CN111808962A - Kit for cervical cancer detection and use method - Google Patents
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Abstract
The invention discloses a kit for detecting cervical cancer, a use method and application. The kit screens and diagnoses cervical cancer by detecting or measuring the methylation state or level of one or more specific genes in the DNA of a test sample. Through experimental tests, the method has the characteristics of high speed, high sensitivity, high specificity and the like, can diagnose the cervical cancer in time at an early stage, enables early and accurate diagnosis of the cervical cancer to be possible, and avoids waste of medical resources. The kit can obviously improve the positive detection rate and specificity (real negative rate) of early cervical cancer.
Description
Technical Field
The invention belongs to the technical field of biotechnology and DNA detection, and particularly relates to a kit for cervical cancer detection, a use method and application thereof, in particular to a kit for cervical cancer detection or screening by using methylation qPCR of cervical cell DNA to determine the methylation state of a target gene target and a use method thereof, and in addition, the invention also relates to application of the detection kit in biomedicine.
Background
Cervical cancer is one of the most common cancers in women, and most commonly occurs at the cervical squamous junction (transition zone). Worldwide, it is estimated that 57 million new cases in 2018 account for 7.5% of all cancer-deceased women. Chinese cervical cancer accounts for over 28 percent of all the world, and the high incidence age is 50-55 years old. In recent years, the incidence and mortality of cervical cancer in china has been on the rise. Of which 85-90% are squamous cell carcinomas, the majority of the remaining 10-15% being adenocarcinomas. The clinical manifestations of cervical cancer are mostly not obvious, or only have symptoms similar to cervicitis, thus easily causing missed diagnosis; once symptoms appear, most progress to the advanced stage, and the optimal therapeutic window is lost.
Multiple large-scale studies indicate that regular acceptance of cervical cancer screening is the best method for preventing cervical cancer. There are two main methods for screening cervical cancer, one is the common cytology of cervix, including the traditional Pap smear and the liquid-based thin-layer cell technology (TCT), which screens the atypical cells possibly cancerated by observing the secretion of cervical part to detect cervical cancer at early stage; the other is human papilloma virus detection (HPV tethering), which is to observe whether infection of high-risk HPV subtypes exists in cervical exfoliated cells. However, the pap smear method is low in sensitivity due to the problems in the processes of material drawing, sheet making and sheet reading, the traditional pap five-grade classification method and the like, so that clinically false negative patients are common; the liquid-based thin-layer cell technology is difficult to popularize due to the high price of related equipment and examination consumables; human papillomavirus detection, although highly sensitive, is prone to high false positives, leading to over-treatment in the clinic and increased patient burden. Therefore, there is a clinically urgent need for the development of an early detection technique for cervical cancer that is immediate, accurate, and inexpensive.
DNA methylation changes are one of the earliest molecular changes in cancer progression and are tissue specific, and the hypermethylation levels of tumor suppressor genes have been identified as important mechanisms for suppressing gene expression and promoting cancer cell growth and expansion. In cervical cancer, hypermethylation of CpG (cytosine (C) followed by guanosine (G)) of some cancer suppressor genes has been considered as one of the biomarkers for cancer. Thus, analysis of the methylation status of one or more of these genes can be used to diagnose the status of cervical cancer. In cervical cancer screening, because epithelial cells of the cervix can be directly collected, early diagnosis, detection or screening of cervical cancer by analyzing gene methylation in cervical cells is easier to handle than tumors occurring on other organs.
Disclosure of Invention
Based on the above data, we find better methylated gene targets for screening early cervical cancer through more extensive and intensive research, and design several sets of primers and probe combinations capable of carrying out methylation detection aiming at the targets, and our scheme can obtain higher detection specificity and sensitivity, and the detection and screening kit for early cervical cancer is prepared by utilizing the newly found gene target detection combination.
The invention aims to provide a polygene joint detection primer probe combination kit for early diagnosis, detection or screening of cervical cancer, so as to overcome the defects of low specificity and sensitivity of a detection method in the prior art.
In a first aspect of the present invention, there is provided a kit for cervical cancer detection, which screens and diagnoses cervical cancer by detecting or measuring the methylation state or level of one or more specific genes in a test sample DNA.
In some embodiments, the kit comprises specific primers and probes that detect or measure the methylation status or level of one or more specific genes in the test sample DNA, including PAX1 and ACTB.
In some embodiments, the specific primers and probes used to detect the methylation state of the PAX1 gene are selected from any one of the following three specific primer and probe combinations: 1-2 specific primers and 3 probes, 4-5 specific primers and 6 probes, 7-8 specific primers and 9 probes.
In some embodiments, the specific primers and probes used to detect methylation status of the ACTB gene are specific primers SEQ ID NO 10-11 and probe SEQ ID NO 12.
In some embodiments, the specific primers SEQ ID NO 1, 2, 4, 5, 7, 8, 10, 11 are phosphorothioate modified and hybridize under stringent conditions to a region of the target gene that is methylated or unmethylated.
In some embodiments, the probes SEQ ID NO 3, 6, 9, 12 are designed based on TaqMan and hybridize under stringent conditions to a region of a target gene, which is methylated or unmethylated.
In some embodiments, the PAX1 gene probe SEQ ID NO 3, 6, 9 nucleotide sequence is labeled with FAM at the 5 'end and QSY at the 3' end; ACTB gene probe SEQ ID NO 12 nucleotide sequence 5 'end labeled VIC, 3' end labeled MGBNFQ.
In some embodiments, the specific primers SEQ ID NO. 1, 2, 4, 5, 7, 8, 10, 11 and the probes SEQ ID NO. 3, 6, 9, 12 are 10-50nt in length.
In some embodiments, the kit further comprises the following components: dNTP mixed solution, MgCl2 solution, DNA polymerase, PCR reaction buffer solution and PCR deionized water.
In some embodiments, the kit further comprises the following components: tissue genome DNA extraction reagent and DNA methylation conversion reagent, preferably, the cervical cell DNA methylation conversion reagent is bisulfite.
In some embodiments, the test sample DNA is whole genome, cell-free DNA, or circulating tumor DNA.
In some embodiments, specific primer and probe sequences are shown in table 1 below:
TABLE 1 specific primers and probes contained in the kit of the invention
A second aspect of the invention provides a method of use of a kit according to the first aspect,
(1) DNA extraction in cervical cells: extracting DNA in cervical cells from a sample to be detected by using a tissue DNA extraction reagent;
(2) DNA methylation conversion: bisulfite treating and subsequently purifying the extracted DNA from the cervical cells with a DNA methylation conversion reagent;
(3) carrying out fluorescent quantitative PCR amplification on the free DNA of the plasma subjected to methylation conversion and purification, setting a Ct threshold value in a linear amplification interval after the PCR reaction is finished, and determining that the amplification with the Ct value less than 40 is positive;
(4) and (4) judging a result: when the detection result of the internal reference site ACTB is positive, at least two of the three repeated amplifications of the PAX1 target point are positive, the result of the target point is determined to be positive;
preferably, in the step (3), the PCR amplification of each template is performed in three repetitions;
preferably, in the step (3), any one of the following methods is selected: methylation specific quantitative PCR, real-time methylation specific PCR, PCR using methylated DNA specific binding proteins.
In some embodiments, 10 μ L of each reaction system contains (0.5-1.5 μ L)1xPCR reaction buffer, 200-.
In some embodiments, the primers for the target of the PAX1 gene region are each 300-500 nM.
In some embodiments, the probes for the PAX1 gene region target are each 200-300 nM.
In some embodiments, the primer for the target of the ACTB gene region is 150-250nM and the probe for the target of the ACTB gene region is 50-150 nM.
In some embodiments, the PCR amplification conditions are: pre-denaturation at 90-100 deg.C for 8-12 min; denaturation at 90-100 deg.C for 10-20s, annealing at 60-70 deg.C and extension for 60-70s, and 40-50 cycles.
The third aspect of the invention provides the medical application of the kit according to the first aspect in preparing a reagent or medical device for detecting cervical cancer.
The invention has the beneficial effects that:
through experimental tests, the method has the characteristics of high speed, high sensitivity, high specificity and the like, can diagnose the cervical cancer in time at an early stage, enables early and accurate diagnosis of the cervical cancer to be possible, and avoids waste of medical resources.
Through experimental tests, the kit can obviously improve the positive detection rate and specificity (true negative rate) of early cervical cancer.
Drawings
FIG. 1 is a diagram of a DNA methylation qPCR amplification curve of a cervical brush sample of a non-cervical cancer control population of a kit according to an embodiment of the present invention;
FIG. 2 is a graph showing the methylation qPCR amplification of the DNA of a cervical brush sample of a cervical cancer patient using the kit according to one embodiment of the present invention;
FIG. 3 is a result of a diagnostic value analysis (ROC curve) of cervical cancer using the kit according to examples 1 and 2 of the present invention.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Definition of
The terms "patient," "individual," or "subject" are used interchangeably herein and may refer to a mammal, particularly a human. The subject may have mild, moderate or severe disease. The patient may be untreated, susceptible to treatment, or refractory. The patient may be an individual in need of treatment or diagnosis based on a particular symptom or family history.
The terms "sample," "patient sample," "biological sample," and the like include various sample types obtained from a patient, individual, or subject, and can be used for diagnostic or monitoring assays. The patient sample may be obtained from a healthy subject, a diseased patient, or a patient with symptoms associated with cervical cancer. Furthermore, the sample obtained from the patient may be segmented and only a portion may be used for diagnosis. In addition, the sample or a portion thereof may be stored under conditions that maintain the sample for later analysis. Specifically included within this definition are blood and other liquid samples of biological origin (including but not limited to peripheral blood, serum, plasma, urine, saliva, sputum, stool, and synovial fluid), solid tissue samples (such as biopsy specimens or tissue cultures or cells derived therefrom and progeny thereof). The definition also includes samples that are manipulated in any manner after being obtained, such as by centrifugation, filtration, precipitation, dialysis, chromatography, reagent treatment, washing, or enrichment for certain cell populations. These terms also include clinical samples, cultured cells, cell supernatants, tissue samples, organs, and the like. The sample may also comprise freshly frozen and/or formalin fixed paraffin embedded tissue blocks, such as blocks prepared by clinical or pathological biopsy, prepared for pathological analysis or by immunohistochemistry studies.
The terms "measuring," "determining," "detecting," or "examining" are used interchangeably throughout and may refer to a method that includes obtaining a patient sample and/or detecting a biomarker methylation status or level in a patient sample. In one embodiment, these terms refer to obtaining a patient sample and detecting the methylation state or level of one or more biomarkers in the sample. In another embodiment, the terms "measuring", "determining" or "detecting" refer to detecting the methylation status or level of one or more biomarkers in a patient sample. Measurement can be accomplished by methods known in the art and further described herein, including but not limited to methylation specific quantitative polymerase chain reaction (qPCR).
The term "methylation" refers to methylation of cytosine at the C5 or N4 position of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. The in vitro amplified DNA is unmethylated because the in vitro DNA amplification method does not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA whose original template was unmethylated or methylated, respectively.
The term "CpG island" refers to a contiguous region of genomic DNA having a high density of CpG.
The term "methylation state" or "methylation level" refers to the presence, absence, and/or amount of methylation at a particular nucleotide or nucleotide in a portion of DNA.
It should be understood that wherever the language "comprising" is used to describe an embodiment, other similar embodiments described in "consisting of …" and/or "consisting essentially of …" are also provided.
Example 1: multiplex qPCR detection of cervical cancer methylation on samples of non-cervical cancer control population by using kit
(I) test materials
1. 10 cervical brushes of the non-cervical cancer control population;
2. tissue genome DNA extraction kit (Beijing Tiangen Biochemical technology Co., Ltd.);
EZ DNAmethation-Lightning kit (available from Zymo Research, Inc.);
4. the kit (comprises a combination 1 primer and probe combination for detecting a target PAX1, and an ACTB amplification primer and probe);
5.AmpliTaq GoldTMDNA polymerase and buffer reagents (available from Thermo Fisher).
(II) Experimental method
1. Cervical tissue and cervical cell brush DNA extraction
Taking 1.0mL of cervical brush samples of clinically confirmed non-cervical cancer control population by pathology to a 1.5mL centrifuge tube, centrifuging for 3-5min at 12000rmp, removing supernatant, and collecting cervical cells; then, DNA was extracted using a tissue genome DNA extraction kit of Beijing Tiangen Biochemical technology Co., Ltd.
DNA methylation transformation
10 parts of the extracted DNA were bisulfite-treated and then purified using EZ DNA Methylation-Lightning kit from Zymo Research.
qPCR amplification
The bisulfite treated DNA was subjected to qPCR amplification using a Thermo Fisher Quant Studio 3 instrument, with three replicates of PCR amplification per template. 10 μ L of each reaction system containing 1xPCR reaction buffer, 400 μm dNTPs, 4mM MgCl22U AmpliTaq Gold DNA polymerase, 30nM ROX dye; 800nM each of the primers for the target of the PAX1 gene region (only one pair of primers was selected for each target), and 500nM each of the probes for the target of the PAX1 gene region (one probe was selected for each target corresponding to the selected primer)A needle); primer 200nM for the target of the ACTB gene region and probe 100nM for the target of the ACTB gene region.
The PCR amplification conditions were: pre-denaturation at 95 ℃ for 10 min;
denaturation at 95 ℃ for 15s, annealing and extension at 65 ℃ for 30s, 45 cycles;
after the PCR reaction was completed, the Ct threshold was set in the linear amplification region (in this experiment,. DELTA.Rn was set to 0.1), and amplification with a Ct value of less than 40 was considered positive.
(III) results of the experiment
The criteria for the results were that the internal reference site (ACTB) test was positive and at least two of the three replicate amplifications at the PAX1 test site/target were positive.
Table 2 below shows the results of testing 10 cervical brush samples obtained from 10 non-cervical cancer control individuals using the multiplex assay of the present invention (PAX1) (fig. 1). As can be seen from Table 2, the detection specificity of this protocol is very high.
TABLE 2 test results of cervical brush samples of non-cervical cancer control population using the kit of the present invention
Example 2: multiplex qPCR detection of cervical cancer methylation on DNA sample of cervical cancer patient by using kit
(I) test materials
1. 30 cervical brushes for cervical cancer patients;
2. tissue genome DNA extraction kit (Beijing Tiangen Biochemical technology Co., Ltd.);
EZ DNA Methylation-Lightning kit (available from Zymo Research Co.);
4. the kit (comprises a combination 1 primer and probe combination for detecting a target PAX1, and an ACTB amplification primer and probe);
5.AmpliTaq GoldTMDNA polymerase and buffer reagents (available from Thermo Fisher).
(II) Experimental method
1. Cervical tissue and cervical cell brush DNA extraction
Taking 1.0mL of cervical brush samples of cervical cancer patients clinically confirmed by pathology to a 1.5mL centrifuge tube, centrifuging for 3-5min at 12000rmp, removing supernatant, and collecting cervical cells; then, DNA was extracted using a tissue genome DNA extraction kit of Beijing Tiangen Biochemical technology Co., Ltd.
DNA methylation transformation
10 parts of the extracted DNA were bisulfite-treated and then purified using EZ DNA Methylation-Lightning kit from Zymo Research.
qPCR amplification
The bisulfite treated DNA was subjected to qPCR amplification using a Thermo Fisher Quant Studio 3 instrument, with three replicates of PCR amplification per template. 10 μ L of each reaction system containing 1xPCR reaction buffer, 400 μm dNTPs, 4mM MgCl22U AmpliTaq Gold DNA polymerase, 30nM ROX dye; 800nM each of the primers for the target of the PAX1 gene region (only one pair of primers was selected for each target), 500nM each of the probes for the target of the PAX1 gene region (one probe was selected for each target corresponding to the selected primer); primer 200nM for the target of the ACTB gene region and probe 100nM for the target of the ACTB gene region.
The PCR amplification conditions were: pre-denaturation at 95 ℃ for 10 min;
denaturation at 95 ℃ for 15s, annealing and extension at 65 ℃ for 30s, 45 cycles;
after the PCR reaction was completed, the Ct threshold was set in the linear amplification region (in this experiment,. DELTA.Rn was set to 0.1), and amplification with a Ct value of less than 40 was considered positive.
(III) results of the experiment
The criteria for the results were that the internal reference site (ACTB) test was positive and at least two of the three replicate amplifications at the PAX1 test site/target were positive.
Table 3 below shows the results of testing 30 cervical brush samples obtained from 30 cervical cancer patients using the multiplex assay of the present invention (PAX1) (fig. 2). As can be seen from Table 3, the positive detection rate of this protocol was very high.
TABLE 3 test results of cervical brush specimens of cervical cancer patients using the kit of the present invention
By combining example 1 and example 2, it can be seen that the cervical cancer screening using the kit of the present invention has a positive detection rate of 90.0%, a specificity (true negative rate) of 100%, and a diagnostic value analysis (ROC curve) of 0.95 (fig. 3).
Different from example 1, the kit comprising the combination 2 primer and probe combination containing the detection target PAX1 and the combination 3 primer and probe combination containing the detection target PAX1 is adopted to detect the samples of the non-cervical cancer control population and the cervical brush samples of the cervical cancer patients, and the results are similar to those of example 1 and example 2.
While the preferred embodiments and examples of the present invention have been described in detail, the present invention is not limited to the embodiments and examples, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
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Claims (10)
1. A kit for cervical cancer detection for screening and diagnosing cervical cancer by detecting or measuring the methylation state or level of one or more specific genes in test sample DNA.
2. The kit of claim 1, wherein the kit comprises specific primers and probes for detecting or measuring the methylation status or level of one or more specific genes in the test sample DNA, wherein the specific genes comprise PAX1 and ACTB.
3. The kit according to claim 1 or 2, wherein the specific primers and probes for detecting the methylation state of the PAX1 gene are selected from any one of the following three specific primer and probe combinations: 1-2 specific primers and 3 probes, 4-5 specific primers and 6 probes, 7-8 specific primers and 9 probes.
4. The kit according to any one of claims 1 to 3, wherein the specific primers and probes for detecting methylation state of ACTB gene are specific primers SEQ ID NO 10-11 and probe SEQ ID NO 12.
5. The kit of any one of claims 1 to 4, wherein the specific primers SEQ ID NOs 1, 2, 4, 5, 7, 8, 10, 11 are phosphorothioate-modified and hybridize under stringent conditions to a region of a target gene that is methylated or unmethylated;
and/or the probes SEQ ID NO 3, 6, 9, 12 are designed based on TaqMan (TM) and hybridize under stringent conditions to a region of a target gene, which is methylated or unmethylated.
6. The kit according to any one of claims 1 to 5, wherein the PAX1 gene probe SEQ ID NO 3, 6, 9 nucleotide sequence is labeled with FAM at the 5 'end and QSY at the 3' end; ACTB gene probe SEQ ID NO 12 nucleotide sequence 5 'end mark VIC, 3' end mark MGBNFQ;
and/or the specific primers SEQ ID NO. 1, 2, 4, 5, 7, 8, 10, 11 and the probes SEQ ID NO. 3, 6, 9, 12 are 10-50nt in length;
and/or, the kit also comprises the following components: dNTP mixed solution, MgCl2 solution, DNA polymerase, PCR reaction buffer solution and PCR deionized water;
and/or, the kit also comprises the following components: a tissue genome DNA extraction reagent and a DNA methylation conversion reagent, wherein the DNA methylation conversion reagent of the cervical cells is preferably bisulfite;
and/or, the test sample DNA is whole genome, cell-free DNA, or circulating tumor DNA.
7. A method of using the kit according to any one of claims 1 to 6, comprising the steps of:
(1) DNA extraction in cervical cells: extracting DNA in cervical cells from a sample to be detected by using a tissue DNA extraction reagent;
(2) DNA methylation conversion: bisulfite treating and subsequently purifying the extracted DNA from the cervical cells with a DNA methylation conversion reagent;
(3) carrying out fluorescent quantitative PCR amplification on the free DNA of the plasma subjected to methylation conversion and purification, setting a Ct threshold value in a linear amplification interval after the PCR reaction is finished, and determining that the amplification with the Ct value less than 40 is positive;
(4) and (4) judging a result: when the detection result of the internal reference site ACTB is positive, at least two of the three repeated amplifications of the target PAX1 are positive, the result of the target is determined to be positive;
preferably, in the step (3), the PCR amplification of each template is performed in three repetitions;
preferably, in the step (3), any one of the following methods is selected: methylation specific quantitative PCR, real-time methylation specific PCR, PCR using methylated DNA specific binding protein;
and/or 10. mu.L of each reaction system containing (0.5-1.5. mu.L) 1xPCR reaction buffer, 200-400. mu.m dNTPs, 3-6mM MgCl2, 1-3U AmpliTaq Gold DNA polymerase, 30-60nM ROX dye.
8. The method as claimed in claim 7, wherein the primers for the target of the PAX1 gene region are 300-500nM each;
and/or, the probes for the PAX1 gene region target point are 200-300nM each;
and/or, the primer of ACTB gene region target point is 150-250nM, and the probe of ACTB gene region target point is 50-150 nM.
9. The method according to any one of claims 7 or 8, wherein the PCR amplification conditions are: pre-denaturation at 90-100 deg.C for 8-12 min; denaturation at 90-100 deg.C for 10-20s, annealing at 60-70 deg.C and extension for 60-70s, and 40-50 cycles.
10. The kit according to any one of claims 1 to 6 for medical use in the preparation of a reagent or medical device for the detection of cervical cancer.
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| CN114540489A (en) * | 2020-11-27 | 2022-05-27 | 广州达健生物科技有限公司 | Cervical cancer early screening and detecting kit and application thereof |
| CN117701718A (en) * | 2024-02-04 | 2024-03-15 | 湖南宏雅基因技术有限公司 | Gene methylation marker for diagnosing cervical cancer, primer pair and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114540489A (en) * | 2020-11-27 | 2022-05-27 | 广州达健生物科技有限公司 | Cervical cancer early screening and detecting kit and application thereof |
| CN114540489B (en) * | 2020-11-27 | 2024-01-30 | 广州达健生物科技有限公司 | Cervical cancer early screening detection kit and application thereof |
| CN114395626A (en) * | 2022-01-04 | 2022-04-26 | 博尔诚（北京）科技有限公司 | Marker and probe composition for cervical cancer screening and application thereof |
| CN114395626B (en) * | 2022-01-04 | 2023-09-08 | 博尔诚（北京）科技有限公司 | Marker for cervical cancer screening, probe composition and application thereof |
| CN117701718A (en) * | 2024-02-04 | 2024-03-15 | 湖南宏雅基因技术有限公司 | Gene methylation marker for diagnosing cervical cancer, primer pair and application thereof |
| CN117701718B (en) * | 2024-02-04 | 2024-05-07 | 湖南宏雅基因技术有限公司 | Gene methylation marker for diagnosing cervical cancer, primer pair and application thereof |
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