CN117265096A - Kit for cervical high-grade lesions and early detection of cervical cancer - Google Patents

Kit for cervical high-grade lesions and early detection of cervical cancer Download PDF

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CN117265096A
CN117265096A CN202311150999.3A CN202311150999A CN117265096A CN 117265096 A CN117265096 A CN 117265096A CN 202311150999 A CN202311150999 A CN 202311150999A CN 117265096 A CN117265096 A CN 117265096A
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gene
seq
primer
methylation
pax1
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朱钧
白冰
张记军
袁耀
金鑫
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Beijing Mokobio Life Science Co ltd
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Beijing Mokobio Life Science Co ltd
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The invention relates to a nucleic acid combination for cervical cell gene methylation detection, which comprises PAX1 gene methylation primers and probes, SOX1 gene methylation primers and probes, FAM19A4 gene methylation primers and probes. The nucleic acid combination of the primer and the probe in the kit has higher specificity and sensitivity to methylation of cervical cell genes, is simple and convenient to operate, and provides wide application prospects for clinical diagnosis.

Description

Kit for cervical high-grade lesions and early detection of cervical cancer
Technical Field
The invention belongs to the technical field of nucleic acid in-vitro diagnosis, and particularly relates to a kit for applying a specific gene methylation marker to cervical high-level lesions and early detection of cervical cancer.
Background
Worldwide, there are nearly 50 thousands of new cases of cervical cancer annually, which has become the third most common malignancy after breast and colon cancer in women. Cervical cancer is caused by HPV (Human Papilloma Virus), and can be effectively prevented by detecting HPV. Many studies have shown that HPV detection is more objective, easier to operate and has higher sensitivity than cytological examination, and thus can be used for cervical cancer primary screening. HPV infection is however common, and in most cases the infected cells do not progress to cancer; thus resulting in over-treatment of false positive patients and wasting medical resources. Along with the wide development of HPV detection projects in China, the requirements of high-specificity detection methods for diversion of positive population after HPV detection primary screening are also increasing. At present, cytological examination is the most dominant diversion method for HPV infection positive population, but cytological detection technology is greatly influenced by subjective factors and the level of doctors, and meanwhile, the consistency between cytology and pathology is greatly different due to the degree of precancerous lesions. Therefore, an objective and high-specificity detection method is found for subsequent management of HPV positive results, which is particularly important for reducing colposcopic referral and saving sanitary and medical resources.
DNA methylation is a common process in epigenetic science, and alterations in host cell gene methylation may affect cell life cycle, contributing to the development and progression of cancer. The hypermethylation of promoters at different stages can cause inactivation of different cancer suppressor genes, and the specific change modes of the promoters are provided at each stage of the occurrence and development of a plurality of tumors (including cervical cancer). The additive effect of epigenetic changes is of great importance in the progression of the cervix from normal tissue-precancerous lesions-cervical cancer. Research shows that there is strong correlation between host cervical epithelial gene methylation and invasive cervical cancer, and has great potential significance in developing new diagnosis technology and therapeutic intervention method.
The method has a higher Positive Predictive Value (PPV) for methylation detection of cervical endothelial cells, and compared with the traditional HPV detection method, the method has a higher Negative Predictive Value (NPV), but the Positive Predictive Value (PPV) is too low, so that people with high risk of cervical cancer can be screened out more accurately to intervene. Compared with cytologic examination such as TCT (Thinprep Cytologic Test) and HPV detection of double-screen positive patients, gene methylation detection can more accurately shunt out high-risk and low-risk cervical cancer people. The pre-cancerous stage of cervical intraepithelial neoplasia (Cervical intraepithelial neoplasia, CIN) still has a high proportion of crowd prognosis, and it is critical to accurately diagnose whether cervical cancer is progressed, and gene methylation detection can be used for monitoring CIN lesions, preventing over-treatment of CIN1/CIN2/CIN3, and providing effective decision basis for selecting treatment time.
In the current diagnosis and treatment process of cervical cancer, CIN2/CIN3 and higher high-grade precancerous lesions are distinguished by histology, the judgment consistency is only 0.36-0.69 due to sampling sites, pathologists and the like, and the gene methylation detection can provide a more objective detection result for a clinician to refer to under the clinical scene. In addition, for patients after cervical conization and circular cutting operations, HPV/TCT and other traditional methodologies cannot judge the risk of further developing cervical cancer of the patients, and the gene methylation detection effectively fills the clinical blank of the part.
Disclosure of Invention
The invention aims to provide a nucleic acid combination for cervical epithelial (or shedding) cell gene methylation, and a primer composition in the nucleic acid combination has better sensitivity and higher specificity on cervical cell gene methylation detection.
The invention also aims to provide a kit for early detection of cervical high-grade lesions and cervical cancer, wherein the nucleic acid combination of the primers in the kit has high sensitivity to methylation of cervical cell genes, and is easy and convenient to operate.
In one aspect of the invention, a set of nucleic acid combinations for cervical cell gene methylation detection is provided, the nucleic acid combinations comprising at least one pair selected from the group consisting of:
(1) As set forth in SEQ ID NO:1, and the forward primer is shown as SEQ ID NO: 2;
(2) As set forth in SEQ ID NO:4, and the forward primer is shown as SEQ ID NO:5, a reverse primer shown in FIG. 5;
(3) As set forth in SEQ ID NO:7, and the forward primer is shown as SEQ ID NO: 8.
In some embodiments, the nucleic acid combination further comprises a sequence selected from the group consisting of SEQ ID NOs: 3. 6 or 9.
In another aspect of the invention, there is provided the use of a nucleic acid combination as described above for the preparation of a kit for detecting cervical cell gene methylation.
In another aspect of the present invention, a kit for cervical high-grade lesions and early detection of cervical cancer is provided, which comprises a PAX1 gene methylation primer, a SOX1 gene methylation primer and a FAM19A4 gene methylation primer.
In some embodiments, the kit further comprises a PAX1 gene probe, a SOX1 gene probe, and a FAM19A4 gene probe.
In some embodiments, the kit further comprises an internal reference gene primer.
In some embodiments, the kit further comprises a reference gene probe.
In some embodiments, the reference gene is a β -actin gene.
In some embodiments, the PAX1 gene probe, SOX1 gene probe, FAM19A4 gene probe, and reference gene probe are each labeled with a fluorescent group.
In some embodiments, the fluorescent substance is selected from the group consisting of fluorescein-based dyes, rhodamine-based dyes, and cyanine dyes.
In some embodiments, the fluorophore on each probe is independently selected from ROX, FAM, or CY5.
In some embodiments, the signals carried by the fluorophores of each probe in the kit can be distinguished under the same reaction system.
The signals carried by the fluorophores that distinguish the probes in the same reaction system can be distinguished from each other by using different fluorescent channels.
In some embodiments, the 5' ends of the PAX1 gene probe, SOX1 gene probe, FAM19A4 gene probe, and internal reference gene probe are labeled with fluorescent groups, which may be the same or different, preferably selected from ROX, FAM, or CY5. The 3' -end of the fluorescent dye is marked with quenching groups which are the same or different, preferably BHQ1 or BHQ2.
In some embodiments, the forward primer of the PAX1 gene methylation primer is set forth in SEQ ID NO:1, the reverse primer is shown as SEQ ID NO: 2.
In some embodiments, the forward primer of the SOX1 gene methylation primer is set forth in SEQ ID NO:4, the reverse primer is shown as SEQ ID NO: shown at 5.
In some embodiments, the forward primer of the FAM19A4 gene methylation primer is set forth in SEQ ID NO:7, the reverse primer is shown as SEQ ID NO: shown at 8.
In some embodiments, the PAX1 gene probe is as set forth in SEQ ID NO: 3.
In some embodiments, the SOX1 gene probe is set forth in SEQ ID NO: shown at 6.
In some embodiments, the FAM19A4 gene probe is as set forth in SEQ ID NO: shown at 9.
In some embodiments, the forward primer of the beta-actin internal reference gene primer is set forth in SEQ ID NO:10, the reverse primer is shown as SEQ ID NO: 11.
In some embodiments, the reference gene probe is set forth in SEQ ID NO: shown at 12.
In some embodiments, the primers and probes comprise sequences that match SEQ ID NO:1-12, has a nucleotide sequence that is greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
In some embodiments, the kit further comprises a PCR reaction solution.
In some embodiments, the PCR reaction solution comprises a PCR enzyme system selected from the group consisting of Mg 2+ And one or more of a PCR reaction buffer.
In some embodiments, the PCR enzyme system comprises Taq DNA polymerase; the concentration of the polymerase is preferably 5U/. Mu.l.
In some embodiments, the PCR reaction buffer comprises dNTPs and Mg 2+ The method comprises the steps of carrying out a first treatment on the surface of the Preferably, the PCR reaction buffer further comprises one or more components selected from DMSO, tris-HCl, betain, EDTA, naCl and K+.
In some embodiments, the final concentration of dNTPs in the PCR reaction buffer is 0.2mM.
In some embodiments, the PCR reaction buffer comprises Mg 2+ The final concentration of (2) mM.
In some embodiments, the kit further comprises a nucleic acid extraction solution.
In some embodiments, the kit comprises a mixture of specific primers and probes for detecting methylation of the human PAX1, SOX1, FAM19A4 genes, and beta-actin internal reference gene primers and probes.
In another aspect of the present invention, a method for detecting cervical high-grade lesions and early detection of cervical cancer in a sample is provided, comprising the steps of:
(1) Extracting sample DNA;
(2) Transforming the DNA extracted in the step (1) to obtain transformed DNA;
(3) Performing PCR amplification on the converted DNA obtained in the step (2) by using a PAX1 gene methylation primer, a SOX1 gene methylation primer and a FAM19A4 gene methylation primer to obtain an amplification product, and detecting methylation levels of different fragments in the amplification product;
preferably, the PCR amplification procedure described in step (4) is as follows:
stage 1: pre-denaturation at 95 ℃ for 5 min, and circulation for 1 time;
stage 2: reacting at 95 ℃ for 10 seconds, reacting at 55 ℃ for 30 seconds, reacting at 68 ℃ for 30 seconds, and circulating for 5 times;
stage 3: the reaction was carried out at 95℃for 10 seconds, at 58℃for 30 seconds, at 68℃for 30 seconds, and the reaction was repeated 40 times.
The method may be for diagnostic or non-diagnostic purposes.
In some embodiments, the DNA extracted in step (1) of step (2) may be converted by chemical or enzymatic methods.
In some embodiments, the chemical method of step (2) is a method that converts unmethylated cytosines in a sample DNA molecule to uracil, while methylated cytosines in the DNA molecule remain.
In some embodiments, the chemical method of step (2) comprises a method of treating a sample DNA molecule with a conversion reagent.
In some embodiments, the conversion reagent is bisulphite.
In some embodiments, step (3) comprises a step of PCR amplifying the converted DNA obtained in step (2) using PAX1 gene methylation primers and PAX1 gene probes, SOX1 gene methylation primers and SOX1 gene probes, FAM19A4 gene methylation primers and FAM19A4 gene probes.
In the above detection method, the forward primer of the PAX1 gene methylation primer is shown in SEQ ID NO:1, the reverse primer is shown as SEQ ID NO:2 is shown in the figure; preferably, the PAX1 gene probe is shown in SEQ ID NO: 3.
The forward primer of the SOX1 gene methylation primer is shown as SEQ ID NO:4, the reverse primer is shown as SEQ ID NO:5 is shown in the figure; preferably, the SOX1 gene probe is as set forth in SEQ ID NO: shown at 6.
The forward primer of the FAM19A4 gene methylation primer is shown as SEQ ID NO:7, the reverse primer is shown as SEQ ID NO: shown as 8; preferably, the FAM19A4 gene probe is as shown in SEQ ID NO: shown at 9.
In some embodiments, during the PCR amplification of step (3), the PAX1 gene methylation primer, the SOX1 gene methylation primer, and the FAM19A4 gene methylation primer are pre-mixed and mixed with the converted DNA substrate obtained in step (2) in the form of a mixed solution for amplification.
In some embodiments, during the PCR amplification of step (3), the PAX1 gene methylation primers and probes, the SOX1 gene methylation primers and probes, and the FAM19A4 gene methylation primers and probes are pre-mixed and mixed with the converted DNA substrate obtained in step (2) in the form of a mixed solution for amplification.
In some embodiments, the mixed solution further comprises an internal reference gene primer and an internal reference gene probe.
In some embodiments, the forward primer of the internal reference gene primer is set forth in SEQ ID NO:10, the reverse primer is shown as SEQ ID NO: 11; the reference gene probe is preferably shown as SEQ ID NO: shown at 12.
In some embodiments, the detection method further comprises the step of performing a result interpretation based on the methylation levels of the different fragments in the amplified product.
Preferably, the result interpretation analysis method is as follows:
a. the internal standard channel is satisfied that the S-type amplification curve exists, the Ct value is less than or equal to 33, the PAX1, SOX1 and FAM19A4 gene channels are free of the S-type amplification curve or the delta Ct value of the PAX1 gene is more than 7.67, the delta Ct value of the SOX1 gene is more than 7.20, and the delta Ct value of the FAM19A4 gene is more than 7.26, and the judgment result is negative;
b. the internal standard channel is satisfied with an S-type amplification curve, the Ct value is less than or equal to 33, the delta Ct value of the PAX1 gene is less than or equal to 7.67, or the delta Ct value of the SOX1 gene is less than or equal to 7.20, or the delta Ct value of the FAM19A4 gene is less than or equal to 7.26, and at least one channel detection of the three genes satisfies the corresponding delta Ct value requirement, and the result is judged to be positive;
c. if the internal standard channel has no S-type amplification curve or Ct value is more than 33, the judgment result is invalid.
Wherein, ct value is the cycle number corresponding to the intersection point of the threshold line and the S-shaped curve.
Delta Ct = test gene Ct value-reference gene Ct value.
In another aspect, the invention provides application of the nucleic acid combination or the kit in preparation of products for detecting cervical high-grade lesions and early detection of cervical cancer or methylation of cervical cancer cells.
In some embodiments, the sample for which the nucleic acid combination, kit, or detection method is directed is selected from cervical cancer tissue or cervical exfoliated cells.
The invention has the beneficial effects that:
1. the primer probe combination has small mutual interference and high amplification efficiency.
2. The primer and the probe provided by the invention can detect various cervical lesions, are accurate and efficient, have the overall sensitivity of 90.22%, and have high specificity.
3. The kit combination can better identify cervical high-grade lesions such as CINII and precancerous lesions and cervical cancer and the like, and realize shunting with low-grade lesions and inflammations.
Drawings
FIG. 1 is a graph of the ROC obtained in example 1.
Detailed Description
I. Definition of the definition
To facilitate an understanding of the present technology, some terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.
The phrase "in one embodiment" as used herein does not necessarily refer to the same embodiment, although it may. Accordingly, as described below, the various embodiments of the present invention may be readily combined without departing from the scope or spirit of the present invention.
As used herein, the term "methylation" refers to methylation of a cytosine at position C5 or N4 of a cytosine, methylation at the N6 position of an adenine, or other types of nucleic acid methylation.
As used herein, the term "PAX1 gene methylation primer" refers to a primer for detecting PAX1 gene methylation, and as such, the term "SOX1 gene methylation primer" refers to a primer for detecting SOX1 gene methylation, and the term "FAM19A4 gene methylation primer" refers to a primer for detecting FAM19A4 gene methylation. The term "PAX1 gene probe" refers to a probe for detecting the PAX1 gene, and the remaining probes are the same.
As used herein, the term "kit" refers to any delivery system that delivers materials. In the case of a reaction assay, such delivery systems include systems that allow for storage, transport of the reaction reagents (e.g., oligonucleotides, enzymes, etc. in a suitable container) and/or support materials (e.g., buffers, written instructions to perform the assay, etc.) or delivery thereof from one location to another.
As used herein, the term "bisulfite" refers to an agent comprising bisulfite, metabisulfite, bisulfite, or a combination thereof, as disclosed herein, that is suitable for distinguishing methylated from unmethylated cytosine dinucleotide sequences. Methods of such treatment are known in the art.
As used herein, the term "methylation assay" refers to any assay for determining the methylation state of one or more CpG dinucleotide sequences within a nucleic acid sequence.
As used herein, the term "primer" refers to a fragment that recognizes a target gene sequence, including forward and reverse primer pairs, preferably primer pairs that provide analytical results with specificity and sensitivity. The nucleic acid sequence of the primer is a sequence that is not identical to a non-target sequence present in the sample, and can impart high specificity when it is a primer that amplifies only a target gene sequence including a complementary primer binding site and does not induce non-specific amplification.
As used herein, the term "probe" refers to a nucleic acid sequence that carries a detectable label and is complementary to the amplification product of interest in a known sequence.
As used herein, the term "sample" refers to any composition that contains or is presumed to contain nucleic acid from an individual. The sample to be tested is a biological sample containing cervical cancer cells.
As used herein, the term "identity" in the context of two or more nucleotide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same.
The primer and probe modification may be performed by a known method. Modified versions of these primer and/or probe sequences can include, by way of non-limiting example, adding one or more nucleotides to the 5 'end, one or more nucleotides to the 3' end, one or more nucleotides to the 5 'and 3' ends, adding tails, shortening the sequence, extending the sequence, shifting the sequence several bases upstream and downstream, or any combination thereof.
As used herein, the term "Ct value" refers to the number of cycles that each reaction tube undergoes when the fluorescent signal within that tube reaches a set threshold.
As used herein, the term "cervical high grade lesions" refers to stage CIN2 and above, including CIN2, CIN3, carcinoma in situ.
Examples II
The invention is further illustrated below with reference to examples. The description of specific exemplary embodiments of the invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the teaching of the present specification. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated.
Example 1: cervical cancer gene methylation detection site exploration
1. Cervical tissue and cervical cell brush DNA extraction
DNA from both cervical tissue and cervical cell brush was extracted using commercial kits, such as QIAamp DNA FFPE Tissue Kit from Qiagen.
2. DNA quantification
The number of extracted molecules of DNA is ensured in the extraction process for subsequent experiments. dsDNA was quantified using commercial kits and double-stranded DNA (dsDNA) concentrations were detected specifically using fluorescent dye technology. For example, invitrogen's Qubit TM dsDNA HS Assay Kit。
3. Methylation-modified transformation of DNA
The extracted DNA was transformed using a commercial kit. After bisulfite treatment, the C base on the CpG island which is methylated in the DNA still keeps the C base, and the C base on the CpG island which is not methylated is changed into the U base, so that the sequence difference between the methylated DNA and the unmethylated DNA is caused, and the methylated DNA can be detected for the subsequent nucleic acid detection method.
The DNA is completely transformed within 3 hours, and the transformed DNA liquid can be immediately used or put into a temperature of minus 20 ℃ for standby, and can be used as a template for the subsequent fluorescent quantitative qPCR reaction. Such as EZ DNA Methylation-Gold Kit (TM) from Zymo company.
4. Fluorescent quantitative detection
The obtained DNA solution was used as a template, and β -actin was used as an internal gene, and the correlation with cervical cancer was examined using a kit containing FAM19A4, TERT, FANCI, GATA3, CDKN2A, JAM, PAX1, and SOX1 gene methylation primer probes, respectively, to examine human cervical exfoliated cell health samples, CINI-III samples, and squamous cell carcinoma and adenocarcinoma (CC) samples. Wherein the components of the qPCR reaction system are shown in Table 1, the qPCR reaction conditions are shown in Table 2, and the nucleic acid sequences of the primers and probes are shown in Table 3. The detection results are shown in tables 4-6, wherein table 4 is delta Ct value obtained by the marker gene Ct-internal reference gene Ct, table 5 is average value calculated by corresponding 3 sample delta Ct values, and table 6 is average value difference value compared with the healthy group and the disease group.
Table 1: qPCR reaction system (20. Mu.L/human)
Table 2: qPCR reaction conditions
Table 3: nucleic acid sequences of primers and probes
Table 4: delta Ct value
Table 5: TABLE 4 sample means
Table 6: TABLE 4 healthy group mean and disease group mean difference
Analysis of results: as can be seen from the experimental results in tables 5 and 6, from health to cancer, the detection results ΔCt of FAM19A4, PAX1 and SOX1 become gradually smaller, and the gene methylation has a positive correlation trend with cervical cancer diseases.
Based on the primary screening result, the inventor researches and discovers that under the nucleic acid combination and detection system of the invention, the three gene methylation combinations have the best detection effect, and the detection result has higher correlation with CINI, CINI-III and cervical carcinoma by adopting a kit containing three gene methylation primer probes of FAM19A4, PAX1 and SOX 1. Other cancer-related methylation genes, such as JAM3, have low correlation with detection results under the detection system of the application, and after the detection results are combined with the FAM19A4, PAX1 or SOX1 methylation gene primers, the interaction among primer probes of different genes can generate serious interference in a multiplex PCR reaction, so that non-specific amplification is generated, false positive of the detection results is caused, and the accuracy of the detection effect is affected.
Example 2: cervical cancer detection using the kit of the invention
1. Cervical tissue and cervical cell brush DNA extraction
DNA from both cervical tissue and cervical cell brush was extracted using commercial kits, such as QIAamp DNA FFPE Tissue Kit from Qiagen.
2. DNA quantification
The number of extracted molecules of DNA is ensured in the extraction process for subsequent experiments. dsDNA was quantified using commercial kits and double-stranded DNA (dsDNA) concentrations were detected specifically using fluorescent dye technology. For example, invitrogen's Qubit TM dsDNA HS Assay Kit。
3. Methylation-modified transformation of DNA
The extracted DNA was transformed using a commercial kit. After bisulfite treatment, the C base on the CpG island which is methylated in the DNA still keeps the C base, and the C base on the CpG island which is not methylated is changed into the U base, so that the sequence difference between the methylated DNA and the unmethylated DNA is caused, and the methylated DNA can be detected for the subsequent nucleic acid detection method.
The GCDNA-rich DNA is completely transformed within 3 hours, and the converted DNA liquid can be immediately used or put into a temperature of minus 20 ℃ for standby use as a template for the subsequent fluorescent quantitative qPCR reaction. Such as EZ DNA Methylation-Gold Kit (TM) from Zymo company.
4. Fluorescent quantitative detection
Taking the DNA solution obtained by transformation as a template and beta-actin as an internal reference gene; the nucleic acid combinations are detected for PCR reactions. The negative reference substance is water, and the positive reference substance is plasmid DNA containing PAX1, SOX1, FAM19A4 and internal reference gene sequences.
The components of the qPCR reaction system are shown in Table 7, the preparation of the primer and probe mixed solution is shown in Table 8, the nucleic acid sequences of the primers and probes are shown in Table 3, and the qPCR reaction conditions are the same as in example 1.
Table 7: qPCR reaction system (20. Mu.L/human)
Table 8: composition table of primer probe mixed solution (100 parts)
Component (A) Addition amount (mu L)
PAX1 gene-F (100. Mu.M) 10
PAX1 gene-R (100. Mu.M) 10
PAX1 Gene-Probe (100. Mu.M) 5
SOX1 Gene-F (100. Mu.M) 10
SOX1 Gene-R (100. Mu.M) 10
SOX1 Gene-Probe (100. Mu.M) 5
FAM19A4 Gene-F (100. Mu.M) 10
FAM19A4 Gene-R (100. Mu.M) 10
FAM19A4 Gene-Probe (100. Mu.M) 5
Beta actin gene-F (100 mu M) 10
Beta actin gene-R (100 mu M) 10
Beta actin gene-Probe (100. Mu.M) 5
Purified water Supplement to 200
5. Analysis of detection results
The target detection signals are ROX, FAM and CY5. The reagent kit reaction system for methylation detection through selecting a clinical sample with definite pathological information determines a positive judgment value through an ROC curve (shown in figure 1), wherein the reagent kit reaction system comprises a human cervical exfoliated cell cervical cancer (adenocarcinoma, squamous carcinoma and the like) sample, a low-level cervical lesion sample (CIN 1), cervical high-level lesion samples (CIN 2 and CIN 3) and an inflammation sample, namely methylation is generated on at least one gene of the PAX1, SOX1 and FAM19A4 genes detected, namely the occurrence of cervical cancer is a high risk. The specific result interpretation method is as follows:
a. the internal standard channel is satisfied that the S-type amplification curve exists, the Ct value is less than or equal to 33, the PAX1, SOX1 and FAM19A4 gene channels are free of the S-type amplification curve or the delta Ct value of the PAX1 gene is more than 7.67, the delta Ct value of the SOX1 gene is more than 7.20, and the delta Ct value of the FAM19A4 gene is more than 7.26, and the judgment result is negative;
b. the internal standard channel is satisfied with an S-type amplification curve, the Ct value is less than or equal to 33, the delta Ct value of the PAX1 gene is less than or equal to 7.67, or the delta Ct value of the SOX1 gene is less than or equal to 7.20, or the delta Ct value of the FAM19A4 gene is less than or equal to 7.26, and at least one channel detection of the three genes satisfies the corresponding delta Ct value requirement, and the result is judged to be positive;
c. if the internal standard channel has no S-type amplification curve or Ct value is more than 33, the judging result is invalid, and the sample is recommended to be extracted again for detection.
A total of 206 samples (Table 10) were tested using the above-described kit reaction system, including 34 cervical cancer samples, 46 low-grade intraepithelial lesions (CIN 1) samples, 58 high-grade intraepithelial lesions (CINII-III) samples, 68 chronic inflammatory samples, and the test results are shown in Table 9. Compared with clinical pathology results, the area of the combined ROC curve of the three genes obtained by using the methylation detection kit is 0.901 (shown in figure 1), the overall specificity is 89.47%, and the overall sensitivity is 90.22%. Wherein, the detection rates of CINII-III group and squamous carcinoma group are 84.48% and 100% respectively; the detection rates for CINI group and chronic inflammation group were 86.96% and 91.18%.
From the results, in view of clinical CINII and above, the kit can better identify cervical high-level lesions and cervical cancer by taking the cervical high-level lesions as colposcopic referral basis, realize shunting with low-level lesions and inflammation, and reduce unnecessary colposcopic referral of patients to a certain extent.
Table 9: statistics of detection results
Table 10: detection results of 206 samples in total by using kit reaction system
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Specific examples are set forth herein to illustrate the invention in detail, and the description of the above examples is only for the purpose of aiding in understanding the core concept of the invention. It should be noted that any obvious modifications, equivalents, or other improvements to those skilled in the art without departing from the inventive concept are intended to be included in the scope of the present invention.

Claims (10)

1. A nucleic acid combination for cervical cell gene methylation detection, characterized in that the nucleic acid combination comprises at least one pair selected from the group consisting of the following primer pairs:
(1) As set forth in SEQ ID NO:1, and the forward primer is shown as SEQ ID NO: 2;
(2) As set forth in SEQ ID NO:4, and the forward primer is shown as SEQ ID NO:5, a reverse primer shown in FIG. 5;
(3) As set forth in SEQ ID NO:7, and the forward primer is shown as SEQ ID NO: 8.
2. The nucleic acid combination of claim 1, further comprising a sequence selected from the group consisting of SEQ ID NOs: 3. 6 or 9.
3. Use of a nucleic acid combination according to claim 1 or 2 for the preparation of a kit for detecting cervical cell gene methylation.
4. The kit for detecting the cervical cell gene methylation is characterized by comprising a PAX1 gene methylation primer, a SOX1 gene methylation primer and a FAM19A4 gene methylation primer;
preferably, the kit further comprises a PAX1 gene probe, a SOX1 gene probe and a FAM19A4 gene probe;
preferably, the kit further comprises an internal reference gene primer;
preferably, the kit further comprises an internal reference gene probe;
preferably, the reference gene is a β -actin gene;
preferably, the PAX1 gene probe, the SOX1 gene probe, the FAM19A4 gene probe and the internal reference gene probe are respectively marked with fluorescent groups;
preferably, the fluorescent group is selected from ROX, FAM or CY5.
5. The kit of claim 4, wherein the forward primer for methylation of the PAX1 gene is set forth in SEQ ID NO:1, the reverse primer is shown as SEQ ID NO:2 is shown in the figure; the forward primer of the SOX1 gene methylation primer is shown as SEQ ID NO:4, the reverse primer is shown as SEQ ID NO:5 is shown in the figure; the forward primer of the FAM19A4 gene methylation primer is shown as SEQ ID NO:7, the reverse primer is shown as SEQ ID NO: shown as 8;
preferably, the PAX1 gene probe is shown in SEQ ID NO:3 is shown in the figure; the SOX1 gene probe is shown as SEQ ID NO:6 is shown in the figure; the FAM19A4 gene probe is shown as SEQ ID NO: shown as 9;
preferably, the forward primer of the beta-actin internal reference gene primer is shown as SEQ ID NO:10, the reverse primer is shown as SEQ ID NO: 11;
preferably, the reference gene probe is as shown in SEQ ID NO: shown at 12.
6. The kit of claim 4 or 5, further comprising a PCR reaction solution;
preferably, the PCR reaction solution comprises a PCR enzyme system and Mg 2+ And one or more of a PCR reaction buffer;
preferably, the PCR enzyme system comprises Taq DNA polymerase;
preferably, the PCR reaction buffer comprises dNTPs and Mg 2+ ；
Preferably, the kit further comprises a nucleic acid extraction solution.
7. The kit according to any one of claims 4 to 6, comprising a mixture of specific primers and probes for detecting methylation of the human PAX1, SOX1, FAM19A4 genes, and beta-actin internal reference gene primers and probes.
8. The detection method for the early detection of the cervical high-grade lesion and the cervical cancer of the biological sample is characterized by comprising the following steps of:
(1) Extracting sample DNA;
(2) Transforming the DNA extracted in the step (1) to obtain transformed DNA;
(3) Performing PCR amplification on the converted DNA obtained in the step (2) by using a PAX1 gene methylation primer, a SOX1 gene methylation primer and a FAM19A4 gene methylation primer to obtain an amplification product, and detecting methylation levels of different fragments in the amplification product;
preferably, the PCR amplification procedure described in step (4) is as follows:
stage 1: pre-denaturation at 95 ℃ for 5 min, and circulation for 1 time;
stage 2: reacting at 95 ℃ for 10 seconds, reacting at 55 ℃ for 30 seconds, reacting at 68 ℃ for 30 seconds, and circulating for 5 times;
stage 3: the reaction was carried out at 95℃for 10 seconds, at 58℃for 30 seconds, at 68℃for 30 seconds, and the reaction was repeated 40 times.
9. The method of claim 8, wherein the forward primer of the PAX1 gene methylation primer is set forth in SEQ ID NO:1, the reverse primer is shown as SEQ ID NO:2 is shown in the figure; the forward primer of the SOX1 gene methylation primer is shown as SEQ ID NO:4, the reverse primer is shown as SEQ ID NO:5 is shown in the figure; the forward primer of the FAM19A4 gene methylation primer is shown as SEQ ID NO:7, the reverse primer is shown as SEQ ID NO: shown as 8;
preferably, the PAX1 gene probe is shown in SEQ ID NO:3 is shown in the figure; the SOX1 gene probe is shown as SEQ ID NO:6 is shown in the figure; the FAM19A4 gene probe is shown as SEQ ID NO: shown as 9;
preferably, the detection method further comprises the step of performing a result interpretation based on the methylation levels of the different fragments in the amplified product;
preferably, the result interpretation analysis method is as follows:
a. the internal standard channel is satisfied that the S-type amplification curve exists, the Ct value is less than or equal to 33, the PAX1, SOX1 and FAM19A4 gene channels are free of the S-type amplification curve or the delta Ct value of the PAX1 gene is more than 7.67, the delta Ct value of the SOX1 gene is more than 7.20, and the delta Ct value of the FAM19A4 gene is more than 7.26, and the judgment result is negative;
b. the internal standard channel is satisfied with an S-type amplification curve, the Ct value is less than or equal to 33, the delta Ct value of the PAX1 gene is less than or equal to 7.67, or the delta Ct value of the SOX1 gene is less than or equal to 7.20, or the delta Ct value of the FAM19A4 gene is less than or equal to 7.26, and at least one channel detection of the three genes satisfies the corresponding delta Ct value requirement, and the result is judged to be positive;
c. if the internal standard channel has no S-type amplification curve or Ct value is more than 33, the judgment result is invalid.
10. Use of a nucleic acid combination according to any one of claims 1-3 or a kit according to any one of claims 4-7 for the preparation of a product for the detection of cervical high grade lesions and early detection of cervical cancer or methylation of cervical cancer cells.
CN202311150999.3A 2023-09-07 2023-09-07 Kit for cervical high-grade lesions and early detection of cervical cancer Pending CN117265096A (en)

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