CN112501306A - Kit for CpG island methylation phenotype detection and application thereof - Google Patents
Kit for CpG island methylation phenotype detection and application thereof Download PDFInfo
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Abstract
The application discloses a kit for CpG island methylation phenotype detection and application thereof. The kit comprises at least one of a CACNA1G gene methylation detection reagent, an IGF2 gene methylation detection reagent, a NEUROG1 gene methylation detection reagent, a RUNX3 gene methylation detection reagent and a SOCS1 gene methylation detection reagent, and an internal reference beta-Actin gene detection reagent. The detection reagents of the five target genes respectively comprise specific upstream and downstream primers, a methylated probe and an unmethylated probe, and the detection reagent of the internal reference beta-Actin gene comprises the specific upstream and downstream primers and the probe of the internal reference gene. The kit can detect the methylation conditions of five target genes, and realizes the methylation phenotype detection of the CpG island with high sensitivity and high accuracy; provides important reference basis for individual accurate medication guidance, drug effect evaluation and dynamic monitoring of targeted therapy of patients.
Description
Technical Field
The application relates to the field of nucleic acid methylation detection, in particular to a kit for CpG island methylation phenotype detection and application thereof.
Background
The methylation level of genomic DNA can determine the expression or inhibition of a class of genes, and has an important effect on the normal function of cells. The phenomena of genome general hypomethylation and local region hypermethylation exist in tumor tissues. Hypermethylation of CpG islands in the promoter region of cancer suppressor genes is an early important event in the development of many tumors, and can cause the expression of related genes to be down-regulated, thereby influencing normal growth and apoptosis in cells and among cells. Hypomethylation of protooncogenes may cause transcriptional activation of protooncogenes.
Recent studies show that the DNA methylation state can be used as a biomarker in the whole course of cancer treatment, such as early diagnosis, pathological typing, disease progress monitoring, treatment effect prediction, prognosis prediction and the like, and particularly has remarkable advantages in the aspect of early diagnosis.
Colorectal cancer is the third most common malignancy in the world. In 1999, Toyota, a japanese scholarly, proposed a CpG Island Methylation Phenotype (CIMP) colorectal cancer, a type of colorectal cancer in which a high proportion of genes in the genome are DNA methylated. Research finds that the pathogenesis of CIMP type colorectal cancer, the response to treatment and the prognosis effect of CIMP type colorectal cancer are greatly different from other types of colorectal cancer, for example, the prognosis is poor, and the CIMP type colorectal cancer is insensitive to chemotherapeutic drugs 5-FU and cetuximab, and the like, so that the research on CIMP has important scientific and clinical significance.
In addition, studies have shown that the CpG island methylation phenotype involves the simultaneous methylation of multiple gene promoters, is tumor specific, and is associated with the occurrence or prognosis of multiple tumors. Therefore, the CpG island methylation phenotype detection has important significance for the research of the occurrence or prognosis of tumors.
At present, CIMP typing is mainly carried out by detecting the methylation state of tumor tissue DNA after operation, and many samples are difficult to obtain tumor tissue clinically, so that molecular typing by using blood free DNA (circulating free DNA, abbreviated as cfDNA) has great clinical significance. However, there is no method that can accurately and effectively detect CpG island methylation phenotypes in cfDNA with high sensitivity and high accuracy.
Disclosure of Invention
The application aims to provide a novel kit for detecting CpG island methylation phenotype and application thereof.
In order to achieve the purpose, the following technical scheme is adopted in the application:
the first aspect of the application discloses a kit for CpG island methylation phenotype detection, which comprises at least one of a CACNA1G gene methylation detection reagent, an IGF2 gene methylation detection reagent, a NEUROG1 gene methylation detection reagent, a RUNX3 gene methylation detection reagent and a SOCS1 gene methylation detection reagent, and an internal reference beta-Actin gene detection reagent; the CACNA1G gene methylation detection reagent comprises a CACNA1G gene amplification primer, a methylation state detection probe and an unmethylated state detection probe, wherein the upstream and downstream primers of the CACNA1G gene amplification primer are respectively sequences shown by SEQ ID No.1 and SEQ ID No.2, the methylation state detection probe is a sequence shown by SEQ ID No.3, and the unmethylated state detection probe is a sequence shown by SEQ ID No. 4; the IGF2 gene methylation detection reagent comprises an IGF2 gene amplification primer, a methylation state detection probe and a non-methylation state detection probe, wherein the upstream and downstream primers of the IGF2 gene amplification primer are respectively sequences shown in SEQ ID NO.5 and SEQ ID NO.6, the methylation state detection probe is a sequence shown in SEQ ID NO.7, and the non-methylation state detection probe is a sequence shown in SEQ ID NO. 8; the NEUROG1 gene methylation detection reagent comprises a NEUROG1 gene amplification primer, a methylation state detection probe and an unmethylated state detection probe, wherein the upstream primer and the downstream primer of the NEUROG1 gene amplification primer are respectively sequences shown in SEQ ID NO.9 and SEQ ID NO.10, the methylation state detection probe is a sequence shown in SEQ ID NO.11, and the unmethylated state detection probe is a sequence shown in SEQ ID NO. 12; the RUNX3 gene methylation detection reagent comprises a RUNX3 gene amplification primer, a methylation state detection probe and a non-methylation state detection probe, wherein the upstream and downstream primers of the RUNX3 gene amplification primer are respectively sequences shown in SEQ ID NO.13 and SEQ ID NO.14, the methylation state detection probe is a sequence shown in SEQ ID NO.15, and the non-methylation state detection probe is a sequence shown in SEQ ID NO. 16; the SOCS1 gene methylation detection reagent comprises an SOCS1 gene amplification primer, a methylation state detection probe and a non-methylation state detection probe, wherein the upstream primer and the downstream primer of the SOCS1 gene amplification primer are respectively sequences shown in SEQ ID NO.17 and SEQ ID NO.18, the methylation state detection probe is a sequence shown in SEQ ID NO.19, and the non-methylation state detection probe is a sequence shown in SEQ ID NO. 20; the internal reference beta-Actin gene detection reagent comprises a beta-Actin gene amplification primer and a detection probe, wherein the upstream primer and the downstream primer of the beta-Actin gene amplification primer are respectively sequences shown as SEQ ID NO.21 and SEQ ID NO.22, and the detection probe is a sequence shown as SEQ ID NO. 23.
The kit of the present application designs multiple detection primers and probes for five target genes and one reference gene for methylation detection of CpG islands, and designs methylation state detection probes and non-methylation state detection probes for five target genes, respectively, so that methylation conditions of the five target genes can be detected, thereby realizing detection of CpG island methylation phenotypes with high sensitivity and high accuracy. In one implementation of the present application, mutation frequencies as low as 1/10,000 (0.01%) can be quantitatively analyzed in conjunction with microdroplet digital PCR (ddPCR) detection techniques.
It will be appreciated that first, the methylation state detection probe and the unmethylated state detection probe of the present application are used in a single reaction, and thus, the fluorophores labeled by the two probes are different, e.g., in one implementation of the present application, the methylation state detection probe is labeled with a FAM fluorophore and the unmethylated state detection probe is labeled with a HEX fluorophore, so long as the fluorophore labels of the two probes can be differentially detected. Secondly, the kit of the present application contains reagents for detecting five target genes and one reference gene, and only some of the reagents can be used to detect some target genes according to requirements, which is not specifically limited herein. Thirdly, the primer and probe sequences defined in the kit of the present application are all the primers and probes specifically used in one implementation manner of the present application, and on this basis, one skilled in the art can add or delete several bases at the 5 'end or 3' end of the primer or probe sequence of the present application according to the design principle of the primers and probes, as long as the specificity of detection is not affected or the amplification efficiency is within the acceptable range of detection.
Preferably, the kit of the present application further comprises at least one of cfDNA extraction reagents, DNA bisulphite treatment reagents and microdroplet digital PCR detection reagents.
It should be noted that, in one usage of the present application, the kit of the present application is developed for cfDNA detection, and with the kit of the present application, CpG island methylation phenotype detection can be performed through cfDNA in peripheral blood of a patient, thereby solving the problem of difficulty in obtaining tumor tissue; the cfDNA extraction reagent can refer to the existing peripheral blood cfDNA extraction reagent, and this application only adds it selectively to the kit of this application for convenience of use. It is understood that the kit of the present application can be used not only for cfDNA detection, but also for conventional tumor tissue DNA detection; in this case, the kit of the present application may also optionally include a tumor tissue DNA increasing agent. Similarly, the DNA bisulfite treatment reagent can be referred to the prior art. In one implementation of the present application, amplification detection and analysis by microdroplet digital PCR are specifically employed, and thus, microdroplet digital PCR detection reagents may be optionally included in the kit of the present application. Of course, if other amplification techniques are used, the kit of the present application may also be added with corresponding detection reagents, and is not particularly limited herein.
The second aspect of the application discloses the application of the kit in preparing a reagent for tumor detection or cancer gene detection, wherein the cancer gene is at least one of CACNA1G gene, IGF2 gene, NEUROG1 gene, RUNX3 gene and SOCS1 gene.
It should be noted that the kit of the present application can be used for detecting CpG island methylation phenotype, and an intermediate reference basis can be provided for tumor detection according to CpG island methylation phenotype, for example, CIMP type colorectal cancer, and therefore, the kit of the present application can be used as a tumor detection reagent. In addition, the kit comprises detection reagents of CACNA1G gene, IGF2 gene, NEUROG1 gene, RUNX3 gene and SOCS1 gene and five target genes; therefore, the kit of the application can also be used for the individual detection of the methylation of five genes, namely the CACNA1G gene, the IGF2 gene, the NEUROG1 gene, the RUNX3 gene or the SOCS1 gene, or the detection of part of target genes.
In a third aspect of the present application, the use of the kit of the present application in screening for a drug for cancer therapy is disclosed.
The kit can judge the methylation change condition of the CpG island before and after administration by detecting the methylation phenotype of the CpG island, thereby evaluating the administration effect. Thus, the kits of the present application may be used for cancer therapy drug screening; can realize individual accurate medication guidance, drug effect evaluation and dynamic monitoring of targeted therapy for patients.
In a fourth aspect of the present application, a method for detecting a methylation phenotype of a CpG island for non-diagnostic therapeutic purposes is disclosed, comprising performing a methylation phenotype detection of a CpG island in a nucleic acid sample to be tested using the kit of the present application.
It should be noted that the detection method of the present application is only used for analyzing and judging the methylation information of the target gene, and provides reliable analysis basis for patient treatment and medication guidance.
Preferably, the detection method further comprises setting a blank control or a negative control equivalent to replace the nucleic acid sample to be detected, and performing a control test; performing data analysis according to the detection signals of the nucleic acid sample to be detected and the control test, and judging the methylation condition of the nucleic acid sample to be detected through the methylation index; methylation index ═ methylation index (detection signal of gene to be detected in nucleic acid sample to be detected: detection signal of β -Actin gene in nucleic acid sample to be detected) ÷ (detection signal of gene to be detected in control test: detection signal of β -Actin gene in control test); if the methylation index is greater than or equal to 0.5%, the nucleic acid sample to be tested is considered to be methylated; wherein the gene to be detected is at least one of CACNA1G gene, IGF2 gene, NEUROG1 gene, RUNX3 gene and SOCS1 gene.
The detection method is characterized in that methylation detection primers and probes of five target genes and one reference gene which are specially designed by the kit are adopted; the determination of methylation by Methylation Index (MI) is only one specific methylation determination method employed in one implementation of the present application, and does not preclude the use of other determinations.
Preferably, in the detection method of the present application, the CpG island methylation phenotype detection and the control test are performed on the nucleic acid sample to be detected by using microdroplet digital PCR; and the methylation index was analyzed and calculated using a droplet analyzer.
Preferably, in the detection method of the present application, the nucleic acid sample to be detected is cfDNA.
Preferably, in one implementation manner of the present application, the detection method specifically includes the following steps,
separating blood cells and plasma from peripheral blood samples;
extracting cfDNA from the separated plasma sample by adopting a two-step purification method;
performing bisulfite treatment on the extracted cfDNA;
methylation index was calculated for bisulfite-treated cfDNA using microdroplet digital PCR for amplification, detection and analysis.
The fourth aspect of the present application discloses the use of the detection method of the present application in cancer gene detection or cancer therapeutic drug screening, wherein the cancer gene is at least one of CACNA1G gene, IGF2 gene, NEUROG1 gene, RUNX3 gene and SOCS1 gene.
Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
the kit can detect the methylation conditions of five target genes, and can realize the methylation phenotype detection of the CpG island with high sensitivity and high accuracy; provides important reference basis for individual accurate medication guidance, drug effect evaluation and dynamic monitoring of targeted therapy of patients.
Detailed Description
Because tumor tissue is difficult to obtain, the application proposes to utilize cfDNA to perform CpG island methylation phenotype detection; however, cfDNA itself is low in blood content, and tumor circulating DNA (abbreviated ctDNA) occupies only a small part of cfDNA, so how to detect CpG island methylation phenotype in cfDNA with high sensitivity and high accuracy is a research focus and difficulty of the present application.
A droplet digital PCR (ddPCR) technology is used as a third-generation PCR technology, a brand-new mode is adopted for quantifying nucleic acid molecules on the basis of a traditional PCR method, compared with real-time fluorescence quantitative PCR, ddPCR does not need to construct a standard curve and is not influenced by amplification efficiency, and the result has higher accuracy, accuracy and sensitivity. The core of droplet digital PCR is the ability to divide a sample into 20,000 nanoliter droplets, each of which contains one to several nucleic acid target molecules to be detected, or no nucleic acid target molecules, and each of which serves as a separate PCR reactor. After PCR amplification, a microdroplet analyzer is adopted to detect each microdroplet one by one, the microdroplet with a fluorescence signal is judged to be1, the microdroplet without the fluorescence signal is judged to be 0, finally, according to the Poisson distribution principle and the proportion of positive microdroplets, analysis software can calculate the concentration and copy number of the target molecules to be detected, and the number of the target molecules can be directly counted in an absolute quantitative mode.
Therefore, circulating DNA detection is the focus, and the ddPCR technology can realize mutation DNA detection with low investment, high sensitivity and high accuracy and can be used for CpG island methylation phenotype detection, so that the pertinence of clinical treatment can be improved, the reasonable use of targeted drugs can be guided, the delay of the patient's condition caused by ineffectiveness or improper treatment can be avoided, and the treatment risk can be reduced.
Based on the research and the recognition, the method creatively provides a real-time, noninvasive, accurate and rapid CpG island methylation phenotype detection scheme by screening a large number of methods and reagents and combining microdroplet digital PCR with a high-specificity primer sequence and a probe sequence, thereby providing a novel kit for CpG island methylation phenotype detection.
The kit and the detection method based on the kit are characterized in that extremely-small ctDNA in peripheral blood is used as a detection object, and after bisulfite treatment is carried out, the influence of an inhibitor is reduced through a droplet emulsification technology, so that background noise is reduced, and the detection sensitivity and accuracy are greatly improved.
The kit and the detection method can assist doctors in selecting proper treatment medicines and formulating individualized treatment schemes according to individual differences of patients, improve the life quality of the patients and prolong the survival time of the patients. In the present application, "blood" and "peripheral blood" are the same concept.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Examples
The detection method mainly comprises the steps of extracting free circulating DNA from blood, carrying out bisulfite treatment by taking cfDNA as a template, carrying out ddPCR amplification on the cfDNA by using specific detection primers and probes of five genes of CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 and an internal reference gene beta-Actin, detecting an amplification product, and analyzing fluorescence signal data to obtain fluorescence information of five genes of CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 and the internal reference gene beta-Actin. Thus analyzing the MI value. In the example, five samples are used for testing, wherein the five samples are clinically CIMP typed samples selected from Shenzhen Helios medical inspection laboratory colorectal cancer project, and are numbered 20190812011-b, 20190813011-a, 20190822011-b, 20190824011-c and 20190811011-b for detection. The specific test is as follows:
method for extracting cfDNA from CIMP typing colorectal cancer patient peripheral blood
1. Blood sample processing
After obtaining blood, the following treatments were carried out in 4 h: centrifuging 10mL of peripheral blood at 1600g at 4 deg.C for 10min, separating into plasma and blood cells, and storing the blood cells at-80 deg.C; centrifuging the supernatant (blood plasma) for the second time at 16000g and 4 deg.C for 10min, transferring the supernatant to a storage tube, and storing at-80 deg.C to obtain blood plasma sample.
cfDNA extraction
In the embodiment, a nucleic acid extraction and purification kit (heavily ancient cooking vessel, ZD-YL-Midi-40) is adopted for cfDNA extraction, and the optimized extraction steps are as follows:
(1) taking a 10mL centrifuge tube, adding 20 mu L of protease K, adding 2mL of the collected plasma sample of 1. blood sample treatment, adding 2mL of solution GH, and mixing by vortex for 15 s; then incubating for 10min at 37 ℃ in a constant-temperature water bath kettle.
(2) Adding 2mL of isopropanol into the mixed solution, and fully mixing by vortexing; then, incubating in a refrigerator at 4 ℃ for 10 min; transferring the mixed solution into a medium-volume centrifugal column sleeved with a collecting pipe, centrifuging at 10000rpm for 1min, and removing waste liquid in the collecting pipe.
(3) Adding 2.5mL of solution W1A into the centrifugal column in the step (2), centrifuging for 3min at 10000rpm, taking out the centrifugal column, removing waste liquid in a collecting pipe, and putting the centrifugal column back; wherein, the solution W1A is added with absolute ethyl alcohol according to the specification.
(4) Adding 4mL of solution W2 into the centrifugal column in the step (3), centrifuging for 3min at 10000rpm, and removing waste liquid; wherein, the solution W2 is added with absolute ethyl alcohol according to the specification.
(5) And (4) adding 2mL of absolute ethyl alcohol into the centrifugal column in the step (4), centrifuging at 10000rpm for 3min, carefully taking out the centrifugal column, transferring to a new 15mL centrifugal tube, uncovering the centrifugal tube, and standing at room temperature for 10min to volatilize residual ethyl alcohol.
(6) 450 μ L of the pre-preheated TE solution was added to the column, and after standing at room temperature for 5min, the column was centrifuged at 10000rpm for 3 min.
(7) Discarding the medium-volume centrifugal column in the 15mL centrifugal tube, adding 450 mu L of solution BK and 450 mu L of absolute ethyl alcohol into the eluent in the tube, uniformly mixing by vortex, and standing for 5-10min at room temperature after uniform mixing.
(8) Taking a micro-centrifugal column containing a collecting pipe, transferring 700 mu L of the mixed solution obtained in the step (7) into the micro-centrifugal column, sealing a cover, centrifuging at 13000rpm for 30s, and discarding waste liquid; repeating the operation until the mixed solution completely passes through the column.
(9)13000rpm, idling for 2min, then standing for 5min at room temperature, and volatilizing residual ethanol.
(10) Taking out the centrifugal column, transferring the centrifugal column into a 1.5mL clean centrifugal tube, adding 43 mu L of solution NF water into the column, standing at room temperature for 5min, and centrifuging at 13000rpm for 2 min; wherein, the solution NF water can be preheated at 50-60 ℃, thereby improving the elution efficiency.
(11) And transferring the centrifuged solution into the centrifugal column again, putting the centrifugal column into a centrifugal tube, opening the cover, standing at room temperature for 2min, and centrifuging at 13000rpm for 3min to obtain filtrate, namely the cfDNA.
The concentration of the extracted cfDNA was determined using a Life Qubit 2.0, and the Agilent 4200 TapeStation nucleic acid analyzer detected the fragment size of the extracted cfDNA. The results are shown in Table 1.
TABLE 1 cfDNA concentration and fragment size
II, bisulfite conversion treatment
(1) Preparation of reagents:
preparing a TET2 Buffer: 100 μ L of TET2 ReactionBuffer was added to a TET2 ReactionBuffer Supplement tube and mixed well, and the prepared buffer was stored at-20 ℃ and used within 4 months.
Fe (II) Solution 1. mu.L at a concentration of 500mM was added to 1249. mu.L of water for dilution, ready to use, and the remainder was discarded.
(2) Oxidation by oxygen
The following system was formulated on ice and added to 29 μ L of DNA sample: 10 μ L of TET2 Reaction Buffer (connected), 1 μ L of Oxidation Supplement, 1 μ L of Oxidation Enhancer, and 4 μ L of TET 2.
After thorough mixing, 5. mu.L of diluted Fe (II) Solution was added. Thoroughly mixed, slightly centrifuged, and then placed in a PCR instrument for reaction at 37 ℃ for 1 hour.
After the reaction is finished, taking out a sample, placing the sample on an ice box, adding 1 mu L of StopReagent, uniformly mixing, placing a PCR instrument for reaction, and carrying out a reaction program: 30 minutes at 37 ℃; 4 ℃ and infinity.
(3) Purification of
mu.L of AMPure XP beads was added for purification, 17. mu.L of Nuclear Water was used for elution, and 16. mu.L of the purified sample was transferred to a new PCR tube.
(4) Denaturation of the material
Preheating a PCR instrument to 85 ℃; add 4. mu.L formamide to 16. mu.L of sample, mix well, centrifuge slightly; putting the sample into a PCR instrument to react for 10 minutes at 85 ℃; then immediately placed on ice.
(5) Deamination
The following system was formulated on ice and added to the 20 μ L sample described above: 68. mu.L of nucleic-free water, 10. mu. L, BSA 1 of APOBEC Reaction Buffer, 1. mu. L, APOBEC 1. mu.L, and 100. mu.L of total volume.
And (3) thoroughly mixing, centrifuging, and placing in a PCR instrument for reaction, wherein the reaction procedure comprises the following steps: at 37 ℃ for 3 h; at 4 deg.C and infinity
(6) Purification of
After the reaction was completed, 100. mu.L of AMPureXPbeads were added for purification, 21. mu.L of nucleowater was used for elution, and 20. mu.L of the purified sample was transferred to a new PCR tube.
Amplification detection of target gene and internal reference beta-Actin gene
The detection of this example uses primers and probes designed for five genes of CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 and the internal reference gene beta-Actin. Specifically, the upstream and downstream primers of the CACNA1G gene amplification primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, the methylation state detection probe is shown as SEQ ID NO.3, and the non-methylation state detection probe is shown as SEQ ID NO. 4; the upstream and downstream primers of the IGF2 gene amplification primer are respectively sequences shown in SEQ ID NO.5 and SEQ ID NO.6, the methylation state detection probe is a sequence shown in SEQ ID NO.7, and the non-methylation state detection probe is a sequence shown in SEQ ID NO. 8; upstream and downstream primers of NEUROG1 gene amplification primer are respectively shown as SEQ ID NO.9 and SEQ ID NO.10, methylation state detection probe is shown as SEQ ID NO.11, and non-methylation state detection probe is shown as SEQ ID NO. 12; the upstream and downstream primers of the RUNX3 gene amplification primer are respectively sequences shown in SEQ ID NO.13 and SEQ ID NO.14, the methylation state detection probe of the RUNX3 gene amplification primer is a sequence shown in SEQ ID NO.15, and the non-methylation state detection probe of the RUNX3 gene amplification primer is a sequence shown in SEQ ID NO. 16; the upstream and downstream primers of the SOCS1 gene amplification primer are respectively sequences shown in SEQ ID NO.17 and SEQ ID NO.18, the methylation state detection probe is a sequence shown in SEQ ID NO.19, and the non-methylation state detection probe is a sequence shown in SEQ ID NO. 20; the upstream and downstream primers of the beta-Actin gene amplification primer are respectively shown as SEQ ID NO.21 and SEQ ID NO.22, and the detection probe is shown as SEQ ID NO. 23. The primers and detection probes used in this example were synthesized by Biotechnology engineering (Shanghai) Inc., and the sequences of the primers and probes are shown in Table 2.
TABLE 2 amplification primers and probes for target and reference genes
Preparing a PCR reaction system: 2 XDddPCR supermix for probes (no dUTP) 10. mu.L, 1. mu.L of Primer-F1 (final concentration 10. mu.M), 1. mu.L of Primer-R1 (final concentration 10. mu.M), 0.5. mu.L of Methlated-probe1 (final concentration 10. mu.M), 0.5. mu.L of Unhlated-probe 2 (final concentration 10. mu.M), 1. mu.L of beta-Actin-F1 (final concentration 10. mu.M), 1. mu.L of beta-Actin-R1 (final concentration 10. mu.M), 0.5. mu.L of beta-Actin-probe (final concentration 10. mu.M), and 4.5. mu.L of bisulfite conversion treated DNA template.
Wherein, the Primer-F1 and the Primer-R1 are upstream and downstream primers of one of five genes of CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1, Methlated-probe1 represents a methylated probe of the gene corresponding to the Primer-F1 and the Primer-R1, Unmethlated-probe2 represents an unmethylated probe of the gene corresponding to the Primer-F1 and the Primer-R1, beta-Actin-F1 and beta-Actin-R1 are upstream and downstream primers of an internal reference beta-Actin gene, and beta-Actin-probe is a detection probe of the internal reference beta-Actin gene.
Mixing the PCR reaction system by a vortex instrument, centrifuging the mixture by a palm centrifuge to remove bubbles, and transferring the reaction system to a ddPCR detection area.
And a ddPCR detection stage, wherein the stage is completed in a ddPCR detection area, and the specific steps are as follows:
(1) preparation of microdroplets
A. Taking out the micro-droplet generation card, assembling according to the operation requirement, and adding Oil into an Oil sample adding hole, wherein the volume of each hole is 70 mu L;
B. 20 μ L of the reaction system prepared above was added to a Sample well, and the seal was closed and reacted in a droplet generator. Since the Oil and Sample wells cannot be emptied when the microdroplets are generated, the blank Oil well is filled with 70. mu.L of Oil and the Sample well is replaced with 25. mu.L of water.
(2) PCR amplification
A. The resulting droplets were slowly pipetted and transferred into wells of a 96-well PCR plate using a pipette gun, which was operated slowly to prevent the formation of air bubbles for about 5 seconds per single operation.
B. Sealing the film by using a preheated PX1 heat sealing instrument, and recommending the operation procedure as follows: 180 ℃ for 5 s.
C. After the membrane sealing is completed, the 96-well plate is placed in a gradient amplification apparatus (T100 Thermal Cycler) to perform PCR reaction under the following conditions:
denaturation at 95 deg.C for 10 min; then 35 cycles were entered: denaturation at 94 deg.C for 30sec, and annealing at 60 deg.C for 1 min; after circulation is finished, extension is carried out for 10min at 72 ℃; storing at 4 ℃.
(3) Droplet detection
Transferring the 96-well plate containing the prepared microdroplets to a QX200 microdroplet analyzer, setting reaction parameters, editing sample information, and performing fluorescence detection on all samples; after the droplet reading is finished, the data is saved and the next analysis is carried out.
Fourthly, reading and numerical analysis of fluorescence signals for gene amplification detection
1. Reading of fluorescent signals
(1) And opening QuantaSoft software, importing sample detection data, and selecting an Analyze module to begin analyzing the data.
(2) It is first confirmed that the number of droplets generated for all sample types is 10000 or more, and only the CV value of the data detected in this range can be controlled within 1.6%. Then 1DAmplitude was selected, the fluorescence threshold was determined, and the probe was thresholded with a blank control.
(3) QuantaSoft software calculates mutations.
2. Analysis of numerical results
The results of the five colorectal cancer samples of this example are shown in tables 3 and 4.
TABLE 3 typing and determination results of colorectal cancer samples
TABLE 4 detailed results of five genes in colorectal cancer samples
In the example, the ctDNA of the sample of the colorectal cancer patient is detected, and the input amount of the template cfDNA is 10 ng; of the five target genes, CIMP typing was considered as if the MI value of one of the five target genes was 0.5 or more. The detection result shows that MI values of 5 samples are all larger than 0.5, and are CIMP types, which are consistent with the expectation.
The CIMP typing detection method of this example has an advantage of high sensitivity by using ctDNA as a detection target, and can detect the methylation state of ctDNA from a trace amount of cfDNA. By adopting the detection method of the embodiment, 15mL of peripheral blood is extracted, the development and change of the cancer are dynamically evaluated through the detection and analysis of ctDNA, an accurate medical scheme is made, the whole detection process is non-invasive, and no negative influence is caused on patients; in addition, the detection method of the embodiment can accurately detect CIMP typing, which provides an important reference basis for individual precise medication guidance.
The detection method of the embodiment combines the international leading gene capture technology to detect the methylation variation condition of the ctDNA in the blood, has the sequencing depth of more than 10,000 multiplied by the number, has the sequencing sensitivity of 0.01 percent, and can provide the most accurate medication guidance information.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
Claims (10)
1. A kit for the detection of a CpG island methylation phenotype, characterized by: comprises at least one of CACNA1G gene methylation detection reagent, IGF2 gene methylation detection reagent, NEUROG1 gene methylation detection reagent, RUNX3 gene methylation detection reagent and SOCS1 gene methylation detection reagent, and reference beta-Actin gene detection reagent;
the CACNA1G gene methylation detection reagent comprises a CACNA1G gene amplification primer, a methylation state detection probe and an unmethylated state detection probe, wherein the upstream and downstream primers of the CACNA1G gene amplification primer are respectively sequences shown by SEQ ID No.1 and SEQ ID No.2, the methylation state detection probe is a sequence shown by SEQ ID No.3, and the unmethylated state detection probe is a sequence shown by SEQ ID No. 4;
the IGF2 gene methylation detection reagent comprises an IGF2 gene amplification primer, a methylation state detection probe and a non-methylation state detection probe, wherein the upstream primer and the downstream primer of the IGF2 gene amplification primer are respectively sequences shown in SEQ ID NO.5 and SEQ ID NO.6, the methylation state detection probe is a sequence shown in SEQ ID NO.7, and the non-methylation state detection probe is a sequence shown in SEQ ID NO. 8;
the NEUROG1 gene methylation detection reagent comprises a NEUROG1 gene amplification primer, a methylation state detection probe and an unmethylated state detection probe, wherein the upstream primer and the downstream primer of the NEUROG1 gene amplification primer are respectively sequences shown in SEQ ID No.9 and SEQ ID No.10, the methylation state detection probe is a sequence shown in SEQ ID No.11, and the unmethylated state detection probe is a sequence shown in SEQ ID No. 12;
the RUNX3 gene methylation detection reagent comprises a RUNX3 gene amplification primer, a methylation state detection probe and an unmethylated state detection probe, wherein the upstream and downstream primers of the RUNX3 gene amplification primer are respectively sequences shown in SEQ ID NO.13 and SEQ ID NO.14, the methylation state detection probe is a sequence shown in SEQ ID NO.15, and the unmethylated state detection probe is a sequence shown in SEQ ID NO. 16;
the SOCS1 gene methylation detection reagent comprises an SOCS1 gene amplification primer, a methylation state detection probe and an unmethylated state detection probe, wherein the upstream primer and the downstream primer of the SOCS1 gene amplification primer are respectively sequences shown in SEQ ID NO.17 and SEQ ID NO.18, the methylation state detection probe is a sequence shown in SEQ ID NO.19, and the unmethylated state detection probe is a sequence shown in SEQ ID NO. 20;
the internal reference beta-Actin gene detection reagent comprises a beta-Actin gene amplification primer and a detection probe, wherein the upstream primer and the downstream primer of the beta-Actin gene amplification primer are respectively sequences shown as SEQ ID NO.21 and SEQ ID NO.22, and the detection probe is a sequence shown as SEQ ID NO. 23.
2. The kit of claim 1, wherein: further comprising at least one of cfDNA extraction reagents, DNA bisulfite treatment reagents, and microdroplet digital PCR detection reagents.
3. Use of the kit according to claim 1 or 2 for preparing a reagent for tumor detection or cancer gene detection, wherein the cancer gene is at least one of CACNA1G gene, IGF2 gene, NEUROG1 gene, RUNX3 gene and SOCS1 gene.
4. Use of a kit according to claim 1 or 2 in the screening of cancer therapeutic drugs.
5. A method for the detection of a methylation phenotype of a CpG island for non-diagnostic therapeutic purposes, comprising: comprising performing CpG island methylation phenotype detection on a nucleic acid sample to be tested by using the kit of claim 1 or 2.
6. The detection method according to claim 5, characterized in that: setting blank control or negative control equivalent to replace the nucleic acid sample to be detected, and carrying out a control test;
performing data analysis according to the detection signals of the nucleic acid sample to be detected and the control test, and judging the methylation condition of the nucleic acid sample to be detected through the methylation index;
methylation index ═ methylation index (detection signal of gene to be detected in nucleic acid sample to be detected: detection signal of β -Actin gene in nucleic acid sample to be detected) ÷ (detection signal of gene to be detected in control test: detection signal of β -Actin gene in control test);
if the methylation index is greater than or equal to 0.5%, the nucleic acid sample to be tested is considered to be methylated;
the gene to be detected is at least one of CACNA1G gene, IGF2 gene, NEUROG1 gene, RUNX3 gene and SOCS1 gene.
7. The detection method according to claim 6, characterized in that: the CpG island methylation phenotype detection and the control test of the nucleic acid sample to be detected are both carried out by adopting microdroplet digital PCR; and analyzing and calculating the methylation index using a droplet analyzer.
8. The detection method according to claim 5, characterized in that: the nucleic acid sample to be detected is cfDNA.
9. The detection method according to any one of claims 5 to 8, characterized in that: the method specifically comprises the following steps of,
separating blood cells and plasma from peripheral blood samples;
extracting cfDNA from the separated plasma sample by adopting a two-step purification method;
performing bisulfite treatment on the extracted cfDNA;
methylation index was calculated for bisulfite-treated cfDNA using microdroplet digital PCR for amplification, detection and analysis.
10. Use of the detection method according to any one of claims 5 to 9 in detection of cancer gene or screening of cancer therapeutic drug, wherein the cancer gene is at least one of CACNA1G gene, IGF2 gene, NEUROG1 gene, RUNX3 gene and SOCS1 gene.
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