CN116640851A - Detection reagent for detecting methylation of colorectal cancer or colorectal adenoma related genes and application thereof - Google Patents
Detection reagent for detecting methylation of colorectal cancer or colorectal adenoma related genes and application thereof Download PDFInfo
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Abstract
The invention discloses a detection reagent for detecting methylation of colorectal cancer or colorectal adenoma related genes and application thereof, wherein the detection reagent is used for detecting methylation of colorectal cancer or colorectal adenoma related genes and is selected from at least one of ADHEE 1 genes and SND1 genes; the detection reagent includes a reagent for detecting methylation of a target region on the ADHEF 1 gene and/or the SND1 gene; the target region for detecting ADHEE 1 gene is selected from the group consisting of the full-length region or a partial region in chr8: 67344553-67344936; the target region for detecting SND1 gene is selected from the group consisting of the full-length region or a partial region in chr7: 128032255-128032597. The detection reagent prepared by the invention applies the methylation detection of ADHEF 1 and SND1 genes to colorectal diagnosis, and can effectively improve the detection rate of adenoma in the advanced stage and the early detection rate of colorectal cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a detection reagent for detecting colorectal cancer or colorectal adenoma related gene methylation and application thereof.
Background
Colorectal cancer (colorectal cancer, CRC) is one of the major cancers threatening the life health of residents, causing a serious social burden, and at the same time, the incidence of colon cancer has increased significantly. Most patients already belong to the middle and late stages at the time of diagnosis.
The occurrence and development of colorectal cancer mostly follow the sequence of 'adenoma-cancer', and the time for the progress from precancerous lesions to cancers generally takes 5-10 years, thereby providing an important time window for early diagnosis and clinical intervention of diseases. Colorectal cancer can be prevented and diagnosed early by screening. Colonoscopy is currently the most common screening method and gold standard. However, colonoscopy is an invasive examination, has the risk of perforation, bleeding, and requires bowel preparation, resulting in poor patient compliance, which is not conducive to its popularization. The fecal gene detection is used as a non-invasive screening technology, and can provide a detection mode which meets personal preference and personalized requirements of patients, thereby increasing compliance of the patients and being more beneficial to popularization and implementation of colorectal cancer screening. The screening and early diagnosis and early treatment guidelines for colorectal cancer in China, 2020, beijing, recommends that multi-target stool FIT-DNA detection can be used for colorectal cancer screening under specific conditions, and has certain screening capability for colorectal cancer and precancerous lesions.
Although colonoscopes have a high diagnostic sensitivity for colorectal cancer, they have limited sensitivity to pre-cancerous lesions. Most post-colonoscopic CRCs are believed to be caused by missed or incompletely resected CRAs, with a significant portion of colorectal lesions missing diagnosis being serrated polyps. Since sessile serrated polyps are typically flat or slightly raised, this makes them more difficult to find by colonoscopy than other polyps. The adenoma detection rate is an important index for measuring colonoscopy quality, and the Chinese colorectal cancer screening and early diagnosis and early treatment guidelines indicate that the adenoma detection rate should be more than 20%, wherein men should be more than 25% and women should be more than 15%. Acceptable colonoscopy is important to reduce the miss rate, and studies have summarized colonoscopy miss rates of 25.7%, 12.9% and 2.1% for <5mm, 5-10 mm and >10mm adenomas, respectively. A2004 study with a virtual colonoscope as a control showed that the colonoscopy omission rate of large adenomas (. Gtoreq.10 mm) was 6% -12%. At present, research shows that the diagnosis rate of cologard to colorectal advanced adenoma is 42%, the diagnosis rate of FIT is 24%, and the detection rate of human fecal SDC2 gene methylation detection kit of Kang Liming company on the market in China is 38%.
Therefore, there is a need to develop a product that can be detected noninvasively and has high detection sensitivity and specificity for colorectal adenoma and early colorectal carcinoma.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. To this end, the invention proposes a detection reagent for detecting methylation of colorectal cancer or colorectal adenoma-associated genes.
The invention also provides a kit containing the detection reagent.
The invention also provides application of the detection reagent and the kit.
In a first aspect of the present invention, a detection reagent for detecting methylation of colorectal cancer or colorectal adenoma-associated genes selected from at least one of ADHFE1 genes and SND1 genes is presented; the detection reagent includes a reagent for detecting methylation of a target region on the ADHEF 1 gene and/or the SND1 gene; the target region for detecting ADHEE 1 gene is selected from the group consisting of the full-length region or a partial region in chr8: 67344553-67344936; the target region for detecting SND1 gene is selected from the group consisting of the full-length region or a partial region in chr7: 128032255-128032597.
In some embodiments of the invention, the reagents for detecting methylation of a target region on the ADHE 1 gene include a forward primer having a sequence shown as SEQ ID NO. 3 and a reverse primer having a sequence shown as SEQ ID NO. 4, a forward primer having a sequence shown as SEQ ID NO. 6 and a reverse primer having a sequence shown as SEQ ID NO. 7, or a forward primer having a sequence shown as SEQ ID NO. 9 and a reverse primer having a sequence shown as SEQ ID NO. 10.
In some embodiments of the invention, the reagents for detecting methylation of a target region on the ADHEF 1 gene include a forward primer having the sequence shown in SEQ ID NO. 6 and a reverse primer having the sequence shown in SEQ ID NO. 7.
In some embodiments of the invention, the reagents for detecting methylation of a target region on an SND1 gene include a forward primer having the sequence shown in SEQ ID NO. 12 and a reverse primer having the sequence shown in SEQ ID NO. 13, or a forward primer having the sequence shown in SEQ ID NO. 15 and a reverse primer having the sequence shown in SEQ ID NO. 16.
In some embodiments of the invention, the reagents for detecting methylation of a target region on an SND1 gene include a forward primer having the sequence shown in SEQ ID NO. 12 and a reverse primer having the sequence shown in SEQ ID NO. 13.
In some embodiments of the invention, the detection reagent further comprises a fluorescent probe sequence having a nucleotide sequence as shown in SEQ ID NO. 5, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 14 and/or SEQ ID NO. 17.
In some embodiments of the invention, the fluorescent probe sequence shown in SEQ ID NO. 5 is a fluorescent sequence that matches the forward primer shown in SEQ ID NO. 3 and the reverse primer shown in SEQ ID NO. 4; the fluorescent probe sequence shown in SEQ ID NO. 8 is a fluorescent sequence matched with a forward primer shown in SEQ ID NO. 6 and a reverse primer shown in SEQ ID NO. 7; the fluorescent probe sequence shown in SEQ ID NO. 11 is a fluorescent sequence matched with a forward primer shown in SEQ ID NO. 9 and a reverse primer shown in SEQ ID NO. 10; the fluorescent probe sequence shown in SEQ ID NO. 14 is a fluorescent sequence matched with a forward primer shown in SEQ ID NO. 12 and a reverse primer shown in SEQ ID NO. 13; the fluorescent probe sequence shown in SEQ ID NO. 17 is a fluorescent sequence matched with a forward primer shown in SEQ ID NO. 15 and a reverse primer shown in SEQ ID NO. 16.
In some embodiments of the invention, the fluorescent probe sequence has a fluorescent group at the 5 'end and a quenching group at the 3' end; the fluorescent group is VIC, ROX, FAM, cy, HEX, TET, JOE, NED or TexasRed; the quenching group is TAMRA, BHQ, MGB or Dabcyl.
In some embodiments of the invention, the detection reagent further comprises an internal reference primer pair and an internal reference probe, wherein the nucleotide sequences of the internal reference primer pair are respectively shown as SEQ ID NO. 18 and SEQ ID NO. 19, and the nucleotide sequence of the internal reference probe is shown as SEQ ID NO. 20.
In some embodiments of the invention, the detection reagent is used to detect sequences of ADHEE 1 gene and SND1 gene modified by the conversion reagent.
The conversion reagent is a reagent that converts unmethylated cytosine in DNA to uracil while leaving 5-MeC substantially unaffected.
In some embodiments of the invention, the conversion reagent comprises one or more of a hydrazine salt, a bisulfite (e.g., sodium bisulfite, etc.), a bisulfite (e.g., sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, etc.), or a compound that can produce a hydrazine salt, a bisulfite under appropriate reaction conditions.
In some embodiments of the invention, the conversion reagent is a bisulfite.
In some embodiments of the invention, the bisulfite conversion in the examples of the invention includes, but is not limited to, conversion using commercial kits, conversion using self-made or purchased bisulfite.
In some embodiments of the invention, the test sample for which the test reagent is directed includes fecal cast-off cell DNA, colorectal cancer tissue cell DNA, colorectal cancer cell DNA, and the like.
In some embodiments of the invention, the detection reagent is used to detect a bisulfite modified sequence.
In a second aspect of the invention there is provided a kit comprising a detection reagent as described above for detecting methylation of a colorectal cancer or colorectal adenoma associated gene.
In some embodiments of the invention, the testThe kit also comprises a PCR reaction liquid, wherein the PCR reaction liquid comprises Taq HS DNA polymerase and Taq HS Buffer (Mg 2+ plus), dNTP Mix, and betaine.
In some embodiments of the invention, the kit further comprises a positive control that is colorectal cancer positive cell line HCT116 cell DNA and a negative control that is intestinal epithelial cell HIEC-6 cell DNA.
In a third aspect of the invention there is provided the use of the above detection reagent or kit in the manufacture of a product for the diagnosis or co-diagnosis of colorectal cancer or colorectal adenoma.
In some embodiments of the invention, the method of using the colorectal cancer or colorectal adenoma diagnostic or auxiliary diagnostic product comprises the steps of:
s1, carrying out bisulphite or hydrazine salt treatment on a sample to be detected to obtain a modified sample to be detected;
s2, detecting methylation of ADHEE 1 genes and/or SND1 genes of the sample to be tested modified in the step S1 by using the colorectal cancer or colorectal adenoma diagnosis or auxiliary diagnosis products.
In some embodiments of the invention, the detection in step S2 is performed using real-time fluorescent quantitative PCR.
In some embodiments of the invention, the amplification procedure of the real-time fluorescent quantitative PCR is:
92-97℃ 8-12min
92-97℃ 13-17s 40-50cycles
56-64 ℃ (collecting fluorescence) 0.5-1.5min 40-50cycles
16-22℃ 1-3min。
In some embodiments of the invention, the real-time fluorescent quantitative methylation specific polymerase chain reaction amplification procedure is;
95℃ 10min
95℃ 15s 45cycles
60 ℃ (collect fluorescence) 1min 45cycles
20℃ 2min。
In some embodiments of the invention, when methylation is detected using the ADHEF 1 gene, the criterion is positive when CT < 37.96 for detection of the ADHEF 1 gene; and the ADHE 1 gene is negative when CT detected by the ADHE 1 gene is more than or equal to 37.96.
In some embodiments of the invention, when methylation is detected using the SND1 gene, the criterion is positive when CT for SND1 gene detection is less than 37.40; and the detection of the SND1 gene is negative when the CT is more than or equal to 37.40.
In some embodiments of the invention, when methylation is detected using the ADHEF 1 gene and the SND1 gene, the criterion is positive when CT for detection of the ADHEF 1 gene is < 37.96 and CT for detection of the SND1 gene is < 37.40; the test result is positive when CT detected by ADHE 1 gene is more than or equal to 37.96 and CT detected by SND1 gene is less than 37.40; the test result is positive when CT of ADHE 1 gene is less than 37.96 and CT of SND1 gene is more than or equal to 37.40; the test is negative when CT detected by ADHE 1 gene is larger than or equal to 37.96 and CT detected by SND1 gene is larger than or equal to 37.40.
According to an embodiment of the invention, at least the following advantages are achieved: the detection reagent for detecting colorectal cancer or colorectal adenoma related gene methylation, which is prepared by the scheme of the invention, applies ADHEE 1 and SND1 gene methylation detection to colorectal diagnosis, and has high sensitivity and high specificity, thereby effectively improving the detection rate of advanced adenoma and the early detection rate of colorectal cancer; meanwhile, the fecal sample is used as a detection sample, so that the sampling is convenient and noninvasive, and the compliance of patients is greatly improved. The matched fecal DNA preservation solution can be preserved for 7 days at normal temperature, and is convenient for sample preservation, so that the coverage crowd for colorectal cancer screening is enlarged, the incidence rate of colorectal cancer in China is reduced, and the health of the whole people is guaranteed.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a graph of ADHEE 1-combination 1-expanded HCT116 cell DNA according to example 1 of the invention;
FIG. 2 is a graph of ADHEE 1-combination 1-amplified HIEC-6 negative cell DNA in example 1 of the present invention;
FIG. 3 is a graph of ADHEE 1-combination 2-expanded HCT116 cell DNA in example 1 of the invention;
FIG. 4 is a graph of ADHEE 1-combination 2-amplified HIEC-6 negative cell DNA in example 1 of the present invention;
FIG. 5 is a graph of ADHEE 1-combination 3-amplified HCT116 cell DNA for example 1 of the invention;
FIG. 6 is a graph of ADHEE 1-combination 3-amplified HIEC-6 negative cell DNA in example 1 of the present invention;
FIG. 7 is a graph of SND 1-combinatorial 1-amplified HCT116 cell DNA in example 1 of the invention;
FIG. 8 is a graph showing DNA of SND 1-combinatorial 1-amplified HIEC-6 negative cells in example 1 of the present invention;
FIG. 9 is a graph of SND 1-combinatorial 2-amplified HCT116 cell DNA of example 1 of the invention;
FIG. 10 is a DNA graph of SND 1-combinatorial 2-amplified HIEC-6 negative cells in example 1 of the present invention.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1A detection reagent for detecting methylation of colorectal cancer or colorectal adenoma-associated Gene
1. Methylation region screening
And (3) carrying out biological information analysis and mining on methylation databases and gene expression data of colorectal cancer in a GEO (gene expression omnibus) database, finding different methylation sites in paired 104 pairs of tumor tissues and control samples, and selecting a site with delta beta of more than 0.4 for Boruta feature screening. The screened loci are further subjected to gene cg locus correlation analysis at the GEO adenoma data, the loci with high-level adenoma AUC >0.85 and low-level adenoma AUC >0.65 are selected, the loci with the correlation >0.9 are subjected to region size screening (> 80 bp), and finally, the TCGA database, the GEO adenoma database and the GEO database are respectively verified, and finally, 11 regions are screened. Sanger sequencing after bisulfite treatment was used to verify the methylation sites of colorectal cancer cell line HCT116 and 10 colorectal cancer positive samples and 10 normal population samples by Sanger sequencing, screening to find that CpG sites in the ADHE 1 gene located in region chr8:67344553-67344936 and the SND1 gene located in region chr7:128032255-128032597 are highly methylated in colorectal cancer positive cell line HCT116 and 10 colorectal cancer positive samples, whereas in normal population, the CpG sites in these regions are unmethylated or hypomethylated.
Region chr8:67344553-67344936 original sequence:
TGCTTTAGTCTGAGTGTAGAACTCAAATTTCTGCAGTATTTAATTTCTACAGTTTCCTTCTGAGCAGAAAAAGAATACCATAAAAATATTTGCAAACATTATATATGGTGATCTAGGGTCACGTCTCCAAGTTCTAAGCTTCTGTGGCTCCACGCACACAGGTTCCCTAATGTTTACTATGCATCTGTCACTGTTCTGTTCTATTAGATTGCCTTAACTATAGGCTTGTCCTAACGTAACCATCCAAACCTAAGGAAGGAGAAACTTAAGTGACTCTGTGAACTTAGTGAAGTGACCAGATTTAGGAAGGCTAAAACCCAGAGGAAATAAAATGGAAAACTTGAAGGTGACAAATTAAAGTGGAAAAATAGATTAATTTTTTTA(SEQ ID NO:1)。
region chr7:128032255-128032597 original sequence:
TCCGGCCGCCCGTCTTCCTCTTTCCTCCTTCCTTCCTCCTCTCTTCCTCTCTCCCCCTCCCCCCTCCCCCCTCCCCGGGCCGCTTCCTCCCCGCCCCCTCTAGACGAGCGGAGCGGGGCCAGGGCGAGCGGGCGAGCGAGCCGGAGTGAGCGTCAAGTGAGGGGCCGCGCGCAAGTCGCAGGCGTTCGCAGCTATTTTGGGCCGGTCGGAGGGAGCAGTTGGGTGGCCGCTGATAGGCTGGAGCGGCGCGGGGCTGCGGCGGGAGAGCCCGCGCCGGCCCCCGCCCTCCGGCCCGCACCCGGGGCCCCCTGGCTCTGAAGAGCGGGGCTCAGGGGCCCGGCGG(SEQ ID NO:2)。
2. design and selection of primer probe sequences
(1) Design of primer probe sequence
3 pairs of primer probes are designed for ADHEE 1 gene region chr8:67344553-67344936, 2 pairs of primer probes are designed for SND1 gene region chr7:128032255-128032597, and specific sequences are shown in Table 1.
TABLE 1 ADHEE 1 Gene and SND1 Gene and internal control Gene primer sequences
(2) Screening of primer probe sequences
The primer probes were screened (template bisulfite converted) using colorectal cancer positive cell line HCT116 cell DNA (cell line purchased from Kunming cell Bank of China academy of sciences; bisulfite Sanger sequencing was methylation positive) and intestinal epithelial cell HIEC-6 cell DNA (cell line purchased from Shanghai Qingqi organism, bisulfite Sanger sequencing was methylation negative). The amplification was performed using the primer probe combinations shown in Table 1, the ADHEE 1 gene amplification system was shown in Table 2, the SND1 gene amplification system was shown in Table 3, and the amplification procedure was shown in Table 4.
TABLE 2 screening and amplification reaction System for ADHOE 1 Gene primer probes
| Reagent name | Additive amount (μL) |
| ADHFE1-1-F1/ADHFE1-2-F1/ADHFE1-3-F1(10uM) | 0.5 |
| ADHFE1-1-R1/ADHFE1-2-R1/ADHFE1-3-R1(10uM) | 0.5 |
| ADHFE1-1-P1/ADHFE1-2-P1/ADHFE1-3-P1(10uM) | 0.5 |
| PCR premixing reaction liquid | 15 |
| Sterile deionized water | 3.5 |
| Template after sulfite conversion | 10 |
| Totalizing | 30 |
TABLE 3 SND1 Gene primer probe screening and amplification reaction System
TABLE 4 Table 4
As shown in the detection results of FIGS. 1-10, it can be seen from the figures that the ADHEE 1 gene primer probe set 2 has an amplification curve for HCT116 cell DNA, the CT value is smaller, the plateau fluorescence value (delta Rn) is higher, and the HIEC-6 negative cell DNA has no amplification curve. Thus ADHEE 1 gene selects primer probe group 2 as primer probe for methylation detection. Compared with the primer probe set 2, the SND1 gene primer probe set 1 has an amplification curve of HCT116 cell DNA, a smaller CT value, a higher plateau fluorescence value (delta Rn) and no amplification curve of HIEC-6 negative cell DNA. Thus, SND1 primer probe set 1 was selected as a primer probe for methylation detection.
Example 2 establishment of fluorescent PCR detection System
1. Fluorescent PCR system establishment for ADHEE 1 gene methylation detection
The embodiment prepares an ADHEF 1 methylation detection kit, which comprises a primer probe combination (ADHEF 1 gene primer probe group 2), PCR premix reaction liquid, sterile deionized water, negative control and positive control.
Wherein the PCR premix reaction solution consists of Taq HS DNA polymerase (Novain), 10×Taq HS Buffer (Mg 2+ plus), dNTP Mix, and betaine (final concentration 0.75M). The negative control is intestinal epithelial cell HIEC-6 cell DNA (cell strain is purchased from Shanghai green flag organism, and the extraction kit is QIAGEN-DNeasy Blood)&Tissue Kit), positive control was colorectal cancer positive cell strain HCT116 cell DNA (cell strain is purchased from Kunming cell bank of China academy of sciences, and the extraction Kit is QIAGEN-DNeasy Blood)&Tissue Kit)。
The ADHEF 1 gene amplification reaction system is shown in Table 5, and the ADHEF 1 gene amplification procedure is shown in Table 6.
TABLE 5 ADHOE 1 Gene amplification reaction System
| Reagent name | Additive amount (μL) |
| ADHFE1-2-F1(10uM) | 0.5 |
| ADHFE1-2-R1(10uM) | 0.5 |
| ADHFE1-2-P1(10uM) | 0.5 |
| bis-ACT-F1(10uM) | 0.5 |
| bis-ACT-R1(10uM) | 0.5 |
| bis-ACT-P1(10uM) | 0.4 |
| 10×Taq HS Buffer(Mg 2+ plus) | 3 |
| dNTP Mix(10mM each) | 0.6 |
| Taq HS DNA polymerase(5U/μl) | 0.6 |
| 5M betaine | 4.5 |
| Sterile deionized water | 8.4 |
| Template after sulfite conversion | 10 |
| Totalizing | 30 |
TABLE 6
2. Establishment of SND1 gene methylation detection PCR system
The SND1 methylation detection kit comprises a primer probe combination (SND 1 gene primer probe set 1), PCR premix reaction liquid, sterile deionized water, negative control and positive control.
Wherein the PCR premix reaction solution consists of Taq HS DNA polymerase (Novain), 10×Taq HS Buffer (Mg 2+ plus), dNTP Mix, and betaine (final concentration 0.75M). The negative control is intestinal epithelial cell HIEC-6 cell DNA (cell strain is purchased from Shanghai green flag organism, and the extraction kit is QIAGEN-DNeasy Blood)&Tissue Kit), positive control was colorectal cancer positive cell strain HCT116 cell DNA (cell strain is purchased from Kunming cell bank of China academy of sciences, and the extraction Kit is QIAGEN-DNeasy Blood)&Tissue Kit)。
The SND1 gene amplification reaction system is shown in Table 7, and the SND1 gene amplification procedure is shown in Table 8.
TABLE 7SND1 Gene amplification reaction System
| Reagent name | Additive amount (μL) |
| SND1-1-F1(10uM) | 0.5 |
| SND1-1-R1(10uM) | 0.5 |
| SND1-1-P1(10uM) | 0.5 |
| bis-ACT-F1(10uM) | 0.5 |
| bis-ACT-R1(10uM) | 0.5 |
| bis-ACT-P1(10uM) | 0.4 |
| 10×Taq HS Buffer(Mg2+plus) | 3 |
| dNTP Mix(10mM each) | 0.6 |
| Taq HS DNA polymerase(5U/μl) | 0.6 |
| 5M betaine | 4.5 |
| Sterile deionized water | 8.4 |
| Template after sulfite conversion | 10 |
| Totalizing | 30 |
TABLE 8
3. Establishment of triple fluorescence PCR (polymerase chain reaction) system for detecting methylation of ADHEF 1 and SND1 genes
The embodiment prepares an ADHEF 1 and SND1 gene methylation detection kit, which comprises a primer probe combination (SND 1 gene primer probe set 1 and ADHEF 1 gene primer probe set 2), PCR premix reaction liquid, sterile deionized water, negative control and positive control.
Wherein the PCR premix reaction solution consists of Taq HS DNA polymerase (Novain), 10×Taq HS Buffer (Mg 2+ plus), dNTP Mix, and betaine (final concentration 0.75M). The negative control is intestinal epithelial cell HIEC-6 cell DNA (cell strain is purchased from Shanghai green flag organism, and the extraction kit is QIAGEN-DNeasy Blood)&Tissue Kit), positive control was colorectal cancer positive cell strain HCT116 cell DNA (cell strain is purchased from Kunming cell bank of China academy of sciences, and the extraction Kit is QIAGEN-DNeasy Blood)&Tissue Kit). The specific reaction system is shown in Table 9, and the detection procedure is shown in Table 10.
TABLE 9 triple amplification reaction System
| Reagent name | 1 person |
| ADHFE1-2-F1 | 0.16uM |
| ADHFE1-2-R1 | 0.16uM |
| ADHFE1-2-P1 | 0.16uM |
| SND1-1-F1 | 0.16uM |
| SND1-1-R1 | 0.16uM |
| SND1-1-P1 | 0.16uM |
| bis-ACT-F1 | 0.16uM |
| bis-ACT-R1 | 0.16uM |
| bis-ACT-P1 | 0.13uM |
| PCR premixing reaction liquid | 15 |
| Sterile deionized water | 2.1 |
| Template after sulfite conversion | 10 |
| Totalizing | 30 |
Table 10
The results of amplification (bisulphite converted templates) of colorectal cancer positive cell line HCT116 cell DNA, intestinal epithelial cell HIEC-6 negative cell DNA, and 5 colorectal cancer positive samples and 5 normal population samples are shown in Table 3. As can be seen from table 3, the triple amplification reaction system of the ADHFE1 and SND1 gene methylation detection kit can amplify positive samples of colorectal cancer positive cell lines HCT116 cell DNA and 5 colorectal cancer positive samples, which are positive samples of ADHFE1 and SND1 gene hypermethylation, better, and have a front CT value; whereas samples without methylation or hypomethylation of the ADHEF 1 and SND1 genes had no amplification or a later CT value for HIEC-6 negative cell DNA and 5 samples from normal population.
TABLE 11 triple amplification reaction System amplification results
Note that: KY001-KY005 is a colorectal cancer positive sample, and KY006-KY0010 is a colorectal cancer negative sample.
Example 3 application of ADHEE 1 and SND1 Gene methylation detection kit in colorectal cancer and adenoma diagnosis
This example tests the ADHEE 1 gene methylation detection kit, SND1 gene methylation detection kit, and the application of ADHEE 1 and SND1 gene methylation detection kit prepared in example 2 in colorectal cancer and adenoma diagnosis.
The specific detection method A comprises the following steps:
1. fecal sample collection
And collecting a proper amount of sample (about 5 g) to 15mL of fecal specimen collection and preservation tube, and enabling the liquid level to be positioned between the lowest scale and the highest scale (20-25 mL) of the tube wall mark.
2. Sample extraction
The method adopts a nucleic acid extraction or purification kit of human and future biotechnology (Changsha) limited company for extraction, and comprises the following specific operation steps:
1. sample pretreatment: sufficiently and uniformly vibrating and mixing the received sample; standing at room temperature overnight; or placing the sample at 60deg.C
Incubation was performed for 1 hour, shaking and mixing once every 20 min.
2. The extraction process comprises the following steps:
1) Taking out the pretreated sample, and centrifuging at 3000rpm for 15min; transferring 1mL of supernatant into a 2mL centrifuge tube (when impurities in the supernatant are more, repeatedly centrifuging to obtain the supernatant once);
2) Adding 400 mu L of lysate and 60 mu L of magnetic beads (uniformly mixed by shaking before use), uniformly mixing by shaking, and placing in a uniformly mixing instrument for incubation at room temperature for 10min;
3) After incubation is completed, performing instantaneous centrifugation, placing the centrifuge tube on a magnetic rack for adsorption for 5min until the centrifuge tube is clarified, and removing supernatant;
4) Adding 600 mu L of washing buffer solution 1, fully oscillating and uniformly mixing, standing for 1min, performing instantaneous centrifugation, placing on a magnetic rack for adsorption until the supernatant is clear, and sucking off the supernatant;
5) Adding 500 mu L of washing buffer solution 2, fully oscillating and uniformly mixing, standing for 1min, performing instantaneous centrifugation, placing on a magnetic rack for adsorption until the mixture is clear, and sucking and discarding the supernatant;
6) Repeating the step 5, and then performing instantaneous centrifugation to suck residual liquid;
7) Standing at room temperature for 5-10 min, adding 60 μl of eluent, shaking thoroughly, mixing well, incubating at 60deg.C for 5min, mixing well for 2-3min, and taking care not to shake the magnetic beads onto the tube cover. And (3) carrying out instantaneous centrifugation, placing the obtained product on a magnetic rack, and sucking the supernatant to obtain a DNA solution.
3. Bisulphite conversion and purification
The extracted DNA was bisulphite converted and purified using the bisulphite conversion kit EZ-96DNA methyl-GoldTM MagPrep (available from ZYMO RESEARCH) as follows:
1. 20. Mu.L of the sample to be converted (less than 20. Mu.L of the sample is made up with sterile deionized water) is added to a 0.2mL octant, then 130. Mu. LLCT Conversion Reagent is added, the 8-octant cap is capped and marked, mixed with careful shaking, and centrifuged instantaneously.
2. The 8-way tube was placed in a PCR instrument and transformed according to the procedure of table 12:
table 12
| Step (a) | Temperature (temperature) | Time |
| 1 | 98℃ | For 10 minutes |
| 2 | 64℃ | 3 hours |
| 3 | 4℃ | ∞ |
3. Prepare 1.5mL centrifuge tube (labeled by sample number), add 600 μ L M-Binding Buffer and 10 μ L M-Binding Beads (shake well before adding), take out 8-tube from PCR instrument after the previous reaction, transfer the liquid in tube to corresponding 1.5mL centrifuge tube, place on vortex instrument for 30s, stand for 5min at room temperature.
4. The centrifuge tube is centrifuged instantaneously, and then placed on a magnetic rack to adsorb for 5min or until the solution is clear, and the supernatant is removed.
5. The sample tube was removed, 400. Mu. L M-Wash Buffer (gently shaking up before use), vortexing for 30s, instantaneous centrifugation, standing on a magnetic rack for 3min or until the solution was clear, and removing the supernatant.
6. Taking off the sample tube, adding 200 mu L M-DesuLphontion Buffer, vortexing for 30s, standing at room temperature for 15min, centrifuging instantaneously, standing on a magnetic rack for adsorption for 3min or until the solution is clear, and removing the supernatant.
7. The sample tube was removed, 400. Mu. L M-Wash Buffer (gently shaking up before use), vortexing for 30s, instantaneous centrifugation, standing on a magnetic rack for 3min or until the solution was clear, and removing the supernatant.
8. And 7, repeating the step 7. (note: the supernatant should be removed again after the suction, the residual liquid is removed)
9. The residual suction sample tube was uncapped and placed on a metal bath for 10-15min at 55deg.C to remove residual liquid and dry magnetic beads (note observation to prevent excessive drying time and cracking of magnetic beads).
10. Adding 25 mu L M-filtration Buffer into a sample tube, swirling to make the magnetic beads suspended, placing the magnetic beads into a metal bath again at 55 ℃ for 4min, standing for instantaneous centrifugation, placing the magnetic beads on a magnetic rack for adsorption for 2min until the solution is clear, and sucking the supernatant into a new 1.5mL centrifuge tube for subsequent detection as soon as possible or freezing the supernatant at-20 ℃.
4. Fluorescent quantitative PCR
10 mu L of DNA subjected to bisulfite conversion and purification is subjected to fluorescence quantitative PCR by using the ADHEE 1 gene methylation detection kit, the SND1 gene methylation detection kit and the ADHEE 1 and SND1 gene methylation detection kit prepared in example 2, and the components and the amounts of a detection system are shown in Table 13.
TABLE 13 detection System
The target genes ADHEE 1 and SND1 respectively correspond to FAM and CY5 channels; the internal standard ACTB selected the VIC/HEX channel and the PCR procedure is shown in Table 14.
TABLE 14PCR reaction procedure
5. Interpretation of results
And after the reaction is finished, automatically storing the result, and analyzing the result by using analysis software matched with an instrument. Baseline setting: automatic Baseline or Baseline Start is 3-10, baseline End is 15-25; the threshold value may be appropriately adjusted according to the curve form.
ADHFE1 gene haplotype read results:
positive when CT detected by ADHEE 1 gene is less than 37.96; and the ADHE 1 gene is negative when CT detected by the ADHE 1 gene is more than or equal to 37.96.
SND1 gene haplotype line interpretation results:
positive when the CT detected by SND1 gene is less than 37.40; and the detection of the SND1 gene is negative when the CT is more than or equal to 37.40.
Multiple system interpretation results:
the result determination is shown in table 15, and the positive determination value is: when the internal standard gene ACTB detection Ct value is less than 38, the ADHEE 1 detection Ct value is less than 37.96 or the SND1 detection Ct value is less than 37.40, and the detection result is positive; when the internal standard gene ACTB detection Ct value is smaller than 38, the ADHEE 1 detection Ct value is larger than or equal to 37.96 or no Ct value, the SND1 detection Ct value is larger than or equal to 37.4 or no Ct value, and the detection result is judged to be negative.
TABLE 15 determination of results
Detection of colorectal cancer and adenoma by A ADHEF 1 and SND1 gene methylation detection kit
115 clinical stool samples, including 30 colorectal cancer samples, were tested using the ADHFE1 and SND1 gene methylation detection kit prepared in example 2; adenoma samples 20; colon polyps, colitis, hemorrhoids and other healthy human samples 65. The specific detection results are shown in Table 16. The detection result shows that the sensitivity of the kit to colorectal cancer is 93.3%, the specificity is 92.3%, and the detection rate to adenoma is 60.0%.
TABLE 16 comparison of fecal sample clinical test results and kit test results
Detection condition of B SND1 gene methylation detection kit in colorectal cancer and adenoma
115 clinical stool samples, including 30 colorectal cancer samples, were tested using the SND1 gene methylation test kit prepared in example 2; adenoma samples 20; colon polyps, colitis, hemorrhoids and other healthy human samples 65. The specific detection results are shown in Table 17. The detection result shows that the sensitivity of the kit to colorectal cancer is 90.0%, the specificity is 92.3%, and the detection rate to adenoma is 60.0%.
Table 17 comparison of SND1 Gene detection results with kit detection results
Detection of colorectal cancer and adenoma by C ADHEF 1 gene methylation detection kit
115 clinical fecal samples, including 30 colorectal cancer samples, were tested using the ADHFE1 gene methylation test kit prepared in example 2; adenoma samples 20; colon polyps, colitis, hemorrhoids and other healthy human samples 65. The specific detection results are shown in Table 18. The detection result shows that the sensitivity of the kit to colorectal cancer is 83.3%, the specificity is 95.4%, and the detection rate to adenoma is 55.0%.
Table 18 comparison of ADHEF 1 Gene detection results with kit detection results
Example 4 sensitivity test
Bisulfite Sanger sequencing was verified to be 100% methylated HCT116 cell DNA and 0% methylated HIEC-6 cell DNA diluted to 10 ng/. Mu.L and then diluted in different ratios to different methylation ratio references, and the specific formulation is shown in Table 19.
TABLE 19 formulation of different methylation ratio references
25ng of the sample was subjected to bisulfite conversion according to example 3, 25. Mu.L of the sample was eluted, and 10. Mu.L (corresponding to 10ng of DNA before conversion) of the sample was subjected to fluorescent PCR using the ADHEE 1 and SND1 gene methylation detection kit prepared in example 2. Specific detection results are shown in the table, and as can be seen from the table 20, the kit can accurately detect 1% of methylation of the target gene under 10ng of background DNA.
TABLE 20 detection CT values for different methylation Rate references
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A detection reagent for detecting methylation of colorectal cancer or colorectal adenoma-associated genes, characterized in that the detection colorectal cancer or colorectal adenoma-associated genes are selected from at least one of ADHFE1 genes and SND1 genes; the detection reagent includes a reagent for detecting methylation of a target region on the ADHEF 1 gene and/or the SND1 gene; the target region for detecting ADHEE 1 gene is selected from the group consisting of the full-length region or a partial region in chr8: 67344553-67344936; the target region for detecting SND1 gene is selected from the group consisting of the full-length region or a partial region in chr7: 128032255-128032597.
2. The detection reagent according to claim 1, wherein the reagent for detecting methylation of a target region on the ADHE 1 gene comprises a forward primer having a sequence shown in SEQ ID NO. 3 and a reverse primer having a sequence shown in SEQ ID NO. 4, a forward primer having a sequence shown in SEQ ID NO. 6 and a reverse primer having a sequence shown in SEQ ID NO. 7, or a forward primer having a sequence shown in SEQ ID NO. 9 and a reverse primer having a sequence shown in SEQ ID NO. 10.
3. The reagent according to claim 1, wherein the reagent for detecting methylation of a target region on an SND1 gene comprises a forward primer having a sequence shown in SEQ ID NO. 12 and a reverse primer having a sequence shown in SEQ ID NO. 13, or a forward primer having a sequence shown in SEQ ID NO. 15 and a reverse primer having a sequence shown in SEQ ID NO. 16.
4. The detection reagent according to claim 1, wherein the detection reagent further comprises a fluorescent probe sequence represented by nucleotide sequences SEQ ID NO. 5, SEQ ID NO. 8, SEQ ID NO. 11, SEQ ID NO. 14 and/or SEQ ID NO. 17; preferably, the fluorescent probe sequence has a fluorescent group at the 5 'end and a quenching group at the 3' end; the fluorescent group is VIC, ROX, FAM, cy, HEX, TET, JOE, NED or TexasRed; the quenching group is TAMRA, BHQ, MGB or Dabcyl.
5. The detection reagent according to claim 1, further comprising an internal reference primer pair and an internal reference probe, wherein the nucleotide sequences of the internal reference primer pair are shown in SEQ ID NO. 18 and SEQ ID NO. 19, respectively, and the nucleotide sequence of the internal reference probe is shown in SEQ ID NO. 20.
6. The test agent according to claim 1, wherein the test sample of the test agent comprises fecal exfoliative cell DNA, colorectal cancer tissue cell DNA, and colorectal cancer cell DNA.
7. A kit comprising a detection reagent according to any one of claims 1 to 6 for detecting methylation of a colorectal cancer or colorectal adenoma-associated gene.
8. The kit of claim 7, further comprising a positive control that is colorectal cancer positive cell line HCT116 cell DNA and a negative control that is intestinal epithelial cell HIEC-6 cell DNA; and/or the kit also comprises a PCR reaction liquid, wherein the PCR reaction liquid comprises Taq HSDNA polymerase and Taq HS Buffer (Mg 2+ plus), dNTP Mix, and betaine.
9. Use of the detection reagent of any one of claims 1-6 or the kit of claim 7 or 8 for the preparation of a product for the diagnosis or auxiliary diagnosis of colorectal cancer or colorectal adenoma.
10. Use according to claim 9, characterized in that the method of use of the colorectal cancer or colorectal adenoma diagnostic or auxiliary diagnostic product comprises the steps of:
s1, carrying out bisulphite or hydrazine salt treatment on a sample to be detected to obtain a modified sample to be detected;
s2, performing methylation detection of ADHE 1 genes and/or SND1 genes on the sample to be tested modified in the step S1 by using the colorectal cancer or colorectal adenoma diagnosis or auxiliary diagnosis product as claimed in claim 9.
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