CN114561465A - Marker composition for detecting colorectal adenoma and early diagnosis reagent thereof - Google Patents
Marker composition for detecting colorectal adenoma and early diagnosis reagent thereof Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology, and particularly relates to a tumor marker combination for detecting colorectal adenocarcinoma at a progressive stage and a reagent for early diagnosis of the colorectal adenoma at the progressive stage. The invention takes feces as a detection sample, takes the DNA methylation of a target gene as a molecular marker, and detects the methylation level of the target gene as one or more of SYNPR, MEGF10, LSM2 and SLC32A1 genes. The result shows that the provided reagent for detecting single or combined genes of SYNPR, MEGF10, LSM2 and SLC32A1 can well distinguish colorectal advanced adenomas from normal human samples, and can be used for screening and early diagnosis of colorectal advanced adenomas. The invention also relates to specific primers and specific probes for detecting methylation of the SYNPR gene, the MEGF10 gene, the LSM2 gene and the SLC32A1 gene.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a reagent for detecting colorectal adenomas in a progressive stage and application thereof, and a reagent kit for detecting the colorectal adenomas in the progressive stage.
Background
Colorectal Cancer (CRC), also known as Colorectal Cancer, is a malignant tumor that is common today.
It is widely believed that CRC needs to pass through the development process of normal mucosal hyperplasia-adenoma-adenocarcinoma, most colorectal cancers originate from colorectal adenoma, the development of adenoma into colorectal cancer needs 10-15 years, the cure rate of colorectal cancer in the early stage can reach more than 90%, and the cure rate of advanced cancer is about 5%.
The traditional colorectal disease screening mainly comprises enteroscopy and stool occult blood tests, and the technologies have certain limitations. Enteroscopy is the gold standard for colorectal cancer diagnosis, but has strong invasiveness, tedious intestinal tract preparation and low compliance of Chinese colonoscopy screening. Fecal occult blood (FIT) is the most widely applied method for colorectal cancer screening at present, but has the defects of high false positive rate and the like. Therefore, there is a need to establish an accurate, noninvasive, simple and convenient method for early screening of colorectal adenomas in the advanced stage.
DNA methylation is an important gene expression regulation mechanism, can regulate the expression and silence of genes, has certain stability, is found to have abnormal DNA methylation in a plurality of cancers, and has close relation with the occurrence of the cancers. Therefore, DNA methylation abnormalities can be used as a biomarker for cancer diagnosis.
The DNA detection of the tumor cast-off cells in the excrement is a non-invasive screening method, and compared with a colonoscope and FIT, the technology has the advantages of non-invasive, painless, safety, convenience, high sensitivity and the like.
Disclosure of Invention
The invention aims to develop a marker combination for screening colorectal adenocarcinoma at the advanced stage, and a reagent and a method for early diagnosis of colorectal adenoma at the advanced stage, so as to realize noninvasive and early diagnosis of colorectal adenoma at the advanced stage.
In order to achieve the purpose, the invention is realized by the following technical scheme:
in a first aspect of the invention, the invention provides a reagent for detecting colorectal advanced adenoma, comprising a reagent for detecting methylation states of a SYNPR gene, an MEGF10 gene, an LSM2 gene and an SLC32A1 gene.
The detection object is a DNA sample obtained by extracting genomic DNA from human feces, and then converting and purifying with hydrogen sulfite.
In a second aspect of the present invention, the present invention provides a kit for detecting colorectal adenomas, where the kit includes the reagent for detecting colorectal adenomas in the first aspect.
The invention provides an application of SYNPR gene DNA methylation as a molecular marker in distinguishing colorectal advanced adenoma, wherein the target gene is positioned at cg09462808, cg04785972 and cg06314761 methylation sites of a 3 # chromosome transcription initiation region, and the CpG sites and DNA base sequences of 200bp upstream and downstream of the CpG sites are shown as SEQ ID NO: 1 is shown.
The invention provides an application of MEGF10 gene DNA methylation as a molecular marker in distinguishing colorectal advanced adenoma, wherein the target gene is positioned at cg08874609, cg04609576 and cg23018092 methylation sites of a transcription initiation region of chromosome 5, and the CpG sites and DNA base sequences of 200bp upstream and downstream of the CpG sites are shown as SEQ ID NO: 2, respectively.
The invention provides an application of LSM2 gene DNA methylation as a molecular marker in distinguishing colorectal advanced adenoma, wherein the target gene is located at cg13806231, cg01937314, cg07935151 and cg07797068 methylation sites of No. 6 chromosome exon regions, and the CpG sites and DNA base sequences of 200bp upstream and downstream of the CpG sites are shown as SEQ ID NO: 3, respectively.
The invention provides an application of SLC32A1 gene DNA methylation as a molecular marker in the judgment of colorectal progression adenoma, wherein the target gene is located at cg07033372, cg25307168 and cg12180703 methylation sites of a 20 # chromosome transcription initiation region, and the CpG sites and DNA base sequences of 200bp upstream and downstream of the CpG sites are shown as SEQ ID NO: 4, respectively.
The evaluation of the efficiency of detecting the colorectal progression adenoma by the DNA methylation markers and the combined markers of the single SYNPR gene, the MEGF10 gene, the LSM2 gene, the SLC32A1 gene shows that the area under the ROC curve (AUC) is 0.863-0.958; the optimal marker composition is SLC32A1-MEGF10, and AUC is 0.948.
In a third aspect of the invention, the invention provides an isolated nucleic acid sequence comprising a primer set and a probe.
In particular, at least one set of specific primer pairs and at least one specific probe for detecting the methylation state or level of the SYNPR gene; the nucleic acid sequence of the primer pair is at least one pair of sequences shown as SEQ ID NO. 5-SEQ ID NO. 14; the nucleic acid sequence of the probe is at least one of the sequences shown in SEQ ID NO. 37-SEQ ID NO. 39.
Specifically, at least one set of specific primer pairs and probes for detecting the methylation state or level of the MEGF10 gene; the nucleic acid sequence of the primer pair is at least one pair of sequences shown as SEQ ID NO. 15-SEQ ID NO. 24; the nucleic acid sequence of the probe is shown as SEQ ID NO. 40.
In particular, at least one set of specific primer pairs and probes for detecting the methylation state or level of the LSM2 gene; the nucleic acid sequence of the primer pair is at least one pair of sequences shown as SEQ ID NO. 25-SEQ ID NO. 34; the nucleic acid sequence of the probe is shown as SEQ ID NO. 41.
In particular, at least one set of specific primer pairs and probes for detecting the methylation status or level of the SLC32a1 gene; the nucleic acid sequence of the primer pair is shown as SEQ ID NO. 35-SEQ ID NO. 36; the nucleic acid sequence of the probe is shown as SEQ ID NO. 42.
Preferably, the probe is labeled with a fluorescence reporter group and a fluorescence quencher group.
More preferably, the probe is labeled with a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end.
Most preferably, the 5 'end labeled fluorescence reporter of the probe is FAM fluorophore, and the 3' end labeled fluorescence quencher is MGB.
Specifically, the kit further comprises: beta-actin detection primers and probes are used as controls.
Preferably, the beta-actin detection primer comprises: the primer comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.43, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 44.
Preferably, the nucleotide sequence of the beta-actin detection probe is shown as SEQ ID NO. 45.
More preferably, the beta-actin detection probe is marked with a fluorescent reporter group and a fluorescent quenching group.
More preferably, the beta-actin detection probe is labeled with a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end.
Most preferably, the fluorescence reporter group marked at the 5 'end of the beta-actin detection probe is a VIC fluorescent group, and the fluorescence quencher group marked at the 3' end is MGB.
In a fourth aspect of the invention, there is provided a method of assessing the colorectal cancer morbidity of a subject, comprising:
1. screening target genes comprising SYNPR, MEGF10, LSM2 and SLC32A1 genes by comparing the methylation degree of the colorectal adenoma sample and the normal sample in the database;
2. assessing the status of the target gene monogene and the combined detection;
3. and evaluating the diseased state of the adenoma in the colon-rectum progressive stage of the subject based on the target gene and the specific primer and probe thereof.
Compared with the prior art, the invention has the following beneficial effects:
(1) the diagnosis method provided by the invention takes the excrement as a detection sample, the sample is easy to obtain, the sampling process is convenient and simple, and any pain and influence on patients can not be caused.
(2) The invention discovers that SEQ ID NO: 1 to SEQ ID NO: 4, the DNA methylation molecular marker can be used for judging the colorectal tissue lesion progress, and the obtained colorectal advanced adenoma differential diagnosis model has better diagnosis efficiency, wherein the AUC is 0.863-0.958; the optimal marker combination was SLC32A1-MEGF10 with an AUC of 0.948.
(3) The DNA methylation molecular marker provided by the invention is used for judging the colorectal advanced adenoma, and has the characteristics of high sensitivity and high specificity. The optimal marker combination is SLC32A1-MEGF10, the detection sensitivity is 92.72%, and the specificity is 81.67%.
(4) The invention adopts fluorescent quantitative PCR to detect methylated DNA, designs specific primers and probes, and selects beta-actin as an internal reference gene, thereby ensuring the specificity and high sensitivity of detection. The provided DNA methylation molecular marker can assist in monitoring the occurrence of colon and rectum advanced adenoma.
The compositions referred to herein do not require that two or more of the materials be present in mixed contact, and reference to a composition is intended to be used in the same environment at the time of use.
In recent years, researches show that DNA methylation is an early event in the occurrence and development of colorectal cancer, so that methylation of specific genes can be used as a molecular marker for early diagnosis of tumors, and sensitivity and specificity of the methylation are obviously superior to those of traditional screening indexes such as blood carcinoembryonic antigen, fecal occult blood and the like. According to the theory of multiple stages of the process of carcinogenesis, CRC occurs through the process of development of adenoma-adenocarcinoma, with the vast majority of colorectal cancers originating from colorectal adenomas. The development of colorectal adenoma to colorectal carcinoma takes 5-10 years, and the early detection of the cure rate can reach more than 90%.
Further research finds that when compared with single gene methylation detection, multiple genes jointly detect colorectal tissue lesion, and detection sensitivity and specificity can be effectively improved.
The invention firstly develops the methylation target aiming at the detection of the adenoma in the colorectal progression stage. The early diagnosis reagent for detecting the methylation genes comprises a SYNPR gene, an MEGF10 gene, an LSM2 gene and an SLC32A1 gene which are used as biomarkers for detecting the colorectal advanced adenoma. The reagent for early diagnosis of colorectal adenoma in the advanced stage can detect the methylation levels of a SYNPR gene, an MEGF10 gene, an LSM2 gene and an SLC32A1 gene in feces by methylation specific quantitative PCR, and achieves the aim of screening colorectal adenoma noninvasively, sensitively, simply and quickly.
The term "primer" refers to a short nucleic acid sequence with a free 3' hydroxyl group that can form a base pair with a complementary template and serve as the origin of replication for the template strand. Under appropriate buffer and temperature conditions, the primers can initiate DNA synthesis in the presence of different nucleoside triphosphates and an agent for polymerization (e.g., DNA polymerase or reverse transcriptase).
The term "probe" refers to a fragment of a polynucleotide, such as RNA or DNA, that is capable of specifically binding to mRNA or complementary DNA (cdna) of a particular gene, and has a length of several to hundreds of base pairs. Since the probe is labeled, the probe can be used to check the presence or absence or expression level of target mRNA or cDNA to be bound.
Drawings
FIG. 1 is a flowchart of the detection for diagnosing colorectal progression adenoma provided in example 3.
FIG. 2 is a ROC plot of the genes SYNPR, MEGF10, LSM2 and SLC32A1 of example 3 tested alone for colorectal adenoma samples.
FIG. 3 is a ROC plot of the combination of any two genes of SYNPR, MEGF10, LSM2 and SLC32A1 in example 3 to detect samples of colorectal adenomas.
FIG. 4 is a ROC plot of samples of colorectal adenomas detected by any three of the combinations of SYNPR, MEGF10, LSM2 and SLC32A1 in example 3.
FIG. 5 is a ROC graph of the combination of four genes of SYNPR, MEGF10, LSM2 and SLC32A1 in example 3 to detect colorectal adenoma samples.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Example 1
The present inventors determined target nucleic acids for colorectal progression adenoma diagnosis according to the following methods and procedures:
step one, download the 450k methylation chip based dataset of colorectal adenomas from the GEO database, merge the matrices, and calculate the difference in the degree of methylation of each methylation site between the adenoma group and the normal group- β value (β value, from 0 to 1, indicates complete demethylation to complete methylation).
And step two, screening a methylation region. And (3) circularly screening by taking each CpG site as a center, wherein the screened methylation sites meet the following conditions:
1. screening at least three differential methylation sites within continuous 20 bp;
2. all methylation sites in each region are significantly different;
3. all methylation sites in each region were collectively enriched in either adenomas or normal groups.
And step three, screening methylation sites. Randomly selecting 2/3 samples from all the data sets in the step 1 as a training set, and selecting 1/3 samples as a verification set; the CpG sites contained in the methylation region selected in step 2 were modeled as a feature. Classifying training sets by using a random forest, sorting the importance of each feature according to the average descending kini coefficient of the feature, screening methylation sites with significant difference (FDR <0.05) in the methylation levels of a normal sample and an adenoma sample, and extracting top50 CpG sites.
And step four, constructing a model. And (3) selecting methylation regions where top50 CpG sites are located in the step three, and modeling all CpG sites in each methylation region as characteristics. The training set of top50 CpG sites, validation set AUC results, and the difference and annotation information of each CpG site were counted and integrated.
And step five, developing and designing a primer probe. And (3) performing gene function annotation on the top50 CpG sites obtained in the step (4), further screening the top50 CpG sites according to the train _ AUC/test _ AUC value, screening 4 genes with high test accuracy, and obtaining 200bp sequence information of the upstream and downstream of the genes. The gene sequence obtained above was developed and designed for methylation primer probes using Beacon Designer software.
TABLE 1 methylation site information
Example 2
First, DNA extraction of fecal samples
The specific extraction method can comprise the following steps:
according to the standard operating procedure of the QIAamp Fast DNA pool Mini Kit:
adding 1mL of Inhibitex Buffer into the sample, swirling for 1min until the sample is sufficiently homogenized, and centrifuging for 1min at 16000 g;
taking 25 mu L of protease K to a new centrifuge tube, sucking 600 mu L of supernatant of the previous step to the protease K tube, adding 600 mu L of Buffer AL, swirling for 15s, and incubating for 10min at 70 ℃;
adding 600 μ L ethanol, mixing by vortex, and centrifuging at 16000g for 1min for three times;
carefully add 500. mu.L Buffer AW1 to the column, centrifuge at 16000g for 1min, remove the liquid in the collection tube;
add 500. mu.L Buffer AW2 to the column, centrifuge at 16000g for 3min, remove the liquid in the collection tube;
centrifugation at 16000g was continued for 3min, and QIAamp spin column was transferred to a new 1.5mL centrifuge tube, 200. mu.L Buffer ATE was added, left at room temperature for 1min, and centrifuged at 16000g for 1min to obtain DNA for subsequent manipulations.
Conversion of di-and sulphite
And (3) carrying out sulfite conversion on the DNA obtained by extraction to obtain sulfite-converted DNA.
A specific sulfite conversion process may comprise the steps of:
zymo Research kit transformation protocol:
adding 130 μ L CT Conversion Reagent (used as ready-prepared) into the extracted 20 μ L DNA sample, mixing uniformly by vortex, and performing Conversion reaction at 98 deg.C, 10min, 64 deg.C, 2.5 hr;
adding 600 mu L M-Binding Buffer into an adsorption column, then adding the converted sample into the system, reversing for several times, uniformly mixing, centrifuging at 10000rpm for 30s, and absorbing and removing the lower layer solution;
adding 100 mu L M-Wash Buffer into the adsorption column, centrifuging at 10000rpm for 1min, and removing the lower layer solution by suction;
placing 200 mu L M-depletion Buffer in an adsorption column, standing at room temperature for 20min, centrifuging at 10000rpm for 1min, and removing the lower layer solution by suction;
adding 200 mu L M-Wash Buffer into the adsorption column, centrifuging at 10000rpm for 1min, and removing the lower layer solution by suction;
repeating the above steps;
the adsorption column was transferred to a new collection tube, 15. mu.L of the dilution Buffer was added, and after standing at room temperature for 5min, centrifugation was carried out at 10000rpm for 1 min.
DNA after sulfite conversion is obtained for subsequent detection.
It is explained here that the host DNA extracted from the feces is methylated according to the principle that sulfite can convert all unmethylated cytosines to uracil, while methylated cytosines are unchanged.
Tri, qMSP detection
The DNA transformed with sulfite was subjected to quantitative methylation PCR.
The detection result judgment standard is as follows:
firstly, ensuring that the sample meets the requirements: the internal reference gene meets the requirement (the internal reference is less than or equal to 35), and one or two target genes meet the condition that the amplification curve is normally exponentially increased and the delta Ct is less than or equal to 12; then, different weights were assigned to the SYNPR gene, the MEGF10 gene, the LSM2 gene, and the SLC32a1 gene, respectively, and the results were determined by a logical operation formula, in which a, b, c, and e were measured by clinical test data distribution.
The logical operation formula is as follows:
Score=ep/(1+ep)
p=aΔCt(SYNPR)+bΔCt(MEGF10)+cΔCt(LSM2)+dΔCt(SLC32A1)+E
then obtaining the Score value of the risk Score of the sample, determining a threshold value according to clinical distribution, and counting positive when the Score is greater than or equal to the threshold value Score; a Score value less than the threshold is considered negative.
(1) The PCR system is shown in Table 2:
TABLE 2 PCR System
Composition (I) | Addition amount (μ L) |
PCR probe primer premix solution | 2 |
Self-prepared 2 XTaqman Taq mix buffer | 15 |
DNA template after sulfite conversion | 13 |
Is totaled | 30 |
(1) The PCR reaction procedure is shown in table 3:
TABLE 3 PCR reaction procedure
Temperature (. degree.C.) | Time | Number of cycles |
95 | |
1 |
95 | 15s | 45 |
60 | 1min | 45 |
Example 3
The inventor uses the reagent for early detection of colorectal adenoma in the advanced stage, which comprises specific primers and probes for specifically detecting methylation of a SYNPR gene, an MEGF10 gene, an LSM2 gene and an SLC32A1 gene, and carries out methylation detection on a sample by the following method, wherein the steps are shown in figure 1 and comprise:
1. collecting samples:
a total of 151 colorectal progressing adenoma samples, 60 healthy human controls were collected.
2. Sample DNA extraction
The collected samples were subjected to DNA extraction using a commercial fecal sample DNA extraction kit. The specific extraction method comprises the following steps: according to the standard operating procedure of the QIAamp Fast DNA pool Mini Kit: adding 1mL of Inhibitex Buffer into the sample, swirling for 1min until the sample is sufficiently homogenized, and centrifuging for 1min at 16000 g; taking 25 mu L of proteinase K to a new centrifuge tube, sucking 600 mu L of supernatant of the previous step to the proteinase K tube, adding 600 mu L of Buffer AL, vortexing for 15s, and incubating for 10min at 70 ℃; adding 600 μ L ethanol, mixing by vortex, and centrifuging at 16000g for 1min for three times of QIAamppin column; carefully add 500. mu.L Buffer AW1 to the column, centrifuge at 16000g for 1min, remove the liquid in the collection tube; add 500. mu.L Buffer AW2 to the column, centrifuge at 16000g for 3min, remove the liquid in the collection tube; centrifugation at 16000g was continued for 3min, and QIAamp spin column was transferred to a new 1.5mL centrifuge tube, 200. mu.L Buffer ATE was added, left at room temperature for 1min, and centrifuged at 16000g for 1min to obtain DNA for subsequent manipulations.
3. Sulfite conversion
DNA was transformed with sulfite using the Zymo Research kit. And (3) carrying out sulfite conversion on the extracted DNA to obtain sulfite-converted DNA. A specific sulfite conversion process may comprise the steps of: zymo Research kit transformation protocol: adding 130 μ L CT Conversion Reagent (used as ready-prepared) into the extracted 20 μ L DNA sample, mixing uniformly by vortex, and performing Conversion reaction at 98 deg.C, 10min, 64 deg.C, 2.5 hr; adding 600 mu L M-Binding Buffer into an adsorption column, then adding the converted sample into the system, reversing for several times, uniformly mixing, centrifuging at 10000rpm for 30s, and absorbing the lower layer solution; adding 100 mu L M-Wash Buffer into the adsorption column, centrifuging at 10000rpm for 1min, and absorbing the lower solution; placing 200 mu L M-depletion Buffer in an adsorption column, standing at room temperature for 20min, centrifuging at 10000rpm for 1min, and removing the lower layer solution by suction; adding 200 mu L M-Wash Buffer into the adsorption column, centrifuging at 10000rpm for 1min, and absorbing the lower solution; repeating the above steps; the adsorption column was transferred to a new collection tube, 15. mu.L of the dilution Buffer was added, and after standing at room temperature for 5min, centrifugation was carried out at 10000rpm for 1 min. DNA after sulfite conversion is obtained for subsequent detection.
4. qMSP detection
In the present embodiment of the present invention,
the reagent for methylation of the SYNPR gene comprises a specific primer and a probe, and the primer group comprises: at least one pair of sequences shown in SEQ ID NO. 5-SEQ ID NO.14, wherein the probe comprises: at least one of the sequences shown in SEQ ID NO. 37-SEQ ID NO. 39;
the reagent for methylation of MEGF10 gene comprises a specific primer and a probe, wherein the primer group comprises: at least one pair of sequences shown in SEQ ID NO. 15-SEQ ID NO.24, wherein the probe comprises: a sequence shown as SEQ ID NO. 40;
the reagent for LSM2 gene methylation comprises specific primers and a probe, wherein the primer group comprises: at least one pair of sequences shown in SEQ ID NO. 25-SEQ ID NO.34, wherein the probe comprises: a sequence shown as SEQ ID NO. 41;
the reagent for methylation of SLC32A1 gene comprises specific primers and a probe, wherein the primer group comprises: SEQ ID NO. 35-36. The probe includes: the sequence shown in SEQ ID NO. 42.
The beta-actin gene detection primer comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.43, the nucleotide sequence of the reverse primer is shown as SEQ ID NO.44, and the nucleotide sequence of the beta-actin gene detection probe is shown as SEQ ID NO. 45.
The collected samples were subjected to fluorescent quantitative PCR assay according to the PCR system and reaction procedure of example 2 above.
TABLE 3 primer set and Probe sequence Listing
5. Analysis of results
And (3) discussing the sensitivity and specificity difference of methylation detection of different combinations such as single gene combination, two gene combination, three gene combination, four gene combination and the like, inspecting the models and ROC curves of different combinations, calculating to obtain corresponding AUC values, and determining the optimal marker combination.
The specific combinations and investigation results are as follows:
(1) single gene methylation assay results
Marker substance | Sensitivity of the probe | Specificity of | Area under subject curve |
SYNPR | 87.42% | 66.67% | 0.879 |
LSM2 | 88.74% | 66.67% | 0.863 |
MEGF10 | 86.75% | 60.00% | 0.902 |
SLC32A1 | 88.74% | 86.67% | 0.927 |
(2) Two genes joint methylation detection results (six combinations in total)
Marker substance | Sensitivity of the probe | Specificity of | Area under subject curve |
LSM2-SYNPR | 90.73% | 78.33% | 0.937 |
MEGF10-SYNPR | 90.73% | 78.33% | 0.939 |
SLC32A1-SYNPR | 92.05% | 86.67% | 0.945 |
MEGF10-LSM2 | 90.73% | 68.33% | 0.927 |
SLC32A1-LSM2 | 91.39% | 85.00% | 0.947 |
SLC32A1-MEGF10 | 92.72% | 81.67% | 0.948 |
(3) Three genes joint methylation detection results (four combinations in total)
(4) Detection result of combined methylation of four genes
When the SLC32A1-MEGF10-LSM2-SYNPR four genes are combined to be used as the biomarker, the sensitivity of detecting the colorectal advanced adenoma is 91.39%, the specificity is 88.33%, and the area under the curve of the subject is 0.958.
Wherein sensitivity is used to indicate the rate of diagnosis of colorectal progressing adenomas by the test, either on a positive sample for the final clinical pathology or on a patient.
Specificity refers to the ratio of samples that are ultimately pathologically normal or patients diagnosed as normal by the test.
The results show that the invention can obtain better detection performance than single gene detection by joint detection of a plurality of genes. The AUC (0.948) of the SLC32A1-MEGF10 combined marker is equivalent to the AUC of SLC32A1-MEGF10-SYNPR, SLC32A1-MEGF10-LSM2, SLC32A1-LSM2-SYNPR and SLC32A1-MEGF10-LSM2-SYNPR (0.954-0.958), and the AUC is larger than 0.9, so that the prompting accuracy is high, and the combination has high sensitivity and specificity in the detection of the colorectal advanced adenoma, but the detection cost of the latter is high. The optimal genome combination with high sensitivity and specificity and lower detection cost is SLC32A1-MEGF10, the AUC is 0.948, the detection sensitivity is 92.72%, the specificity is 81.67%, and the kit has higher diagnostic value on colorectal advanced adenoma.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and equivalent substitutions or changes according to the technical solution and the inventive concept of the present invention should be covered by the scope of the present invention.
Sequence listing
<110> Shanghai Sharp next Biotech Co., Ltd
<120> a marker composition for detecting colorectal adenoma and an early diagnostic reagent thereof
<130> 2010
<141> 2021-12-20
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<213> Artificial Sequence (Artificial Sequence)
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Claims (10)
1. An agent for detecting a colorectal-progression adenoma, comprising an agent for detecting a methylation state or level of at least one of a SYNPR gene, an meg 10 gene, an LSM2 gene, and an SLC32a1 gene.
2. The reagent for detecting adenoma in the colorectal progression according to claim 1, wherein the detection of methylation status or level is obtained by detecting at least one specific methylation site of SYNPR gene, MEGF10 gene, LSM2 gene, SLC32a1 gene in the sample, specifically as follows:
the specific methylation sites of the SYNPR gene are cg09462808, cg04785972 and/or cg06314761 methylation sites located in the transcription initiation region of chromosome 3, and the CpG sites and the DNA base sequences of 200bp upstream and downstream of the CpG sites are shown in SEQ ID NO: 1 is shown in the specification;
the specific methylation sites of the MEGF10 gene are cg08874609, cg04609576 and/or cg23018092 methylation sites positioned in the transcription initiation region of chromosome 5, and the CpG sites and the DNA base sequences of 200bp upstream and downstream of the CpG sites are shown in SEQ ID NO: 2 is shown in the specification;
the specific methylation sites of the LSM2 gene are cg13806231, cg01937314, cg07935151 and/or cg07797068 methylation sites located in exon regions of chromosome 6, and the CpG sites and DNA base sequences of 200bp upstream and downstream of the CpG sites are shown as SEQ ID NO: 3 is shown in the specification;
the specific methylation sites of the SLC32A1 gene are cg07033372, cg25307168 and/or cg12180703 methylation sites of a 20 chromosome transcription initiation region, and the CpG sites and the DNA base sequences of 200bp upstream and downstream of the CpG sites are shown in SEQ ID NO: 4, respectively.
3. The reagent for detecting advanced colorectal adenoma as claimed in claim 1, comprising: at least one set of specific primer pairs and at least one specific probe for detecting methylation status or level of the SYNPR gene;
the nucleic acid sequence of the first primer pair is shown as SEQ ID NO: 5 and SEQ ID NO: as shown in figure 6, the flow of the gas,
the nucleic acid sequence of the second primer pair is shown as SEQ ID NO: 7 and SEQ ID NO: as shown in figure 8, the flow of air,
the nucleic acid sequence of the third primer pair is shown as SEQ ID NO: 9 and SEQ ID NO: as shown in figure 10 of the drawings,
the nucleic acid sequence of the fourth primer pair is shown as SEQ ID NO: 11 and SEQ ID NO: as shown in figure 12 of the drawings,
the nucleic acid sequence of the fifth primer pair is shown as SEQ ID NO: 13 and SEQ ID NO: as shown in figure 14, the first and second,
the nucleic acid sequence of the probe is shown as SEQ ID NO: 37. SEQ ID NO: 38 or SEQ ID NO: shown at 39.
4. The reagent for detecting advanced colorectal adenoma as claimed in claim 1, comprising: at least one set of specific primer pairs and probes for detecting the methylation state or level of the MEGF10 gene;
the nucleic acid sequence of the sixth primer pair is shown as SEQ ID NO: 15 and SEQ ID NO: as shown at 16, the flow of the gas,
the nucleic acid sequence of the seventh primer pair is shown as SEQ ID NO: 17 and SEQ ID NO: as shown at 18, the flow of air is,
the nucleic acid sequence of the eighth primer pair is shown as SEQ ID NO: 19 and SEQ ID NO: as shown at 20, the flow of the gas,
the nucleic acid sequence of the ninth primer pair is shown as SEQ ID NO: 21 and SEQ ID NO: as shown at 22, the flow of air is,
the nucleic acid sequence of the tenth primer pair is shown as SEQ ID NO: 23 and SEQ ID NO: as shown at 24, the flow of the gas,
the nucleic acid sequence of the probe is shown as SEQ ID NO: shown at 40.
5. The reagent for detecting advanced colorectal adenoma as claimed in claim 1, comprising: at least one set of specific primer pairs and probes for detecting methylation status or level of LSM2 gene;
the nucleic acid sequence of the eleventh primer pair is shown as SEQ ID NO: 25 and SEQ ID NO: as shown at 26, the flow of the gas,
the nucleic acid sequence of the twelfth primer pair is shown as SEQ ID NO: 27 and SEQ ID NO: as shown at 28, the flow of the gas,
the nucleotide sequence of the thirteenth primer pair is shown as SEQ ID NO: 29 and SEQ ID NO: as shown at 30, the flow of the gas,
the nucleic acid sequence of the fourteenth primer pair is shown as SEQ ID NO: 31 and SEQ ID NO: as shown at 32, the flow of the gas,
the nucleic acid sequence of the fifteenth primer pair is shown as SEQ ID NO: 33 and SEQ ID NO: as shown at 34, the first and second side walls of the container,
the nucleic acid sequence of the probe is shown as SEQ ID NO: shown at 41.
6. The reagent for detecting advanced colorectal adenoma as claimed in claim 1, comprising: specific primer pairs and probes for detecting the methylation state or level of the SLC32a1 gene;
the nucleic acid sequence of the eleventh primer pair is shown as SEQ ID NO: 35 and SEQ ID NO: as shown at 36, the flow of gas is,
the nucleic acid sequence of the probe is shown as SEQ ID NO: shown at 42.
7. The reagent for detecting advanced adenoma in the colorectal cancer according to any one of claims 2 to 6, wherein cytosine in CpG is converted into 5' -methylcytosine by DNA methyltransferase catalysis; the probe is marked with a fluorescence reporter group and a fluorescence quenching group.
8. The reagent for detecting advanced adenoma in the colorectal region of claim 1, comprising a reagent for detecting the methylation status or level of SLC32A1 gene and MEGF10 gene simultaneously.
9. Use of a reagent according to any one of claims 1 to 8 for the detection of colorectal adenomas in the advanced stage of the colorectal adenoma for the preparation of an early diagnostic product for the detection of colorectal adenomas in the advanced stage of the colorectal adenoma.
10. A kit for detecting colorectal adenomas in advanced stage, comprising the reagent for detecting colorectal adenomas in any one of claims 1 to 8.
Priority Applications (1)
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US20110318738A1 (en) * | 2008-12-23 | 2011-12-29 | University Of Utah Research Foundation | Identification and regulation of a novel dna demethylase system |
US20140045915A1 (en) * | 2010-08-31 | 2014-02-13 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
US20130338020A1 (en) * | 2010-09-13 | 2013-12-19 | Commonwealth Scientific And Industrial Research Organisation | Epigenetic markers of colorectal cancers and diagnostic methods using the same |
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US20130065228A1 (en) * | 2011-06-01 | 2013-03-14 | University Of Southern California | Genome-scale analysis of aberrant dna methylation in colorectal cancer |
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