CN110643709A - Nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3 methylation detection kit - Google Patents
Nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3 methylation detection kit Download PDFInfo
- Publication number
- CN110643709A CN110643709A CN201911101925.4A CN201911101925A CN110643709A CN 110643709 A CN110643709 A CN 110643709A CN 201911101925 A CN201911101925 A CN 201911101925A CN 110643709 A CN110643709 A CN 110643709A
- Authority
- CN
- China
- Prior art keywords
- nfat1
- dact1
- shisa3
- nasopharyngeal carcinoma
- pcr reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/6876—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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a methylation detection kit for nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3, which comprises an alkaline solution and a fluorescent PCR reaction solution, wherein the alkaline solution and the fluorescent PCR reaction solution are packaged in the same PCR amplification tube by layers by a hot-melt material; the alkaline solution is NaOH solution with the pH value of 12.5-13.3 and the concentration of 50-100 mmol/L, and the volume of the alkaline solution is not more than 50% of that of the PCR reaction solution; the fluorescent PCR reaction solution comprises primers and probes aiming at DACT1, NFAT1 and SHISA3 genes and UDG enzyme. The invention effectively applies UDG enzyme by carrying out systematic and step-by-step heat treatment before the fluorescent PCR reaction, effectively solves the problem of PCR pollution in methylation detection, and has higher detection sensitivity and specificity and higher detection flux.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a detection kit for nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3 methylation.
Technical Field
Nasopharyngeal carcinoma is a malignant tumor originating from the nasopharyngeal epithelium, is highly locally invasive and can produce distant metastases, with a high incidence in southern china and southeast asia (Torre LAet al, CACancer J Clin,2015,65, 87-108). In recent years, the application of intensive-setting radiotherapy and chemotherapy has greatly improved the prognosis of nasopharyngeal carcinoma, especially early nasopharyngeal carcinoma, but about 30% of patients with nasopharyngeal carcinoma eventually develop recurrence and/or distant metastasis (Lai SZ et al, Int J Radiation Oncology Biol Phys,2011,80, 661-. Thus, the major challenge in nasopharyngeal cancer treatment remains the lack of effective biomarkers to develop more accurate diagnostic, prognostic, therapeutic and prophylactic methods.
Like other types of cancer, nasopharyngeal carcinoma is associated with a variety of genetic mutations and epigenetic abnormalities; among them, abnormal DNA methylation of promoter CpG islands is the basis for the development and progression of nasopharyngeal carcinoma (Jiang W et al, Asian Pac Jcancer Prev,2015,16, 8059-8065). In nasopharyngeal carcinoma epithelial cells, many genes are primarily silenced by DNA methylation. Therefore, the identification of the DNA methylation differential gene is helpful for understanding the pathogenesis of the DNA methylation differential gene, and provides an effective biomarker for early diagnosis of the nasopharyngeal carcinoma, recurrence/metastasis of the nasopharyngeal carcinoma, optimization and personalized treatment of the nasopharyngeal carcinoma.
DACT1 is a tumor-associated gene that plays an important role in regulating tumor Cell growth, proliferation, invasion and metastasis (Cheyette BNR, et al, development Cell,2002,2, 449-461). The DACT1 gene contains 6 exons and maps to human chromosome 14q 23.1. Recent studies found that the DACT1 gene is highly methylated in 71% of patients with nasopharyngeal carcinoma, and that the incidence of methylation of the DACT1 gene is higher, up to 84%, in patients with lymph node metastasis nasopharyngeal carcinoma (Yang jhe al., Biological Research,2019,52, 31). Further studies have shown that methylation of the DACT1 gene may down-regulate DACT1 expression, thereby promoting invasion and metastasis of nasopharyngeal carcinoma cells (Yang JH et al, Biological Research,2019,52, 31). The research shows that DACT1 gene methylation may be an important index for the development and prognosis of nasopharyngeal carcinoma in the future.
NFAT1, also known as NFATC2, NFATP, is one of the nuclear factor family members of activated T cells. It regulates cell transformation by regulating the expression of cell cycle related protein, and exerts tumor inhibiting effect by reversing the transformation phenotype of tumor cells. The gene contains 13 exons and is located on human chromosome 20q 13.2. One genome-wide microarray data showed that NFAT1 is one of the most methylated transcription factor genes in nasopharyngeal carcinoma tissues (Ren X et al, nature communications,2017,8, 14053). Compared with normal tissues and normal nasopharyngeal epithelial cell lines, the methylation level of NFAT1 in nasopharyngeal carcinoma tissues and nasopharyngeal carcinoma cell lines is obviously higher (Zhang J et al, Neopalasia, 2019,21, 311-321). In nasopharyngeal carcinoma, NFAT1 promoter methylation leads to NFAT1 epigenetic silencing, while NFAT1 epigenetic silencing promotes nasopharyngeal carcinoma epithelial-mesenchymal transition and metastasis by activating ITGA6 transcription (Zhang J et al, Neoplasia,2019,21, 311-321). It can be seen that NFAT1 gene methylation can be a potential biomarker for nasopharyngeal carcinoma metastasis/progression.
SHISA3 is a cancer suppressor gene, which contains 2 exons and is located on human chromosome 4p 13. Hypermethylation of the SHISA3 promoter is a common phenomenon of various tumors, including nasopharyngeal carcinoma; SHISA3 methylation levels were significantly higher in nasopharyngeal carcinoma tissues and nasopharyngeal carcinoma cell lines than those of normal tissues and normal nasopharyngeal epithelial cell lines (Zhang J et al, Cancer Res,2019,79, 747-759). In nasopharyngeal carcinoma, hypermethylation of the SHISA3 promoter leads to down-regulation of SHISA3 expression, ectopic SHISA3 expression inhibits nasopharyngeal carcinoma cell migration, invasion and metastasis, while down-regulation of SHISA3 expression increases nasopharyngeal carcinoma and normal nasopharyngeal epithelial cell migration and invasion capacity by decreasing SGSM1 stability (Zhang J et al, Cancer Res,2019,79, 747-. It can be seen that SHISA3 exerts its cancer suppressing effect mainly by suppressing metastasis, while SHISA3 promoter hypermethylation is a potential indicator of nasopharyngeal carcinoma metastasis.
In view of the role of methylation of DACT1, NFAT1 and SHISA3 genes in nasopharyngeal carcinoma, it is necessary to detect the methylation states of the three genes in nasopharyngeal carcinoma patients in order to provide reference information for early diagnosis of nasopharyngeal carcinoma, recurrence/metastasis of nasopharyngeal carcinoma, optimization and personalized treatment of nasopharyngeal carcinoma.
The methylation detection method of the CpG island of the gene which is most widely applied at present is a bisulfite conversion method. The method adopts bisulfite to treat a DNA sample to be detected, and converts cytosine which is not methylated into uracil, while the methylated cytosine remains unchanged; after PCR amplification, uracil is converted into thymine, and methylated cytosine is converted into unmethylated cytosine, so that the difference between methylation and non-methylation on the genome is converted into the difference of cytosine/thymine base, and whether the gene is methylated or not can be determined by detecting the state of the cytosine/thymine base on the target site.
Methylation detection methods based on bisulfite conversion are many, such as bisulfite sequencing, pyrosequencing, methylation-specific PCR, fluorescent PCR, and the like. In the methods, the target gene is subjected to PCR amplification through a specific primer, and then subsequent analysis and detection are carried out. However, PCR is particularly susceptible to contamination due to its high sensitivity, and very small amounts of contamination can result in false positive results. The cause of PCR contamination may be: cross contamination between samples; contamination of PCR reagents; PCR amplification product pollution; fourthly, aerosol pollution; and the like. In addition, non-specific amplification may also cause inaccuracy in PCR detection results. In order to eliminate contamination of PCR products and to prevent non-specific amplification, uracil-DNA-glycosylase (UDG enzyme) was added to the PCR system. The UDG enzyme can crack N-glycosyl bonds between uracil bases and sugar phosphate frameworks in PCR amplification products, and the UDG enzyme is used for treating PCR reaction liquid before PCR amplification to eliminate PCR product pollution and prevent non-specific amplification, so that false positive results are prevented, and the detection result is accurate and reliable.
However, the use of UDG enzymes is limited in PCR systems for detecting gene methylation. Since unmethylated cytosine in a DNA sample is converted to uracil by bisulfite conversion, if UDG enzyme is added to the PCR system, it can be degraded by the UDG enzyme, and the target fragment cannot be amplified. In the bisulfite conversion process, cytosine is first converted to 6-sulfonyl-uracil, which is then converted to uracil by NaOH desulfurization. The conversion intermediate containing 6-sulfonyl-uracil is neither degraded by UDG enzyme nor amplified by Taq DNA polymerase.
Therefore, the problems of PCR contamination, nonspecific amplification, etc. in the detection of gene methylation by PCR technology remain to be solved. In addition, no kit product for detecting methylation of nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3 exists in the market at present.
Disclosure of Invention
In order to solve the problems, the invention provides a methylation detection kit for nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3, which is based on a specially designed fluorescent PCR system and takes DNA converted by bisulfite but not desulfurized as a template for methylation detection, can effectively reduce PCR pollution and nonspecific amplification, and has higher detection sensitivity and specificity.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the kit for detecting methylation of nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3 comprises an alkaline solution and a fluorescent PCR reaction solution, wherein the alkaline solution and the fluorescent PCR reaction solution are packaged in the same PCR amplification tube layer by a hot-melt material; the alkaline solution is NaOH solution with the pH value of 12.5-13.3 and the concentration of 50-100 mmol/L, and the volume of the alkaline solution is not more than 50% of that of the PCR reaction solution; the fluorescent PCR reaction solution comprises PCR buffer solution, dNTP, primers and probes aiming at DACT1, NFAT1 and SHISA3 genes, DNA polymerase, nuclease-free water and UDG enzyme (uracil-DNA-glycosylase).
In one embodiment, the alkaline solution is NaOH solution with pH value of 12.5-12.8 and concentration of 70-80 mmol/L, and the volume of the NaOH solution is not more than 50% of the volume of the PCR reaction solution.
In one embodiment, the hot melt material is a hydrophobic material that is immiscible with the alkaline solution, the PCR reaction solution, and the UDG enzyme; preferably, the hot melt material is paraffin with a melting point of 54-56 ℃.
In one embodiment, the primers and probes comprise: SEQ ID NO.1 to SEQ ID NO.3 for the DACT1 gene, SEQ ID NO.4 to SEQ ID NO.6 for the NFAT1 gene, and/or SEQ ID NO.7 to SEQ ID NO.9 for the SHISA3 gene. The primers are all designed as Locked Nucleic Acid (LNA) modified primers; the LNA is modified at least one of the 3 bases on the outermost side of the 3 'end of the primer, preferably the 2 nd to last or 3 rd to last base among the 3 bases on the outermost side of the 3' end of the primer. The primer with the 3' end modified by the LNA can improve the mismatch recognition capability in the specific PCR amplification process, and can ensure the amplification specificity while ensuring the amplification efficiency. The probe is designed into a competitive fluorescent probe and has a secondary structure, a base sequence of a site to be detected is placed at the 5 'end of the fluorescent probe, 3-5 bases which are not matched with the probe completely are added near the 3' end of the fluorescent probe to form a semi-ring structure with hydrolyzability, 3-5 bases which are complementary with the 5 'end are added at the 3' end, and the specificity of the probe is improved. The 5 'end of the modified fluorescent probe is provided with a fluorescent reporter group, and the 3' end of the modified fluorescent probe is provided with a fluorescent quenching group; the fluorescent reporter group comprises FAM, HEX, ROX or CY 5; the fluorescence quenching group is BHQ.
Preferably, the 5 ' end of the fluorescent probe is a base sequence of a site to be detected, the 3 ' end is additionally provided with 3 bases which are complementary with the 5 ' end of the fluorescent probe, and the 3 ' end and the 5 ' end of the fluorescent probe compete together with a human genome template, so that the specificity of a reaction system is improved. When the human genome template has a site to be detected, the 5' end of the fluorescent probe is combined with the site to be detected and hydrolyzed to release a fluorescent signal in the extension stage of the fluorescent PCR; when the human genome template does not have a target detection site, the 5 'end of the fluorescent probe is not combined with the human genome template, but is combined with the 3' end of the fluorescent probe, so that the fluorescent probe cannot emit fluorescence, and the human genome methylation is detected.
Preferably, 4 bases which are not complementary with the probe are additionally added at the 3' end of the probe, and the bases are not complementary with the template genome, so that the hydrolysis efficiency of the probe is improved, and the sensitivity of a reaction system is improved.
In one embodiment, the fluorescent PCR reaction solution further includes a blocker primer.
The Blocker primers sequentially comprise sequences shown as SEQ ID 13 to SEQ ID16, are designed aiming at a gene fragment which is not methylated or a gene fragment which is not transformed by bisulfite, and the 3' ends of the Blocker primers are all marked with fluorescent dye CY3 so as to prevent the amplification of the Blocker primers. In the PCR amplification process, the target gene amplification primer can be combined with a gene segment which is not methylated or a gene segment which is not converted by bisulfite, so that a non-specific amplification product is generated, and the interpretation of the final detection result is influenced; and a Blocker primer is introduced into a PCR amplification system and is specifically combined with other gene fragments except the methylated fragment, so that the target gene amplification primer is only combined with the methylated fragment, the non-specific amplification of the target gene amplification primer is reduced, and the PCR amplification sensitivity and the amplification specificity are improved.
In one embodiment, the kit further comprises primers and probes of reference genes shown in SEQ ID 10 to SEQ ID 12.
In one embodiment, the UDG enzyme is used at a concentration of 5U/. mu.l in an amount of 2. + -. 0.1. mu.l.
In one embodiment, the method further comprises a negative quality control product and a positive quality control product. The negative quality control consists of BSA and bisulphite-converted, but not desulphated, unmethylated human genomic DNA.
The positive quality control consists of BSA, bisulphite-converted non-methylated human genomic DNA without desulphation treatment and bisulphite-converted methylated human genomic DNA without desulphation treatment.
The fluorescent PCR reaction system also introduces UDG enzyme which is used for eliminating PCR pollution possibly existing in a methylation amplification system. In general, bisulfite conversion products are all required to be desulfurized to be recognized by Taq DNA polymerase, and then can be used as an effective template for PCR amplification; however, uracil produced after desulfurization can be degraded by UDG enzyme, which limits the application of UDG enzyme in methylation detection systems. The invention fully utilizes the characteristic that the UDG enzyme can not degrade the 6-sulfonyl-uracil, optimizes the methylation detection process, and can effectively eliminate PCR pollution and non-specific amplification in a methylation amplification system by skillfully combining a hot melting material and reaction conditions and matching with reasonable design of pH value, and sequentially finishing UDG enzyme digestion, 6-sulfonyl-uracil desulfurization and methylation PCR amplification in a closed PCR amplification tube by using a DNA sample which is converted by bisulfite and is not subjected to desulfurization treatment as an initial template.
Another object of the present invention is to provide a method for preparing the above-mentioned detection kit.
The preparation method of the nasopharyngeal carcinoma related gene DACT1, NFAT1 and SHISA3 methylation detection kit comprises the following steps: 1) adding the alkaline solution to the bottom of the PCR amplification tube, and sealing the alkaline solution at the bottom of the PCR amplification tube by using a hot-melt material with a proper melting point; 2) then, the fluorescent PCR reaction solution and UDG enzyme were added to the PCR amplification tube.
Another object of the present invention is to provide a method for using the above-mentioned detection kit.
The technical scheme for achieving the purpose is as follows.
The use method of the nasopharyngeal carcinoma related gene DACT1, NFAT1 and SHISA3 methylation detection kit comprises the following steps:
(1) obtaining a bisulfite converted, but not desulfurized, DNA sample as a template
(2) And (3) fluorescent PCR reaction: adding the template into a fluorescent PCR reaction system, and sequentially reacting on a fluorescent PCR instrument under the following reaction conditions:
UDG enzymatic digestion: incubation at 37 ℃ for 10 min, 1 cycle;
UDG enzyme inactivation, desulfurization and pre-denaturation: incubation at 95 ℃ for 10 min, 1 cycle;
and (3) PCR amplification: 95 ℃ temperature in 30 seconds, 60 ℃ temperature in 30 seconds, 45 cycle.
The invention has the beneficial effects that:
(1) the fluorescent PCR reaction system fully utilizes the characteristic that the UDG enzyme can not degrade the 6-sulfonyl-uracil, uses a hot melting material with a proper melting point to separate an alkaline solution, the PCR reaction solution and the UDG enzyme in the same PCR amplification tube, and uses a DNA sample which is converted by bisulfite but not desulfurized as an initial template to realize the processes of sequentially finishing the digestion of the UDG enzyme, the 6-sulfonyl-uracil and the methylation PCR amplification in the same PCR amplification tube. The design can eliminate PCR pollution of the UDG enzyme under the condition of not degrading a DNA template, thereby preventing the generation of false positive results and ensuring the accuracy and reliability of detection results.
(2) The 3' end of the primer for PCR amplification is introduced with LNA, which can improve the mismatch recognition capability in the specific PCR amplification process, and can improve the amplification efficiency and the amplification stability and specificity.
(3) The fluorescent probe adopts a specially designed competitive probe, and 3-5 bases complementary to the 5 'end of the fluorescent probe are additionally arranged at the 3' end, so that the non-specific combination of the fluorescent probe and an amplification product is prevented, and the specificity of a reaction system is improved; and 3-5 bases which are not complementary with the probe are additionally arranged at the 3' end, so that the hydrolysis efficiency of the probe is improved, and the sensitivity of a reaction system is improved.
(4) The invention adopts quadruple fluorescent PCR reaction, can detect the methylation state of three genes of DACT1, NFAT1 and SHISA3 by one-time reaction in a single tube, greatly saves reagent consumables and shortens detection time. The quadruple fluorescent PCR reaction can detect the methylation states of three genes of DACT1, NFAT1 and SHISA3 by one-time reaction in a single tube, and has relatively high flux.
(5) Furthermore, the invention introduces a Blocker primer into the amplification system to remove the interference of non-methylated gene segments or gene segments which are not converted by bisulfite and strictly control non-specific amplification, thereby further improving the sensitivity and specificity of detection and ensuring the accuracy and reliability of detection results.
(6) Furthermore, the invention is designed with a negative quality control product and a positive quality control product, which can better prevent the generation of false positive results and false negative results, thereby ensuring the accuracy and reliability of the detection result.
Drawings
FIG. 1 is an agarose gel electrophoresis diagram of the PCR amplification product of LNA modified primers and common primers, wherein, 1: NFAT1 gene (140bp) amplified by LNA modified primer; 2: the gene (174bp) of SHISA3 amplified by the LNA modified primer; 3: internal reference gene (108bp) amplified by LNA modified primer; 4: DACT1 gene (118bp) amplified by LNA modified primer; 5: NFAT1 gene amplified by traditional primer; 6: the gene of SHISA3 amplified by traditional primers; 7: an internal reference gene amplified by a traditional primer; 8: negative control; 9: DACT1 gene amplified by traditional primer.
FIG. 2 is a fluorescent PCR curve of the negative quality control of the present invention, curve 1: the internal reference ACTB gene (CY5 channel).
FIG. 3 is a fluorescent PCR curve of the positive quality control of the present invention, wherein the curve 1: NFAT1 gene (HEX channel), curve 2: DACT1 gene (FAM channel), curve 3: SHISA3 gene (ROX channel), curve 4: internal reference genes (CY5 channel).
Detailed Description
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, cell biology, immunology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and maniotis, molecular cloning, a laboratory manual, 3 rd edition (2002). The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The present invention will be further illustrated with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
Example 1: preparation of kit for detecting methylation of nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3
The kit for detecting methylation of nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3 comprises a fluorescent PCR reaction system, a negative quality control product and a positive quality control product. The preparation of the kit comprises the following steps:
(1) preparing an alkaline solution: preparing a solution with the concentration of 75mM and the pH value of 12.5 by taking a proper amount of NaOH, namely an alkaline solution, and storing for later use.
(2) Designing and preparing primers and probes: a plurality of groups of primers and probes are respectively designed aiming at CpG islands of DACT1, NFAT1 and SHISA3 genes and ACTB genes, the primers and the probes are subjected to pre-experiments, the performances such as sensitivity, specificity and the like are compared, and finally four groups of LNA modified primers for methylation detection and specially designed competitive fluorescent probes are preferably selected, specifically shown as SEQID 1-SEQ ID 12. The preferred primers and probes are stored as 100. mu.M stock solution and prepared as 20. mu.M working solution as required for use.
(3) Designing and preparing a Blocker primer: blocker primers were designed for the DACT1, NFAT1 and SHISA3 gene fragments that were not methylated or for the gene fragments that were not transformed with bisulfite, specifically as shown in SEQ ID 13 to SEQ ID 16. The Blocker primers were stored in 100. mu.M stock solution and prepared as 20. mu.M working solution as required.
The primer, probe and Blocker primer sequences of the kit are specifically shown in the following table 1.1:
in the above sequences, "bold and italicized" indicates that the base at the 3' end is an LNA modified base, and the underlined sequence in the probe indicates the sequence to be added.
(4) Preparing a fluorescent PCR reaction solution: preparing fluorescent PCR reaction solution according to the preparation scheme of the fluorescent PCR reaction solution, and storing. The preparation scheme of the fluorescent PCR reaction solution is as follows:
| name of reagent | Per reaction (ul) |
| |
10 |
| dNTP(10mM) | 4 |
| Primer (20. mu.M) | Adding 0.5 μ l each |
| Probe (20 μ M) | Adding 0.5 μ l each |
| Blocker primer (20. mu.M) | Adding 0.5 μ l each |
| DNA polymerase (5U/. mu.l) | 0.4 |
| Nuclease-free water | Adding water to 28 μ l |
| |
28 |
The above formulation systems are merely illustrative and in practice the system volume and the content of the components therein may be scaled up or down.
(5) Preparing a fluorescent PCR reaction system: and (2) uniformly mixing the alkaline solution prepared in the step (1), adding 10 mu l of the alkaline solution to the bottom of a PCR amplification tube, sealing the bottom of the PCR amplification tube by using paraffin with a melting point of 54-56 ℃, cooling and solidifying the paraffin, adding 28 mu l of the fluorescent PCR reaction solution prepared in the step (4) to the upper surface of solid paraffin in the PCR amplification tube, and then adding 2 mu l of UDG enzyme with the concentration of 5U/mu l into the fluorescent PCR reaction solution to form a fluorescent PCR reaction system. The fluorescent PCR reaction system can also be prepared in a PCR octal tube.
(6) Preparing a negative quality control product and a positive quality control product: preparing negative quality control products by BSA and non-methylated human genome DNA converted by bisulfite but not subjected to desulfurization treatment; positive quality controls were formulated with BSA, bisulfite converted non-methylated human genomic DNA without desulfurization, and bisulfite converted methylated human genomic DNA without desulfurization.
(7) Subpackaging and assembling the kit: the specification of the kit is 24 persons/box, and the split charging and assembling scheme is as follows:
the whole detection process of the kit comprises the following steps:
(1) and (3) fluorescent PCR reaction: adding 10 μ l of DNA sample converted by bisulfite but not desulfurized into the upper layer (containing fluorescent PCR reaction solution and UDG enzyme) of the fluorescent PCR reaction system, placing the DNA sample on a PCR instrument, and sequentially carrying out UDG enzyme digestion, 6-sulfonyl-uracil desulfurization and PCR amplification, wherein the reaction conditions are as follows: incubation at 37 ℃ for 10 min, 1 cycle (UDG enzymatic digestion); incubation at 95 ℃ for 10 min, 1 cycle (UDG enzyme inactivation, desulfurization and pre-denaturation); 95 ℃ temperature in 30 seconds, 60 ℃ temperature in 30 seconds, 45 cycles (degeneration and annealing). The fluorescence channel is selected from FAM, HEX, ROX or CY5 channel, and the fluorescence signal is collected at 60 deg.C, and the fluorescence signal is collected once per cycle.
(2) Interpretation of the detection results: and interpreting the detection result according to the fluorescence signal detected by the fluorescence PCR instrument. Detecting the fluorescence intensity of FAM, HEX, ROX and CY5 of the reaction system, and indicating that the DNA sample loading amount is in an allowable range when CY5 reaches a set threshold value, wherein the FAM, HEX and ROX signal results are reliable; taking Ct values required when FAM, HEX and ROX reach threshold values as negative and positive judgment standards, wherein the Ct values are 0 or more than or equal to 45: negative, Ct value less than 45: and (4) positive. The specific test result judgment table is as follows:
example 2: effectiveness verification experiment of fluorescent PCR reaction system for detection of non-desulfurated sample
(1) Purpose of experiment
In this example, the detection results of the desulfurized sample and the non-desulfurized sample are compared with those of the conventional fluorescent PCR system and the fluorescent PCR system of the present invention without adding UDG enzyme, so as to verify the effectiveness of the fluorescent PCR reaction system of the present invention in detecting the non-desulfurized sample.
(2) Experimental methods
In this embodiment, methylated human genomic DNA is used, and after bisulfite conversion, the methylated human genomic DNA is divided into two groups, one of which is subjected to NaOH desulfurization and purified to obtain a desulfurized DNA sample, and the other is directly purified without desulfurization to obtain a non-desulfurized DNA sample. The two DNA samples were used as templates, and the detection was carried out using a conventional fluorescent PCR reaction system, a fluorescent PCR reaction system of the present invention without UDG enzyme, and a fluorescent PCR reaction system (containing UDG enzyme) described in example 1 of the present invention, respectively, with a loading amount of 10. mu.l, each for 3 replicates. The reaction conditions for each set of fluorescent PCR systems were the same and were performed according to the reaction conditions in the detection step of example 1.
The conventional fluorescent PCR system consists of:
| name of reagent | Per reaction (ul) |
| |
10 |
| dNTP(10mM) | 4 |
| Primer (20. mu.M) | Adding 0.5 μ l each |
| Probe (20 μ M) | Adding 0.5 μ l each |
| Blocker primer (20. mu.M) | Adding 0.5 μ l each |
| DNA polymerase (5U/. mu.l) | 0.4 |
| Nuclease-free water | Adding water to 40 μ l |
| Template to be detected | 10 |
| |
50 |
The fluorescence PCR system of the invention as described in example 1 without UDG enzyme comprises a lower alkaline solution, an upper fluorescence PCR reaction solution and an intermediate paraffin isolation layer, and specifically comprises the following components:
the fluorescent PCR system of embodiment 1 of the invention comprises a lower alkaline solution, an upper fluorescent PCR reaction solution, UDG enzyme and an intermediate paraffin isolation layer, and specifically comprises the following components:
(3) the experimental results and the analytical test results are shown in the following table.
From the detection results in the table, the conventional PCR system can effectively amplify the desulfurization sample, but the amplification efficiency of the desulfurization-free sample is low, and the Ct value is obviously higher than that of the desulfurization sample; by adopting the fluorescent PCR system without adding the UDG enzyme, both the desulfurization sample and the non-desulfurization sample can be effectively amplified, which shows that in the system, the non-desulfurization sample is treated by incubating at 95 ℃ for 10 minutes, the desulfurization step can be effectively completed, and the non-desulfurization sample is converted into a DNA template which can be identified by DNA polymerase; in the fluorescent PCR system, the non-desulfurated sample can be effectively amplified, and the desulfurated sample can not completely detect an amplification signal (Ct value is more than 45), which indicates that the UDG enzyme in the fluorescent PCR system can degrade uracil in the desulfurated sample at the incubation period of 10 minutes at 37 ℃, so that the desulfurated sample can not be amplified; while the 6-sulfonyl-uracil in the non-desulfurated samples was not degraded by the UDG enzyme and was therefore not affected by the UDG enzyme. The above experiment results show that the fluorescent PCR system can effectively perform methylation amplification on a non-desulfurated sample in the presence of UDG enzyme.
Example 3: effect analysis experiment for eliminating PCR pollution by fluorescent PCR reaction system
(1) Purpose of experiment
In this example, the effect of eliminating PCR contamination by the fluorescent PCR reaction system of the present invention was analyzed by comparing the detection results of samples containing or not containing PCR contamination with the fluorescent PCR system of the present invention without UDG enzyme.
(2) Experimental methods
In this example, 4 samples containing or not containing PCR contaminants were prepared by selecting non-methylated human genomic DNA that was bisulfite converted but not desulfated, and PCR contaminants. Wherein the PCR contaminants are amplification products of methylated human genomic DNA. The formulation of the 4 samples with or without PCR contaminants is specified in the table below.
The 4 samples were used as templates, and the detection was carried out using the fluorescent PCR reaction system of the present invention without the addition of UDG enzyme and the fluorescent PCR reaction system of the present invention (containing UDG enzyme), respectively, with a loading of 10. mu.l, each of 3 replicates. The two sets of fluorescent PCR systems were formulated identically to the corresponding systems described in example 2. The reaction conditions for both sets of fluorescent PCR systems were identical and were performed according to the reaction conditions in the detection procedure of example 1.
(3) Results and analysis of the experiments
The results of the measurements are shown in the following table.
From the detection results in the table, in the fluorescent PCR system without UDG enzyme, the negative sample N1 without the pollutant can not detect the methylated fluorescent amplification signal, and the negative sample N2 with 10000 copies of the pollutant can detect a strong methylated fluorescent amplification signal; methylated fluorescence amplification signals are detected in both positive samples, but the Ct value of amplification of the positive sample P1 without the pollutants is slightly higher than that of the positive sample P2 with the pollutants; the result shows that in a reaction system without UDG enzyme, PCR pollutants obviously influence the amplification of target genes, thereby influencing the accuracy of a sample methylation detection result. In the fluorescent PCR reaction system added with the UDG enzyme, methylated fluorescent amplification signals cannot be detected by both the negative sample N1 containing no pollutants and the negative sample N2 containing pollutants, methylated fluorescent amplification signals are detected by both the positive sample P1 containing no pollutants and the positive sample P2 containing pollutants, and the amplification Ct values have no obvious difference; therefore, PCR pollutants in the reaction system added with the UDG enzyme have no obvious influence on the amplification of the target gene. The results show that in the fluorescent PCR reaction system, the UDG enzyme can effectively eliminate PCR pollution and ensure the accuracy of methylation detection results.
Example 4: verification experiment for detection effect of LNA modified primer
(1) Purpose of experiment
In the present embodiment, the detection effect of the LNA modified primer of the present invention is verified by comparing the detection result with that of a common primer not modified by LNA.
(2) Experimental methods
Methylated human genomic DNA was selected as the test sample in this example. 3 parts of the same concentration of bisulfite-converted methylated human genomic DNA without desulfurization were each detected using the kit described in example 1 prepared from LNA modified primers and a fluorescent PCR reaction system set up with common primers (same base composition as the primers described in example 1, but without LNA modification). Meanwhile, 4 pairs of LNA modified primers and 4 pairs of corresponding common primers are respectively used for carrying out PCR amplification on methylated human genome DNA which is converted by bisulfite and desulfurized by DACT1, NFAT1, SHISA and reference genes, and PCR amplification products are subjected to agarose gel electrophoresis detection. The fluorescent PCR reaction system established by the common primers is as follows:
PCR amplification reaction system adopted in agarose gel electrophoresis detection
| Name of reagent | Per reaction (ul) |
| |
10 |
| dNTP(10mM) | 4 |
| Upstream primer (20. mu.M) | 0.5 |
| Downstream primer (20. mu.M) | 0.5 |
| DNA polymerase (5U/. mu.l) | 0.4 |
| Nuclease-free water | Adding water to 40 μ l |
| Template to be detected | 10 |
| |
50 |
(3) Results and analysis of the experiments
The results of the measurements are shown in the following table and in FIG. 1.
As can be seen from the above-mentioned fluorescence PCR detection results, the detection results of the LNA modified primers are consistent with those of the common primers, and the detection results of all methylated human genome DNA samples are methylation positive; however, the Ct values detected by the LNA modified primers are lower than those of the common primers, which indicates that the LNA modified primers have better amplification efficiency and better detection effect. As can be seen from the agarose gel electrophoresis detection result of FIG. 1, the LNA modified primer and the traditional primer of the DACT1 gene can amplify a target band with a size of about 118bp, the LNA modified primer and the traditional primer of the NFAT1 gene can amplify a target band with a size of about 140bp, the LNA modified primer and the traditional primer of the SHISA3 gene can amplify a target band with a size of about 174bp, and the LNA modified primer and the traditional primer of the reference gene can amplify a target band with a size of about 108 bp; however, the target band amplified by the LNA modified primer is brighter than that of the common primer, and non-specific amplification does not exist, while the common primer has a weak non-specific band and non-specific amplification exists (for example, non-specific amplification exists in the SHISA3 gene and the internal reference gene in FIG. 1); the specific amplification efficiency of the LNA modified primer is better than that of the common primer, and the occurrence of non-specific amplification is avoided. In summary, the above results demonstrate that the LNA modified primers used in the present invention are superior to the common primers.
Example 5: competitive probe detection effect verification experiment
(1) Purpose of experiment
In this example, the detection effect of the competitive probe of the present invention was verified by comparing the detection results with those of a common fluorescent probe.
(2) Experimental methods
In this example, 3 identical concentrations of bisulfite converted methylated human genomic DNA that has not been desulfurated were detected using the kit described in example 1 prepared from competitive probes and a fluorescent PCR reaction set up with a common fluorescent probe (base composition as described in example 1, but not including the underlined additional sequence segments in the probe). Meanwhile, 9 repeated fluorescent PCR reactions were performed using the kit described in example 1 prepared from competitive probes and a fluorescent PCR reaction system established from common fluorescent probes, using nuclease-free water as a template. The fluorescent PCR reaction system established by the common fluorescent probe is as follows:
(3) the experimental results and the analytical test results are shown in the following table.
From the above table of the results of the fluorescence PCR detection of the competitive probe and the ordinary fluorescent probe for detecting methylated DNA, it can be seen that the detection results of the competitive probe and the ordinary fluorescent probe for methylated human genomic DNA converted by bisulfite without desulfurization are methylation positive, but the Ct value detected by the competitive probe is lower than that of the ordinary fluorescent probe, which indicates that the sensitivity of the competitive probe is higher. According to the results of repeated fluorescence PCR reaction of the competitive probe and the common fluorescent probe on nuclease-free water, the competitive probe generates endogenous false positive amplification from the 7 th repeated PCR reaction, the Ct value is in a slow reduction trend along with the increase of the repetition times, the common fluorescent probe generates endogenous false positive amplification from the 3 rd repeated PCR reaction, the endogenous false positive amplification is more serious along with the increase of the repetition times, the Ct value is gradually reduced, all detection sites generate endogenous amplification when the 8 th to 9 th repeated PCR reactions are carried out, and the Ct value is obviously lower than that of the competitive probe; indicating that the competitive probes of the present invention are more specific. In conclusion, the results prove that the competitive probe adopted by the invention has high sensitivity and good specificity, and is beneficial to improving the sensitivity and specificity of a reaction system, thereby ensuring the accuracy and reliability of the detection result.
Example 6: comparison experiment of detection effects of fluorescent PCR reaction system and fluorescent PCR reaction system without Blocker primer
(1) Purpose of experiment
In the present example, the effect of the Blocker primer in the fluorescent PCR reaction system of the present invention was verified by comparing the detection results with those of the fluorescent PCR reaction system without the Blocker primer.
(2) Experimental methods
In this example, unmethylated human genomic DNA that was bisulfite-converted but not desulfurized and methylated human genomic DNA were selected as samples to be tested. The 2 samples were used as templates, and the samples were separately detected using the fluorescent PCR reaction system of the present invention containing a Blocker primer and the fluorescent PCR reaction system without a Blocker primer, and were loaded in an amount of 10. mu.l, each for 3 replicates. The reaction conditions for both sets of fluorescent PCR systems were identical and were performed according to the reaction conditions in the detection procedure of example 1.
The fluorescent PCR reaction system without the Blocker primer is as follows:
(3) results and analysis of the experiments
The results of the measurements are shown in the following table.
As can be seen from the above-mentioned results, the detection results of the fluorescent PCR reaction system containing the Blocker primers of the present invention are consistent with those of the fluorescent PCR reaction system without the Blocker primers, the detection results of unmethylated DNAs are all methylation negative, and the detection results of methylated DNAs are all methylation positive. However, the Ct value detected by the fluorescent PCR reaction system containing the Blocker primer is lower than that detected by the fluorescent PCR reaction system without the Blocker primer, which indicates that the fluorescent PCR reaction system containing the Blocker primer has better detection effect. That is, the fluorescent PCR reaction system of the present invention to which the Blocker primer is added is more advantageous than the fluorescent PCR reaction system without the Blocker primer.
Example 7: sensitivity analysis experiment
(1) Purpose of experiment
In this example, the sensitivity of the present invention was analyzed by detecting methylated DNA samples at different concentrations.
(2) Experimental methods
In this example, DNA samples prepared from bisulfite-converted, non-desulfurized methylated human genomic DNA at concentrations of 1ng/ml, 100pg/ml, 10pg/ml, 1pg/ml and 0.1pg/ml were selected as test samples, and the methylation detection of nasopharyngeal carcinoma-associated genes DACT1, NFAT1 and SHISA3 described in example 1 was performed according to the detection procedure in example 1 with a loading of 10. mu.l, each of 2 replicates.
(3) Results and analysis of the experiments
The results of the measurements are shown in the following table.
As can be seen from the above table detection results, when the sample concentration of the methylated DNA is as low as 1pg/ml, amplification signals exist in all four fluorescence channels CY5, FAM, HEX and ROX (Ct value of CY5 is less than 36, Ct values of the other channels are less than 45), and the detection results are all positive for methylation; that is, the invention can accurately detect DNA samples with the concentration as low as 1pg/ml, proves that the invention has high detection sensitivity and can improve the positive detection rate of the detection result of the sample with low methylated DNA content.
Example 8: experiment for analysis of specificity
(1) Purpose of experiment
The specificity of the present invention was analyzed in this example by detecting DNA samples that were determined to be methylation negative by bisulfite sequencing, and positive by DACT1, NFAT1 or SHISA3 methylation.
(2) Experimental methods
In this example, 3 each of 7 DNA samples determined to be methylation negative by bisulfite sequencing and 3 DNA samples determined to be methylation positive by bisulfite sequencing as DACT1, NFAT1, or SHISA3 were selected, and were directly purified by bisulfite conversion without desulfurization treatment to serve as samples to be tested, and the detection was performed according to the detection procedure in example 1 using the kit for methylation detection of nasopharyngeal carcinoma-related genes DACT1, NFAT1, and SHISA3 described in example 1.
(3) Results and analysis of the experiments
The results are shown in the following table (N1-N7 represent DNA samples with negative methylation, P1-P3, P4-P6 and P7-P9 represent DNA samples with positive methylation, such as DACT1, NFAT1 and SHISA3, respectively).
From the above-mentioned detection results, it can be seen that the detection results of 7 DNA samples determined to be methylation negative by the bisulfite sequencing method are all methylation negative, the detection results of 3 DNA samples determined to be methylation positive by the DACT1 by the bisulfite sequencing method are all methylation positive by DACT1, the detection results of 3 DNA samples determined to be methylation positive by NFAT1 by the bisulfite sequencing method are all methylation positive by NFAT1, and the detection results of 3 DNA samples determined to be methylation positive by shiisa 3 by the bisulfite sequencing method are all methylation positive by shiisa 3, which proves that the detection specificity of the invention is good, the negative coincidence rate is 100%, and the positive coincidence rate is 100%.
Example 9: comparative experiment of similar products
(1) Purpose of experiment
In the embodiment, the accuracy of the kit is verified by comparing the detection result with the detection result of a methylation detection 'gold standard' bisulfite sequencing method.
(2) Experimental methods
In this example, 100 total DNA samples of nasopharyngeal carcinoma patients were collected as samples to be tested, and appropriate amounts of the samples were tested using the kit and the sulfite hydrogen sequencing method described in example 1 of the present invention. The detection process of the kit of the present invention was carried out according to the detection procedure in example 1. Bisulfite sequencing is entrusted to the megi organism.
(3) Results and analysis of the experiments
The results of the measurements are shown in the following table.
The detection results in the table show that the coincidence rate of the detection result of the kit and the detection result of the bisulfite sequencing method is 100%, which proves the detection accuracy of the invention.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Yishan Biotechnology Ltd
<120> nasopharyngeal carcinoma related gene DACT1, NFAT1 and SHISA3 methylation detection kit
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgggatagta gtagtcggc 19
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cgctaaaact acgaccgcg 19
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tcgttagcgt cgcgtttcgt tatttggcga 30
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttcgtaggga cgtacgtcg 19
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cgcctccccg aactacaac 19
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gcggcggcgg cgacggcggc gcgagagcgc 30
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tagtttcggg cgttatggt 19
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cataatacgc gaaacgaat 19
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gcgcgttttt cgcgcgggga gcgtctccgc 30
<210> 10
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gttgttatag gaagttttt 19
<210> 11
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
aaaacaatac tatcacctc 19
<210> 12
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ttaaggagaa tggtttagtt ggggttaa 28
<210> 13
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gtagtagttg gtggttgtgt gtag 24
<210> 14
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tggggatggt gtgtttggtg tagt 24
<210> 15
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ttgggtgaag ggtggtagtg atag 24
<210> 16
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gccaccccac ttctctctaa ggag 24
Claims (10)
1. The kit for detecting methylation of nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3 is characterized by comprising an alkaline solution and a fluorescent PCR reaction solution, wherein the alkaline solution and the fluorescent PCR reaction solution are packaged in the same PCR amplification tube by hot melt materials in a layering manner; the alkaline solution is NaOH solution with the pH value of 12.5-13.3 and the concentration of 50-100 mmol/L, and the volume of the alkaline solution is not more than 50% of that of the PCR reaction solution; the fluorescent PCR reaction solution comprises PCR buffer solution, dNTP, primers and probes aiming at DACT1, NFAT1 and SHISA3 genes, DNA polymerase, nuclease-free water and UDG enzyme.
2. The kit for detecting methylation of DACT1, NFAT1 and SHISA3 genes related to nasopharyngeal carcinoma according to claim 1, wherein the alkaline solution is NaOH solution with pH value of 12.5-12.8 and concentration of 70-80 mmol/L, and the volume of the alkaline solution is not more than 50% of the volume of the PCR reaction solution.
3. The kit for detecting methylation of genes DACT1, NFAT1 and SHISA3 related to nasopharyngeal carcinoma according to claim 1, wherein the hot-melt material is a hydrophobic material which is not miscible with alkaline solution, PCR reaction solution and UDG enzyme; preferably, the hot melt material is paraffin with a melting point of 54-56 ℃.
4. The kit for detecting methylation of genes DACT1, NFAT1 and SHISA3 related to nasopharyngeal carcinoma according to claim 1, wherein the primers and probes comprise: SEQ ID NO.1 to SEQ ID NO.3 for the DACT1 gene, SEQ ID NO.4 to SEQ ID NO.6 for the NFAT1 gene, and/or SEQ ID NO for the SHISA3 gene.
7 to SEQ ID NO. 9.
5. The kit for detecting methylation of genes DACT1, NFAT1 and SHISA3 related to nasopharyngeal carcinoma according to claim 1, wherein at least one of the outermost 3 bases at 3' end of the primer has LNA modification; preferably, the penultimate 2 or penultimate 3 base of the outermost 3 bases of the 3' end of the primer has an LNA modification.
6. The kit for detecting methylation of genes DACT1, NFAT1 and SHISA3 related to nasopharyngeal carcinoma according to claim 1, wherein the fluorescent probe is modified to have a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end; preferably, the fluorescent reporter group comprises FAM, HEX, ROX or CY 5; the fluorescence quenching group is BHQ.
7. The kit for detecting methylation of DACT1, NFAT1 and SHISA3 genes associated with nasopharyngeal carcinoma according to claim 1, further comprising primers and probes of reference genes shown in SEQ ID 10 to SEQ ID 12.
8. The kit for detecting methylation of nasopharyngeal carcinoma-associated genes DACT1, NFAT1 and SHISA3 according to any one of claims 1 to 7, wherein the fluorescent PCR reaction solution further comprises a blocker primer; preferably, the blocker primer is SEQ ID No.13 for the DACT1 gene, SEQ ID No.14 for the NFAT1 gene, SEQ ID No.15 for the shasa 3 gene, and/or SEQ ID No.16 for an internal reference gene.
9. The kit for detecting methylation of genes DACT1, NFAT1 and SHISA3 associated with nasopharyngeal carcinoma according to any one of claims 1 to 7, wherein said UDG enzyme is used in a concentration of 5U/μ l and in an amount of 2 ± 0.1 μ l.
10. The methylation detection kit for nasopharyngeal carcinoma related genes DACT1, NFAT1 and SHISA3 according to any one of claims 1 to 7, further comprising a negative quality control product and a positive quality control product, wherein preferably the negative quality control product comprises BSA and non-methylated human genomic DNA which is converted by bisulfite but not desulfurized;
the positive quality control consists of BSA, bisulphite-converted non-methylated human genomic DNA without desulphation treatment and bisulphite-converted methylated human genomic DNA without desulphation treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911101925.4A CN110643709B (en) | 2019-11-12 | 2019-11-12 | Nasopharyngeal carcinoma related gene DACT1, NFAT1, SHISA3 methylation detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911101925.4A CN110643709B (en) | 2019-11-12 | 2019-11-12 | Nasopharyngeal carcinoma related gene DACT1, NFAT1, SHISA3 methylation detection kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110643709A true CN110643709A (en) | 2020-01-03 |
| CN110643709B CN110643709B (en) | 2022-11-22 |
Family
ID=69014500
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911101925.4A Active CN110643709B (en) | 2019-11-12 | 2019-11-12 | Nasopharyngeal carcinoma related gene DACT1, NFAT1, SHISA3 methylation detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110643709B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115851921A (en) * | 2021-09-24 | 2023-03-28 | 圣湘生物科技股份有限公司 | Primer probe combination product, kit and application of primer probe combination product and kit in nasopharyngeal carcinoma methylation detection |
| CN116064798A (en) * | 2022-08-29 | 2023-05-05 | 广州达健生物科技有限公司 | Endometrial cancer gene methylation detection reagent and application thereof |
| CN115851921B (en) * | 2021-09-24 | 2024-06-21 | 圣湘生物科技股份有限公司 | Primer probe combination product, kit and application thereof in nasopharyngeal carcinoma methylation detection |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967515A (en) * | 2010-03-16 | 2011-02-09 | 中国人民解放军第二军医大学 | Methylation quantitative detection method of PCDH8 gene |
| US20130157879A1 (en) * | 2011-12-16 | 2013-06-20 | The Chinese University Of Hong Kong | Biomarker dact1 for gastric cancer |
| CN105385760A (en) * | 2015-11-30 | 2016-03-09 | 宁波市医疗中心李惠利医院 | Kit capable of being used for detecting SHISA3 gene promotor region methylation level related to laryngocarcinoma and application thereof |
| CN106011292A (en) * | 2016-07-27 | 2016-10-12 | 北京鑫诺美迪基因检测技术有限公司 | Kit for detecting methylation of lung cancer-associated gene SHOX2 (short stature homebox2) |
| CN106244724A (en) * | 2016-10-26 | 2016-12-21 | 北京鑫诺美迪基因检测技术有限公司 | The primer of detection septin9 gene methylation and test kit |
| CN107488721A (en) * | 2017-09-15 | 2017-12-19 | 基因科技（上海）股份有限公司 | Eliminate the methylated amplification method and its application of PCR primer pollution |
| CN108048566A (en) * | 2017-12-19 | 2018-05-18 | 益善生物技术股份有限公司 | A kind of Nucleic acid combinations and kit for detecting septin9 gene methylations |
| CN109609638A (en) * | 2019-01-05 | 2019-04-12 | 敬善生物科技江苏有限公司 | A kind of kit applied and its application in nasopharyngeal carcinoma detection |
| CN109811056A (en) * | 2019-02-28 | 2019-05-28 | 苏州唯善生物科技有限公司 | For colorectal cancer and its primed probe group and kit of precancerous lesion early diagnosis, detection or screening |
| CN110283913A (en) * | 2019-07-21 | 2019-09-27 | 广州奥百阕谱生物科技有限公司 | RASSF1A gene methylation state detection kit and its application |
-
2019
- 2019-11-12 CN CN201911101925.4A patent/CN110643709B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967515A (en) * | 2010-03-16 | 2011-02-09 | 中国人民解放军第二军医大学 | Methylation quantitative detection method of PCDH8 gene |
| US20130157879A1 (en) * | 2011-12-16 | 2013-06-20 | The Chinese University Of Hong Kong | Biomarker dact1 for gastric cancer |
| CN103627785A (en) * | 2011-12-16 | 2014-03-12 | 香港中文大学 | Biomarker DACT1 for gastric cancer |
| CN105385760A (en) * | 2015-11-30 | 2016-03-09 | 宁波市医疗中心李惠利医院 | Kit capable of being used for detecting SHISA3 gene promotor region methylation level related to laryngocarcinoma and application thereof |
| CN106011292A (en) * | 2016-07-27 | 2016-10-12 | 北京鑫诺美迪基因检测技术有限公司 | Kit for detecting methylation of lung cancer-associated gene SHOX2 (short stature homebox2) |
| CN106244724A (en) * | 2016-10-26 | 2016-12-21 | 北京鑫诺美迪基因检测技术有限公司 | The primer of detection septin9 gene methylation and test kit |
| CN107488721A (en) * | 2017-09-15 | 2017-12-19 | 基因科技（上海）股份有限公司 | Eliminate the methylated amplification method and its application of PCR primer pollution |
| CN108048566A (en) * | 2017-12-19 | 2018-05-18 | 益善生物技术股份有限公司 | A kind of Nucleic acid combinations and kit for detecting septin9 gene methylations |
| CN109609638A (en) * | 2019-01-05 | 2019-04-12 | 敬善生物科技江苏有限公司 | A kind of kit applied and its application in nasopharyngeal carcinoma detection |
| CN109811056A (en) * | 2019-02-28 | 2019-05-28 | 苏州唯善生物科技有限公司 | For colorectal cancer and its primed probe group and kit of precancerous lesion early diagnosis, detection or screening |
| CN110283913A (en) * | 2019-07-21 | 2019-09-27 | 广州奥百阕谱生物科技有限公司 | RASSF1A gene methylation state detection kit and its application |
Non-Patent Citations (4)
| Title |
|---|
| YANG等: "Efects of DACT1 methylation status on invasion and metastasis of nasopharyngeal carcinoma", 《BIOLOGICAL RESEARCH》 * |
| ZHANG等: "Hypermethylation of SHISA3 Promotes Nasopharyngeal Carcinoma Metastasis by Reducing SGSM1 Stability", 《CANCER RESEARCH》 * |
| ZHANG等: "NFAT1 Hypermethylation Promotes Epithelial-Mesenchymal Transition and Metastasis in Nasopharyngeal Carcinoma by Activating ITGA6 Transcription", 《NEOPLASIA》 * |
| 孙建海等: "《临床肿瘤诊断与治疗》", 31 March 2014 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115851921A (en) * | 2021-09-24 | 2023-03-28 | 圣湘生物科技股份有限公司 | Primer probe combination product, kit and application of primer probe combination product and kit in nasopharyngeal carcinoma methylation detection |
| CN115851921B (en) * | 2021-09-24 | 2024-06-21 | 圣湘生物科技股份有限公司 | Primer probe combination product, kit and application thereof in nasopharyngeal carcinoma methylation detection |
| CN116064798A (en) * | 2022-08-29 | 2023-05-05 | 广州达健生物科技有限公司 | Endometrial cancer gene methylation detection reagent and application thereof |
| CN116064798B (en) * | 2022-08-29 | 2024-02-27 | 广州达健生物科技有限公司 | Endometrial cancer gene methylation detection reagent and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110643709B (en) | 2022-11-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3194624B1 (en) | Methods for diagnosis, prognosis and monitoring of breast cancer and reagents therefor | |
| US10544467B2 (en) | Solid tumor methylation markers and uses thereof | |
| US11396678B2 (en) | Breast and ovarian cancer methylation markers and uses thereof | |
| US20190345560A1 (en) | Lung cancer methylation markers and uses thereof | |
| US20190300965A1 (en) | Liver cancer methylation markers and uses thereof | |
| US20190300964A1 (en) | Colon cancer methylation markers and uses thereof | |
| EA018010B1 (en) | Methods and nucleic acids for analyses of cell proliferative disorders | |
| CN110484621B (en) | Early warning method for liver cancer | |
| Mori et al. | S100A11 gene identified by in-house cDNA microarray as an accurate predictor of lymph node metastases of gastric cancer | |
| CN110669831B (en) | Human SGIP1, SCAND3 and MYO1G gene methylation detection kit | |
| CN110643709B (en) | Nasopharyngeal carcinoma related gene DACT1, NFAT1, SHISA3 methylation detection kit | |
| CN104818340B (en) | Detect primer, kit and its PCR method of JAK2 gene V617F loci polymorphisms | |
| CN104846108B (en) | Detect primer, kit and its PCR method in C-KIT gene D816V mutational sites | |
| US20180223367A1 (en) | Assays, methods and compositions for diagnosing cancer | |
| CN105463096B (en) | MGMT gene promoter methylation detection primer probe system and kit thereof | |
| CN113493835A (en) | Method and kit for screening large intestine tumor by detecting methylation state of BCAN gene region | |
| CN108060228A (en) | A kind of detection primer, kit and method for detecting BRCA1 and BRCA2 genetic mutations | |
| CN111961723A (en) | Tumor marker for noninvasive detection of early ovarian cancer diagnosis and kit | |
| CN114891886B (en) | Nucleic acid product, kit and application for diagnosing bladder cancer | |
| US20090181397A1 (en) | Predictive and diagnostic methods for cancer | |
| US8828662B2 (en) | Method and kit for detection of microsatellite instability-positive cell | |
| KR101498640B1 (en) | Method for Identifying Body-Fluid by Using DNA Methylation Marker, Composition For Identifying Body-Fluid and Kit Comprising the Same | |
| CN104846107B (en) | Detect primer, kit and its PCR method of RET gene M 918T site mutations | |
| CN115651980A (en) | Human RNF180 gene methylation detection kit and method for extracting DNA to be detected | |
| CN105441557A (en) | Primer probe system for MLH1 (MutL homolog1) gene methylation detection and kit adopting primer probe system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder |
Address after: No. 29, Helix 3 Road, Guangzhou International Biological Island, Huangpu District, Guangzhou City, Guangdong Province, 510663 Patentee after: SUREXAM BIO-TECH Co.,Ltd. Address before: 5 / F, area C, Guangzhou innovation base, No. 80, guanyue Road, Science City, Guangzhou, Guangdong Province Patentee before: SUREXAM BIO-TECH Co.,Ltd. |
|
| CP02 | Change in the address of a patent holder |