CN115838798A - Application of reagent for detecting methylation level in preparation of colorectal cancer diagnosis product - Google Patents
Application of reagent for detecting methylation level in preparation of colorectal cancer diagnosis product Download PDFInfo
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Abstract
The invention relates to an application of an agent for detecting methylation level of a target region in preparing a colorectal cancer diagnosis product, wherein GRCh38.P14 is taken as a reference genome, and the target region comprises full-length or partial regions of a positive strand and/or a negative strand of Chr7: 142796895-142797358. By detecting the methylation level of the target region, colorectal cancer patients and healthy people can be effectively distinguished, the detection sensitivity and specificity are high, and the detection rate of colorectal cancer is improved.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of a reagent for detecting methylation level in preparing a colorectal cancer diagnosis product.
Background
Colorectal cancer is the third most common malignancy worldwide. Despite the improvements of current treatment, prognosis for patients with advanced stage colorectal cancer remains poor, e.g., 5-year survival rates for patients with stage iv colorectal cancer are only about 14%, and most patients with colorectal cancer are already in mid-advanced stage when first diagnosed. In fact, it takes about 5 to 15 years to develop from adenomatous polyps to carcinoma, and the 5-year survival rate of stage i colorectal cancer patients is still as high as 91%, which provides sufficient time for early detection of colorectal cancer. Therefore, early screening of patients with pre-cancerous lesions and colorectal cancer is one of the key strategies to improve their quality of life and reduce mortality.
At present, the gold standard for colorectal cancer diagnosis is colorectal endoscopy and pathological biopsy, but as an invasive procedure, a great deal of intestinal tract preparation is required before examination, and the compliance of the enteroscope in the majority of people is low. In addition, some fecal-based assays, such as Fecal Occult Blood Test (FOBT) and Fecal Immunochemical Test (FIT), have low sensitivity for detecting early stage colorectal cancer, e.g., 27% and 53% sensitivity for the fecal immunochemical assay for diagnosing advanced adenomas and stage i colorectal cancer, respectively.
Disclosure of Invention
The researchers of the invention find that the methylation characteristics of the full-length or partial region of the positive strand and/or the negative strand of Chr7:142796895-142797358 are taken as biomarkers of colorectal cancer, and the defect of low sensitivity of the traditional detection products to the colorectal cancer can be improved by detecting the methylation level of the colorectal cancer.
Based on the above, the invention provides an application of a reagent for detecting the methylation level of a target area in the preparation of a colorectal cancer diagnosis product.
In addition, a nucleic acid product and a detection kit with high sensitivity for diagnosing the colorectal cancer are also provided.
Use of an agent for detecting the methylation level of a target region, said target region comprising the full length or partial region of the plus strand and/or minus strand of Chr7:142796895-142797358, in the preparation of a diagnostic product for colorectal cancer, using grch38.P14 as the reference genome.
By detecting the methylation level of the target region, colorectal cancer patients and healthy people can be effectively distinguished, the detection sensitivity and specificity are high, and the detection rate of colorectal cancer is improved.
In one embodiment, the target region comprises a full length or partial region of at least one of:
the positive strand of Chr7: 142796895-142797041;
the positive strand of Chr7: 142797056-142797207;
the plus strand of Chr7: 142797214-142797358;
the minus strand of Chr7: 142797358-142797249;
negative strand of Chr7:142797252-142797118 and
the minus strand of Chr7: 142797038-142796907.
In one embodiment, the target region further comprises a full-length or partial region of the minus strand of Chr8: 71843685-71843533.
A nucleic acid product for diagnosing colorectal cancer, which nucleic acid product is capable of detecting the level of methylation of a region of interest, including the full length or a partial region of the positive and/or negative strand of Chr7:142796895-142797358, using grch38.P14 as a reference genome.
In one embodiment, the nucleic acid product comprises at least one of the following pairs of detection primers:
a first detection primer pair for detecting the methylation level of the full-length or partial region of the positive strand of Chr7: 142796895-142797041;
a second detection primer pair for detecting the methylation level of the full-length or partial region of the positive strand of Chr7: 142797056-142797207;
a third detection primer pair for detecting the methylation level of the full-length or partial region of the plus strand of Chr7: 142797214-142797358;
a fourth detection primer pair for detecting the methylation level of the full-length or partial region of the minus strand of Chr7: 142797358-142797249;
a fifth detection primer pair for detecting the methylation level of the full-length or partial region of the minus strand of Chr7: 142797252-142797118; and
a sixth detection primer pair for detecting the methylation level of the full-length or partial region of the minus strand of Chr7: 142797038-142796907.
In one embodiment, the nucleotide sequence of the first detection primer pair is shown as SEQ ID NO. 23-24; and/or the nucleotide sequence of the second detection primer pair is shown as SEQ ID NO. 26-27; and/or the nucleotide sequence of the third detection primer pair is shown as SEQ ID NO. 29-30; and/or the nucleotide sequence of the fourth detection primer pair is shown as SEQ ID NO. 32-33; and/or the nucleotide sequence of the fifth detection primer pair is shown as SEQ ID NO. 35-36; and/or the nucleotide sequence of the sixth detection primer pair is shown in SEQ ID NO. 38-39.
In one embodiment, the nucleic acid product further comprises a detection probe corresponding to the detection primer pair.
In one embodiment, the nucleotide sequence of the first detection probe corresponding to the first detection primer pair is shown as SEQ ID NO. 25; and/or the nucleotide sequence of a second detection probe corresponding to the second detection primer is shown as SEQ ID NO. 28; and/or the nucleotide sequence of a third detection probe corresponding to the third detection primer is shown as SEQ ID NO. 31; and/or the nucleotide sequence of a fourth detection probe corresponding to the fourth detection primer is shown as SEQ ID NO. 34; and/or the nucleotide sequence of a fifth detection probe corresponding to the fifth detection primer pair is shown as SEQ ID NO. 37; and/or the nucleotide sequence of a sixth detection probe corresponding to the sixth detection primer pair is shown as SEQ ID NO. 40.
In one embodiment, the nucleic acid product further comprises a seventh detection primer pair for detecting the methylation level of the full-length or partial region of the negative strand of Chr8: 71843685-71843533.
In one embodiment, the nucleotide sequence of the seventh detection primer pair is shown in SEQ ID NO. 46-47.
In one embodiment, the nucleic acid product further comprises a detection probe corresponding to the seventh pair of detection primers.
In one embodiment, the nucleotide sequence of the detection probe corresponding to the seventh detection primer pair is shown as SEQ ID NO. 48.
A kit for the detection of colorectal cancer, the kit comprising reagents for detecting the level of methylation in a target region, the target region comprising a target region as described in the use according to any one of the preceding examples.
In one embodiment, the test kit detects the methylation level of the target region 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 a target region comprises the nucleic acid product of any one of the embodiments above.
In one embodiment, the reagent for detecting the methylation level of the target region 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.
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" includes any and all combinations of one or more of the associated listed items.
"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 an application of an agent for detecting methylation level of a target region in preparing a colorectal cancer diagnosis product, wherein GRCh38.P14 is taken as a reference genome, and the target region comprises the full-length or partial region of a positive strand and/or a negative strand of Chr7: 142796895-142797358.
The researchers of the invention find that the methylation characteristics of the full-length or partial region of the positive strand and/or the negative strand of Chr7:142796895-142797358 are taken as biomarkers of colorectal cancer, and the defect of low sensitivity of the traditional detection products to the colorectal cancer can be improved by detecting the methylation level of the colorectal cancer.
By detecting the methylation level of the target region, colorectal cancer patients and healthy people can be effectively distinguished, the detection sensitivity and specificity are high, and the detection rate of colorectal cancer is improved.
Optionally, the target region comprises a full length or partial region of at least one of:
the positive strand of Chr7: 142796895-142797041; the positive strand of Chr7: 142797056-142797207; the plus strand of Chr7: 142797214-142797358; the minus strand of Chr7: 142797358-142797249; the minus strand of Chr7:142797252-142797118 and the minus strand of Chr7: 142797038-142796907.
Further, the target region also includes the full length or partial region of the minus strand of Chr8: 71843685-71843533.
It is understood that the DNA on the chromosome is a double-stranded structure composed of a positive strand and a negative strand. In this context, a region, if it is not specified as a positive strand or a negative strand of DNA, means that it may be the positive strand or the negative strand of DNA of the region or both the positive and negative strands of DNA of the region. For example, if the region Chr7:142796895-142797358 is described as "Chr7:142796895-142797358", it indicates that it may be the positive strand of DNA in the region Chr7:142796895-142797358, the negative strand of DNA in the region Chr7:142796895-142797358, or both the positive and negative strands of DNA in the region Chr7: 142796895-142797358. If the region Chr7:142796895-142797358 is described as "the plus strand in the region Chr7:142796895-142797358" or "the plus strand of Chr7:142796895-142797358", it means the plus strand of DNA in the region Chr7: 142796895-142797358; if the region Chr7:142796895-142797358 is described as the "minus strand in the region Chr7:142796895-142797358" or "minus strand of Chr7:142796895-142797358," this indicates the minus strand of DNA in the region Chr7: 142796895-142797358. In addition, "Chr7:142797358-142796895" and "the minus strand of Chr7:142797358-142796895" both represent the minus strand of DNA of the region Chr7: 142796895-142797358.
Based on the above, one embodiment of the present application further provides a nucleic acid product for diagnosing colorectal cancer, which is capable of detecting the methylation level of a target region, including the full length or partial region of the positive strand and/or negative strand of Chr7:142796895-142797358, with grch38.P14 as a reference genome.
In one embodiment, the nucleic acid product comprises at least one of the following pairs of detection primers: a first detection primer pair for detecting the methylation level of the full-length or partial region of the positive strand of Chr7: 142796895-142797041; a second detection primer pair for detecting the methylation level of the full-length or partial region of the plus strand of Chr7: 142797056-142797207; a third detection primer pair for detecting the methylation level of the full-length or partial region of the plus strand of Chr7: 142797214-142797358; a fourth detection primer pair for detecting the methylation level of the full-length or partial region of the minus strand of Chr7: 142797358-142797249; a fifth detection primer pair for detecting the methylation level of the full-length or partial region of the minus strand of Chr7: 142797252-142797118; and a sixth detection primer pair for detecting the methylation level of the full-length or partial region of the negative strand of the Chr7: 142797038-142796907.
Optionally, the nucleotide sequence of the first detection primer pair is shown as SEQ ID NO. 23-24; and/or the nucleotide sequence of the second detection primer pair is shown as SEQ ID NO. 26-27; and/or the nucleotide sequence of the third detection primer pair is shown as SEQ ID NO. 29-30; and/or the nucleotide sequence of the fourth detection primer pair is shown as SEQ ID NO. 32-33; and/or the nucleotide sequence of the fifth detection primer pair is shown as SEQ ID NO. 35-36; and/or the nucleotide sequence of the sixth detection primer pair is shown in SEQ ID NO. 38-39.
In one embodiment, the nucleic acid product further comprises a detection probe corresponding to the detection primer pair.
Optionally, the nucleotide sequence of the first detection probe corresponding to the first detection primer pair is shown as SEQ ID NO. 25; and/or the nucleotide sequence of a second detection probe corresponding to the second detection primer is shown as SEQ ID NO. 28; and/or the nucleotide sequence of a third detection probe corresponding to the third detection primer is shown as SEQ ID NO. 31; and/or the nucleotide sequence of a fourth detection probe corresponding to the fourth detection primer is shown as SEQ ID NO. 34; and/or the nucleotide sequence of a fifth detection probe corresponding to the fifth detection primer is shown as SEQ ID NO. 37; and/or the nucleotide sequence of a sixth detection probe corresponding to the sixth detection primer pair is shown as SEQ ID NO. 40.
In one embodiment, the nucleic acid product further comprises a seventh detection primer pair for detecting the methylation level of the full-length or partial region of the negative strand of Chr8: 71843685-71843533.
Optionally, the nucleotide sequence of the seventh detection primer pair is shown in SEQ ID NO. 46-47.
In one embodiment, the nucleic acid product further comprises a detection probe corresponding to the seventh detection primer pair.
Alternatively, the nucleotide sequence of the detection probe corresponding to the seventh detection primer pair is shown in SEQ ID NO. 48.
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 methylation detection 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 the actual situation. In an alternative specific example, the methylation of the target region and the detection of the reference gene are performed in the same reaction well, and a plurality of fluorescence reporters and fluorescence quenchers are selected to be combined, so that the detection result is not influenced.
Based on the above, an embodiment of the present application further provides a colorectal cancer detection kit, which includes a reagent for detecting the methylation level of a target region, wherein the target region includes the target region in the application as described in any one of the above examples.
In one embodiment, the above-mentioned detection kit detects the methylation level of the target region 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 a target region comprises a nucleic acid product of any of the embodiments above.
In one embodiment, the reagent for detecting the methylation level of the target region 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 embodiment, the PCR reaction 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 sample to which the above-described test kit is applied includes, but is not limited to, a stool sample, a tissue sample, or a blood sample.
Proved by verification, the detection sensitivity of the colorectal cancer detection kit can reach 87.79%, the specificity can reach 94.58%, and early diagnosis of colorectal cancer is facilitated.
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. Sample collection
A total of 280 tissue samples of different disease courses confirmed by enteroscopy and pathological biopsy were collected and the sample information was as follows: 84 colorectal cancer tissue samples and 84 paracarcinoma normal tissue samples corresponding to the colorectal cancer tissue samples, 56 advanced adenoma tissue samples and 56 adjacent normal tissue samples matched with the advanced adenoma tissue samples. Of the colorectal cancer tissue samples, 38 samples with stage I pathological stages were observed, and the remaining 46 samples had stage II, III or IV pathological stages. All samples 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.
2. Extraction of sample DNA
DNA in the Tissue sample was extracted using the QIAamp DNA FFPE Tissue Kit (Cat: 56404), and the detailed procedures were performed according to the Kit instructions.
3. Bisulfite conversion
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. Methylation specific quantitative PCR (qMSP)
In order to ensure the amplification efficiency of the fluorescent quantitative PCR, the DNA fragment at the position of Chr7:142796524-142797358 (taking GRCh38.P14 as a reference genome) is divided into a plurality of short DNA regions for methylation quantitative PCR detection. After bisulfite conversion of the DNA molecule, unmethylated cytosines are deaminated to uracil, while methylated cytosines remain unchanged, and during subsequent PCR amplification, all uracils are converted to thymine. It follows that, after undergoing bisulfite conversion, unmethylated cytosines in one single strand of a double-stranded DNA molecule are no longer complementary to guanine in its reverse complement strand, i.e., the sense and antisense strands of the converted DNA molecule are no longer perfectly complementary paired, and it is understood that the methylation levels of the sense or antisense strand of the converted DNA molecule are different. Thus, in the present invention, the level of methylation was detected for both the sense and the antisense strands of the DNA molecule at positions Chr7: 142796524-142797358.
Detection primer pairs and probes were then designed for the qMSP reaction, and bisulfite converted target and unconverted DNA sequences detected by the detection primer pairs and probes and their corresponding chromosomal locations are shown in Table 1. In order to ensure the efficiency and specificity of the qMSP amplification, a large number of detection primer pairs and probes were screened, and the detection primer pairs and probes finally used are shown in Table 2. The sites of methylated cytosines recognizable by the detection primer pairs and the probes for detecting each region at Chr7:142796524-142797358 are shown in Table 3.
TABLE 1
TABLE 2
TABLE 3
The bisulfite converted sample DNA was subjected to a qMSP reaction to detect the methylation status of various regions of Chr7:142796524-142797358 in each sample. When methylation fluorescent quantitative PCR reaction is carried out, necessary components such as reaction buffer solution, dNTP, DNA polymerase, template and the like, a detection primer pair and a detection probe corresponding to a detection region, and a detection primer pair and a detection probe of an internal reference gene ACTB are added into one PCR tube. The detection probe used here is a Taqman probe, the 5 'end of the detection probe in the target region has a reporter group FAM, the 3' end of the detection probe is MGB, the 5 'end of the detection probe of ACTB gene has a reporter group VIC, and the 3' end of the detection probe of ACTB gene has a quenching group BHQ1. Invitrogen Platinum II Taq hot start DNA polymerase (Invitrogen, cat: 14966005) was used for PCR amplification, and the PCR reaction solution preparation system was as shown in Table 4, and PCR amplification was carried out according to the amplification procedure shown in Table 5.
TABLE 4
TABLE 5
Negative and positive controls: when the detection is carried out on each region of the Chr7:142796524-142797358 positions, the detection is synchronously carried out on the negative control and the positive control. The negative control was TE buffer. The preparation method of the positive control template comprises the following steps: and (3) artificially synthesizing a sequence which is completely converted by the bisulfite and corresponds to the amplified region of the ACTB, and cloning the sequence onto a vector to form an artificially synthesized plasmid. The sequences (SEQ ID NO: 9-16) converted by bisulfite were artificially synthesized and cloned to vectors, respectively, to form artificially synthesized plasmids. The positive control of the 1-8 areas of the Chr7:142796524-142797358 is 10 3 Copy/microliter ACTB Artificial Synthesis plasmid and 10 3 Copies/microliter of the synthetic plasmids 1 of regions 1-8 after transformation were mixed, as for region 1 the positive control was 10 3 Copy/microliter ACTB synthetic plasmids and 10 3 Copy/microliter of the synthetic plasmid 1 containing SEQ ID NO 9 mixed together.
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.
Analyzing the performance of intestinal cancer tissue samples diagnosed by a methylation fluorescence quantitative PCR method. The sensitivity is the proportion of methylation positive in the sample with positive enteroscopy result, and the specificity is the proportion of methylation negative in the sample with negative enteroscopy result. The method for analyzing the methylation state of each tissue sample in each region of Chr7:142796524-142797358 and the sensitivity and specificity of detecting colorectal cancer and precancerous lesion are shown in Table 6.
TABLE 6
As can be seen from Table 6, the effect of using the methylation fluorescent quantitative PCR method to diagnose colorectal cancer and precancerous lesion tissue samples by detecting the methylation level of each region at the Chr7:142796524-142797358 positions is different. First, the diagnostic performance of region 1 (SEQ ID NO: 1) and region 2 (SEQ ID NO: 2) is poor, the sensitivity for detecting colorectal cancer tissue samples is less than 30%, while the diagnostic performance of regions 3 to 8 (SEQ ID NO:3 to 8) is better, the sensitivity for detecting colorectal cancer tissue samples is greater than or equal to 75%, the sensitivity for detecting colorectal adenoma tissue samples is greater than 44%, i.e., the locations of Chr7:142796895-142797358 in the region of Chr7:142796524-142797358 distinguish cancerous or precancerous tissue samples from normal tissue samples better. Then, for the regions 3-8, the colorectal adenoma tissue sample can be detected by detecting the methylation level of the tissue sample, wherein the sensitivity of detecting the colorectal adenoma in the region 3 can reach 62.50%. The effect of detecting the colorectal cancer in the areas 3-8 is also good, the sensitivity range of detecting early colorectal cancer is 63.16% -78.95%, the sensitivity range of detecting middle and late colorectal cancer is 84.78% -95.65%, and the specificity of detecting a normal tissue sample beside the cancer is higher than 83.33%. Therefore, the methylation levels of SEQ ID NO 3-8 can be used to effectively distinguish colorectal cancer/precancerous lesion and paracancerous normal sample.
Example 2
1. Collection of samples
In total, 156 stool samples were collected from colorectal cancer patients diagnosed by enteroscopy and pathological biopsy, and 185 healthy persons who had undergone routine physical examination. The mass of each stool sample 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.
2. Extraction of sample DNA
DNA in the fecal 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
The specific procedure of this step is the same as in step 3 of example 1.
4. Methylation specific quantitative PCR (qMSP)
The specific procedure of this step is the same as in step 4 of example 1.
5. Analysis of results
The specific process of this step is substantially the same as that of step 5 of example 1, except that:
the method for analyzing the methylation state of each type of fecal sample in each region of Chr7:142796524-142797358 positions and the sensitivity and specificity for detecting colorectal cancer and precancerous lesion are shown in Table 7.
TABLE 7
In particular, as the tendency to examine tissue samples was the same, the detection performance of region 1 (SEQ ID NO: 1) and region 2 (SEQ ID NO: 2) was not good. However, the methylation level of the Chr7:142796895-142797358 (i.e., region 3 to region 8, SEQ ID NO. The sensitivity range of the fecal samples of the colorectal cancer patients detected by the areas 3-8 is 73.08% -86.54%, wherein the detection sensitivity of the area 3 is the highest. The specificity of detecting the feces samples of healthy people in the areas 3-8 is higher than 92.4%, wherein the detection specificity of the areas 3 and 4 is as high as 96.22%. In conclusion, the feces samples of colorectal cancer patients and healthy people can be effectively distinguished by using the methylation levels of SEQ ID NO. 3-8, and the diagnostic effect of SEQ ID NO. 3 is the best.
Example 3
1. Collection of samples
139 blood samples of patients with advanced colorectal adenoma, 145 blood samples of patients with stage I colorectal carcinoma, 158 blood samples of patients with stage II, III and IV colorectal carcinoma, and 270 blood samples of healthy persons were collected together. The volume of each blood sample was greater than 8mL. All samples were collected under examination and approval by the ethics committee, all volunteers signed informed consent, and all samples were processed anonymously.
2. Extraction of sample DNA
The blood plasma cfDNA was extracted using a magnetic bead method serum/blood plasma free DNA (cfDNA) extraction kit (cat # DP 709) from Tiangen Biochemical technology (Beijing) Ltd, according to the kit instructions.
3. Bisulfite conversion
The specific procedure of this step is the same as in step 3 of example 1.
4. Methylation specific quantitative PCR (qMSP)
The specific procedure of this step is the same as in step 4 of example 1.
5. Analysis of results
The Ct values were read and the internal quality control was judged in the same manner as in example 1.
If the Ct values of the negative control, the positive control and the reference gene all meet the experimental requirements, judging the sample to be detected according to the following standards: for the plasma sample, if the Ct value of the sample to be detected in a certain detection area is less than or equal to 45, the area in the sample is considered to be methylation positive, and if the Ct value of the sample to be detected in the certain detection area is greater than 45, the area in the sample is considered to be methylation negative. And (3) according to the detected Ct value, counting methylation negative and positive results of the sample to be detected in each region, and calculating the sensitivity and specificity of detecting the methylation level of each region of the Chr7:142796895-142797358DNA by using a qMSP method to diagnose the blood sample of the colorectal cancer and the precancerous lesion patient, wherein the results are shown in a table 8.
TABLE 8
As can be seen from Table 8, the blood samples from patients with colorectal cancer or precancerous lesions further have better performance by detecting the methylation level of the Chr7:142796895-142797358DNA regions (i.e., region 3-region 8, SEQ ID NO. For the blood sample of the advanced adenoma patient, the detection sensitivity range of the areas 3-8 is 41.73-56.12%; the detection sensitivity range of the areas 3-8 is 61.38% -74.48% for the blood sample of the early colorectal cancer patients, and the detection sensitivity range of the areas 3-8 is 75.95% -91.14% for the blood sample of the middle and late colorectal cancer patients. In addition, the specificity of detecting blood samples of healthy people in the areas 3-8 is higher than 92.2%. In conclusion, better diagnostic results can be achieved by detecting the methylation level of one region in SEQ ID NO. 3-8, and the diagnostic performance of SEQ ID NO. 3 and SEQ ID NO. 8 is better.
Example 4
1. Collection of samples
The specific procedure of this step is the same as in step 1 of example 3.
2. Extraction of sample DNA
The specific procedure of this step is the same as in step 2 of example 3.
3. Bisulfite conversion
The specific procedure of this step is the same as in step 3 of example 1.
4. Methylation specific quantitative PCR (qMSP)
The DNA sequences of SEQ ID NOS.3 to 8, the bisulfite converted sequences and the detection primer pairs and probes for the respective regions are shown in Table 1 and Table 2 in example 1, the DNA sequences of SEQ ID NO:44 and the bisulfite converted sequences are shown in Table 9, the primer pairs and probes for detecting the methylation level of SEQ ID NO:44 are shown in Table 10, and the cytosine sites methylated in SEQ ID NO:44 detectable by the detection primer pairs and probes are shown in Table 11.
TABLE 9
Watch 10
TABLE 11
The DNA converted by bisulfite was subjected to methylation fluorescent quantitative PCR reaction to detect the methylation state of any region of SEQ ID NO. 3-8 and the composition of SEQ ID NO. 44 in each sample. In a PCR tube, in addition to necessary reaction components and templates, a detection primer pair and a probe for any region of SEQ ID NOS: 3 to 8 and SEQ ID NO:44 are added at the same time, and a detection primer pair and a probe for an internal reference gene ACTB are also added. The probe of the detection target region is a Taqman probe, the report group at the 5 'end of the detection probe in any region of SEQ ID NO. 3-8 is FAM, and the quenching group at the 3' end is MGB; 44, the reporter group at the 5 'end of the detection probe is ROX, and the quenching group at the 3' end is MGB; the ACTB probe has VIC as the 5 'end reporter group and BHQ1 as the 3' end quenching group. Invitrogen Platinum II Taq hot start DNA polymerase was used for PCR amplification, the PCR reaction solution configuration system is shown in Table 12, and PCR amplification was performed according to the amplification procedure shown in Table 5.
TABLE 12
Negative and positive controls: when detecting blood samples, a negative control and a positive control also need to be detected simultaneously, and the DNA template of the negative control tube is TE buffer solution. In addition, the sequence of the region SEQ ID NO. 45 was artificially synthesized and cloned into a vector to form an artificially synthesized plasmid. The DNA templates for the positive control tubes were: 10 3 Copies/microliter of the artificially synthesized plasmid containing the transformed ACTB, 10 3 Copies/microliter of the synthetic plasmid of region (any of regions) 11 to 16 of SEQ ID NO and 10 3 Copies/microliter of the synthetic plasmid from the region of SEQ ID NO:45 were mixed in equal volumes. After the completion of the quantitative PCR reaction, the Ct value was read and the internal quality control was determined in the same manner as in example 1.
5. Analysis of results
If the Ct values of the negative control, the positive control and the reference gene all meet the experimental requirements, judging according to the following standards: for a blood sample, if the Ct value of a certain region amplified is less than or equal to 45, the region in the sample is considered to be methylation positive, and if the Ct value of a certain region amplified is more than 45, the region in the sample is considered to be methylation negative. If a sample to be detected is methylation positive in any region of SEQ ID NO. 3-8 and/or region of SEQ ID NO. 44, the sample is a colorectal cancer or precancerous lesion positive sample, and the sample is a non-cancerous sample only if the sample to be detected is methylation negative in any region of SEQ ID NO. 3-8 and is methylation negative in region of SEQ ID NO. 44. The methylation status, detection sensitivity and specificity of the combination of any region of SEQ ID NO. 3 to 8 and SEQ ID NO. 44 in each type of plasma samples are shown in Table 13.
Watch 13
As can be seen from Table 13, if the methylation levels of any region of SEQ ID NOS 3 to 8 and the composition of SEQ ID NO 44 are detected simultaneously, the colorectal cancer and precancerous lesion diagnosis performance is better improved compared with that of a single marker, and particularly, after the combination of SEQ ID NO 4 and SEQ ID NO 6 with SEQ ID NO 44 respectively, the detection sensitivity is greatly improved compared with that of the original respective detection sensitivity, and the detection specificity is also higher. For blood samples of advanced adenoma patients, the detection sensitivity of the composition of any region of SEQ ID NO. 3-8 and the region of SEQ ID NO. 44 is higher than 53.9%; for blood samples of stage I colorectal cancer patients, the detection sensitivity is higher than 70.3%; the detection sensitivity of the blood samples of middle and late colorectal cancer patients is higher than 85.4%; the specificity of detection is still higher than 91.5% for blood samples of healthy people. In addition, the combination of SEQ ID NO 3 and SEQ ID NO 44, and the combination of SEQ ID NO 8 and SEQ ID NO 44 have the best diagnostic performance, and have higher detection rates on advanced colorectal adenomas and early colorectal carcinoma.
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 various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the 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 (12)
1. Use of an agent for detecting the methylation level of a target region, said target region comprising the full length or partial region of the plus strand and/or minus strand of Chr7:142796895-142797358, in the preparation of a diagnostic product for colorectal cancer, using grch38.P14 as the reference genome.
2. The use of claim 1, wherein the target region comprises a full length or partial region of at least one of:
the positive strand of Chr7: 142796895-142797041;
the positive strand of Chr7: 142797056-142797207;
the plus strand of Chr7: 142797214-142797358;
the minus strand of Chr7: 142797358-142797249;
negative strand of Chr7:142797252-142797118 and
the minus strand of Chr7: 142797038-142796907.
3. The use of any one of claims 1-2, wherein the target region further comprises the full length or partial region of the minus strand of Chr8: 71843685-71843533.
4. A nucleic acid product for the diagnosis of colorectal cancer, wherein the nucleic acid product is capable of detecting the level of methylation of a region of interest, including the full length or a partial region of the positive and/or negative strand of Chr7:142796895-142797358, using grch38.P14 as the reference genome.
5. The nucleic acid product of claim 4, wherein the nucleic acid product comprises at least one of the following pairs of detection primers:
a first detection primer pair for detecting the methylation level of the full-length or partial region of the positive strand of Chr7: 142796895-142797041;
a second detection primer pair for detecting the methylation level of the full-length or partial region of the positive strand of Chr7: 142797056-142797207;
a third detection primer pair for detecting the methylation level of the full-length or partial region of the plus strand of Chr7: 142797214-142797358;
a fourth detection primer pair for detecting the methylation level of the full-length or partial region of the minus strand of Chr7: 142797358-142797249;
a fifth detection primer pair for detecting the methylation level of the full-length or partial region of the minus strand of Chr7: 142797252-142797118; and
a sixth detection primer pair for detecting the methylation level of the full-length or partial region of the minus strand of Chr7: 142797038-142796907.
6. The nucleic acid product of claim 5, wherein the nucleotide sequence of the first detection primer pair is shown as SEQ ID NO. 23-24; and/or the nucleotide sequence of the second detection primer pair is shown as SEQ ID NO. 26-27; and/or the nucleotide sequence of the third detection primer pair is shown as SEQ ID NO. 29-30; and/or the nucleotide sequence of the fourth detection primer pair is shown as SEQ ID NO: 32-33; and/or the nucleotide sequence of the fifth detection primer pair is shown as SEQ ID NO. 35-36; and/or the nucleotide sequence of the sixth detection primer pair is shown in SEQ ID NO 38-39.
7. The nucleic acid product of claim 6, further comprising a detection probe corresponding to the detection primer pair;
further, the nucleotide sequence of the first detection probe corresponding to the first detection primer pair is shown as SEQ ID NO. 25; and/or the nucleotide sequence of a second detection probe corresponding to the second detection primer is shown as SEQ ID NO. 28; and/or the nucleotide sequence of a third detection probe corresponding to the third detection primer is shown as SEQ ID NO. 31; and/or the nucleotide sequence of a fourth detection probe corresponding to the fourth detection primer is shown as SEQ ID NO. 34; and/or the nucleotide sequence of a fifth detection probe corresponding to the fifth detection primer pair is shown as SEQ ID NO. 37; and/or the nucleotide sequence of a sixth detection probe corresponding to the sixth detection primer pair is shown as SEQ ID NO. 40.
8. The nucleic acid product according to any one of claims 4 to 7, further comprising a seventh detection primer pair for detecting the methylation level of the full length or partial region of the minus strand of Chr8: 71843685-71843533;
optionally, the nucleotide sequence of the seventh detection primer pair is shown in SEQ ID NO. 46-47.
9. The nucleic acid product of claim 8, further comprising a detection probe corresponding to the seventh pair of detection primers;
optionally, the nucleotide sequence of the detection probe corresponding to the seventh detection primer pair is shown as SEQ ID NO. 48.
10. A kit for the detection of colorectal cancer comprising reagents for detecting the methylation level of a target region, said target region comprising the target region as claimed in any one of claims 1 to 3 for use.
11. The test kit of claim 10, wherein the test kit detects the methylation level of the target region 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 a target region comprises the nucleic acid product according to any one of claims 4 to 9.
12. The detection kit according to any one of claims 10 to 11, wherein the reagent for detecting the methylation level of the target region 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.
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