CN112410425A - Gene methylation digital PCR kit for early screening of liver cancer and application thereof - Google Patents
Gene methylation digital PCR kit for early screening of liver cancer and application thereof Download PDFInfo
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Abstract
The invention relates to a gene methylation digital PCR kit for early screening of liver cancer and application thereof. The invention provides a gene methylation digital PCR kit for early screening of liver cancer, which comprises a specific primer and a first specific probe, wherein the specific primer and the first specific probe are used for detecting the methylation of a BMPR1A gene; a specific primer and a second specific probe for detecting methylation of the ARHGAP25 gene; a specific primer and a third specific probe for detecting methylation of the KLF3 gene; and a specific primer and a fourth specific probe for detecting the methylation of the ATXN2 gene. The method accurately detects the methylation sites of the BMPR1A gene, the ARHGAP25 gene, the KLF3 gene and the ATXN2 gene by combining a methylation specificity PCR detection method and a microdroplet digital PCR detection method, has the advantages of absolute quantification, high specificity, high accuracy and sensitivity, convenient and quick detection and accurate and reliable result, and can be used for screening the early liver cancer.
Description
Technical Field
The invention relates to the technical field of medical inspection, in particular to a gene methylation digital PCR kit for early screening of liver cancer and application thereof.
Background
Liver cancer, a malignant tumor of the liver, can be divided into primary and secondary types. The primary liver malignant tumor originates from the epithelium or mesenchymal tissue of the liver, and the former is called primary liver cancer, which is a malignant tumor with high incidence and great harm in China.
According to recent statistics, the global incidence rate is over 62.6 ten thousand per year, and the number 6 of malignant tumors is: death was approximately 60 million/year, and was at the 4 th of tumor-associated deaths. In 2018, 841,080 new cases and 781,631 deaths will occur globally. China is a well-known major liver cancer country, and about 50% of new liver cancers and liver cancer deaths occur worldwide each year in china. Liver cancer is seriously threatening the health and life of people in China. The clinical manifestations of primary liver cancer are extremely atypical, and the symptoms are usually much less obvious, especially in the early stage of the disease course. Although the treatment technique for liver cancer is changing day by day, the symptoms of liver cancer are not obvious in the early stage, and even are not unknown for a long time after the disease. When the disease condition develops to a certain extent, symptoms such as liver pain, appetite decrease, fatigue and weakness, gradual emaciation and the like are gradually produced. In late stage, jaundice, ascites, hematemesis, coma, etc. may occur. Huge swelling is often felt in the upper abdomen of a liver cancer patient, but the liver cancer patient reaches the middle and late stage and even has metastasized to the lung and the like, and the optimal treatment opportunity is missed. About 15% of liver cancer patients in China are in early stage, which means that 85% of initial diagnosis liver cancer patients in China are in middle and late stage. For tumors, surgical resection has always been the treatment with the best prognosis and the best efficacy in reducing recurrence. However, because of late findings, only 25% of patients with initial liver cancer can undergo surgical resection in our country. The 5-year survival rate of liver cancer patients in China is only about 12.5 percent. Research shows that if radical resection operation is performed on liver cancer in the early stage, the five-year survival rate exceeds 95%. Therefore, early diagnosis and early surgery of liver cancer are one of the most effective methods for improving 5-year survival rate and reducing mortality rate of liver cancer patients. However, no very effective early detection method exists clinically at present, so that establishment of a high-sensitivity, economic and simple molecular technology screening method is particularly important.
Epigenetic (mainly including DNA methylation and micro RNAs) abnormality is an important sign of liver cancer, and occurrence and development of liver cancer can be effectively monitored. Among them, abnormal methylation (hypomethylation and hypermethylation) of DNA plays an extremely important role in the process of somatic canceration, and a tumor-specific DNA methylation marker is a promising marker for early warning and clinical diagnosis of tumors.
miRNAs can play an important role in the pathogenesis of liver cancer by regulating and controlling key genes in a carcinogenic pathway. In recent years, related researches show that the abnormal methylation of DNA can change the expression of miRNAs in tumor cells, thereby having an effect on the occurrence and development of tumors. The detection of the methylation state of specific genes in samples such as tissues, serum/plasma and the like is an effective way for promoting the early diagnosis of liver cancer and improving prognosis.
BMPR1A belongs to the receptor (BMPR) family of BMPs, and Bone Morphogenetic Proteins (BMPs) are a member of the transforming growth factor beta (TGF-beta) superfamily. Researches show that the compound can regulate the differentiation, proliferation and apoptosis of cells and plays an important role in the growth and development of organisms. There are 3 important components in the BMPs signaling pathway, BMPs receptor (BMPR), and Smads proteins, respectively. Recent studies have shown that bone morphogenetic protein receptor 1(BMPR1A) is expressed in many malignant tumors including lung, liver, stomach and colon cancers. The BMPR1A gene has great influence on the expression of downstream gene products, so that the hypermethylation of the BMPR1A gene can influence the expression of a plurality of proteins, thereby promoting the canceration of cells. The ARHGAP25 gene is also called KAIA0053 or HEL-S-308 gene, and the gene encodes a negative regulator of Rho protein family, and the protein family is involved in processes such as actin remodeling, cell polarity and cell migration. Methylation of the gene has certain influence on the function of the encoded protein product, so that the processes of migration and the like of cancer cells are influenced. The KLF3 gene, also known as basic KLF factor (bKLF), is a member of the KLF family. KLF family members are involved in regulating a variety of physiological processes in cells, including differentiation, development, cell proliferation, apoptosis, and the like. And the KLF family is involved in regulating the proliferation, invasion and metastasis of tumor cells. Research shows that KLF3 may be involved in the metabolic process of tumor, may be an oncosuppressor gene, and acts as KRAS signal channel, so as to inhibit the growth and transformation of tumor cells and promote the apoptosis of tumor cells. The ATXN2 gene is located at 12q24, and the coded protein is Ataxin-2, and Ataxin-2 regulates the translation process of mRNA through the interaction with poly (A) binding protein, and is also involved in the formation of stress particles and P-body, and the P-body plays a certain role in RNA regulation. Mutations in the ATXN2 gene can cause spinocerebellar ataxia type 2 (SCA2), and abnormalities in the ATXN2 gene also play an important role in cancer.
The Liquid Biopsy (Liquid Biopsy) technique is a rapid and simple cancer blood detection method, is one of the important ways of precise medical treatment, and is a non-invasive and repeatable way to extract tumor samples. The liquid biopsy technology is applied to realize the prevention, diagnosis and prognosis of cancers, thereby having wide prospects in the aspects of selection of anti-cancer drugs, evaluation of drug efficacy, detection of drug resistance and the like. Existing fluid biopsy techniques primarily involve Circulating Tumor Cells (CTCs) and circulating tumor dna (ctdna) and exosome detection.
Circulating tumor DNA (ctdna) refers to a class of DNA released into the circulation system after shedding or apoptosis of tumor cells, and is a characteristic biomarker of tumor cells. ctDNA also contains free DNA released to blood from other tumor parts, such as tumor metastasis parts, free DNA released by clonal hematopoietic cells and the like, and the ctDNA detection result provides panoramic data of tumors in a patient body, so that the application of ctDNA detection in the late stage of tumor metastasis has the advantages of high feasibility, complete data, good effect and the like, and has wide application potential in the clinical detection of cancer. The ctDNA gene detection technology can be used for noninvasive tumor early screening, auxiliary diagnosis and typing, medication guidance, prognosis judgment, dynamic monitoring and the like. Methylation of CpG sites in DNA molecules is an important regulatory factor for gene expression, and previous researches show that compared with normal cells, tumor cells show the tendency that the methylation level of the whole genome is reduced and the methylation level of CpG islands of cancer suppressor gene promoters is increased. It was found that stable and consistent characteristic changes in DNA methylation can be detected in the same type of tumor cells at an early stage of tumorigenesis, which often leads to inactivation of the corresponding tumor suppressor genes, and thus can be used as an ideal marker for early diagnosis of tumors. The research detects the genome methylation levels of 6 normal liver tissues and 30 liver cancer and paracancer tissues, and the result shows that the DNA methylation level of the liver cancer tissues is obviously lower than that of the paracancer and normal liver tissues, in addition, the related research detects the c-myc of the cancer tissues and the paracancer tissues of 24 liver cancer patients, the methylation degree of the c-myc of the cancer tissues is obviously lower than that of the paracancer tissues, the paracancer tissues also show different degrees of hypomethylation, and the hypomethylation of the 3 rd exon of the c-myc possibly plays an important role in the occurrence of the liver cancer. Research results show that the CpG methylation detection has great value in early diagnosis of liver cancer.
As a brand-new nucleic acid detection method, the digital PCR (polymerase chain reaction) technology is used, wherein a reaction system is divided into a plurality of reaction units to independently perform PCR, and the number of nucleic acids is calculated according to Poisson distribution and a positive proportion. Compared with the traditional PCR technology, the dPCR has the advantages of high sensitivity, high accuracy, high tolerance and absolute quantification. At present, dPCR is mainly divided into a droplet type and a chip type. The micro-Drop Digital PCR (DDPCR) is a sensitive method for quantitative detection, can detect only a very small amount of samples, does not need to refer to a standard substance or endogenous control, has high sensitivity and small required sample amount, and greatly meets the requirements of micro-detection of target sequences in clinical precious samples such as puncture samples, hydrothorax, ascites and peripheral blood. The ddPCR technology has the advantages of low cost, strong practicability, accuracy, high efficiency, convenience and the like, and has a good application prospect in the clinical field.
At present, the diagnosis of cancer is mainly based on clinical symptoms, imaging detection, histopathological examination and the like, but the clinical symptoms of many liver cancer patients appear later, and the biopsy detection is difficult. The alpha-fetoprotein assay is one of the most specific methods for diagnosing hepatocellular carcinoma at present, but liver cancer can be diagnosed only if the alpha-fetoprotein is positive. For the confirmation of liver cancer, the change of alpha-fetoprotein content must be dynamically observed for a long time. The isozymes of gamma-glutamyltranspeptidase, alkaline phosphatase and lactate dehydrogenase in the serum of liver cancer patients can be higher than normal, but the hematology and enzymology examination is mostly used as auxiliary diagnosis due to the lack of specificity. Radionuclide liver scan: the positive coincidence rate of liver cancer diagnosis is 85-90%, but tumors with the diameter less than 3 cm are not easy to show on a scanning image. High resolution, can detect early liver cancer about 1.0 cm in diameter, and is helpful for hemangioma identification by applying enhanced scanning. The diagnosis coincidence rate for liver cancer is as high as 90%. But at a high cost. Have not been generally applicable.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a gene methylation digital PCR kit for early screening of liver cancer.
The invention provides a gene methylation digital PCR kit for early screening of liver cancer, which is used for screening early liver cancer patients by jointly detecting methylation conditions of free DNA BMPR1A gene, ARHGAP25 gene, KLF3 gene and ATXN2 gene promoter region in blood of liver cancer high-risk people by adopting a droplet type digital PCR absolute quantitative technical means, and assisting in early diagnosis of liver cancer. The invention combines a methylation-specific PCR detection method with a microdroplet digital PCR detection method, simultaneously selects plasma as a specimen, and provides a new way which is simple, convenient, accurate, reliable and rapid to operate for early diagnosis of liver cancer.
The invention provides a gene methylation digital PCR kit for early screening of liver cancer, which comprises a specific primer and a first specific probe, wherein the specific primer and the first specific probe are used for detecting the methylation of a BMPR1A gene; a specific primer and a second specific probe for detecting methylation of the ARHGAP25 gene; a specific primer and a third specific probe for detecting methylation of the KLF3 gene; and a specific primer and a fourth specific probe for detecting the methylation of the ATXN2 gene.
The invention provides application of the kit in preparation of a reagent for early screening of liver cancer.
Specifically, the present invention proposes the following technical solutions.
In a first aspect, the invention provides a gene methylation digital PCR kit for early screening of liver cancer, which comprises a specific primer and a first specific probe for detecting the methylation of a BMPR1A gene;
a specific primer and a second specific probe for detecting methylation of the ARHGAP25 gene;
a specific primer and a third specific probe for detecting methylation of the KLF3 gene; and
a specific primer and a fourth specific probe for detecting the methylation of the ATXN2 gene.
Preferably, the specific primer for detecting the methylation of the BMPR1A gene comprises a primer 1 and a primer 2, wherein the sequence of the primer 1 is shown as SEQ ID NO. 1, and the sequence of the primer 2 is shown as SEQ ID NO. 2.
Preferably, the sequence of the first specific probe for detecting the methylation of the BMPR1A gene is shown as SEQ ID NO. 3.
Preferably, the specific primer for detecting the methylation of the ARHGAP25 gene comprises a primer 3 and a primer 4, wherein the sequence of the primer 3 is shown as SEQ ID NO. 4, and the sequence of the primer 4 is shown as SEQ ID NO. 5.
Preferably, the sequence of the second specific probe for detecting the methylation of the ARHGAP25 gene is shown as SEQ ID NO. 6.
Preferably, the specific primer for detecting methylation of the KLF3 gene comprises a primer 5 and a primer 6, wherein the sequence of the primer 5 is shown as SEQ ID NO. 7, and the sequence of the primer 6 is shown as SEQ ID NO. 8.
Preferably, the sequence of the third specific probe for detecting the methylation of the KLF3 gene is shown as SEQ ID NO. 9.
Preferably, the specific primer for detecting the methylation of the ATXN2 gene comprises a primer 7 and a primer 8, wherein the sequence of the primer 7 is shown as SEQ ID NO. 10, and the sequence of the primer 8 is shown as SEQ ID NO. 11.
Preferably, the sequence of the fourth specific probe for detecting the methylation of the ATXN2 gene is shown as SEQ ID NO. 12.
Preferably, the kit comprises a microdroplet generating oil.
Preferably, the kit also comprises a specific primer and a fifth specific probe for detecting methylation of the beta-actin internal reference gene.
Preferably, the specific primer for detecting methylation of the beta-actin reference gene comprises a primer 9 and a primer 10, wherein the sequence of the primer 9 is shown as SEQ ID NO. 13, and the sequence of the primer 10 is shown as SEQ ID NO. 14.
Preferably, the sequence of the fifth specific probe for detecting methylation of the reference gene of beta-actin is shown as SEQ ID NO. 15.
Preferably, the kit also comprises a positive quality control product.
Preferably, the positive quality control product comprises methylated fragments of the BMPR1A gene, the ARHGAP25 gene, the KLF3 gene and the ATXN2 gene.
Preferably, the kit also comprises a negative quality control product.
Preferably, the negative quality control product comprises non-methylated fragments of BMPR1A gene, ARHGAP25 gene, KLF3 gene and ATXN2 gene.
Preferably, the kit further comprises nuclease-free water.
In a second aspect, the invention provides an application of the gene methylation digital PCR kit in preparing a reagent for screening liver cancer at an early stage.
The reagent for screening the liver cancer at the early stage is used by the following method: adding a sample DNA, a positive quality control product and a negative quality control product into a specific primer and a first specific probe for detecting BMPR1A gene methylation, a specific primer and a second specific probe for detecting ARHGAP25 gene methylation, a specific primer and a third specific probe for detecting KLF3 gene methylation, a specific primer and a fourth specific probe for detecting ATXN2 gene methylation, microdroplet generation oil, a specific primer and a fifth specific probe for detecting beta-actin internal reference gene methylation, mixing and preparing into microdroplets, and then carrying out fluorescent quantitative PCR reaction.
Preferably, the fluorescent quantitative PCR reaction conditions are: pre-denaturation at 94-95 ℃ for 5-10 min for 1 cycle; 94-95 ℃ for 15-30 s, 58-60 ℃ for 30-45 s, and 40-50 cycles.
The invention has the following beneficial effects:
the kit provided by the invention adopts a means of Taqman-MGB probe detection combined with droplet type digital PCR detection to accurately detect the methylation sites of the BMPR1A gene, the ARHGAP25 gene, the KLF3 gene and the ATXN2 gene, and has the advantages of absolute quantification, high specificity, high accuracy and sensitivity, convenience and rapidness in detection, accurate and reliable results, and capability of being used for screening liver cancer at an early stage.
Detailed Description
As described above, the present invention provides a gene methylation digital PCR kit for early screening of liver cancer, which comprises a specific primer and a first specific probe for detecting the methylation of BMPR1A gene; a specific primer and a second specific probe for detecting methylation of the ARHGAP25 gene; a specific primer and a third specific probe for detecting methylation of the KLF3 gene; and a specific primer and a fourth specific probe for detecting the methylation of the ATXN2 gene.
In a preferred embodiment of the invention, the specific primer for detecting the methylation of the BMPR1A gene comprises a primer 1 and a primer 2, wherein the sequence of the primer 1 is shown as SEQ ID NO. 1, and the sequence of the primer 2 is shown as SEQ ID NO. 2; preferably, the sequence of the first specific probe for detecting the methylation of the BMPR1A gene is shown as SEQ ID NO. 3.
Preferably, the sequence of the primer 1 is 5'-CAGGAGGGATCGTGGAAGAA-3';
the sequence of the primer 2 is 5'-GACGGATCACTCGGTACCATGT-3';
the sequence of the first specific probe is 5 '-FAM-ACCAATTGTCATATTAC-BHQ-3'.
In a preferred embodiment of the invention, the specific primer for detecting methylation of the ARHGAP25 gene comprises a primer 3 and a primer 4, wherein the sequence of the primer 3 is shown as SEQ ID NO. 4, and the sequence of the primer 4 is shown as SEQ ID NO. 5; preferably, the sequence of the second specific probe for detecting the methylation of the ARHGAP25 gene is shown as SEQ ID NO. 6.
Preferably, the sequence of the primer 3 is 5'-CAGCTCTCAGGCGGAGATG-3';
the sequence of the primer 4 is 5'-CAGGGTGTGCCAGCAACTCT-3';
the sequence of the second specific probe is 5 '-FAM-AGTGGGTTAAATTCC-BHQ-3'.
In a preferred embodiment of the invention, the specific primer for detecting methylation of the KLF3 gene comprises a primer 5 and a primer 6, wherein the sequence of the primer 5 is shown as SEQ ID NO. 7, and the sequence of the primer 6 is shown as SEQ ID NO. 8; preferably, the sequence of the third specific probe for detecting the methylation of the KLF3 gene is shown as SEQ ID NO. 9.
Preferably, the sequence of the primer 5 is 5'-GGACTGTGACCGCAGCTTCT-3';
the sequence of the primer 6 is 5'-CAGGGTGTGCCAGCAACTCT-3';
the sequence of the third specific probe is 5 '-FAM-TTCTGACCATCTTGTC-BHQ-3'.
In a preferred embodiment of the invention, the specific primer for detecting methylation of ATXN2 gene comprises a primer 7 and a primer 8, wherein the sequence of the primer 7 is shown as SEQ ID NO. 10, and the sequence of the primer 8 is shown as SEQ ID NO. 11; preferably, the sequence of the fourth specific probe for detecting the methylation of the ATXN2 gene is shown as SEQ ID NO. 12.
Preferably, the sequence of the primer 7 is 5'-GCATGTCCCAAATTACCATACAAC-3';
the sequence of the primer 8 is 5'-CCGTGGAAATGGCAAAGTAGA-3';
the sequence of the fourth specific probe is 5 '-FAM-AGGAGACAAGTCCTTC-BHQ-3'.
In a preferred embodiment of the invention, the kit further comprises a specific primer and a fifth specific probe for detecting methylation of the beta-actin internal reference gene, preferably, the specific primer for detecting methylation of the beta-actin internal reference gene comprises a primer 9 and a primer 10, the sequence of the primer 9 is shown as SEQ ID NO. 13, and the sequence of the primer 10 is shown as SEQ ID NO. 14; preferably, the sequence of the fifth specific probe for detecting methylation of the reference gene of beta-actin is shown as SEQ ID NO. 15.
Preferably, the sequence of the primer 9 is 5'-AAGATAGTGTTGTGGGTGTAGGT-3';
the sequence of the primer 10 is 5'-CCTACTTAATACACACTCCAAAAC-3';
the sequence of the fifth specific probe is 5 '-HEX-ACACCAACCTCATAACCTTATCACAC-BHQ-3'.
In a preferred embodiment of the present invention, the kit further comprises a microdroplet generating oil for making the methylated DNA template and a premix comprising primers and probes for methylation of different detection genes and primers and probes for methylation of a β -actin internal reference gene into microdroplets in a microdroplet generator.
In a preferred embodiment of the present invention, the kit further comprises a positive quality control substance, preferably, the positive quality control substance comprises methylated fragments of BMPR1A gene, ARHGAP25 gene, KLF3 gene and ATXN2 gene, preferably, the methylated fragments are artificially synthesized fragments.
In a preferred embodiment of the invention, the kit further comprises a negative quality control product, and preferably, the negative quality control product comprises non-methylated fragments of BMPR1A gene, ARHGAP25 gene, KLF3 gene and ATXN2 gene.
In a preferred embodiment of the present invention, the kit further comprises nuclease-free water, and preferably, the kit further comprises a template-free control, wherein the template-free control refers to a reaction system without human genomic DNA.
The invention provides application of the kit in preparation of a reagent for early screening of liver cancer.
In a preferred embodiment of the present invention, the reagent for screening liver cancer at an early stage is used as follows: adding a sample DNA, a positive quality control product and a negative quality control product into a specific primer and a first specific probe for detecting BMPR1A gene methylation, a specific primer and a second specific probe for detecting ARHGAP25 gene methylation, a specific primer and a third specific probe for detecting KLF3 gene methylation, a specific primer and a fourth specific probe for detecting ATXN2 gene methylation, microdroplet generation oil, a specific primer and a fifth specific probe for detecting beta-actin internal reference gene methylation, mixing and preparing into microdroplets, and then carrying out fluorescent quantitative PCR reaction.
Preferably, the DNA is methylated DNA, more preferably, the methylated DNA is obtained by centrifuging peripheral blood, extracting free DNA with a DNA extraction reagent, and then modifying with sulfite.
In a preferred embodiment of the present invention, the fluorescent quantitative PCR reaction conditions are: pre-denaturation at 94-95 ℃ for 5-10 min for 1 cycle; 94-95 ℃ for 15-30 s, 58-60 ℃ for 30-45 s, and 40-50 cycles.
Preferably, the fluorescent quantitative PCR reaction conditions are: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, performing 40 cycles, and finally performing PCR amplification by extension at 98 ℃ for 10 min.
The invention adopts a Berle QX200TM microdroplet digital PCR detection system to carry out detection and analysis, and the reaction comprises 4 steps of configuring a system, generating microdroplets, amplifying cycles and reading signals. The microdroplet digital PCR system was 20. mu.l containing 10. mu.l of 2 XDd PCR Master Mix, 10. mu. mol/L of each of the forward and reverse primers 0.8. mu.l, probe 0.4. mu.l, and DNA template 2. mu.l. The generation of droplets required the use of a specialized droplet generation card and droplet generator, and the droplets were generated by adding 40. mu.l of the PCR system and 70. mu.l of the droplet-generating oil to the droplet-generating card, covering the specialized gel pad and then placing into the droplet generator.
Microdroplet generation is a key step of ddPCR, and the system can count positive and negative microdroplets only when the number of microdroplets is more than 10000, and the distribution of DNA molecules can accord with the statistical principle of Poisson distribution.
The determination method of the detection result comprises the following steps: (1) positive control: 20 + -5 copies; (2) negative control: <1 copy; and (3) judging the result of the sample to be detected: (1) positive: the specimen detection result is more than or equal to 1 copy. (2) Negative: specimen test results <1 copy.
The primer and the probe used in the present invention were both synthesized by England Weiji (Shanghai) trade Co., Ltd.
The type of the droplet generator used in the invention is QX200, the type of the droplet generating card is DG8TM, and the manufacturer is Bole Life medical products (Shanghai) limited;
the KIT for modifying sulfite used in the present invention is EZ DNA Methylation-direct KIT (D5020) provided by ZYMO RESEARCH Bio.
Example 1
The kit comprises a specific primer and a first specific probe for detecting the methylation of a BMPR1A gene, a specific primer and a second specific probe for detecting the methylation of an ARHGAP25 gene, a specific primer and a third specific probe for detecting the methylation of a KLF3 gene, a specific primer and a fourth specific probe for detecting the methylation of an ATXN2 gene, a specific primer and a fifth specific probe for detecting the methylation of a beta-actin internal reference gene, micro-droplet generation oil, a positive quality control product, a negative quality control product, a template-free control product and nuclease-free water;
the specific primer for detecting the methylation of the BMPR1A gene comprises a primer 1 and a primer 2, wherein the sequence of the primer 1 is shown as SEQ ID NO. 1, the sequence of the primer 2 is shown as SEQ ID NO. 2, and the sequence of the first specific probe for detecting the methylation of the BMPR1A gene is shown as SEQ ID NO. 3;
the specific primer for detecting methylation of the ARHGAP25 gene comprises a primer 3 and a primer 4, wherein the sequence of the primer 3 is shown as SEQ ID NO. 4, the sequence of the primer 4 is shown as SEQ ID NO. 5, and the sequence of the second specific probe for detecting methylation of the ARHGAP25 gene is shown as SEQ ID NO. 6;
the specific primer for detecting methylation of the KLF3 gene comprises a primer 5 and a primer 6, wherein the sequence of the primer 5 is shown as SEQ ID NO. 7, the sequence of the primer 6 is shown as SEQ ID NO. 8, and the sequence of the third specific probe for detecting methylation of the KLF3 gene is shown as SEQ ID NO. 9;
the specific primer for detecting the methylation of the ATXN2 gene comprises a primer 7 and a primer 8, wherein the sequence of the primer 7 is shown as SEQ ID NO. 10, the sequence of the primer 8 is shown as SEQ ID NO. 11, and the sequence of the fourth specific probe for detecting the methylation of the ATXN2 gene is shown as SEQ ID NO. 12;
the specific primer for detecting methylation of the beta-actin internal reference gene comprises a primer 9 and a primer 10, wherein the sequence of the primer 9 is shown as SEQ ID NO. 13, the sequence of the primer 10 is shown as SEQ ID NO. 14, and the sequence of the fifth specific probe for detecting methylation of the beta-actin internal reference gene is shown as SEQ ID NO. 15;
the positive quality control product comprises methylated fragments of a BMPR1A gene, an ARHGAP25 gene, a KLF3 gene and an ATXN2 gene;
the negative quality control product comprises non-methylated fragments of a BMPR1A gene, an ARHGAP25 gene, a KLF3 gene and an ATXN2 gene;
the no-template control is a reaction system that does not contain human genomic DNA.
EXAMPLE two methods of Using the kit
(1) Plasma separation: collecting 5-10 mL of peripheral blood of a human by using an EDTA (ethylene diamine tetraacetic acid) anticoagulant tube, centrifuging a blood sample for 1600 g and 10min, carefully transferring the collected plasma sample to a 2mL centrifuge tube, centrifuging the plasma sample at 4 ℃, 16000 g and 10min, carefully transferring the collected plasma sample to a new 2mL centrifuge tube, and storing the collected plasma sample at-20 ℃;
(2) extraction of free DNA: extracting peripheral blood free DNA by using a DNA extraction reagent, monitoring the DNA quality, and selecting free DNA with OD260/OD280 of 1.6-2.0;
(3) DNA modification: performing sulfite modification on the free DNA obtained in the step (2) by using an EZ DNA Methylation-DirectTM KIT (D5020) instruction of a ZYMO RESEARCH biological company KIT, so that unmethylated 5 'cytosine in the DNA is converted into uracil, and methylated 5' cytosine is not changed, thereby obtaining Bis-cfDNA;
(4) preparing a microdroplet digital PCR reaction system: preparing 20 mu L of microdroplet digital PCR reaction system by taking the Bis-cfDNA in the step (3) as a template, wherein the microdroplet digital PCR reaction system comprises 18 mu L of ddPCR probe method premixed solution and 2 mu L of Bis-cfDNA, the ddPCR probe method premixed solution for detecting the methylation of the BMPR1A gene comprises 500 mu L of 2X ddPCR Supermix for probes, specific primers 1 and 2 for detecting the methylation of the BMPR1A gene, the primer amounts are respectively 60 mu L, the first specific probe for detecting the methylation of the BMPR1A gene is 20 mu L, specific primers 9 and 10 for detecting the methylation of the beta-actin internal reference gene are respectively 30 mu L, the fifth specific probe for detecting the methylation of the beta-actin internal reference gene is 15 mu L and RNase dH2O 185μl；
The ddPCR probe method premix solution for detecting methylation of the ARHGAP25 gene comprises 500 mu L of 2X ddPCR Supermix for probes, 60 mu L of a specific primer 3 and a specific primer 4 for detecting methylation of the ARHGAP25 gene, 20 mu L of a second specific probe for detecting methylation of the ARHGAP25 gene, 30 mu L of a specific primer 9 and a specific primer 10 for detecting methylation of a beta-actin internal reference gene, 15 mu L of a fifth specific probe for detecting methylation of the beta-actin internal reference gene and RNase Free dH2O 185μl；
The pre-mixed solution of ddPCR probe method for detecting methylation of KLF3 gene comprises 500 mu L of 2X ddPCR Supermix for probes, 60 mu L of specific primer 5 and primer 6 for detecting methylation of KLF3 gene, 20 mu L of third specific probe for detecting methylation of KLF3 gene, 30 mu L of specific primer 9 and primer 10 for detecting methylation of beta-actin internal reference gene, and fifth specific probe for detecting methylation of beta-actin internal reference geneThe probe was 15. mu.l and RNase Free dH2O185μl；
The ddPCR probe method premix solution for detecting the methylation of the ATXN2 gene comprises 500 mu L of 2X ddPCR Supermix for probes, 60 mu L of a specific primer 7 and a specific primer 8 for detecting the methylation of the ATXN2 gene, 20 mu L of a fourth specific probe for detecting the methylation of the KLF3 gene, 30 mu L of a specific primer 9 and a primer 10 for detecting the methylation of the beta-actin internal reference gene, 15 mu L of a fifth specific probe for detecting the methylation of the beta-actin internal reference gene and RNase Free dH2O185μl；
Wherein the concentration of the primer and the concentration of the probe are both 10 mu M;
(5) preparing microdroplets: adding 20 μ l of PCR reaction solution prepared in step (4) into a corresponding hole on a DG8TM microdroplet generation card, covering a microdroplet generation card sealing pad with 70 μ l of microdroplet generation special oil, and putting the microdroplet generation card sealing pad into a QX200 microdroplet generator for reaction; then slowly transferring the treated microdroplets to a 96-well plate, then placing the 96-well plate on a heat sealing instrument, covering a heat sealing film for sealing the film to obtain microdroplet reaction liquid;
(6) digital PCR amplification: denaturing the microdroplet reaction solution obtained in the step (5) at 95 ℃ for 5min, then denaturing at 95 ℃ for 15sec, and annealing at 60 ℃ for 30sec for 40 cycles; finally, performing PCR amplification at the step of extending for 10min at 98 ℃;
(7) signal collection and data analysis: the PCR plate reacted in step (6) was transferred to a microdroplet analyzer (Bio-rad Laboratories USA) for fluorescence detection, and concentration calculation was performed using Quanta software, and methylation of the gene was judged if amplification (droplet distribution in the FAM fluorescence signal region) was present under the premise that amplification was controlled (droplet distribution in the HEX fluorescence signal region).
Example 3
The kit of the first embodiment is used for detecting 20 samples according to the using method of the second embodiment, wherein 10 samples are blood samples of normal liver disease-free volunteers, and 10 samples are blood samples of volunteers diagnosed with liver cancer. The detection result obtained by using the kit in the first embodiment is consistent with the fact. The kit provided by the invention can be applied to liver cancer screening, and the detection result is accurate and reliable.
The foregoing is considered as illustrative and not restrictive in character, and that various modifications, equivalents, and improvements made within the spirit and principles of the invention are intended to be included within the scope of the invention.
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Claims (13)
1. A gene methylation digital PCR kit for early screening of liver cancer is characterized by comprising a specific primer and a first specific probe for detecting the methylation of a BMPR1A gene;
a specific primer and a second specific probe for detecting methylation of the ARHGAP25 gene;
a specific primer and a third specific probe for detecting methylation of the KLF3 gene; and
a specific primer and a fourth specific probe for detecting the methylation of the ATXN2 gene.
2. The gene methylation digital PCR kit according to claim 1, wherein the specific primers for detecting the methylation of the BMPR1A gene comprise primer 1 and primer 2,
the sequence of the primer 1 is shown as SEQ ID NO. 1, and the sequence of the primer 2 is shown as SEQ ID NO. 2;
preferably, the sequence of the first specific probe for detecting the methylation of the BMPR1A gene is shown as SEQ ID NO. 3.
3. The gene methylation digital PCR kit according to claim 1 or 2, wherein the specific primers for detecting methylation of the ARHGAP25 gene comprise a primer 3 and a primer 4,
the sequence of the primer 3 is shown as SEQ ID NO. 4, and the sequence of the primer 4 is shown as SEQ ID NO. 5;
preferably, the sequence of the second specific probe for detecting the methylation of the ARHGAP25 gene is shown as SEQ ID NO. 6.
4. The gene methylation digital PCR kit according to any one of claims 1 to 3, wherein the specific primers for detecting methylation of the KLF3 gene comprise primer 5 and primer 6,
the sequence of the primer 5 is shown as SEQ ID NO. 7, and the sequence of the primer 6 is shown as SEQ ID NO. 8;
preferably, the sequence of the third specific probe for detecting the methylation of the KLF3 gene is shown as SEQ ID NO. 9.
5. The gene methylation digital PCR kit according to any one of claims 1 to 4, wherein the specific primers for detecting methylation of the ATXN2 gene comprise primer 7 and primer 8,
the sequence of the primer 7 is shown as SEQ ID NO. 10, and the sequence of the primer 8 is shown as SEQ ID NO. 11;
preferably, the sequence of the fourth specific probe for detecting the methylation of the ATXN2 gene is shown as SEQ ID NO. 12.
6. The gene methylation digital PCR kit according to any one of claims 1 to 5, wherein the kit comprises a microdroplet generating oil.
7. The gene methylation digital PCR kit according to any one of claims 1 to 6, wherein the kit further comprises a specific primer and a fifth specific probe for detecting methylation of a β -actin reference gene,
preferably, the specific primers for detecting methylation of the beta-actin reference gene comprise a primer 9 and a primer 10,
the sequence of the primer 9 is shown as SEQ ID NO. 13, and the sequence of the primer 10 is shown as SEQ ID NO. 14;
preferably, the sequence of the fifth specific probe for detecting methylation of the reference gene of beta-actin is shown as SEQ ID NO. 15.
8. The gene methylation digital PCR kit according to any one of claims 1 to 7, wherein the kit further comprises a positive quality control,
preferably, the positive quality control product comprises methylated fragments of the BMPR1A gene, the ARHGAP25 gene, the KLF3 gene and the ATXN2 gene.
9. The gene methylation digital PCR kit according to any one of claims 1 to 8, wherein the kit further comprises a negative quality control,
preferably, the negative quality control product comprises non-methylated fragments of BMPR1A gene, ARHGAP25 gene, KLF3 gene and ATXN2 gene.
10. The gene methylation digital PCR kit according to any one of claims 1 to 9, wherein the kit further comprises nuclease-free water.
11. Use of the gene methylation digital PCR kit according to any one of claims 1 to 10 in the preparation of a reagent for screening liver cancer at an early stage.
12. The use of claim 11, wherein the reagent for screening liver cancer at an early stage is used by the following method: adding a sample DNA, a positive quality control product and a negative quality control product into a specific primer and a first specific probe for detecting BMPR1A gene methylation, a specific primer and a second specific probe for detecting ARHGAP25 gene methylation, a specific primer and a third specific probe for detecting KLF3 gene methylation, a specific primer and a fourth specific probe for detecting ATXN2 gene methylation, microdroplet generation oil, a specific primer and a fifth specific probe for detecting beta-actin internal reference gene methylation, mixing and preparing into microdroplets, and then carrying out fluorescent quantitative PCR reaction.
13. The use of claim 12, wherein the fluorescent quantitative PCR reaction conditions are: pre-denaturation at 94-95 ℃ for 5-10 min for 1 cycle; 94-95 ℃ for 15-30 s, 58-60 ℃ for 30-45 s, and 40-50 cycles.
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