CN114507740B - Biomarkers, nucleic acid products and kits for gastrointestinal cancer diagnosis - Google Patents
Biomarkers, nucleic acid products and kits for gastrointestinal cancer diagnosis Download PDFInfo
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Abstract
The present invention relates to a biomarker, a nucleic acid product and a kit for gastrointestinal cancer diagnosis. The biomarker is selected from at least two of ADHFE1 gene, FAM19A4 gene and VSTM2B gene, and gastrointestinal cancer is diagnosed by detecting the methylation level of the biomarker.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a biomarker, a nucleic acid product and a kit for gastrointestinal cancer diagnosis.
Background
Because early symptoms of gastrointestinal tumors are mostly unobvious and the onset of the gastrointestinal tumors is mostly hidden, most patients have the obvious symptoms which belong to the middle and late stages and are mostly accompanied by lymph node metastasis. Wherein: 80% of early stage gastric cancer is asymptomatic, some patients may have dyspepsia, and the most common symptoms of advanced gastric cancer are weight loss (about 60%) and upper abdominal pain (50%), as well as anemia, anorexia, hypodynamia, and the like. The incidence of colorectal cancer increases markedly from 50 years, peaks at 75-80 years, and then declines slowly, but not rarely in the young bowel cancer under 30 years. Intestinal cancer is also hidden, and only fecal occult blood is positive in the early stage, and then defecation habit, fecal character change, abdominal pain, rectal mass during finger examination, anemia, low fever, progressive emaciation, cachexia, abdominal dropsy and other symptoms can appear. When the above symptoms appear, the disease is not early, and most gastrointestinal cancer patients are diagnosed after the symptoms appear in clinic. Therefore, the survival rate of the gastrointestinal tumor patients is lower at present.
If the patient can actively screen for gastrointestinal cancer in the asymptomatic state, the early detection rate is obviously improved, and the prognosis is better. Therefore, gastrointestinal cancer screening is of considerable importance. However, the traditional clinical diagnosis mainly refers to the imaging examination and tumor markers for gastrointestinal cancer early screening, has low diagnostic sensitivity and is not suitable for early screening and early diagnosis.
Disclosure of Invention
Therefore, the need is directed to providing a biomarker for gastrointestinal cancer, which can detect the methylation level of the biomarker in blood to diagnose the gastrointestinal cancer, can improve the problem of low sensitivity of the traditional gastrointestinal cancer diagnosis product, and is beneficial to early screening and early diagnosis of the gastrointestinal cancer.
In addition, a nucleic acid product and a diagnostic kit for diagnosing gastrointestinal cancer are provided.
A biomarker for gastrointestinal cancer selected from at least two of the ADHFE1 gene, FAM19a4 gene and VSTM2B gene, the gastrointestinal cancer being diagnosed by detecting the methylation level of the biomarker.
Proved by verification, at least two of ADHFE1 gene, FAM19A4 gene and VSTM2B gene are selected as biomarkers for diagnosing gastrointestinal cancer, and the gastrointestinal cancer is diagnosed by detecting the methylation level of the markers, so that the sensitivity is high, and the specificity is good.
A nucleic acid product comprising a pair of detection primers comprising at least two of the following pairs of primers:
an ADHFE1 primer pair for detecting the methylation level of the ADHFE1 gene, a FAM19A4 primer pair for detecting the methylation level of the FAM19A4 gene and a VSTM2B primer pair for detecting the methylation level of the VSTM2B gene.
In one embodiment, the nucleic acid product further comprises a detection probe corresponding to the detection primer pair, and the detection probe corresponding to the detection primer pair is connected with a fluorescent group.
In one embodiment, the ADHFE1 primer pair is designed for the promoter-to-first exon region of the ADHFE1 gene, the FAM19a4 primer pair is designed for the promoter-to-first exon region of the FAM19a4 gene, and the VSTM2B primer pair is designed for the promoter-to-first exon region of the VSTM2B gene.
In one embodiment, the nucleotide sequence of the ADHFE1 primer pair is as set forth in SEQ ID No: 1-2;
and/or the nucleotide sequence of the FAM19A4 primer pair is shown as SEQ ID No: 3-4;
and/or the nucleotide sequence of the VSTM2B primer pair is shown as SEQ ID No: 5 to 6.
In one embodiment, the nucleotide sequence of the detection probe corresponding to the ADHFE1 primer pair is as shown in SEQ ID No: 7 is shown in the specification;
and/or the nucleotide sequence of a detection probe corresponding to the FAM19A4 primer pair is shown as SEQ ID No: 8 is shown in the specification;
and/or the nucleotide sequence of a detection probe corresponding to the VSTM2B primer pair is shown as SEQ ID No: shown at 9.
In one embodiment, the nucleic acid product further comprises an internal reference primer pair and an internal reference probe corresponding to the internal reference primer pair, the internal reference primer pair comprising at least one of the following primer pairs:
an ACTB primer pair designed for the ACTB gene, a GAPDH primer pair designed for the GAPDH gene, an ALDOA primer pair designed for the ALDOA gene, and a PGK1 primer pair designed for the PGK1 gene;
the internal reference probe is connected with a fluorescent group different from the detection probe.
In one embodiment, the ACTB primer pair has the nucleotide sequence set forth in SEQ ID No: 10-11, wherein the nucleotide sequence of the internal reference probe corresponding to the ACTB primer pair is shown as SEQ ID No: shown at 12.
A kit for gastrointestinal cancer diagnosis comprising a reagent for detecting the methylation level of a biomarker of said gastrointestinal cancer.
In one embodiment, the gastrointestinal cancer is gastric cancer, colorectal cancer, and/or adenomatous polyps.
In one embodiment, the nucleic acid product described above is included.
In one embodiment, the kit further comprises at least one of nucleic acid extraction reagents, methylation conversion reagents, quality control reagents, PCR reaction reagents and sequencing reagents.
Drawings
FIG. 1 shows the results of the detection of positive reference samples in each group in example 1;
FIG. 2 shows the results of the detection of negative reference samples in each group in example 1;
FIG. 3 shows the results of detection of each set of reference samples with detection sensitivity in example 1;
FIG. 4 is a graph showing the results of testing each group of samples of healthy persons in example 1;
FIG. 5 is a graph showing the results of examination of samples from each group of patients with gastric cancer in example 1;
FIG. 6 shows the results of examination of samples of intestinal cancer patients in each group of example 1.
Detailed Description
The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The term "and/or" includes any and all combinations of one or more of the associated listed items.
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.
The research of the inventor finds that the sensitivity and the specificity for diagnosing the gastrointestinal cancer are good by detecting the methylation levels of at least two genes of the ADHFE1 gene, the FAM19A4 gene and the VSTM2B gene, and the early diagnosis of the gastrointestinal cancer is facilitated. Specifically, in the NCBI database, the Gene ID of the ADHFE1 Gene: 137872, Gene ID of FAM19A4 Gene: 151647, Gene ID of VSTM2B Gene: 342865.
in some embodiments, the gastrointestinal cancer is gastric cancer, colorectal cancer, and/or adenomatous polyps.
In this embodiment, the sample to be detected is blood. It is understood that in other embodiments, other biological samples are also possible. Such as one or more of cell lines, histological sections, tissue biopsies/paraffin-embedded tissues, body fluids, stool, colonic effluents, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood.
Circulating tumor DNA (ctDNA) is free DNA from tumor cells in the blood of a patient with a tumor, and after necrosis or apoptosis of the tumor cells, the DNA in the cells is released into the circulatory system and is free in the blood. By detecting the methylation state of the related gene of ctDNA, the in-vivo tumor tissue information can be obtained, thereby providing a basis for tumor diagnosis. In an alternative specific example, the sample for detection is plasma. The free DNA in the plasma can be used for detecting tumors and has the characteristics of little harm to patients, good specificity and the like. However, the content of the compound in plasma is extremely low, and thus, there is a problem of low sensitivity. The detection reagent can be used for detecting free DNA in plasma as a sample, and has high sensitivity and accuracy.
In some embodiments, the biomarker for gastrointestinal cancer is a combination of the ADHFE1 gene and the FAM19a4 gene. In one embodiment, the biomarker for gastric cancer is a combination of ADHFE1 gene and FAM19a4 gene.
In some embodiments, the biomarker for gastrointestinal cancer is a combination of FAM19a4 gene and VSTM2B gene.
In some embodiments, the biomarker for gastrointestinal cancer is a combination of the ADHFE1 gene and the VSTM2B gene.
In some embodiments, the biomarker for gastrointestinal cancer is a combination of ADHFE1 gene, FAM19a4 gene, and VSTM2B gene.
Gastrointestinal cancer can be diagnosed by detecting the methylation level of at least two genes selected from the ADHFE1 gene, FAM19A4 gene and VSTM2B gene as biomarkers of gastrointestinal cancer. An embodiment of the present application also provides a kit for diagnosis of gastrointestinal cancer. The kit comprises reagents for detecting the methylation levels of at least two genes of ADHFE1 gene, FAM19A4 gene and VSTM2B gene. The detection result of the kit can be used for diagnosing gastrointestinal cancer.
In some embodiments, the kit detects the methylation level of at least two of the ADHFE1 gene, FAM19a4 gene, and VSTM2B gene using methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation fluorescence, methylation-specific high performance liquid chromatography, or digital PCR. It is understood that, in other embodiments, the method of detecting the methylation level of the gene by the kit is not limited to the above method, and other methods can be used.
In some embodiments, the kit comprises a chip capable of detecting the methylation level of at least two of the ADHFE1 gene, FAM19a4 gene, and VSTM2B gene. The chip is provided with a detection reagent for detecting the corresponding gene.
In this embodiment, the kit detects the methylation level of at least two genes selected from the group consisting of ADHFE1 gene, FAM19A4 gene and VSTM2B gene by a methylation fluorescence PCR method. Specifically, the kit comprises a nucleic acid product for detecting the methylation level of at least two genes selected from the group consisting of ADHFE1 gene, FAM19A4 gene, and VSTM2B gene.
Specifically, the nucleic acid product comprises a detection primer pair, and the detection primer pair comprises at least two groups of the following primer pairs: an ADHFE1 primer pair for detecting the methylation level of the ADHFE1 gene, a FAM19A4 primer pair for detecting the methylation level of the FAM19A4 gene and a VSTM2B primer pair for detecting the methylation level of the VSTM2B gene.
In some embodiments, the ADHFE1 primer pair is designed for the promoter-to-first exon region of the ADHFE1 gene, the FAM19a4 primer pair is designed for the promoter-to-first exon region of the FAM19a4 gene, and the VSTM2B primer pair is designed for the promoter-to-first exon region of the VSTM2B gene. It should be noted that the region from the promoter to the first exon includes the promoter, the region between the promoter and the first exon, and the first exon; it is understood that the promoter may be a partial region where the promoter is located, or may be a region where the entire promoter is located; the first exon may be a partial region of the first exon, or may be the entire region of the first exon.
In one embodiment, the nucleotide sequence of the ADHFE1 primer pair is as set forth in SEQ ID No: 1 to 2. In one embodiment, the nucleotide sequence of FAM19a4 primer pair is as set forth in SEQ ID No: 3 to 4. In one embodiment, the nucleotide sequence of VSTM2B primer pair is as set forth in SEQ ID No: 5 to 6.
It is understood that, in other embodiments, the primer pairs for detecting methylation levels of the ADHFE1 gene, the FAM19A4 gene and the VSTM2B gene are not limited to the above, and may be other primer pairs designed for other sections of the above genes or the above sections.
In some embodiments, the nucleic acid product further comprises a detection probe corresponding to the detection primer pair, and the detection probe corresponding to the detection primer pair has a fluorophore attached thereto.
In one embodiment, the nucleotide sequence of the detection probe corresponding to the primer pair ADHFE1 is as shown in SEQ ID No: shown at 7. In one embodiment, the nucleotide sequence of the detection probe corresponding to the primer pair FAM19a4 is as shown in SEQ ID No: shown in fig. 8. In one embodiment, the nucleotide sequence of the detection probe corresponding to the VSTM2B primer pair is as set forth in SEQ ID No: shown at 9. It is understood that in other embodiments, the detection probe corresponding to the detection primer pair is not limited to the above, and may be other nucleic acid fragments.
In some embodiments, the nucleic acid product further comprises an internal reference primer pair and an internal reference probe corresponding to the internal reference primer pair, the internal reference primer pair comprising at least one of the following primer pairs: an ACTB primer pair designed for the ACTB gene, a GAPDH primer pair designed for the GAPDH gene, an ALDOA primer pair designed for the ALDOA gene, and a PGK1 primer pair designed for the PGK1 gene; the internal reference probe is connected with a fluorescent group different from the detection probe. In an alternative embodiment, the nucleotide sequence of the ACTB primer pair is as set forth in SEQ ID No: 10-11, and the nucleotide sequence of the internal reference probe corresponding to the ACTB primer pair is shown as SEQ ID No: shown at 12. The above-mentioned reference primer pair is designed with ACTB gene, GAPDH gene, ALDOA gene and/or PGK1 gene. It is understood that, in other embodiments, other genes can be selected as the reference gene, and in this case, the reference primer pair and the reference probe are designed correspondingly.
In some embodiments, the detection probe and the internal reference probe have a fluorophore and a quencher attached thereto. Alternatively, the fluorescent group is located at the 5 'end of the probe and the quencher group is located at the 3' end of the probe. Optionally, the detection probe and the internal reference probe are respectively and independently connected with one of FAM, HEX, VIC, CY5, ROX, Texsa Red, JOE and Quasar 705, wherein the fluorescent groups are respectively and independently connected with the internal reference probe and the detection probe. Of course, when two or more reference probes are present in the same reaction system, the fluorophores attached to the different reference probes are different. It is understood that the fluorescent group attached to the detection probe and the reference probe is not limited to the above, and may be other fluorescent groups.
In some embodiments, each detection probe and the internal reference probe are each attached to a different fluorophore. In this case, each detection probe and the internal reference probe can be placed in a single system for PCR reaction, and different amplification products can be distinguished by fluorescence emitted from the fluorophore connected to the detection probe.
In some embodiments, the kit further comprises at least one of a nucleic acid extraction reagent, a methylation conversion reagent, a quality control reagent, a PCR reaction reagent, and a sequencing reagent. The nucleic acid extraction reagent is used for extracting nucleic acid; the methylation conversion reagent is used for converting cytosine without methylation in DNA into uracil by deamination, and methylated cytosine is kept unchanged; the quality control reagent is used for quality control; the PCR reaction reagent is used for constructing a PCR amplification reaction system; nucleic acid sequencing reagents were used for sequencing.
In one embodiment, the methylation conversion reagent is a sulfite conversion reagent or an enzymatic conversion reagent.
In one embodiment, the PCR reaction reagent comprises PCR buffer, dNTP, MgCl 2 And TaqDNA polymerase.
In one embodiment, the quality control reagent comprises a positive reference substance and a negative reference substance. Alternatively, the positive reference is human methylated genomic DNA and the negative reference is human unmethylated genomic DNA. Optionally, the quality control reagent further comprises a sensitivity reference. Specifically, the sensitivity reference is 10ng/mL human unmethylated genomic DNA containing 0.1% human methylated genomic DNA.
In addition, the embodiment of the application also provides a method for detecting the methylation state of at least one gene of ADHFE1 gene, FAM19A4 gene and VSTM2B gene. The method comprises the following steps:
(1) extracting sample DNA;
(2) carrying out methylation conversion on the extracted DNA to obtain a converted DNA template;
(3) the obtained DNA template was used to detect the methylation levels of the ADHFE1 gene, FAM19A4 gene and VSTM2B gene using any of the above-described nucleic acid products.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer. In the following examples, the nucleic acid extraction reagent and the methylation transformation kit were the reagents developed by the same company; PCR buffer, dNTP, TaqDNA polymerase were purchased from Takara; MgCl 2 Purchased from Sigma company; the primer probe is synthesized by Shanghai Biotech, Inc.; the fluorescent quantitative PCR instrument is ABI 7500.
Example 1
First, entrustment company synthesizes the following primer pairs and probes
The nucleotide sequence for detecting the methylation state of the ADHFE1 gene is shown as SEQ ID No: 1 and SEQ ID No: 2, and the nucleotide sequence of the primer pair of ADHFE1 shown in SEQ ID No: 7, an ADHFE1 probe;
the nucleotide sequence for detecting the methylation state of the FAM19A4 gene is shown as SEQ ID No: 3 and SEQ ID No: 4, and the nucleotide sequence of the FAM19A4 primer pair is shown as SEQ ID No: FAM19A4 probe shown in FIG. 8;
the nucleotide sequence for detecting the methylation state of the VSTM2B gene is shown as SEQ ID No: 5 and SEQ ID No: 6, and the nucleotide sequence of the VSTM2B primer pair is shown as SEQ ID No: FAM19A4 probe shown in FIG. 9;
the nucleotide sequence for detecting the reference gene ACTB is shown as SEQ ID No: 10 and SEQ ID No: 11 and the nucleotide sequence is shown as SEQ ID No: ACTB probe shown in FIG. 12.
Wherein, the 5 'end of the ADHFE1 probe is marked with a report fluorescent group FAM, and the 3' end is marked with a quenching fluorescent group BHQ; the 5 'end of the FAM19A4 probe is marked with a report fluorescent group ROX, and the 3' end is marked with a quenching fluorescent group BHQ; the 5 'end of the VSTM2B probe is marked with a reporter fluorophore CY5, and the 3' end is marked with a quenching fluorophore BHQ; the ACTB probe is labeled with a reporter fluorophore JOE at the 5 'end and a quencher fluorophore BHQ at the 3' end. Specifically, the results are shown in Table 1.
TABLE 1
The positive reference substance adopts human methylated genomic DNA, the negative reference substance adopts human non-methylated genomic DNA, and the sensitivity reference substance is that the human non-methylated genomic DNA of 10ng/mL contains 0.1 percent of human methylated genomic DNA; the samples to be treated were 1 plasma sample from a patient with confirmed diagnosis of gastric cancer, 1 plasma sample from a patient with confirmed diagnosis of intestinal cancer, and 1 plasma sample from a normal healthy person.
Third, DNA extraction
The DNA extraction method of each sample includes but is not limited to the following steps:
1. adding 2mL of sample, 5mL of nucleic acid lysis adsorption solution and 100 muL of magnetic beads into a 15mL centrifuge tube, uniformly mixing by vortex, and placing the centrifuge tube at 56 ℃ for 10 minutes.
2. Placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 1mL of washing liquid A, and uniformly mixing to ensure that the magnetic beads are thoroughly resuspended;
3. placing the centrifugal tube in a magnetic rack for magnetic attraction for 2min, sucking away all the waste liquid, removing residual liquid as much as possible by using a 10-100 mu L gun head, moving the centrifugal tube to a nonmagnetic test tube rack, opening a tube cover, and drying at room temperature for 5 min;
4. adding 40 mu L of eluent, covering a tube cover, whirling and uniformly mixing the eluent and the heavy suspension magnetic beads, and incubating the centrifuge tube for 5 minutes at 56 ℃;
5. place the centrifuge tube in a magnetic rack for magnetic attraction for 2min and move all eluents to a new 0.2mL PCR tube.
IV, sulfite conversion
The steps for DNA sulfite conversion of each sample include, but are not limited to, the following:
1. adding 110 muL of sulfite solution into a 0.2mL PCR tube of 40 muL of DNA, covering the centrifuge tube, performing vortex mixing, performing short-time centrifugation, and placing the centrifuge tube in a common PCR instrument for reaction under the conditions of 95 ℃ for 5 minutes, 60 ℃ for 10 minutes, 95 ℃ for 5 minutes and 60 ℃ for 10 minutes;
2. transferring the DNA solution after the reaction to a new 1.5mL centrifuge tube, adding 600 muL of binding solution and 10 muL of magnetic beads, uniformly mixing in a vortex manner, and standing at room temperature for 5 minutes;
3. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 mu L of washing liquid A, and performing vortex mixing to ensure that the magnetic beads are thoroughly resuspended;
4. placing the centrifugal tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 200 mu L of desulfonation liquid, performing vortex mixing to ensure that the magnetic beads are thoroughly resuspended, and standing at room temperature for 15 min;
5. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 mu L of washing liquid B, and performing vortex mixing to ensure that the magnetic beads are thoroughly resuspended;
6. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 muL of washing liquid C, and performing vortex mixing to ensure that the magnetic beads are thoroughly resuspended;
7. Placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 mu L of washing liquid D, and performing vortex mixing to ensure that the magnetic beads are thoroughly resuspended;
8. placing the centrifugal tube in a magnetic rack for magnetic attraction for 2min, sucking away all the waste liquid, removing residual liquid as much as possible by using a 10-100 mu L gun head, moving the centrifugal tube to a nonmagnetic test tube rack, opening a tube cover, and drying at room temperature for 5 min;
9. adding 50 muL of eluent, covering a tube cover, whirling and uniformly mixing the mixture, and incubating the centrifuge tube for 5 minutes at 56 ℃;
10. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, and transferring all the eluates to a new centrifuge tube for later use.
Fifth, PCR reaction
The PCR reaction of each sample includes, but is not limited to, the following steps:
preparation of PCR reaction solution
According to the experimental amount, an ADHFE1 gene methylation reaction system is prepared according to Table 2, FAM19A4 gene methylation reaction solution is prepared according to Table 3, VSTM2B gene methylation reaction solution is prepared according to Table 4, ADHFE1+ FAM19A4 gene methylation reaction solution is prepared according to Table 5, ADHFE1+ VSTM2B gene methylation reaction solution is prepared according to Table 6, FAM19A4+ VSTM2B gene methylation reaction solution is prepared according to Table 7, and VSTM2B gene + FAM19A4 gene + ADHFE1 gene methylation reaction solution is prepared according to Table 8. It should be noted that tables 2 to 8 are all single reaction systems, that is, systems for performing PCR reaction on a computer. In table 2, the concentrations of the components are the concentrations at the time of using the components.
TABLE 2
TABLE 3
TABLE 4
TABLE 5
TABLE 6
TABLE 7
TABLE 8
2. Sample application
After 15. mu.L of each PCR reaction solution was added to each prepared PCR reaction tube, 10. mu.L of the sample DNA was added, and the tube cap was closed, followed by instantaneous low-speed centrifugation. Of course, the sample adding amount of the reference substance in the negative quality control and the positive quality control is the same as that of the sample.
3. Fluorescent quantitative PCR detection
1) Fluorescence channel selection: each sample selected 4 channels of FAM, JOE, ROX, CY 5. The Reference fluorescence (Passive Reference) is set to none;
2) the reaction conditions were set as shown in Table 9 (the reaction volume was set to 25. mu.L):
TABLE 9
Sixthly, result analysis
Analysis of PCR results
And (3) automatically storing the result after the reaction is finished, automatically analyzing the result by using instrument matched software, and calculating the Ct value of any channel in the PCR amplification if the channel has an amplification curve, wherein the Ct value reflects the relative content of the detected gene. The QPCR results are shown in FIGS. 1 to 6.
2. Determination of detection result
Taking the result when the Ct of the ACTB gene is less than or equal to 32 as an effective result, indicating that the DNA content in the sample is sufficient, otherwise, taking the result as an ineffective result; results were positive when Ct ≦ 43 for FAM or ROX or CY5 channel, indicating high risk for gastrointestinal cancer, and negative when Ct >43 or n.d. for FAM and ROX and CY5 channel, indicating low risk for gastrointestinal cancer, where the abbreviation for "Not Detected" for n.d. means "Not Detected".
3. The result of the detection
(1) Methylation of the ADHFE1 gene was not detected in both the negative reference sample and the normal sample, and the result was negative, while the methylation of the ADHFE1 gene was detected in all the positive reference sample, the sensitivity reference sample, and the gastrointestinal cancer sample, and the result was positive. Specific results are shown in table 10 below.
(2) FAM19A4 gene methylation was not detected in both negative reference and normal samples, and the results were negative, and positive results were detected in all positive reference, sensitivity reference and gastrointestinal cancer samples. The specific results are shown in Table 11 below.
TABLE 11
(3) The methylation of the VSTM2B gene is not detected in a negative reference product and a normal sample, the result is negative, and the result is positive when the methylation of the VSTM2B gene is detected in a positive reference product, a sensitivity reference product and a gastrointestinal cancer sample. Specific results are shown in table 12 below.
TABLE 12
(4) Methylation of the ADHFE1+ FAM19A4 gene was not detected in both the negative reference sample and the normal sample, and was negative, while it was detected in all the positive reference sample, the sensitivity reference sample, and the gastrointestinal cancer sample, and was positive. The results are shown in Table 13.
Watch 13
(5) Methylation of the ADHFE1+ VSTM2B gene was not detected in both the negative reference sample and the normal sample, and the result was negative, and was detected in the positive reference sample, the sensitivity reference sample, and the gastrointestinal cancer sample, and the result was positive. The specific results are shown in Table 14.
TABLE 14
(6) FAM19A4+ VSTM2B gene methylation was not detected in both negative reference and normal samples, and the results were negative, and positive in all positive reference, sensitivity reference and gastrointestinal cancer samples. The results are shown in Table 15.
Watch 15
(7) Methylation of ADHFE1+ FAM19A4+ VSTM2B gene was not detected in both negative reference and normal samples, and was negative, and was detected in all positive reference, sensitivity reference and gastrointestinal cancer samples, and was positive. The results are shown in Table 16.
TABLE 16
From the above-mentioned results, it was found that the primer set ADHFE1, FAM19A4, and VSTM2B and the probes thereof can be used for gastrointestinal cancer detection.
Example 2
Clinical plasma samples were collected in several, of which: 82 patients with primary gastric cancer, 124 patients with primary colorectal cancer, 108 patients with adenomatous polyp, 75 patients with enteritis, 62 patients with gastritis and 150 healthy human plasma samples. The primer pairs and corresponding probes of example 1 were used to test the clinical samples, the specific steps are shown in example 1, and the test results are shown in tables 17-23. Table 17 shows the results of methylation detection of ADHFE1 gene, Table 18 shows the results of methylation detection of FAM19A4 gene, and Table 19 shows the results of methylation detection of VSTM2B gene; table 20 shows the results of detecting methylation of ADHFE1 gene + FAM19A4 gene; table 21 shows the results of detecting methylation of ADHFE1 gene + VSTM2B gene; table 22 shows the results of methylation detection of FAM19A4 gene + VSTM2B gene; table 23 shows the results of detecting methylation of ADHFE1 gene, FAM19A4 gene, and VSTM2B gene. In tables 17-23, the sensitivity is characterized by the positive rates of gastric cancer, colorectal cancer and adenomatous polyps, and (%) = number of positive samples/number of total detected samples; specificity was characterized by enteritis, gastritis and negative rate of healthy persons, specificity (%) = number of negative samples/number of total test samples.
TABLE 17
Watch 19
TABLE 21
TABLE 22
TABLE 23
From the results in tables 17 to 23, it can be seen that the sensitivity of detecting the methylation level of the ADHFE1 gene alone to gastrointestinal cancer is 73.17%, the sensitivity to colorectal cancer is 73.39%, and the specificity is 98.39% to 99.33%. The sensitivity of detecting the methylation level of the FAM19A4 gene independently to gastric cancer is 80.49%, the sensitivity to colorectal cancer is 88.71%, and the specificity is 96.77% -99.33%. The sensitivity of detecting the methylation level of the VSTM2B gene independently on gastric cancer is 68.29%, the sensitivity on colorectal cancer is 85.48%, and the specificity is 97.33% -99.33%. And when the methylation levels of the ADHFE1 gene, the FAM19A4 gene and the VSTM2B gene are detected, the sensitivity to gastrointestinal cancer is 92.68%, the sensitivity to colorectal cancer is 95.97%, the sensitivity to adenoma polyp is 84.26%, and the specificity is 96.77-99.33%.
From the above, the detection of the methylation levels of the ADHFE1 gene, the FAM19A4 gene and the VSTM2B gene can achieve the sensitivity of more than 80% to gastrointestinal cancer and the specificity of more than 95%, and can provide reference for early screening and diagnosis of gastrointestinal cancer. Compared with the conventional gastrointestinal cancer diagnosis method, the method of the embodiment can be used for screening and diagnosing the gastrointestinal cancer in an early non-invasive way by using the DNA extraction, methylation conversion and QPCR associated technologies.
All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, which is convenient for specific and detailed understanding of the technical solutions of the present invention, but the present invention should not be construed as being limited to 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. It should be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the present patent shall be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.
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Claims (8)
1. A detection primer pair and probe combination of gastrointestinal cancer biomarkers, wherein the biomarkers are ADHFE1 gene, FAM19A4 gene and VSTM2B gene, and the gastrointestinal cancer is diagnosed by detecting the methylation level of a promoter of the biomarkers to a first exon region;
the detection primer pair and the probe combination comprise a detection primer pair, and the detection primer pair comprises the following primer pairs: an ADHFE1 primer pair for detecting the methylation level of the ADHFE1 gene, a FAM19A4 primer pair for detecting the methylation level of the FAM19A4 gene and a VSTM2B primer pair for detecting the methylation level of the VSTM2B gene, wherein the ADHFE1 primer pair is designed from a promoter to a first exon region of the ADHFE1 gene, the FAM19A4 primer pair is designed from the promoter to the first exon region of the FAM19A4 gene, and the VSTM2B primer pair is designed from the promoter to the first exon region of the VSTM2B gene; the nucleotide sequence of the ADHFE1 primer pair is shown as SEQ ID No: 1-2; the nucleotide sequence of the FAM19A4 primer pair is shown as SEQ ID No: 3-4; the nucleotide sequence of the VSTM2B primer pair is shown as SEQ ID No: 5-6;
The detection primer pair and probe combination also comprises a detection probe corresponding to the detection primer pair, and the detection probe corresponding to the detection primer pair is connected with a fluorescent group; the nucleotide sequence of a detection probe corresponding to the ADHFE1 primer pair is shown as SEQ ID No: 7 is shown in the specification; the nucleotide sequence of a detection probe corresponding to the FAM19A4 primer pair is shown as SEQ ID No: 8 is shown in the specification; the nucleotide sequence of a detection probe corresponding to the VSTM2B primer pair is shown as SEQ ID No: shown at 9.
2. The detection primer pair and probe combination according to claim 1, further comprising an internal reference primer pair and an internal reference probe corresponding to the internal reference primer pair, wherein the internal reference primer pair comprises at least one of the following primer pairs:
an ACTB primer pair designed for the ACTB gene, a GAPDH primer pair designed for the GAPDH gene, an ALDOA primer pair designed for the ALDOA gene, and a PGK1 primer pair designed for the PGK1 gene;
the internal reference probe is connected with a fluorescent group different from the detection probe.
3. The detection primer pair and probe combination of claim 2, wherein the ACTB primer pair has a nucleotide sequence as set forth in SEQ ID No: 10-11, wherein the nucleotide sequence of the internal reference probe corresponding to the ACTB primer pair is shown as SEQ ID No: shown at 12.
4. The detection primer pair and probe combination according to any one of claims 1 to 3, wherein the fluorescent group attached to the detection probe is selected from one of FAM, HEX, VIC, CY5, ROX, Texsa Red, JOE and Quasar 705.
5. A kit for gastrointestinal cancer diagnosis, comprising the pair of detection primers according to any one of claims 1 to 4 in combination with a probe.
6. The kit of claim 5, wherein the gastrointestinal cancer is gastric cancer, colorectal cancer, and/or adenomatous polyps.
7. The kit of claim 5 or 6, further comprising at least one of a nucleic acid extraction reagent, a methylation conversion reagent, a quality control reagent, a PCR reaction reagent, and a sequencing reagent.
8. The kit of claim 7, wherein the PCR reagents comprise PCR buffer, dNTP, MgCl 2 And TaqDNA polymerase.
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