CN111440872A - Kit for detecting methylation of gastric cancer related genes and application of kit - Google Patents
Kit for detecting methylation of gastric cancer related genes and application of kit Download PDFInfo
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Abstract
The invention relates to a kit for detecting methylation of gastric cancer related genes, which is simple and convenient to operate, noninvasive, and higher in sensitivity and specificity, and application thereof, wherein the kit comprises a nucleic acid separation and purification reagent, a DNA sulfite conversion reagent, SDC2 and a TERT gene methylation detection reagent; wherein the nucleic acid separation and purification reagent is used for separating and purifying human source DNA in the excrement sample; DNA sulfite conversion reagents were used to sulfite convert purified portions of human DNA, SDC2 and TERT gene methylation detection reagents were used for subsequent detection of SDC2 and TERT gene methylation levels. The invention has the advantages of convenient sampling, no wound, convenience, no environmental interference, capability of realizing full coverage of stomach pathological changes, and suitability for popularization in popular physical examination and people needing to screen abnormal stomach diseases.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a kit for detecting methylation of gastric cancer related genes, which is simple and convenient to operate, has no wound and has higher sensitivity and specificity, and an application thereof.
Background Art Artificial sequence
Gastric cancer is one of common digestive tract tumors and seriously threatens the life and health of human beings. In recent decades, the incidence of gastric cancer generally decreases with the improvement of living conditions, the formation of good eating habits, the eradication of Helicobacter Pylori (HP), and other factors, but the incidence is high in the fourth place of male tumor and the mortality is the third place in the world.
The main stomach cancer screening methods at home and abroad are divided into two types: serological screening and endoscopic screening. Serological screening markers include pepsinogen, gastrin, Hp infection, MG7 antigen, carbohydrate antigen, and the like, and endoscopic screening includes electronic gastroscope, magnetic control capsule gastroscope, and the like. The serological screening method is simple and convenient to operate, has certain sensitivity and specificity, but is not high enough and not accurate enough; endoscopy and pathological analysis are the gold standards for diagnosing gastric cancer, but rely on equipment and endoscopic physician resources, have certain pain, and are not high in patient compliance, so that large-scale screening cannot be carried out.
TERT promoter methylation is a potential marker for gastrointestinal cancer. According to literature reports, in a clinical test consisting of stool samples of 69 gastrointestinal cancer patients (35 gastric cancers and 34 intestinal cancers) and 62 healthy adults, the sensitivity and specificity of combined detection of gastrointestinal cancer at two methylation sites on a TERT gene promoter are 52.2% and 90%, respectively; the sensitivity and specificity for detecting gastric cancer are 54.3% and 90%, respectively, and the sensitivity and specificity for detecting intestinal cancer are 50% and 90%, respectively.
In addition, SDC2 gene methylation is also an important marker for gut tumors. According to literature reports, SDC2 is a potential marker of colorectal cancer with significantly higher methylation levels in colorectal cancer tissues than in normal tissues. Through clinical verification, the sensitivity of detecting colorectal cancer by using the methylation of the faecal SDC2 gene is 81.1% (159/196), the sensitivity of detecting adenoma is 58.2% (71/122), and the total specificity is 93.3% (167/179). Meanwhile, the methylation detection of the SDC2 in the feces is also approved by NMPA and can be used for the auxiliary diagnosis of colorectal cancer. In addition to colorectal cancer, the methylation level of SDC2 gene was also significantly higher in gastric cancer tissues than in normal tissues. The methylation level of the SDC2 gene promoter in gastric cancer tissues is obviously higher than that of normal tissues through searching a MethHC database, and the gene is a potential marker for screening gastric cancer.
In addition, the methylation of RASSF2 and SFRP2 gene promoters can be used for detecting gastrointestinal tumors. In a clinical trial consisting of stool samples of 21 gastric cancers, 152 intestinal cancers, 10 patients with non-tumor or inflammatory lesions and 113 normal controls, the methylation of the RASSF2 and SFRP2 gene promoters combined detected gastric cancer at 57.1% sensitivity, colorectal cancer at 75% sensitivity, and advanced colorectal adenoma at 44.4% sensitivity, with specificity around 90%.
In conclusion, the purpose of screening gastrointestinal tumors can be realized by detecting gene methylation markers in the DNA of the excrement.
Disclosure of Invention
The invention aims to solve the problems of the existing gastric cancer screening method and provides a kit for detecting gastric cancer and a using method thereof, namely, the gastric cancer is detected by collecting excrement samples of detected persons and detecting the levels of two gene methylation markers in a combined manner, the operation is simple and convenient, the detection is noninvasive, and the kit has higher sensitivity and specificity.
In order to achieve the purpose, the invention adopts the following technical scheme:
a kit for detecting methylation of a gastric cancer-associated gene, the kit comprising a nucleic acid isolation and purification reagent, a DNA sulfite conversion reagent, SDC2 and a TERT gene methylation detection reagent; wherein the nucleic acid separation and purification reagent is used for separating and purifying human source DNA in the excrement sample; DNA sulfite conversion reagents were used to perform sulfite conversion of purified portions of human DNA, SDC2 and TERT gene methylation detection reagents were used for subsequent detection of SDC2 and TERT gene methylation levels.
Preferably, the SDC2 and TERT gene methylation detection reagents comprise a premixed reagent, water, and primers and probes for two target genes and an internal reference gene.
Preferably, the two target genes are SDC2 and TERT, respectively, and the reference gene is GAPDH.
Preferably, the sequences of the target gene and the reference gene are respectively:
SDC2 forward primer: aataagtgagagggcgtcgc
SDC2 reverse primer: aaactcgaactcgaaactcg
SDC2 probe: aacgctcgcttcctcctcctacgc
TERT forward primer: gtttagattttcgggttcgttcg
TERT reverse primer: gtctatacccgcgaatccactaa
TERT probe: cgacctaaccccgacaacgcaa
GAPDH forward primer: ggaggtaattaggatggtgtggtt
GAPDH reverse primer: catcrccccctaattttaaaaa
GAPDH probe: tgggtatatggtaattttgt are provided.
The application of the kit for detecting the methylation of the gastric cancer related gene comprises the following steps:
1) collecting and processing a fecal sample, and collecting a sample for gene detection;
2) extracting DNA of the fecal sample, and separating nucleic acid by a magnetic bead method by adopting a nucleic acid separation and purification reagent;
3) transforming the separated and purified nucleic acid by using bisulfite, and detecting the methylation level of the SDC2 and TERT gene in the sample by using a fluorescence quantitative PCR method;
4) and (6) analyzing results.
Preferably, the result analysis is to set a fluorescence threshold according to the amplification curve after the PCR reaction is finished, so as to obtain Ct values of different channels.
Preferably, the Ct value of the reference gene is less than or equal to 35, which indicates that the detection is qualified; the Ct value of the target gene SDC2 is positive when less than 40, and is negative when no Ct value is more than 40; the target gene TERT is positive if Ct value is less than 35 and is negative if Ct value is more than or equal to 35.
Preferably, if any target gene methylation marker is detected to be positive, the final result is judged to be positive, and a gastroscopy is required to be carried out subsequently to confirm the diagnosis.
Preferably, the SDC2 and TERT gene methylation detection reagents comprise a premixed reagent, water, and primers and probes for two target genes and an internal reference gene.
Preferably, the sequences of the target gene and the reference gene are respectively:
SDC2 forward primer: AATAAGTGAGAGGGCGTCGC
SDC2 reverse primer: AAACTCGAACTCGAAACTCG
SDC2 probe: AACGCTCGCTTCCTCCTCCTACGC
TERT forward primer: GTTTAGATTTTCGGGTTCGTTCG
TERT reverse primer: GTCTATACCCGCGAATCCACTAA
TERT probe: CGACCTAACCCCGACAACGCAA
GAPDH forward primer: GGAGGTAATTAGGATGGTGTGGTT
GAPDH reverse primer: CATCRCCCCACTTAATTTTAAAAA
GAPDH probe: TGGGTATATGGTAATTTTGT are provided.
The invention has the beneficial effects that:
1. the sensitivity of the kit for detecting the gastric cancer can reach about 80%, the specificity exceeds 80%, and the kit can realize high-sensitivity and high-specificity detection of the gastric cancer;
2. the invention has convenient sampling, no wound, convenience and no environmental interference, can realize the full coverage of stomach pathological changes, and is more suitable for popularization in the popular physical examination and the population needing to screen abnormal stomach diseases.
The kit for detecting gastric cancer related gene methylation and the use method thereof can be used for noninvasive and convenient screening of gastric cancer, have high specificity and high accuracy, and can be clinically used as an auxiliary detection index for early prevention and screening of tumorigenesis.
Drawings
FIG. 1 is a flow chart of the detection of the kit of the present invention;
FIG. 2 is a graph showing a positive result of the gene methylation detection;
FIG. 3 is a graph showing the negative results of the gene methylation assay.
Detailed Description
Embodiments of the invention are described in further detail below with reference to the accompanying drawings: materials and reagents required in the examples of the present invention are commercially available unless otherwise specified.
Example 1
1. Collection of fecal samples
Immediately collecting 2-5g of excrement sample after human excreting excrement, putting the excrement sample into a collecting tube, wherein 10-15m L excrement storage liquid is filled in the collecting tube, and shaking up the collecting tube to fully and uniformly mix the excrement sample and the excrement storage liquid;
the feces retaining liquid comprises the following components: 0.3-0.6M EDTA, 3-6M NaCl, 0.3-1.5M Tris;
2. fecal DNA extraction
The fecal DNA extraction is divided into the following steps: (1) precipitating the cells; (2) lysing the cells to release the DNA; (3) precipitating impurities; (4) removing impurities and purifying DNA; (5) binding magnetic beads with DNA; (6) magnetic bead washing and DNA elution.
For specific steps, please refer to the patent "a human fecal DNA extraction method" published in 2018, 11/23, application No.: CN201810812474.4, which will not be described herein.
3. DNA bisulfite conversion
The method is characterized in that after a thermal denaturation method is adopted for 10-20 minutes at 95 ℃, the DNA is immediately frozen for 5-10 minutes to prevent renaturation, then the DNA is converted for 45-60 minutes at 75 ℃, and the converted DNA is purified by a magnetic bead method and a binding solution containing PEG6000/8000, so that the methylated DNA with high conversion rate and high quality can be obtained, the standardization and full-automatic operation of methylation detection is facilitated, and the method is used for the methylation level analysis of genes in human DNA in subsequent fecal samples.
For the specific steps, please refer to the patent "a human feces total DNA sulfite transformation and recovery purification" published by the applicant at 2018, 12, month and 7, with the application numbers: CN201810814365.6, which will not be described herein.
4. Fluorescent quantitative PCR
SDC2 and TERT gene methylation levels are detected by a fluorescent quantitative PCR method, detection is carried out by an ABI7500 device, the total reaction system is 35ul, and 17.5ul of PCR premixed reagent, 2.5ul of reaction liquid and 15ul of DNA after bisulfite conversion are included. The PCR premixed reagent is a commercial quantitative PCR reaction mixed reagent, the reaction solution consists of a probe (100uM, 0.105ul) (synthesized by professional probe synthesis company), a primer (positive and negative primers, 100uM, 0.315ul 2) (synthesized by professional primer synthesis company), and water (1.765ul), and each reaction solution of SDC2, TERT and a reference Gene (GAPDH).
The reaction parameters are shown in table 1:
table 1: PCR reaction parameters
And after the reaction is finished, setting a proper fluorescence threshold value according to the amplification curve to obtain Ct values of different channels.
5. Analysis of detection results
(1) Reagent validity determination
Weak positive control: the Ct value of the VIC channel of the reference gene is less than or equal to 35; FAM channel Ct values for target genes (SDC2 and TERT) were less than 40 and 35, respectively; the amplification curve has obvious exponential growth period;
blank control: the FAM channels of the internal reference gene VIC channel and the target genes (SDC2 and TERT) have no amplification curve or the amplification curve is a straight line or a slightly oblique line and has no obvious exponential growth period.
(2) Sample validity determination
The Ct value of the internal reference gene in the sample detection result is less than or equal to 35, and the amplification curve has an obvious exponential amplification period.
(3) Determination of results of single-Gene methylation detection
Table 2: determination of results
6. Determination of results
And (3) the Ct value of the internal reference gene is less than or equal to 35, which indicates that the detection is qualified, and the result of the detected sample is comprehensively judged according to the detection levels of the methylation markers of the SDC2 gene and the TERT gene, namely the Ct value, so as to evaluate whether the detected person is a high-risk group with gastric cancer.
Specific judgment criteria are as follows:
if the detection result of any target gene methylation marker is positive, the final result is judged to be positive, the high risk of gastric cancer is met, and follow-up gastroscopy is recommended.
Limitations of the detection method:
a. the results of the sample testing are related to the quality of the sample collection, handling and storage, where any error will result in inaccurate test results. If the quality of the DNA is not well controlled during sample processing, false negative results may occur.
b. The result is only for reference and can not be directly used as evidence for determining gastric cancer, and patients with positive detection result need to be subjected to gastroscopy for determining the gastric cancer.
Example 2
Clinical sample test results
A total of 27 gastric cancer samples and 54 non-gastric cancer samples were collected. Then two genes in the fecal sample are respectively detected
(SDC2 and TERT) methylation levels, and then all clinical samples were classified by the positive decision criteria in example one, the sensitivity of the final model to differentiate gastric cancer (high risk) samples reached 81.5% (22/27) and the specificity reached 85.2% (46/54).
Table 3: example II partial sample pathological information and model determination results
| Sample numbering | Type of pathology | GAPDH | Methylation of SDC2 | TERT methylation | The result of the judgment |
| T1 | Stomach cancer | 28.2 | 33.9 | 33.7 | Positive/high risk |
| T2 | Stomach cancer | 32.3 | 38.9 | 36.6 | Positive/high risk |
| T3 | Stomach cancer | 32.9 | 41.2 | 33.1 | Positive/high risk |
| T4 | Stomach cancer | 33.5 | 38.3 | 35.4 | Positive/high risk |
| T5 | Stomach cancer | 33.4 | 40.5 | 38.4 | Negative/low risk |
| N1 | Non-gastric cancer | 31.6 | 42.0 | 38.9 | Negative/low risk |
| N2 | Non-gastric cancer | 32.9 | 41.1 | 40.3 | Negative/low risk |
| N3 | Non-gastric cancer | 33.2 | 40.7 | 37.3 | Negative/low risk |
| N4 | Non-gastric cancer | 34.6 | 41.8 | 39.2 | Negative/low risk |
| N5 | Non-gastric cancer | 33.3 | 38.5 | 41.5 | Positive/high risk |
Patients who have a positive test result need to be gastroscopically examined to confirm the diagnosis.
The sensitivity for detecting the gastric cancer can reach about 80 percent, the specificity exceeds 80 percent, and the high-sensitivity and high-specificity detection of the gastric cancer can be realized.
The invention provides a kit for detecting gastric cancer, which comprises a nucleic acid separation and purification reagent, a DNA sulfite conversion reagent, SDC2 and a TERT gene methylation detection reagent; wherein the nucleic acid separation and purification reagent is used for separating and purifying human source DNA in the excrement sample; DNA sulfite conversion reagents were used to sulfite convert purified portions of human DNA for subsequent detection of SDC2 and TERT gene methylation levels.
The invention has the advantages of convenient sampling, no wound, convenience, no environmental interference, capability of realizing full coverage of stomach pathological changes, and suitability for popularization in popular physical examination and people needing to screen abnormal stomach diseases.
The kit for detecting gastric cancer related gene methylation and the use method thereof can be used for noninvasive and convenient screening of gastric cancer, have high specificity and high accuracy, and can be clinically used as an auxiliary detection index for early prevention and screening of tumorigenesis.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
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<120> kit for detecting methylation of gastric cancer related genes and application thereof
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Claims (10)
1. A kit for detecting methylation of a gastric cancer-associated gene, the kit comprising a nucleic acid isolation and purification reagent, a DNA sulfite conversion reagent, SDC2 and a TERT gene methylation detection reagent; wherein the nucleic acid separation and purification reagent is used for separating and purifying human source DNA in the excrement sample; DNA sulfite conversion reagents are used to sulfite convert purified portions of human DNA characterized by SDC2 and TERT gene methylation detection reagents for subsequent detection of SDC2 and TERT gene methylation levels.
2. The kit for detecting methylation of gastric cancer-associated genes according to claim 1, wherein the SDC2 and TERT gene methylation detection reagents comprise premixed reagents, water, and primers and probes for two target genes and an internal reference gene.
3. The kit for detecting methylation of gastric cancer-associated genes according to claim 2, wherein the two target genes are SDC2 and TERT, and the reference gene is GAPDH.
4. The kit for detecting methylation of gastric cancer-associated genes according to claim 2 or 3, wherein the sequences of the target gene and the reference gene are respectively:
SDC2 forward primer: AATAAGTGAGAGGGCGTCGC
SDC2 reverse primer: AAACTCGAACTCGAAACTCG
SDC2 probe: AACGCTCGCTTCCTCCTCCTACGC
TERT forward primer: GTTTAGATTTTCGGGTTCGTTCG
TERT reverse primer: GTCTATACCCGCGAATCCACTAA
TERT probe: CGACCTAACCCCGACAACGCAA
GAPDH forward primer: GGAGGTAATTAGGATGGTGTGGTT
GAPDH reverse primer: CATCRCCCCACTTAATTTTAAAAA
GAPDH probe: TGGGTATATGGTAATTTTGT are provided.
5. Use of the kit for detecting methylation of gastric cancer-associated genes according to any one of claim 1, wherein the use method comprises the following steps:
1) collecting and processing a fecal sample, and collecting a sample for gene detection;
2) extracting DNA of the fecal sample, and separating nucleic acid by a magnetic bead method by adopting a nucleic acid separation and purification reagent;
3) transforming the separated and purified nucleic acid by using bisulfite, and detecting the methylation level of the SDC2 and TERT gene in the sample by using a fluorescence quantitative PCR method;
4) and (6) analyzing results.
6. The application of the kit for detecting methylation of gastric cancer-related genes according to claim 5, wherein the result analysis is to set a fluorescence threshold according to an amplification curve after the PCR reaction is finished, so as to obtain Ct values of different channels.
7. The application of the kit for detecting methylation of gastric cancer-related genes according to claim 6, wherein the Ct value of the reference gene is less than or equal to 35, which indicates that the detection is qualified; the Ct value of the target gene SDC2 is positive when less than 40, and is negative when no Ct value is more than 40; the target gene TERT is positive if Ct value is less than 35 and is negative if Ct value is more than or equal to 35.
8. The use of the kit according to claim 7, wherein the detection result of any one target gene methylation marker is positive, and the final result is determined to be positive, and a gastroscopy is required for confirmation.
9. The application of the kit for detecting the methylation of the gastric cancer-associated genes according to claim 5, wherein the SDC2 and the TERT gene methylation detection reagent comprise a premixed reagent, water, and primers and probes of two target genes and an internal reference gene.
10. The use of the kit according to claim 9, wherein the sequences of the target gene and the reference gene are respectively:
SDC2 forward primer: AATAAGTGAGAGGGCGTCGC
SDC2 reverse primer: AAACTCGAACTCGAAACTCG
SDC2 probe: AACGCTCGCTTCCTCCTCCTACGC
TERT forward primer: GTTTAGATTTTCGGGTTCGTTCG
TERT reverse primer: GTCTATACCCGCGAATCCACTAA
TERT probe: CGACCTAACCCCGACAACGCAA
GAPDH forward primer: GGAGGTAATTAGGATGGTGTGGTT
GAPDH reverse primer: CATCRCCCCACTTAATTTTAAAAA
GAPDH probe: TGGGTATATGGTAATTTTGT are provided.
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| CN202010377370.2A CN111440872A (en) | 2020-05-07 | 2020-05-07 | Kit for detecting methylation of gastric cancer related genes and application of kit |
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| CN202010377370.2A CN111440872A (en) | 2020-05-07 | 2020-05-07 | Kit for detecting methylation of gastric cancer related genes and application of kit |
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Cited By (1)
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| WO2023071889A1 (en) * | 2021-10-25 | 2023-05-04 | 广州市基准医疗有限责任公司 | Methylation biomarker related to detection of gastric cancer lymph node metastasis, or combination thereof and use thereof |
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Application publication date: 20200724 |