art to readily practice the invention in light of the present disclosure. The embodiments described below are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless specifically stated otherwise, reagents, methods and apparatus employed in the present invention are those conventional in the art, and experimental methods without specifying specific conditions are generally carried out under conventional conditions or conditions suggested by the manufacturer.
According to the invention, a bisulphite modification method is adopted to carry out bisulphite conversion on a cervical cancer nucleic acid sample to be detected, a fluorescent quantitative PCR technology is combined, a literature research result, a TCGA methylation chip database and a transcriptome sequencing expression spectrum are comprehensively analyzed, cervical cancer hypermethylation candidate genes are screened through multiple data filtering analysis, a specific gene methylation detection primer and a specific gene are designed, a DNA sample to be detected which is modified by the bisulphite is amplified, the methylation condition of a target gene in the sample to be detected is determined according to a PCR amplification result, and early screening and diagnosis of cervical cancer are realized.
The gene methylation detection site of the cervical cancer gene methylation detection kit provided by the invention comprises one or more of PCDH10 and LMX1A, ZNF 582.
The PCR primer probe combination of the cervical cancer gene methylation detection kit provided by the invention comprises one or more of the following nucleic acid sequence combinations shown in 1) -3):
1) The PCR primer and the probe for PCDH10 methylation detection comprise one or more of a primer probe combination 1, a primer probe combination 2 and a primer probe combination 3, wherein the primer probe combination 1 comprises an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2 and a fluorescent probe shown as SEQ ID NO.3, the primer probe combination 2 comprises an upstream primer shown as SEQ ID NO.4, a downstream primer shown as SEQ ID NO.5 and SEQ ID NO.6 and a fluorescent probe shown as SEQ ID NO.7, and the primer probe combination 3 comprises an upstream primer shown as SEQ ID NO.8, a downstream primer shown as SEQ ID NO.9 and SEQ ID NO.10 and a fluorescent probe shown as SEQ ID NO. 11;
2) The PCR primer and the probe for LMX1A methylation detection comprise one or more of a primer probe combination 4, a primer probe combination 5 and a primer probe combination 6, wherein the primer probe combination 4 comprises an upstream primer shown as SEQ ID NO.12, a downstream primer shown as SEQ ID NO.13 and a fluorescent probe shown as SEQ ID NO.14, the primer probe combination 5 comprises an upstream primer shown as SEQ ID NO.15, a downstream primer shown as SEQ ID NO.16 and a fluorescent probe shown as SEQ ID NO.17, and the primer probe combination 6 comprises an upstream primer shown as SEQ ID NO.18, a downstream primer shown as SEQ ID NO.19 and a fluorescent probe shown as SEQ ID NO. 20.
3) The PCR primer and the probe for the ZNF582 methylation detection comprise one or more of a primer probe combination 7, a primer probe combination 8 and a primer probe combination 9, wherein the primer probe combination 7 comprises an upstream primer shown as SEQ ID NO.21, a downstream primer shown as SEQ ID NO.22 and a fluorescent probe shown as SEQ ID NO.23, the primer probe combination 8 comprises an upstream primer shown as SEQ ID NO.24, a downstream primer shown as SEQ ID NO.25 and a fluorescent probe shown as SEQ ID NO.26, and the primer probe combination 9 comprises an upstream primer shown as SEQ ID NO.27, a downstream primer shown as SEQ ID NO.28 and a fluorescent probe shown as SEQ ID NO. 29.
In an embodiment of the invention, the PCR primer probe combination for methylation detection of cervical cancer genes further comprises a PCR primer for detecting an internal reference gene GAPDH and a probe, wherein the primer comprises an upstream primer shown as SEQ ID NO.30 and a downstream primer shown as SEQ ID NO.31, and the probe comprises a fluorescent probe shown as SEQ ID NO. 32.
Preferably, the 5' end of the fluorescent probe comprises a fluorescent reporter group, including any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY and CY 5.
Preferably, the 3' end of the fluorescent probe comprises a fluorescence quenching group, including any one of MGB, BHQ-1, BHQ-2 and BHQ-3.
Further preferably, the fluorescence quenching group is MGB.
The cervical cancer gene methylation detection kit provided by the invention detects one of tissues and cells.
The cervical cancer gene methylation detection kit provided by the invention judges and reads detection results, and comprises the following steps:
1) Threshold setting: the device can automatically output according to the instrument, or manually adjust the base line according to the use instruction of the instrument, set the threshold value in the linear part of the fluorescence value logarithmic graph, derive data from software and read CT value.
2) And (3) judging the effectiveness of the kit:
the internal reference gene of the negative quality control product is amplified, the CT value is less than or equal to 25, and the methylation detection site of the gene is not amplified; the positive quality control internal reference gene and the gene methylation detection site are amplified, and the CT value is less than or equal to 25.
3) Sample validity determination:
a) The internal reference gene is amplified and the CT value is less than or equal to 25, so that the analysis can be continued;
b) The CT value of the internal reference gene is more than or equal to 25 or no amplification, but the CT value of the methylation detection site of the gene is less than or equal to 25, so that the analysis can be continued;
c) The CT value of the reference gene is >25 or no amplification, the methylation detection site of the gene is no amplification or amplification but the CT value is >25, the analysis cannot be continued, repeated detection is needed, and resampling detection is needed if the CT value of the reference gene is >25 or no amplification is detected repeatedly.
4) Determination of methylation detection result (Δct value=gene methylation detection site CT value-internal reference gene CT value)
a) The CT value of the gene methylation detection site is less than or equal to 25, if the CT value of the gene methylation detection site is less than the CT value of the reference gene or the reference gene is not amplified, the result is positive; if the CT value of the gene methylation detection site is larger than the CT value of the internal reference gene, the delta CT value is less than or equal to 5, and the result is judged to be positive;
b) The gene methylation detection site is not amplified, or the CT value of the gene methylation detection site is larger than that of the reference gene and delta CT value is more than 5, and the result is negative.
In order to more clearly demonstrate the technical solution of the present invention, the present invention will be further described below with reference to specific examples.
Example 1: sample DNA extraction and bisulfite conversion
1. Treatment of cervical exfoliated cell sample and extraction of DNA
1) Sample collection:
cervical exfoliated cell sample collection procedure was as follows: a medical staff exposes the cervical by using a vaginal speculum, and the secretion with excessive cervical opening is wiped by using a cotton swab. Gong Gengshua is placed at cervical orifice, and rotated for 5 times in one direction to obtain sufficient epithelial cell sample, then the cervical brush head is placed in sample tube containing cell preservation solution, the cervical brush handle is broken along the crease of the brush handle, the brush head is left in the sample tube, and the tube cover is screwed to make sample mark
2) DNA extraction:
cervical exfoliated cell DNA was extracted by using a nucleic acid extraction or purification reagent (general type) produced by anhuida medical science and technology limited, specifically as follows:
(a) Centrifuging a cervical exfoliated cell preservation solution sample to be detected at 4000rpm for 15min, sucking the waste supernatant by a sample-adding gun, and adding 500 mu L of cell preservation solution;
(b) Taking the cell sample obtained in the step a, adding 500 mu L of lysate A,30 mu L of proteinase K into each tube, and performing cleavage at 70 ℃ for 40min;
(c) Centrifuging briefly, adding 200 μl of isopropanol into each tube, mixing thoroughly, and centrifuging briefly;
(d) C, adding all the liquid in the step c into an adsorption column, and centrifuging for 30s at full speed;
(e) Washing once with 600. Mu.L of rinse solution I, centrifuging at full speed for 30s;
(f) Rinsing once with 600. Mu.L of rinse solution II, centrifuging at full speed for 30s;
(g) Rinsing with 600 μl of rinsing liquid II once again, and centrifuging at full speed for 2min;
(h) The centrifuge column was dried in the air in a suction cabinet for 3min, 60. Mu.L of eluent preheated at 70℃was added in suspension, left standing at room temperature for 5min, centrifuged at 13000rpm for 2min, and DNA was collected into a centrifuge tube and stored at-20 ℃.
2. Bisulfite conversion:
the genomic DNA obtained in the step was subjected to bisulfite conversion by using a nucleic acid extraction or purification reagent (centrifugation column) produced by Anhuida medical science, inc., having the steps of:
(a) 45. Mu.L of the DNA sample to be tested is taken in a new 1.5mL centrifuge tube and 5. Mu.L of conversion buffer is added and incubated in a metal bath at 37℃for 15min.
(b) After the incubation is completed, 100 mu L of the prepared conversion solution is added into each sample, the mixture is uniformly mixed and centrifuged for a short time, and the metal bath is incubated for 12 to 16 hours at 50 ℃ in a dark place
(c) The sample is placed on ice (0-4 ℃) for incubation for 10min
(d) Placing the adsorption column in a collecting tube, adding 400 μL of binding solution into the adsorption column
(e) Adding the sample obtained in step c into adsorption column (containing binding solution), covering the tube, mixing, centrifuging at full speed (14000 rpm) for 30s, and discarding the waste liquid
(f) Adding 100 μl of the rinse solution into the adsorption column, centrifuging at full speed for 30s, and discarding the waste liquid
(g) Adding 200 mu L of desulfonation liquid into an adsorption column, incubating for 20min at room temperature (20-30 ℃), centrifuging for 30s at full speed, and discarding waste liquid
(h) Adding 200 μl of the rinse solution into the adsorption column, centrifuging at full speed for 30s, repeatedly adding 200 μl of the rinse solution, centrifuging at full speed for 30s, discarding the waste liquid, and collecting the tube
(i) Placing the adsorption column into a 1.5mL sterile centrifuge tube, suspending and dripping 30 mu L of eluent into the middle part of the adsorption film, eluting and transforming DNA, centrifuging at full speed for 1min, collecting Bis-DNA, and preserving at-20 ℃.
Example 2: cervical cancer hypermethylation candidate gene, specific primer and probe screening
1. Screening of cervical cast cell hypermethylation candidate genes of cervical cancer patients
And comprehensively analyzing a literature research result, a TCGA methylation chip database and a transcriptome sequencing expression profile, screening methylation sites with obvious differences, and finally screening and determining PCDH10 and LMX1A, ZNF582 as cervical cancer hypermethylation candidate genes through multiple data filtering analysis.
2. Primer probe combination screening for methylation detection of cervical cancer
1) Specific primer and probe screening:
according to the nucleic acid sequences of PCDH10 and LMX1A, ZNF582, methylation primers and probes are designed on Methyl primerExpress v 1.0.0 software, PCR probes and primers for related gene methylation are obtained through screening by repeated design and push of the applicant, and the designed primers and probes are sent to Beijing Rui Boxing family biotechnology Co., ltd for synthesis, and specific sequences are shown in the following table:
meanwhile, a specific primer and a probe aiming at the reference gene GAPDH are arranged, and the specific sequence is as follows:
name of the name Sequence (5 '-3')
MethyGAPDH-F AAGTTAGGTTAGTTTGGTAGGGAAGTT(SEQ ID NO.30)
Methy-GAPDH-R AACCCTAAACCACCTCCCC(SEQ ID NO.31)
Methy-GAPDH-P TTTGGGTTTTTTTGGGGGTAAGGAGATGT(SEQ ID NO.32)
Wherein the 5 'end of the probe sequence is modified with a fluorescent group selected from any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY and CY5, and the 3' end is marked with a fluorescence quenching group selected from any one of MGB, BHQ-1, BHQ-2 and BHQ-3.
Example 3: methylation fluorescent quantitative PCR (polymerase chain reaction) amplification detection of cervical cancer hypermethylation candidate gene
1. The reaction system of methylation fluorescent quantitative PCR is as follows: 2 XPCR reaction premix solution 7.5. Mu.L, 10. Mu.M GAPDH primer and probe each 0.1. Mu.L, 10. Mu.M primer in the primer probe combinations each 0.5. Mu.L, probe each 0.2. Mu.L, 3. Mu.L Bis-DNA, and water was added to 15. Mu.L.
2. Reaction conditions for methylation fluorescent quantitative PCR
3. Detection result interpretation of methylation fluorescent quantitative PCR
1) Threshold setting: the device can automatically output according to the instrument, or manually adjust the base line according to the use instruction of the instrument, set the threshold value in the linear part of the fluorescence value logarithmic graph, derive data from software and read CT value.
2) And (3) judging the effectiveness of the kit:
the internal reference gene of the negative quality control product is amplified, the CT value is less than or equal to 25, and the methylation detection site of the gene is not amplified; the positive quality control internal reference gene and the gene methylation detection site are amplified, and the CT value is less than or equal to 25.
3) Sample validity determination:
a) The internal reference gene is amplified and the CT value is less than or equal to 25, so that the analysis can be continued;
b) The CT value of the internal reference gene is more than or equal to 25 or no amplification, but the CT value of the methylation detection site of the gene is less than or equal to 25, so that the analysis can be continued;
c) The CT value of the reference gene is >25 or no amplification, the methylation detection site of the gene is no amplification or amplification but the CT value is >25, the analysis cannot be continued, repeated detection is needed, and resampling detection is needed if the CT value of the reference gene is >25 or no amplification is detected repeatedly.
4) Determination of methylation detection result (Δct value=gene methylation detection site CT value-internal reference gene CT value)
a) The CT value of the gene methylation detection site is less than or equal to 25, if the CT value of the gene methylation detection site is less than the CT value of the reference gene or the reference gene is not amplified, the result is positive; if the CT value of the gene methylation detection site is larger than the CT value of the internal reference gene, the delta CT value is less than or equal to 5, and the result is judged to be positive;
b) The gene methylation detection site is not amplified, or the CT value of the gene methylation detection site is larger than that of the reference gene and delta CT value is more than 5, and the result is negative.
Example 4: clinical sample detection and verification kit effect
1. Cervical exfoliated cell sample detection
According to the experimental procedures described in the above examples 1, 2 and 3, the clinical cervical exfoliated cell samples were subjected to detection verification of the effect of the kit, and in order to verify the detection effect of the primer probe combinations for methylation detection of cervical cancer genes, 22 cervical exfoliated cell samples and 8 normal female cervical exfoliated cell samples were detected by using the primer probe combinations in the above example 2, respectively, wherein numbers 1 to 22 are cervical exfoliated cell samples, 23 to 30 are normal female cervical exfoliated cell samples, and under the condition that the negative quality control and the positive quality control meet the validity of the kit, typical methylation detection result amplification diagrams are shown in fig. 1, 2 and 3, the detailed results are shown in the following table, "+" represents detection positive, "-" represents detection negative:
based on the above results, the statistical analysis is as follows:
from the results, the detection sensitivity of the single primer probe combination in the cervical cancer gene methylation detection kit provided by the invention is 72.7-86.4%, and the specificity is 87.5-100%.
In order to further improve the performance of the kit, by combining the primer probe combinations described above, it is desirable to improve the detection effect of the kit in a multiplex assay, preferably the following combinations:
meanwhile, the combined detection effect is carried out by respectively combining three groups of primer probes of PCDH10, LMX1A or ZNF582 as follows:
the result is that the single-tube detection analysis based on the cervical cancer single-gene single detection site is obtained, the detection mode needs to consume more reagents, meanwhile, the operation of experimenters is increased, the experiment cost is high, the reagent consumption can be reduced to a great extent, the consumable cost is reduced, meanwhile, the operation of experimenters is reduced, and the labor cost is reduced. Therefore, the invention establishes the preferential combination of single-tube multiplex gene methylation detection site joint inspection for further screening cervical cancer gene methylation detection kit, takes cervical cancer cervical exfoliated cell samples and DNA samples of cervical cancer cervical exfoliated cell samples as templates, tests the detection effect of single-tube multiplex gene methylation detection site joint inspection, and results are as follows:
the results show that the detection sensitivity and the specificity of part of single-tube multiplex gene methylation detection site joint detection are reduced to a certain extent, so that the primer probe combinations 2, 4 and 8, the primer probe combinations 2, 6 and 7, the primer probe combinations 3, 4 and 7 and the primer probe combinations 1, 2 and 3 are preferable for further verification.
2. Tissue sample detection
By detecting the tissue sample with the above preferred combination, the detection effect thereof on the tissue sample is detected, with the following results:
the result shows that the cervical cancer gene methylation detection kit provided by the invention has the detection sensitivity of 90-100%, the specificity of 100%, and higher detection sensitivity and detection specificity when detecting cervical cancer tissue sample gene methylation.
In conclusion, the cervical cancer gene methylation detection kit provided by the invention has higher detection sensitivity and detection specificity, and is ideal for diagnosis and early screening of cervical cancer, and early diagnosis and early treatment of cervical cancer are assisted.
While the foregoing description illustrates and describes several preferred embodiments of the invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the invention described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
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Claims (8)

1. The PCR primer probe combination for detecting cervical cancer gene methylation is characterized by comprising the following components:
1) The PCR primer and probe for PCDH10 methylation detection comprises a primer probe combination 2 or a primer probe combination 3, wherein the primer probe combination 2 comprises an upstream primer shown as SEQ ID NO.4, a downstream primer shown as SEQ ID NO.5 and SEQ ID NO.6 and a fluorescent probe shown as SEQ ID NO.7, and the primer probe combination 3 comprises an upstream primer shown as SEQ ID NO.8, a downstream primer shown as SEQ ID NO.9 and SEQ ID NO.10 and a fluorescent probe shown as SEQ ID NO. 11;
the PCR primer and probe for LMX1A methylation detection comprises a primer probe combination 4 or a primer probe combination 6, wherein the primer probe combination 4 comprises an upstream primer shown as SEQ ID NO.12, a downstream primer shown as SEQ ID NO.13 and a fluorescent probe shown as SEQ ID NO.14, and the primer probe combination 6 comprises an upstream primer shown as SEQ ID NO.18, a downstream primer shown as SEQ ID NO.19 and a fluorescent probe shown as SEQ ID NO. 20;
the PCR primer and the probe for the ZNF582 methylation detection comprise a primer probe combination 7 or a primer probe combination 8, wherein the primer probe combination 7 comprises an upstream primer shown as SEQ ID NO.21, a downstream primer shown as SEQ ID NO.22 and a fluorescent probe shown as SEQ ID NO.23, and the primer probe combination 8 comprises an upstream primer shown as SEQ ID NO.24, a downstream primer shown as SEQ ID NO.25 and a fluorescent probe shown as SEQ ID NO. 26;
or (b)
2) The PCR primer and probe for PCDH10 methylation detection comprises a primer probe combination 1, a primer probe combination 2 and a primer probe combination 3, wherein the primer probe combination 1 comprises an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2 and a fluorescent probe shown as SEQ ID NO.3, the primer probe combination 2 comprises an upstream primer shown as SEQ ID NO.4, a downstream primer shown as SEQ ID NO.5 and SEQ ID NO.6 and a fluorescent probe shown as SEQ ID NO.7, and the primer probe combination 3 comprises an upstream primer shown as SEQ ID NO.8, a downstream primer shown as SEQ ID NO.9 and SEQ ID NO.10 and a fluorescent probe shown as SEQ ID NO. 11.
2. The PCR primer probe combination for methylation detection of cervical cancer genes according to claim 1, further comprising a PCR primer for detecting GAPDH of the internal reference gene and a probe, wherein the primer comprises an upstream primer shown as SEQ ID No.30 and a downstream primer shown as SEQ ID No.31, and the probe comprises a fluorescent probe shown as SEQ ID No. 32.
3. The PCR primer probe set for methylation detection of cervical cancer according to claim 1 or claim 2, wherein the 5' end of the fluorescent probe contains a fluorescent reporter group comprising any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY, CY 5.
4. The PCR primer probe set for methylation detection of cervical cancer according to claim 1 or claim 2, wherein the 3' end of the fluorescent probe contains a fluorescence quenching group comprising any one of MGB, BHQ-1, BHQ-2, BHQ-3.
5. A cervical cancer gene methylation detection kit, which is characterized by comprising the PCR primer probe combination according to claim 1, and further comprising a positive quality control and a negative quality control.
6. The cervical cancer gene methylation detection kit according to claim 5, wherein the final concentration composition of the cervical cancer gene methylation detection kit reaction system comprises: 0.1-1. Mu.M PCR primer and 0.1-1. Mu.M probe.
7. The detection method for the purpose of non-disease diagnosis and treatment for methylation detection of cervical cancer genes is characterized by comprising the following steps of:
1) Isolating nucleic acid of a target gene in a biological sample to be tested;
2) Subjecting the nucleic acid obtained in the step 1) to bisulfite conversion treatment to obtain bisulfite converted DNA (Bis-DNA);
3) Detecting the methylation state of the Bis-DNA obtained in the step 2) by adopting the PCR primer probe combination and methylation fluorescence quantitative PCR technology according to claim 1.
8. The use of the PCR primer probe combination for methylation detection of cervical cancer genes according to claim 1 in the preparation of a kit for detection of cervical cancer.
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