CN115838799A - Application of reagent for detecting gene methylation in preparation of product for diagnosing colorectal cancer - Google Patents
Application of reagent for detecting gene methylation in preparation of product for diagnosing colorectal cancer Download PDFInfo
- Publication number
- CN115838799A CN115838799A CN202210799765.0A CN202210799765A CN115838799A CN 115838799 A CN115838799 A CN 115838799A CN 202210799765 A CN202210799765 A CN 202210799765A CN 115838799 A CN115838799 A CN 115838799A
- Authority
- CN
- China
- Prior art keywords
- primer pair
- region
- detecting
- methylation
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention relates to an application of a reagent for detecting the methylation level of a CpG island region of an AGRN gene and a CpG island region of an MSC gene in the preparation of a product for diagnosing colorectal cancer. The research of researchers of the invention finds that the detection sensitivity and specificity of colorectal cancer can be improved by detecting the methylation levels of the CpG island region of the AGRN gene and the CpG island region of the MSC gene, so that the early detection rate of colorectal cancer is improved, the treatment time is strived for patients, and the detection process causes less pain to the patients.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of a reagent for detecting gene methylation in preparation of a product for diagnosing colorectal cancer.
Background
Worldwide, colorectal cancer is the third most common malignancy, which ranks second among cancer-related death statistics. According to statistics, about 190 million new cases occur globally in 2020, and although colorectal cancer progresses slowly and takes as long as 8-10 years to develop from an adenoma to malignant canceration, the early screening and diagnosis effect of colorectal cancer is not ideal, most patients are in the middle and late stage of cancer at the first diagnosis, and the optimal treatment time is missed.
Colorectal screening using fecal occult blood testing and intestinal endoscopy has been reported to be effective in reducing colorectal cancer mortality. However, whether the enteroscopy is directly performed or the fecal occult blood test is performed first and then the enteroscopy is performed again, the invasive operation is required, the pain degree of the patient is high, and the compliance is poor. However, no detection means with good patient compliance and high sensitivity exists at present.
Disclosure of Invention
Based on the detection result, the invention provides an application of a reagent for detecting the methylation level of the CpG island region of the AGRN gene and the CpG island region of the MSC gene in the preparation of a product for diagnosing colorectal cancer. In addition, the nucleic acid product and the kit for diagnosing the colorectal cancer can realize high-sensitivity diagnosis of the colorectal cancer and have small wound to patients.
In one example, the reagent is capable of detecting the methylation level of the full length or partial region of the region Chr1:1033941-1033829 and the region Chr8:71843999-71843823, using GRCh.38p14 as the reference genome.
In one embodiment, the reagent is capable of detecting the methylation level of a full length or partial region of at least one of the regions Chr1:1033914-1033829, chr1:1033937-1033844 and Chr1:1033941-1033844, and the methylation level of a full length or partial region of at least one of the regions Chr8:71843952-71843823, chr8:71843970-71843842 and Chr8: 71843999-71390845.
A nucleic acid product for diagnosing colorectal cancer, the nucleic acid product being capable of detecting the methylation level of a CpG island region of the agnn gene and a CpG island region of the MSC gene.
In one embodiment, the nucleic acid product is capable of detecting methylation levels in all or a portion of the regions Chr1:1033941-1033829 and Chr8: 71843999-71843823.
In one embodiment, the nucleic acid product comprises a primer pair capable of detecting the methylation level of a CpG island region of an AGRN gene and a primer pair capable of detecting the methylation level of a CpG island region of an MSC gene; wherein:
the primer pair capable of detecting the methylation level of the CpG island region of the AGRN gene comprises at least one of a first primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033914-1033829 region, a second primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033937-1033844 region, and a third primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033941-1033844 region; and/or the presence of a catalyst in the reaction mixture,
the primer pair capable of detecting the methylation level of the CpG island region of the MSC gene comprises at least one of a fourth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843952-71843823 region, a fifth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843970-71843842 region, and a sixth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843999-71843905 region.
In one embodiment, the nucleotide sequence of the first primer pair is shown as SEQ ID NO 1-2; and/or the nucleotide sequence of the second primer pair is shown as SEQ ID NO. 4-5; and/or the nucleotide sequence of the third primer pair is shown as SEQ ID NO. 7-8; and/or the nucleotide sequence of the fourth primer pair is shown as SEQ ID NO 10-11; and/or the nucleotide sequence of the fifth primer pair is shown as SEQ ID NO 13-14; and/or the nucleotide sequence of the sixth primer pair is shown as SEQ ID NO 16-17.
In one embodiment, the nucleic acid product further comprises a detection probe corresponding to the primer pair.
In one embodiment, the nucleotide sequence of the first detection probe corresponding to the first primer pair is shown as SEQ ID NO. 3; and/or the nucleotide sequence of a second detection probe corresponding to the second primer pair is shown as SEQ ID NO. 6; and/or the nucleotide sequence of a third detection probe corresponding to the third primer pair is shown as SEQ ID NO. 9; and/or the nucleotide sequence of a fourth detection probe corresponding to the fourth primer pair is shown as SEQ ID NO. 12; and/or the nucleotide sequence of a fifth detection probe corresponding to the fifth primer pair is shown as SEQ ID NO. 15; and/or the nucleotide sequence of a sixth detection probe corresponding to the sixth primer pair is shown as SEQ ID NO. 18.
A kit for diagnosing colorectal cancer, comprising a reagent for detecting a methylation level of a CpG island region of an agnn gene and a CpG island region of an MSC gene.
In one embodiment, the kit detects the methylation level of the CpG island region of the agnn gene and the CpG island region of the MSC gene by at least one of the following methods: methylation-specific PCR, fluorescent quantitative PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve, and methylation-sensitive restriction endonuclease.
In one embodiment, the reagent for detecting the methylation level of the CpG island region of the agnn gene and the CpG island region of the MSC gene comprises the nucleic acid product of any of the above embodiments.
In one embodiment, the reagent for detecting the methylation level of the CpG island region of the agnn gene and the CpG island region of the MSC gene 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.
Detailed Description
The present invention will be described in detail with reference to the following embodiments in order to make the aforementioned objects, features and advantages of the invention more comprehensible. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, as those skilled in the art will recognize without departing from the spirit and scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" and/or "includes any and all combinations of one or more of the associated listed items. The terms "preferably" and "preferred" are used herein only to describe preferred embodiments or examples, and it should be understood that the scope of the present invention is not limited thereto. The word "further" or "still further" is used for descriptive purposes to indicate a difference in content, but should not be construed to limit the scope of the present invention.
"diagnosis" as used herein includes the aspects of aiding diagnosis, assessing risk of recurrence, assessing risk and degree of carcinogenesis, and determining prognosis.
The methylation is a form of chemical modification of DNA, and can change genetic expression without changing DNA sequence. DNA methylation refers to the covalent attachment of a methyl group at the cytosine position 5 of a genomic CpG dinucleotide, under the influence of DNA methyltransferase. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.
The term "methylation level" refers to whether or not a cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/ratio/percentage of methylation, and represents both qualitative and quantitative concepts. In practical application, different detection indexes can be adopted to compare DNA methylation levels according to actual conditions. As in some cases, comparisons can be made based on Ct values detected for the samples; in some cases, the proportion of gene methylation in the sample, i.e., the number of methylated molecules/(the number of methylated molecules + the number of unmethylated molecules) × 100, may be calculated and then compared; in some cases, statistical analysis and integration are also performed on each index to obtain a final judgment index.
The "primer" refers to an oligonucleotide that can be used in an amplification method (e.g., polymerase chain reaction PCR) to amplify a target sequence based on a polynucleotide sequence corresponding to a target gene or a partial region thereof. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence specific for that polynucleotide sequence. The exact length of the primer depends on many factors, including temperature, source of primer, and method used, among others. For example, for diagnostic and prognostic applications, oligonucleotide primers typically contain at least 10, 15, 20, 25 or more nucleotides, but may contain fewer nucleotides, depending on the complexity of the target sequence.
The "primer pair" refers to a pair of primers that can hybridize to the double strand of the target DNA molecule or to the regions of the target DNA molecule flanking the nucleotide sequence to be amplified.
The TaqMan probe is an oligonucleotide sequence containing a 5 'end fluorescence reporter group and a 3' end fluorescence quenching group. When the probe binds to the corresponding site on the DNA, the probe does not fluoresce because of the presence of a quencher in the vicinity of the fluorophore. During amplification, if the probe binds to the amplified strand, the 5'-3' exonuclease activity of the DNA polymerase (e.g., taq enzyme) digests the probe and the fluorescent moiety is away from the quencher and its energy is not absorbed, i.e., a fluorescent signal is generated. The fluorescent signal also has a synchronous exponential growth process as the target fragment every PCR cycle.
One embodiment of the application provides application of a reagent for detecting methylation levels of a CpG island region of an AGRN gene and a CpG island region of an MSC gene in preparation of a product for diagnosing colorectal cancer.
The research of researchers of the invention finds that the detection sensitivity and specificity of colorectal cancer can be improved by detecting the methylation levels of the CpG island region of the AGRN gene and the CpG island region of the MSC gene, so that the early detection rate of colorectal cancer is improved, the treatment time is strived for patients, and the detection process causes less pain to the patients.
In one example, the reagent is capable of detecting the methylation level of the whole or partial region of the region Chr1:1033941-1033829 and the region Chr8:71843999-71843823, using GRCh.38p14 as the reference genome.
Further, the above-mentioned reagent is capable of detecting the methylation level of the whole length or a part of at least one of the regions Chr1:1033914-1033829, chr1:1033937-1033844 and Chr1:1033941-1033844, and the methylation level of the whole length or a part of at least one of the regions Chr8:71843952-71843823, chr8:71843970-71843842 and Chr8: 71843999-71390845.
Preferably, the above reagent is capable of detecting the methylation level of the region Chr1:1033914-1033829 and the region Chr8: 71843999-71843905.
The detection of the methylation level of the Chr1:1033914-1033829 region and the Chr8:71843999-71843905 region has been researched to realize the optimal diagnostic sensitivity and specificity for the advanced adenoma and the colorectal cancer.
It is understood that the DNA on the chromosome is a double-stranded structure composed of a positive strand and a negative strand. It should be noted that, in this document, the regions "Chr1:1033941-1033829" and "the minus strand of Chr1:1033941-1033829" both represent the minus strand of the DNA located at position "1033829-1033941" on chromosome 1. The other regions are written in the same way, and GRCh.38p14 is used as reference genome.
Based on the above, an embodiment of the present application also provides a nucleic acid product for diagnosing colorectal cancer, which is capable of detecting the methylation level of the CpG island region of the agnn gene and the CpG island region of the MSC gene.
In one embodiment, the nucleic acid product is capable of detecting methylation levels in all or a portion of the regions Chr1:1033941-1033829 and Chr8: 71843999-71843823.
Further, the above-mentioned nucleic acid product is capable of detecting the methylation level of the full length or a part of at least one of the regions Chr1:1033914-1033829, chr1:1033937-1033844 and Chr1:1033941-1033844, and the methylation level of the full length or a part of at least one of the regions Chr8:71843952-71843823, chr8:71843970-71843842 and Chr8: 71843999-71390845.
In one embodiment, the nucleic acid product comprises a primer pair capable of detecting the methylation level of a CpG island region of an agnn gene and a primer pair capable of detecting the methylation level of a CpG island region of an MSC gene.
In one embodiment, the primer pair capable of detecting the methylation level of the CpG island region of the agnn gene comprises at least one of a first primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033914-1033829 region, a second primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033937-1033844 region, and a third primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033941-1033844 region.
In one embodiment, the primer pair capable of detecting the methylation level of the CpG island region of the MSC gene includes at least one of a fourth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843952-71843823 region, a fifth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843970-71843842 region, and a sixth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843999-71843905 region.
Preferably, the primer pair capable of detecting the methylation level of the CpG island region of the AGRN gene comprises at least one of a first primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033914-1033829 region, a second primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033937-1033844 region, and a third primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033941-1033844 region; and, the primer pair capable of detecting the methylation level of the CpG island region of the MSC gene includes at least one of a fourth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843952-71843823 region, a fifth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843970-71843842 region, and a sixth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843999-71843905 region.
More preferably, the primer pair capable of detecting the methylation level of the CpG island region of the AGRN gene includes a first primer pair for detecting the methylation level of the full length or partial region of Chr1:1033914-1033829 region, and the primer pair capable of detecting the methylation level of the CpG island region of the MSC gene includes a sixth primer pair for detecting the methylation level of the full length or partial region of Chr8:71843999-71843905 region.
In one embodiment, the nucleotide sequence of the first primer pair is shown as SEQ ID NO. 1-2; and/or the nucleotide sequence of the second primer pair is shown as SEQ ID NO. 4-5; and/or the nucleotide sequence of the third primer pair is shown as SEQ ID NO. 7-8; and/or the nucleotide sequence of the fourth primer pair is shown as SEQ ID NO. 10-11; and/or the nucleotide sequence of the fifth primer pair is shown as SEQ ID NO. 13-14; and/or the nucleotide sequence of the sixth primer pair is shown as SEQ ID NO 16-17.
Further, the nucleic acid product also comprises a detection probe corresponding to the primer pair.
In one embodiment, the nucleotide sequence of the first detection probe corresponding to the first primer pair is set forth in SEQ ID NO. 3; and/or the nucleotide sequence of a second detection probe corresponding to the second primer pair is shown as SEQ ID NO. 6; and/or the nucleotide sequence of a third detection probe corresponding to the third primer pair is shown as SEQ ID NO. 9; and/or the nucleotide sequence of a fourth detection probe corresponding to the fourth primer pair is shown as SEQ ID NO. 12; and/or the nucleotide sequence of a fifth detection probe corresponding to the fifth primer pair is shown as SEQ ID NO. 15; and/or the nucleotide sequence of a sixth detection probe corresponding to the sixth primer pair is shown as SEQ ID NO. 18.
In one embodiment, the detection probe is a TaqMan fluorescent probe. The 5 'end of the detection probe is connected with a fluorescence reporter group, and the 3' end is connected with a fluorescence quenching group. The fluorescence reporter group may be one selected from FAM, TET, VIC, JOE, HEX, cy3, cy3.5, cy5, cy5.5, NED and Texas Red, and the fluorescence quencher group may be one selected from TAMRA, BHQ and MGB. It is understood that the detection of methylation of the target region and the detection of the reference gene can be performed in the same reaction well or different reaction wells, and the fluorescent group of the detection probe is appropriately selected according to actual conditions.
Based on the above, an embodiment of the present application also provides a kit for diagnosing colorectal cancer, which includes a reagent for detecting the methylation level of the CpG island region of the agnn gene and the CpG island region of the MSC gene.
In one embodiment, the kit detects the methylation level of the CpG island region of the agnn gene and the CpG island region of the MSC gene by at least one of the following methods: methylation-specific PCR, fluorescent quantitative PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high resolution solubility curves, and methylation-sensitive restriction endonuclease.
In one embodiment, the reagent for detecting the methylation level of the CpG island region of the AGRN gene and the CpG island region of the MSC gene comprises a nucleic acid product as described in any of the above embodiments.
In one embodiment, the reagent for detecting the methylation level of the CpG island region of the agnn gene and the CpG island region of the MSC gene 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.
In one embodiment, the methylation conversion reagent is a sulfite conversion reagent or an enzymatic conversion reagent.
In one of the two casesIn the embodiment, the PCR reagent comprises PCR buffer, dNTP, mgCl 2 And a DNA polymerase.
In one embodiment, the quality control reagent comprises a positive reference substance and a negative reference substance.
In some embodiments, the kit is suitable for samples including, but not limited to, stool samples, tissue samples, or blood samples.
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.
Example 1
1. Collection of samples
280 tissue samples of different disease courses confirmed by enteroscopy and pathological biopsy were collected, all of which were formalin-soaked and paraffin-embedded tissue samples. All samples were collected under examination and approval by the ethics committee, all volunteers signed informed consent, and all samples were processed anonymously. The information for the tissue samples is shown in table 1:
TABLE 1
2. Extraction of sample DNA
DNA was extracted from the Tissue sample using QIAamp DNA FFPE Tissue Kit (Cat: 56404), and the specific procedures were performed according to the Kit instructions.
3. Bisulfite conversion of sample DNA
And (3) carrying out bisulfite conversion on the extracted sample DNA, wherein the nucleic acid conversion kit is a nucleic acid conversion reagent (20200843, ehan instruments) of Wuhan Amison Life technologies, inc., and the specific experimental operation is shown in the kit specification.
4. Quantitative methylation specific PCR (qMSP)
A plurality of pairs of methylation primer pairs and detection probes are respectively designed for DNA molecules in a Chr1:1033941-1033829 region and a Chr8:71843999-71843823 region, 3 pairs of primer pairs and probes with high amplification efficiency and high specificity are respectively reserved for each gene after screening, and the nucleotide sequences of the primer pairs and the probes are shown in Table 2. The bisulfite converted target sequence and the unconverted DNA sequence detected by each primer pair and probe pair and their corresponding chromosomal location are shown in Table 3. The methylated cytosine sites detectable for each primer pair and probe are shown in Table 4. It is understood that within a segment of a DNA molecule, the methylation degree of cytosine in CpG dinucleotides at different positions is different, and in a region where CpG dinucleotides are dense, if the amplicon is changed, the position of the CpG dinucleotides detected by the amplicon is also changed, and the methylation degree of the region is also changed. Therefore, when PCR amplification is carried out, the researchers of the present invention try to carry out combined screening of the primer pair and the probe of the Chr1:1033941-1033829 region and the Chr8:71843999-71843823 region, and the specific combination mode is shown in Table 5.
TABLE 2
TABLE 3
TABLE 4
TABLE 5
And carrying out qMSP reaction on the sample DNA after bisulfite conversion to detect the methylation state of each sample to be detected in a Chr1:1033941-1033829 area and a Chr8:71843999-71843823 area. When PCR reaction is carried out, in a PCR tube, necessary components such as reaction buffer solution, dNTP, DNA polymerase, template and the like are added; then a combination of a primer pair and a probe is added, for example, one of the primer pair and the probe 1-3 for detecting the methylation level of a Chr1:1033941-1033829 region (a CpG island region of AGRN gene) and one of the primer pair and the probe 4-6 for detecting the methylation level of a Chr8:71843999-71843823 region (a CpG island region of MSC gene) are added; in addition, a detection primer pair and a detection probe (SEQ ID NO. 19-21) of the reference gene ACTB are also required to be added. The detection probe used here is a Taqman probe, the reporter group at the 5 'end of the detection probe in the AGRN gene target region is FAM, the quenching group at the 3' end is MGB, the reporter group at the 5 'end of the detection probe in the MSC target region is ROX, the quenching group at the 3' end is MGB, the reporter group at the 5 'end of the detection probe in the ACTB gene is VIC, and the quenching group at the 3' end is BHQ1. PCR amplification was carried out using Invitrogen Platinum II Taq hot start DNA polymerase (Invitrogen, cat: 14966005) and the PCR reaction solution preparation system was as shown in Table 6, following the amplification procedure shown in Table 7.
TABLE 6
TABLE 7
When PCR detection is performed according to different primer pairs and probe combinations, detection of a negative control and detection of a positive control should be performed simultaneously. For the same combination of primer pairs and probes, the negative control and the positive control are different from other sample wells to be tested in that different templates are added. The negative control was TE buffer. The positive control template was prepared as follows: artificially synthesizing target regions of AGRN genes such as SEQ ID No.23, 25 and 27, and respectively cloning to a vector to form artificially synthesized plasmids; artificially synthesizing target regions of the MSC gene such as SEQ ID NO.29, 31 and 33, and respectively cloning to a vector to form artificially synthesized plasmids; and (3) artificially synthesizing a sequence which is completely converted by the bisulfite and corresponds to the amplified region of the ACTB gene, and cloning the sequence to a vector to form an artificially synthesized plasmid. During PCR detection, a template to be added to the positive control wells is selected according to the combination mode in Table 5, and the DNA templates of the positive control wells are as follows: 10 3 Copies/microliter of the artificially synthesized plasmid containing the transformed ACTB, 10 3 Copies/microliter of a synthetic plasmid comprising one of SEQ ID No.23, 25 and 27, 10 3 Copy/microliter of an artificially synthesized plasmid comprising one of SEQ ID nos. 29, 31 and 33, 1.
5. Analysis of results
1) Ct value reading: and after the PCR is finished, adjusting a base line, setting a fluorescence value of the sample in the primary PCR before the minimum Ct value is advanced by 1-2 cycles as a base line value, and setting a threshold value at an inflection point of an S-shaped amplification curve to obtain the Ct value of each gene of the sample.
2) Quality control: negative control needs no amplification, positive control needs obvious exponential increase period, and positive control Ct value should be between 26-30. The Ct value of the reference gene of the sample to be detected is less than or equal to 35, and the negative control, the positive control and the reference gene all meet the requirements, which shows that the experiment is effective and the next sample result can be judged. Otherwise, when the experiment is invalid, the detection is required to be carried out again.
3) Results analysis and interpretation methods:
regarding the tissue sample to be detected, if the Ct value of the tissue sample in a certain detection area is less than or equal to 38, the tissue sample is considered to be methylation positive in the area, and if the Ct value of the tissue sample to be detected in the certain detection area is greater than 38, the tissue sample is considered to be methylation negative in the area.
According to the combination mode of the detection primer pairs and the probes in the table 5, if at least one of the 2 target regions detected by the sample to be detected is methylation positive, the sample is a positive sample of colorectal cancer or precancerous lesion. And if and only if the sample to be detected is methylation negative in the 2 target areas detected at the same time, the sample is a colorectal cancer negative sample. The performance of the intestinal tissue samples was diagnosed by analyzing the qMSP method based on the Ct value, and the results are shown in table 8, where the sensitivity is the proportion of methylation positive in the samples with a positive enteroscopy result, and the specificity is the proportion of methylation negative in the samples with a negative enteroscopy result.
TABLE 8
As can be seen from Table 8, methylation detection of the region Chr1:1033941-1033829 and the region Chr8:71843999-71843823 by using the qMSP method has good effect on distinguishing colorectal cancer precancerous lesion from cancerous tissue sample. All the primer pairs and the probes are combined in a certain detection rate on the advanced adenoma tissue sample, and have a good detection effect on the colorectal cancer tissue sample. The sensitivity range of the primer pair and probe combination modes A-I for detecting the advanced adenoma tissue sample is 50% -64.29%, and the specificity of the primer pair and probe combination modes in the tissue beside the adenoma is higher than 85.7%; the sensitivity range of the kit for detecting the colorectal cancer tissue sample is 75-86.9%, and the specificity of the kit for detecting the tissue sample beside the cancer is higher than 82.14%. In addition, the combination mode of the primer pair and the probe A-I also has better detection rate on the colorectal cancer at the first stage, and the sensitivity range of the early stage colorectal cancer tissue sample diagnosis is 65.79-78.95%. Furthermore, the primer pair and the probe (which can simultaneously detect the DNA methylation levels of the Chr1:1033914-1033829 region and the Chr8:71843999-71843905 region) in the combination mode C have the best diagnosis performance on the tissue samples of the advanced adenoma and the colorectal cancer.
Example 2
1. Collection of samples
A total of 156 stool samples from enteroscopy and pathology biopsy confirmed colorectal cancer patients and 185 healthy persons who had undergone a routine physical examination were collected. The mass of each stool specimen is greater than 1g. All samples were collected under examination and approval by the ethics committee, all volunteers signed informed consent, and all samples were processed anonymously. The information of the stool sample is shown in table 9.
TABLE 9
2. Extraction of sample DNA
DNA in the feces sample is extracted by using nucleic acid extraction kit (Cat: AA 07) of Wuhan Amisen Life technologies, inc., and the specific operation is carried out according to the kit instruction.
3. Bisulfite conversion of sample DNA
And (3) carrying out bisulfite conversion on the extracted sample DNA, wherein the nucleic acid conversion kit is a nucleic acid conversion reagent (20200843, ehan instruments) of Wuhan Amison Life technologies, inc., and the specific experimental operation is shown in the kit specification.
4. Quantitative methylation specific PCR (qMSP)
The qMSP reaction was performed on bisulfite converted sample DNA using the same procedure, primers and probes used, and detection sites as in step 4 of example 1.
5. Analysis of results
The Ct value reading method and the quality control method for the result of the qMSP reaction are the same as those of step 5 in example 1.
For the stool sample to be detected, if the Ct value of the stool sample in a certain detection area is less than or equal to 38, the sample is considered to be methylation positive in the area, and if the Ct value of the stool sample to be detected in the certain detection area is greater than 38, the sample is considered to be methylation negative in the area.
According to the combination mode of the detection primer pairs and the probes in the table 5, if at least one of the 2 target regions detected by the sample to be detected is methylation positive, the sample is a positive sample of colorectal cancer or precancerous lesion. And if and only if the sample to be detected is methylation negative in the 2 target areas detected at the same time, the sample is a colorectal cancer negative sample. The qMSP method was used to diagnose the performance of stool samples based on Ct value analysis, and the results are shown in Table 10, where the sensitivity is the proportion of methylation positivity in samples with a positive enteroscopy result, and the specificity is the proportion of methylation negativity in samples with a negative enteroscopy result.
TABLE 10
As can be seen from Table 10, stool samples from colorectal cancer patients and healthy persons can also be distinguished by measuring the methylation levels in the region Chr1:1033941-1033829 and in the region Chr8:71843999-71843823 by means of qMSP. In general, the sensitivity of the fecal samples of the colorectal cancer patients detected by the combination mode of different primer pairs and probes is higher, the minimum is 74.36%, and the maximum can reach 85.9%; the specificity of the human excrement sample detected by the kit is higher than 89.19 percent and can reach 95.68 percent at most. And the primer pair and probe combination A-I also has better detection rate on the stool sample of the early colorectal cancer patient, and the sensitivity range of the stool sample for diagnosing the early colorectal cancer patient is 72-82.67 percent. Similar to the tissue samples, composition C was the most effective diagnostic.
Example 3
1. Collection of samples
139 blood samples of patients with advanced colorectal adenoma confirmed by enteroscopy and pathological biopsy were collected, and the specific information is shown in table 11. Among them, 303 blood samples of patients suffering from colorectal cancer and 295 blood samples of healthy persons were obtained. The volume of each blood sample was greater than 8mL. All samples were collected under examination and approval by the ethical committee, all volunteers signed informed consent, and all samples were processed anonymously.
TABLE 11
2. Extraction of sample DNA
The blood plasma cfDNA extraction was carried out using a magnetic bead method serum/blood plasma free DNA (cfDNA) extraction kit (Cat: DP 709) from Tiangen Biochemical technology (Beijing) Ltd, the specific procedures were carried out according to the kit instructions.
3. Bisulfite conversion of sample DNA
And (3) carrying out bisulfite conversion on the extracted sample DNA, wherein the nucleic acid conversion kit is a nucleic acid conversion reagent (20200843, ehan instruments) of Wuhan Amison Life technologies, inc., and the specific experimental operation is shown in the kit specification.
4. Quantitative methylation specific PCR (qMSP)
The qMSP reaction was performed on bisulfite converted sample DNA using the same procedure, primers and probes used, and detection sites as in step 4 of example 1.
5. Analysis of results
The Ct value reading method and the quality control method for the result of the qMSP reaction are the same as those of step 5 in example 1.
Regarding a blood sample to be detected, if the Ct value of the blood sample in a certain detection area is less than or equal to 45, the sample is considered to be methylation positive in the area, and if the Ct value of the blood sample to be detected in the certain detection area is greater than 45, the sample is considered to be methylation negative in the area.
According to the combination mode of the detection primer pairs and the probes in the table 5, if at least one of the 2 target regions detected by the sample to be detected is methylation positive, the sample is a positive sample of colorectal cancer or precancerous lesion. And if and only if the sample to be detected is methylation negative in the 2 target areas detected at the same time, the sample is a colorectal cancer negative sample. The qMSP method was used to diagnose the performance of blood samples based on Ct value analysis, and the results are shown in Table 12, where the sensitivity is the proportion of methylation positivity in samples with a positive enteroscopy result, and the specificity is the proportion of methylation negativity in samples with a negative enteroscopy result.
TABLE 12
As can be seen from Table 12, the blood samples from the patients with colorectal adenoma, colorectal cancer and healthy people were further diagnosed to be good by detecting the methylation levels of the Chr1:1033941-1033829 region and the Chr8:71843999-71843823 region by the qMSP method. The sensitivity range of the blood sample of the patient with colorectal adenoma detected by different primer pairs and probes is 46.76-56.83%; the sensitivity range of the kit for detecting the blood sample of the early colorectal cancer patient is 68.97-80.69%, the total sensitivity of the kit for detecting the blood sample of the colorectal cancer patient is higher than 76.24% and can reach 86.47% at most, and the specificity of the kit for detecting the blood sample of the healthy person is higher than 89.49% and can reach 95.25% at most. Similarly, primer set and probe set C gave the best diagnostic results.
As can be seen from the above examples, although the combination of different primer pairs and probes has slightly different effects on detecting precancerous lesions and canceration, it can be found that the primer pairs and probes for detecting the Chr1:1033941-1033829 region and the Chr8:71843999-71843823 region can both significantly improve the early detection rate of colorectal cancer, and the optimal combination mode can be selected for clinically diagnosing colorectal cancer or precancerous lesions, improving the life quality of patients and prolonging the life.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification 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, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. 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 patent of the invention is subject to the appended claims, and the description can be used for explaining the contents of the claims.
Claims (11)
1. Application of a reagent for detecting the methylation level of a CpG island region of an AGRN gene and a CpG island region of an MSC gene in preparing a product for diagnosing colorectal cancer.
2. The use according to claim 1, characterized in that said reagent is capable of detecting the methylation level of the whole or partial region of the region Chr1:1033941-1033829 and of the region Chr8:71843999-71843823, using grch.38p14 as reference genome.
3. The use according to claim 2, wherein the reagent is capable of detecting the methylation level of all or part of at least one of the regions Chr1:1033914-1033829, chr1:1033937-1033844 and Chr1:1033941-1033844, and all or part of at least one of the regions Chr8:71843952-71843823, chr8: 71843970-71388442 and Chr8: 71843999-71843905.
4. A nucleic acid product for diagnosing colorectal cancer, wherein the nucleic acid product is capable of detecting the methylation level of a CpG island region of an agnn gene and a CpG island region of an MSC gene.
5. The nucleic acid product of claim 4, wherein the nucleic acid product is capable of detecting the methylation level of all or part of the Chr1:1033941-1033829 and Chr8:71843999-71843823 regions.
6. The nucleic acid product of claim 4 or 5, wherein the nucleic acid product comprises a primer pair capable of detecting the methylation level of a CpG island region of an AGRN gene and a primer pair capable of detecting the methylation level of a CpG island region of an MSC gene; wherein:
the primer pair capable of detecting the methylation level of the CpG island region of the AGRN gene comprises at least one of a first primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033914-1033829 region, a second primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033937-1033844 region, and a third primer pair for detecting the methylation level of the full-length or partial region of the Chr1:1033941-1033844 region; and/or the presence of a catalyst in the reaction mixture,
the primer pair capable of detecting the methylation level of the CpG island region of the MSC gene comprises at least one of a fourth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843952-71843823 region, a fifth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843970-71843842 region, and a sixth primer pair for detecting the methylation level of the full-length or partial region of the Chr8:71843999-71843905 region.
7. The nucleic acid product of claim 6, wherein the nucleotide sequence of the first primer pair is shown as SEQ ID NO. 1-2; and/or the nucleotide sequence of the second primer pair is shown as SEQ ID NO. 4-5; and/or the nucleotide sequence of the third primer pair is shown as SEQ ID NO. 7-8; and/or the nucleotide sequence of the fourth primer pair is shown as SEQ ID NO. 10-11; and/or the nucleotide sequence of the fifth primer pair is shown as SEQ ID NO. 13-14; and/or the nucleotide sequence of the sixth primer pair is shown as SEQ ID NO 16-17.
8. The nucleic acid product of claim 7, further comprising a detection probe corresponding to the primer pair;
further, the nucleotide sequence of the first detection probe corresponding to the first primer pair is shown as SEQ ID NO. 3; and/or the nucleotide sequence of a second detection probe corresponding to the second primer pair is shown as SEQ ID NO. 6; and/or the nucleotide sequence of a third detection probe corresponding to the third primer pair is shown as SEQ ID NO. 9; and/or the nucleotide sequence of a fourth detection probe corresponding to the fourth primer pair is shown as SEQ ID NO. 12; and/or the nucleotide sequence of a fifth detection probe corresponding to the fifth primer pair is shown as SEQ ID NO. 15; and/or the nucleotide sequence of a sixth detection probe corresponding to the sixth primer pair is shown as SEQ ID NO. 18.
9. A kit for diagnosing colorectal cancer, comprising a reagent for detecting the methylation level of a CpG island region of an agnn gene and a CpG island region of an MSC gene.
10. The kit of claim 9, wherein the kit detects the methylation level of the CpG island region of the agnn gene and the CpG island region of the MSC gene by at least one of the following methods: methylation specificity PCR method, fluorescent quantitative PCR method, bisulfite sequencing method, methylation specificity microarray method, whole genome methylation sequencing method, pyrosequencing method, methylation specificity high performance liquid chromatography, digital PCR method, methylation specificity high resolution solubility curve method and methylation sensitivity restriction endonuclease method;
further, the reagent for detecting the methylation level of the CpG island region of AGRN gene and the CpG island region of MSC gene comprises the nucleic acid product of any one of claims 4 to 8.
11. The kit of claim 9 or 10, wherein the reagents for detecting the methylation level of the CpG island region of the agnn gene and the CpG island region of the MSC gene further comprise 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210799765.0A CN115838799A (en) | 2022-07-08 | 2022-07-08 | Application of reagent for detecting gene methylation in preparation of product for diagnosing colorectal cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210799765.0A CN115838799A (en) | 2022-07-08 | 2022-07-08 | Application of reagent for detecting gene methylation in preparation of product for diagnosing colorectal cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115838799A true CN115838799A (en) | 2023-03-24 |
Family
ID=85574716
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210799765.0A Pending CN115838799A (en) | 2022-07-08 | 2022-07-08 | Application of reagent for detecting gene methylation in preparation of product for diagnosing colorectal cancer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115838799A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852974A (en) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | Compositions and methods for treating and diagnosing cancer |
| US20130022974A1 (en) * | 2011-06-17 | 2013-01-24 | The Regents Of The University Of Michigan | Dna methylation profiles in cancer |
| CN106232833A (en) * | 2014-01-30 | 2016-12-14 | 加利福尼亚大学董事会 | The haplotyping that methylates (MONOD) for non-invasive diagnostic |
| US20210115519A1 (en) * | 2018-04-19 | 2021-04-22 | The University Of Chicago | Methods and kits for diagnosis and triage of patients with colorectal liver metastases |
-
2022
- 2022-07-08 CN CN202210799765.0A patent/CN115838799A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852974A (en) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | Compositions and methods for treating and diagnosing cancer |
| US20130022974A1 (en) * | 2011-06-17 | 2013-01-24 | The Regents Of The University Of Michigan | Dna methylation profiles in cancer |
| CN106232833A (en) * | 2014-01-30 | 2016-12-14 | 加利福尼亚大学董事会 | The haplotyping that methylates (MONOD) for non-invasive diagnostic |
| US20210115519A1 (en) * | 2018-04-19 | 2021-04-22 | The University Of Chicago | Methods and kits for diagnosis and triage of patients with colorectal liver metastases |
Non-Patent Citations (2)
| Title |
|---|
| LUDOVIC BARAULT等: "Discovery Of Methylated Circulating DNA Biomarkers For Comprehensive Non-Invasive Monitoring Of Treatment Response In Metastatic Colorectal Cancer", GUT., vol. 67, no. 11, pages 1995 - 2005, XP093024614, DOI: 10.1136/gutjnl-2016-313372 * |
| 张威;叶颖江;任仙文;黄晶;申占龙;: "利用转录组二代测序探索直肠癌术前放化疗的敏感性分子特征", 北京大学学报(医学版), no. 03 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114277135B (en) | Gastric cancer lymph node metastasis related methylation biomarker and combination and detection kit thereof | |
| CN111100866A (en) | Gene segment for identifying benign and malignant thyroid nodules and application thereof | |
| CN113430272B (en) | Reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesion and application of reagent and kit | |
| WO2018211404A1 (en) | Composite epigenetic biomarkers for accurate screening, diagnosis and prognosis of colorectal cancer | |
| CN115838799A (en) | Application of reagent for detecting gene methylation in preparation of product for diagnosing colorectal cancer | |
| CN115786502B (en) | Use of an agent for detecting the methylation level of a target region for the preparation of a diagnostic product for bladder cancer | |
| CN114959030B (en) | Application of reagent for detecting HCG9 gene methylation in preparation of product for diagnosing bladder cancer | |
| CN115094139B (en) | Application of reagent for detecting methylation level in preparation of bladder cancer diagnosis product and bladder cancer diagnosis kit | |
| CN115838798A (en) | Application of reagent for detecting methylation level in preparation of colorectal cancer diagnosis product | |
| CN117344010B (en) | DNA methylation biomarker for diagnosing gastric cancer, kit and application | |
| CN115948561B (en) | Reagent and detection kit for esophageal squamous carcinoma diagnosis or auxiliary diagnosis and application thereof | |
| EP2922973A1 (en) | A method of detecting methylation | |
| CN117363729A (en) | Kit for in vitro detection of liver cancer and application thereof | |
| CN117587125A (en) | Application of reagent for detecting methylation in preparation of pancreatic cancer diagnosis product | |
| CN115927610A (en) | Application of reagent for detecting methylation level of target region in FOXO6 gene in preparation of bladder cancer diagnosis product | |
| CN117604095A (en) | Methylation detection reagent and kit for esophageal cancer diagnosis | |
| CN116904598A (en) | Nucleic acid combination, reagent and kit for detecting primary liver cancer and application of nucleic acid combination | |
| CN116121371A (en) | Application of reagent for detecting methylation level of target region in TLX1 gene in preparation of esophageal cancer diagnosis product | |
| CN117568480A (en) | Reagent and kit for breast cancer or precancerous lesion detection and application | |
| CN116083586A (en) | Nucleic acid product, kit and application for diagnosing esophageal cancer | |
| CN116463417A (en) | Application of reagent for detecting methylation level of target region in preparation of liver cancer diagnosis product | |
| CN117248021A (en) | Nucleic acid combination, kit and application for detecting urothelial cancer or precancerous lesions | |
| CN117344010A (en) | DNA methylation biomarker for diagnosing gastric cancer, kit and application | |
| WO2023083348A1 (en) | Methylation detection reagent and kit for esophageal cancer diagnosis | |
| CN116814779A (en) | Biomarker for gastric cancer, nucleic acid product and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |