阅读说明：本技术 用于***检测的DNA甲基化qPCR试剂盒及其使用方法 (DNA methylation qPCR kit for cervical cancer detection and use method thereof ) 是由 王志强 万季 潘晓新 夏迪 金泰庆 蔡雪儿 罗海燕 王一杏 关建洪 王奕 宋麒 于 2020-07-23 设计创作，主要内容包括：本发明公开了一种用于宫颈癌检测的DNA甲基化qPCR试剂盒及其使用方法、应用。该试剂盒通过检测或测量受试样品DNA中一种或多种特定基因的甲基化状态或水平来筛查和诊断宫颈癌。经实验测试,本发明的方法具有快速、灵敏度高和特异性高等特点,能够在早期及时对人宫颈癌进行诊断,使得早期准确诊断宫颈癌成为可能,避免了医疗资源的浪费。本发明试剂盒能够显著提高早期宫颈癌的阳性检出率和特异性(真实阴性率)。(The invention discloses a DNA methylation qPCR kit for cervical cancer detection and a use method and application thereof. 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.)

1. A DNA methylation qPCR kit for cervical cancer detection, which is used 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, and comprises specific primers and probes for detecting or measuring the methylation state or level of one or more specific genes in the test sample DNA, wherein the specific genes comprise JAM3 and ACTB.

2. The kit according to claim 1, characterized in that the specific primers and probes for detecting the methylation state of JAM3 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.

3. The kit according to claim 1 or 2, 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.

4. The kit of any one of claims 1 to 3, wherein 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 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.

5. The kit according to any one of claims 1 to 4, wherein the nucleotide sequences of JAM3 gene probe SEQ ID NO 3, 6 and 9 are labeled with Cy5 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.

6. A method of using the kit according to any one of claims 1 to 5, 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, determining that the result of the target point is positive if at least two of the three repeated amplifications of the target point JAM3 are 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.

7. The method of claim 6, wherein each reaction system comprises 10 μ L of (0.5-1.5 μ L)1xPCR reaction buffer, 200-400 μm dNTPs, 3-6mM MgCl2, 1-3U AmpliTaq Gold DNA polymerase, and 30-60nM ROX dye.

8. The method according to claim 6 or 7, wherein the primer for the target of JAM3 gene region is 300-500nM each, and the probe for the target of JAM3 gene region is 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 6 to 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 5 for medical use in the preparation of a reagent or medical device for the detection of cervical cancer.

Technical Field

The invention belongs to the technical field of biotechnology and DNA detection, and particularly relates to a DNA methylation qPCR kit for cervical cancer detection, a using method and application thereof, in particular to a kit for determining the methylation state of a target gene target by methylation qPCR by using cervical cell DNA for cervical cancer detection or screening, and a using method thereof.

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.

The first aspect of the invention provides a DNA methylation qPCR kit for cervical cancer detection, and the DNA methylation qPCR kit is used 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.

In some embodiments, the kits comprise specific primers and probes that detect or measure the methylation status or level of one or more specific genes in test sample DNA, including JAM3 and ACTB.

In some embodiments, the specific primers and probes used to detect the methylation status of JAM3 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 JAM3 gene probe SEQ ID NO 3, 6, 9 nucleotide sequence is labeled with Cy5 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, determining that the result of the target point is positive if at least two of the three repeated amplifications of the target point JAM3 are 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 JAM3 gene region are each 300-500 nM.

In some embodiments, the probes for the target of the JAM3 gene region 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.