CN114561465B - Marker composition for detecting colorectal adenoma and early diagnosis reagent thereof - Google Patents

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a tumor marker combination for detecting adenoma in colorectal progression and a reagent for early diagnosis of adenoma in colorectal progression. The invention takes faeces as a detection sample and DNA methylation of a target gene as a molecular marker, wherein the target gene is methylated into one or more of SYNPR, MEGF10, LSM2 and SLC32A1 genes, and the methylation level of the target gene is detected. The results show that the provided reagent for detecting SYNPR, MEGF10, LSM2 and SLC32A1 genes can well distinguish colorectal adenoma from normal human samples, and can be used for screening and early diagnosis of colorectal adenoma in the progressive stage. The invention also relates to a specific primer and a specific probe for detecting methylation of SYNPR genes, MEGF10 genes, LSM2 genes and SLC32A1 genes.

Description

Marker composition for detecting colorectal adenoma and early diagnosis reagent thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a reagent for detecting adenoma in the colorectal progression stage and application thereof, and a kit for detecting adenoma in the colorectal progression stage.

Background

Colorectal cancer (Colorectal Cancer, CRC), also known as carcinoma of large intestine, is a common malignancy at present.

It is generally considered that CRC occurs through the development process of normal mucosa hyperplasia-adenoma-adenocarcinoma, most colorectal cancers originate from colorectal adenoma, the evolution from adenoma to colorectal cancer takes 10-15 years, the early detection cure rate of colorectal cancer can reach more than 90%, and the early detection cure rate of colorectal cancer is about 5%.

Traditional colorectal disease screening is mainly performed by enteroscopy and fecal occult blood test, and the technologies have certain limitations. Enteroscopy is a gold standard for colorectal cancer diagnosis, but has strong invasiveness and complicated intestinal preparation, and has low compliance in Chinese colonoscopy screening. Fecal occult blood (FIT) is the most widely used method for screening colorectal cancer at present, but has the defects of higher false positive rate and the like. Thus, there is a need to establish an accurate, noninvasive, simple method for early screening of colorectal progressive adenomas.

DNA methylation is an important gene expression control mechanism, can regulate the expression and silencing of genes, has certain stability, and can be found to have abnormal DNA methylation in many cancers, thus having close relation with the occurrence of cancers. Thus, DNA methylation abnormalities can be used as a biomarker for cancer diagnosis.

The detection of tumor cell DNA in feces is a non-invasive screening method, and compared with colonoscopy and FIT, the technology has the advantages of no wound, no pain, safety, convenience, high sensitivity and the like.

Disclosure of Invention

The invention aims to develop a marker combination for screening colorectal adenoma in the progressive stage, and a reagent and a method for diagnosing the colorectal adenoma in the progressive stage in early stage so as to realize noninvasive and early-stage diagnosis of the colorectal adenoma in the progressive stage.

In order to achieve the above purpose, the present invention is realized by the following technical scheme:

in a first aspect, the invention provides a reagent for colorectal advanced adenoma detection comprising reagents for detecting methylation status of the SYNPR gene, the MEGF10 gene, the LSM2 gene and the SLC32A1 gene.

The detection object is a DNA sample obtained by extracting genome DNA from human feces, then converting and purifying the genome DNA by using hydrogen sulfite.

In a second aspect, the present invention provides a kit for detection of colorectal progressive adenoma, the kit comprising the reagent for colorectal progressive adenoma detection of the first aspect described above.

The invention provides an application of SYNPR gene DNA methylation as a molecular marker in judging colorectal adenoma in the progressive stage, wherein the target gene is positioned at cg09462808, cg04785972 and cg06314761 methylation sites of a transcription initiation region of chromosome 3, and the CpG sites and 200bp DNA base sequences upstream and downstream of the CpG sites are shown as SEQ ID NO: 1.

The invention provides an application of MEGF10 gene DNA methylation as a molecular marker in judging colorectal adenoma in the progressive stage, wherein the target gene is positioned at cg08874609, cg04609576 and cg23018092 methylation sites of a chromosome 5 transcription initiation region, and the CpG sites and 200bp DNA base sequences upstream and downstream of the CpG sites are shown as SEQ ID NO: 2.

The invention provides application of LSM2 gene DNA methylation as a molecular marker in judging adenomas in colorectal progression, wherein the target genes are positioned at cg13806231, cg01937314, cg07935151 and cg07797068 methylation sites in the exon region of No. 6, and the CpG sites and 200bp DNA base sequences upstream and downstream of the CpG sites are shown as SEQ ID NO: 3.

The invention provides application of SLC32A1 gene DNA methylation as a molecular marker in identifying colorectal adenoma in the advanced stage, wherein the target gene is positioned at cg07033372, cg25307168 and cg12180703 methylation sites of a 20 # chromosome transcription initiation region, and the CpG sites and 200bp DNA base sequences at the upstream and downstream of the CpG sites are shown as SEQ ID NO: 4.

By evaluating the DNA methylation markers and the combined markers of the single SYNPR gene, the MEGF10 gene, the LSM2 gene and the SLC32A1 gene, the area under the ROC curve (AUC) of the single SYNPR gene is 0.863-0.958; the optimal marker composition was SLC32A1-MEGF10 with an AUC of 0.948.

In a third aspect of the invention, the invention provides an isolated nucleic acid sequence comprising a primer set and a probe.

Specifically, 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 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 sequences shown as SEQ ID NO. 37-SEQ ID NO. 39.

Specifically, at least one set of specific primer pairs and probes for detecting the methylation status 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.

Specifically, at least one set of specific primer pairs and probes for detecting the methylation status 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.

Specifically, 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 fluorescent reporter group and a fluorescent quenching group.

More preferably, the probe has a fluorescent reporter group at the 5 'end and a fluorescent quenching group at the 3' end.

Most preferably, the 5 '-end-labeled fluorescent reporter group of the probe is a FAM fluorescent group and the 3' -end-labeled fluorescent quenching group is MGB.

Specifically, the kit further comprises: beta-actin detection primer and probe are used as contrast.

Preferably, the beta-actin detection primer comprises: 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 fluorescence report group and a fluorescence quenching group.

More preferably, the 5 'end of the beta-actin detection probe is marked with a fluorescent report group, and the 3' end is marked with a fluorescent quenching group.

Most preferably, the fluorescent reporter group marked at the 5 'end of the beta-actin detection probe is a VIC fluorescent group, and the fluorescent quenching group marked at the 3' end is an MGB.

In a fourth aspect of the invention, the invention provides a method of assessing the condition of colorectal cancer in a subject comprising:

1. screening target genes including SYNPR, MEGF10, LSM2 and SLC32A1 genes by comparing methylation degrees of colorectal adenoma samples and normal samples in a database;

2. evaluating the single gene of the target gene and the state of joint detection;

3. based on the target gene and the specific primer and probe thereof, the adenoma disease state of the subject in the development stage of the colon straight intestine is estimated.

Compared with the prior art, the invention has the following beneficial effects:

(1) The diagnosis method provided by the invention uses the feces as a detection sample, the sample is easy to obtain, the sampling process is convenient and simple, and no pain and influence are caused to patients.

(2) The invention discovers SEQ ID NO for the first time: 1-SEQ ID NO:4 can be used for judging the pathological change progress of colorectal tissues, and the obtained colorectal advanced adenoma differential diagnosis model has better diagnosis efficiency and AUC of 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 distinguishing adenoma in the colorectal development stage, 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 the methylated DNA, designs specific primers and probes, selects beta-actin as an internal reference gene, and ensures the specificity and high sensitivity of detection. The provided DNA methylation molecular marker can assist in monitoring the occurrence of adenomas in the progression stage of the colon.

The compositions mentioned herein do not require that two or more substances must be present in mixed contact, and the compositions mentioned are intended to be applied in the same environment at the time of use.

In recent years, research shows that DNA methylation is an early event in colorectal cancer occurrence and development, so that methylation of specific genes can be used as a molecular marker for early diagnosis of tumors, and the sensitivity and the specificity of the methylation are obviously superior to those of traditional screening indexes such as carcinoembryonic antigen, fecal occult blood and the like. According to the multistage theory of the cancerous process, CRC occurs through the progression of adenoma-adenocarcinomas, the vast majority of colorectal cancers originate from colorectal adenomas. Colorectal adenoma takes 5-10 years to develop to colorectal cancer, and the early detection cure rate can reach more than 90%.

Further research shows that compared with single gene methylation detection, the multi-gene combined detection of colorectal tissue lesions can effectively improve detection sensitivity and specificity.

The invention aims at methylation target development for colorectal adenoma detection in the advanced stage for the first time. The early diagnosis reagent for detecting methylation genes including SYNPR gene, MEGF10 gene, LSM2 gene and SLC32A1 gene is used as a biomarker for detecting colorectal adenoma in the progressive stage. The early diagnosis reagent for colorectal adenoma in the progressive stage can detect methylation levels of SYNPR gene, MEGF10 gene, LSM2 gene and SLC32A1 gene in feces through methylation specific quantitative PCR, so that the aim of noninvasively, sensitively, simply and rapidly screening colorectal adenoma is fulfilled.

The term "primer" refers to a short nucleic acid sequence with a free 3' hydroxyl group that can form base pairs with a complementary template and serve as an origin of replication of the template strand. Under appropriate buffer and temperature conditions, the primers may initiate DNA synthesis in the presence of different nucleoside triphosphates and a polymerization reagent (e.g., DNA polymerase or reverse transcriptase).

The term "probe" refers to a fragment of a polynucleotide, such as RNA or DNA, capable of specifically binding mRNA or complementary DNA (cDNA) of a particular gene, and having a length of several to hundreds of base pairs. Since the probe is labeled, the probe can be used to check for the presence or expression level of the target mRNA or cDNA to be bound.

Drawings

FIG. 1 is a flow chart of the test provided in example 3 for the diagnosis of colorectal progressive adenoma.

FIG. 2 is a ROC graph of SYNPR, MEGF10, LSM2 and SLC32A1 gene alone for colorectal adenoma samples of example 3.

FIG. 3 is a ROC graph of a sample of colorectal adenoma detected by any two of the gene combinations SYNPR, MEGF10, LSM2 and SLC32A1 of example 3.

FIG. 4 is a ROC graph of a sample of colorectal adenoma detected by any three of the combinations of genes SYNPR, MEGF10, LSM2 and SLC32A1 of example 3.

FIG. 5 is a ROC graph of a sample of colorectal adenoma detected by a combination of four genes of SYNPR, MEGF10, LSM2 and SLC32A1 of example 3.

Detailed Description

The invention is further illustrated below in connection with specific embodiments.

Example 1

The inventors determined the target nucleic acid for diagnosis of colorectal advanced adenoma according to the following methods and steps:

step one, downloading the 450k methylation chip-based dataset of colorectal adenomas from the GEO database, combining the matrices, and calculating the differential- β value (β value, from 0 to 1, representing complete demethylation to complete methylation) of the degree of methylation at each methylation site between the adenoma group and the normal group.

Step two, screening methylation areas. And circularly screening by taking each CpG site as a center, wherein the screened methylation site needs to meet the following conditions:

1. screening at least three different methylation sites within 20 bp;

2. all methylation sites in each region are significantly different;

3. all methylation sites in each region are enriched together in adenomas or in the normal group.

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 taking 1/3 samples as a verification set; modeling the CpG sites contained in the methylation area screened in the step 2 as characteristics. The training set is classified by using random forests, the importance of each feature is ordered according to the average decreasing coefficient of the feature, methylation sites with obvious difference (FDR < 0.05) in methylation level of normal samples and adenoma samples are screened, and top50 CpG sites are extracted.

And fourthly, constructing a model. And (3) screening methylation areas where the obtained top50 CpG sites are located in the step (III), and modeling all CpG sites in each methylation area as characteristics. The training set, validation set AUC results for top50 CpG sites were counted and integrated as well as the difference and annotation information for each CpG site.

And fifthly, developing and designing a primer probe. And (3) carrying out gene function annotation according to the top50 CpG sites obtained in the step (4), further screening the top50 CpG sites according to the train_AUC/test_AUC values, screening 4 genes with high verification test accuracy, and obtaining 200bp sequence information on the upstream and downstream of the genes. The methylation primer probe was developed and designed for the gene sequence obtained above using Beacon designer software.

TABLE 1 methylation site information

Example 2

1. DNA extraction of fecal samples

The specific extraction method can comprise the following steps:

the standard operation procedure of the QIAamp Fast DNA Stool Mini Kit kit is as follows:

adding 1mL InhibitEX Buffer to 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 from the previous step to the proteinase K tube, adding 600 mu L of Buffer AL, swirling for 15s, and placing at 70 ℃ for incubation for 10min;

600 μl of ethanol was added and vortexed and mixed well, divided into three times QIAampspin column, and centrifuged at 16000g for 1min each time;

500. Mu.L Buffer AW1 was carefully added to the column and centrifuged at 16000g for 1min to remove liquid from the collection tube;

500. Mu.L Buffer AW2 was added to the column, and the mixture was centrifuged at 16000g for 3min to remove the liquid in the collection tube;

continue centrifugation at 16000g for 3min, transfer QIAamp spin column to a new 1.5mL centrifuge tube, add 200. Mu.L Buffer ATE, leave at room temperature for 1min, and centrifuge at 16000g for 1min to obtain DNA for subsequent manipulation.

2. Sulfite conversion

And (3) performing sulfite conversion on the DNA obtained by extraction to obtain the DNA subjected to sulfite conversion.

The specific sulfite conversion process may comprise the steps of:

zymo Research kit transformation procedure:

adding 130 mu L CT Conversion Reagent (for preparation) into the extracted 20 mu L DNA sample, and carrying out a conversion reaction according to the conditions of (98 ℃,10min;64 ℃ and 2.5 h) after vortex mixing uniformly;

adding 600 mu L M-Binding Buffer into the adsorption column, then adding the converted sample into the system, reversing for mixing for several times, 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 removing the lower layer solution;

placing in an adsorption column with 200 mu L M-Desulphonation Buffer at room temperature for 20min, centrifuging at 10000rpm for 1min, and removing the lower layer solution;

adding 200 mu L M-Wash Buffer into the adsorption column, centrifuging at 10000rpm for 1min, and removing the lower layer solution;

repeating the steps;

the adsorption column was transferred to a new collection tube, added 15 μ L MElution Buffer, left at room temperature for 5min, and centrifuged at 10000rpm for 1min.

The sulfite-converted DNA was obtained for subsequent detection.

It is described herein that the methylation of host DNA extracted from feces is performed according to the principle that "sulfite is capable of converting all unmethylated cytosine to uracil, while methylated cytosine is unchanged".

3. qMSP detection

Methylation quantitative PCR was performed on the sulfite-converted DNA.

The detection result judging standard is as follows:

firstly, ensuring that the sample meets the requirements: the reference genes meet the requirement (the reference is less than or equal to 35), and one or two target genes meet the condition that the amplification curve normally grows exponentially and the delta Ct is less than or equal to 12; then, SYNPR gene, MEGF10 gene, LSM2 gene and SLC32A1 gene are respectively given different weights, and the result is judged through a logic operation formula, wherein a, b, c and e are determined through clinical test data distribution.

The logical operation formula is:

Score＝e ^p /(1+e ^p )

p＝aΔCt(SYNPR)+bΔCt(MEGF10)+cΔCt(LSM2)+dΔCt(SLC32A1)+E

then the risk Score value of the sample can be obtained, and positive is counted when the risk Score value is greater than or equal to the threshold Score value according to the determination threshold value determined by clinical distribution; score values less than the threshold value were counted as negative.

(1) The PCR system is shown in Table 2:

TABLE 2 PCR System

Composition of the components	Addition amount (mu L)
		PCR probe primer premix	2
Self-assembled 2X Taqman Taq mix buffer	15
		Sulfite converted DNA template	13
Totalizing	30

(1) The PCR reaction procedure is shown in Table 3:

TABLE 3 PCR reaction procedure

Temperature (. Degree. C.)	Time	Cycle number
			95	15min	1
95	15s	45
			60	1min	45

Example 3

The inventor uses the reagent for early detection of colorectal adenoma in progressive stage, comprising specific primers and probes for specifically detecting methylation of SYNPR gene, MEGF10 gene, LSM2 gene and SLC32A1 gene, and the methylation detection is carried out on a sample by the following method, wherein the steps are shown in figure 1, and the reagent comprises the following steps:

1. sample collection:

a total of 151 colorectal advanced adenoma samples and 60 healthy human controls were collected.

2. Sample DNA extraction

DNA extraction was performed on the collected samples using commercial fecal sample DNA extraction kits. The specific extraction method comprises the following steps: the standard operation procedure of the QIAamp Fast DNA Stool Mini Kit kit is as follows: adding 1mL InhibitEX Buffer to 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 from the previous step to the proteinase K tube, adding 600 mu L of Buffer AL, vortexing for 15s, and incubating at 70 ℃ for 10min; 600 μl of ethanol was added and vortexed and mixed well, divided into three times QIAampspin column, and centrifuged at 16000g for 1min each time; 500. Mu.L Buffer AW1 was carefully added to the column and centrifuged at 16000g for 1min to remove liquid from the collection tube; adding 500 mu L Buffer AW2 to the column, centrifuging at 16000g for 3min, and removing liquid in the collecting tube; continue centrifugation at 16000g for 3min, transfer QIAamp spin column to a new 1.5mL centrifuge tube, add 200. Mu.L Buffer ATE, leave at room temperature for 1min, and centrifuge at 16000g for 1min to obtain DNA for subsequent manipulation.

3. Sulfite conversion

The DNA was sulfite converted using the Zymo Research kit. And (3) performing sulfite conversion on the extracted DNA to obtain sulfite converted DNA. The specific sulfite conversion process may comprise the steps of: zymo Research kit conversion procedure: adding 130 mu L CT Conversion Reagent (for preparation) into the extracted 20 mu L DNA sample, and carrying out a conversion reaction according to the conditions of (98 ℃,10min;64 ℃ and 2.5 h) after vortex mixing uniformly; adding 600 mu L M-Binding Buffer into the adsorption column, adding the converted sample into the system, reversing for mixing for several times, 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 removing the lower layer solution; placing in an adsorption column with 200 mu L M-Desulphonation Buffer at room temperature for 20min, centrifuging at 10000rpm for 1min, and removing the lower layer solution; adding 200 mu L M-Wash Buffer into the adsorption column, centrifuging at 10000rpm for 1min, and removing the lower layer solution; repeating the steps; the adsorption column was transferred to a new collection tube, added 15 μ L MElution Buffer, left at room temperature for 5min, and centrifuged at 10000rpm for 1min. The sulfite-converted DNA was obtained for subsequent detection.

4. qMSP detection

In the present embodiment of the present invention, in the present embodiment,

reagents for methylation of the SYNPR gene comprise specific primers and probes, the primer set comprising: 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 to SEQ ID No. 39;

reagents for the methylation of the MEGF10 gene comprise specific primers and probes, the primer set comprising: at least one pair of sequences shown in SEQ ID No. 15-SEQ ID No.24, wherein the probe comprises: SEQ ID NO. 40;

the LSM2 gene methylation reagent comprises specific primers and probes, wherein the primer group comprises: at least one pair of sequences shown in SEQ ID No.25 to SEQ ID No.34, wherein the probe comprises: SEQ ID NO. 41;

the reagent for methylation of the SLC32A1 gene comprises a specific primer and a probe, wherein the primer group comprises: SEQ ID NO. 35-SEQ ID NO. 36. The probe includes: 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 detection probe is shown as SEQ ID NO. 45.

The collected samples were subjected to fluorescent quantitative PCR detection according to the PCR system and the reaction procedure of example 2 described above.

TABLE 3 primer set and probe sequence listing

5. Analysis of results

Sensitivity and specificity differences of methylation detection of different combinations such as single gene, two gene combinations, three gene combinations, four gene combinations and the like are discussed, models and ROC curves of the different combinations are examined, corresponding AUC values are obtained through calculation, and the optimal marker combination is determined.

The specific combination and investigation results are as follows:

(1) Single gene methylation detection results

Marker(s)	Sensitivity of	Specificity (specificity)	Area under the curve of the subject
				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 were combined with methylation detection results (six combinations in total)

Marker(s)	Sensitivity of	Specificity (specificity)	Area under the curve of the subject
				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 combined methylation detection results (four combinations in total)

(4) Four genes combined methylation detection results

When four genes of SLC32A1-MEGF10-LSM2-SYNPR are combined to serve as biomarkers, the sensitivity of detecting colorectal adenoma is 91.39%, the specificity is 88.33%, and the area under the curve of a subject is 0.958.

Wherein sensitivity is used to indicate the ratio of positive samples diagnosed by detection with colorectal advanced adenomas for the final clinical pathology or colorectal advanced adenomas for the patient.

Specificity refers to the ratio of samples or patients diagnosed as normal to the final clinical pathology diagnosis by detection.

The results show that the invention can obtain better detection performance than single gene detection through the combined detection of a plurality of genes. The AUC (0.948) of the SLC32A1-MEGF10 combined marker is equal to the AUC of the SLC32A1-MEGF10-SYNPR, the SLC32A1-MEGF10-LSM2, the SLC32A1-LSM2-SYNPR and the SLC32A1-MEGF10-LSM2-SYNPR (0.954-0.958), which are all larger than 0.9, the prompt accuracy is higher, and the combination has higher sensitivity and specificity in detecting adenoma in the colorectal progression period, but the detection cost of the latter is higher. The optimal gene combination with high sensitivity and specificity and lower detection cost is SLC32A1-MEGF10, AUC is 0.948, detection sensitivity is 92.72%, specificity is 81.67%, and the kit has higher diagnostic value for adenoma in colorectal progressive stage.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art who is skilled in the art to which the present invention pertains should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof within the scope of the present invention.

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<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tacaccaata tctataaacc ctacctcg 28

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gggaaggttg ttattggtta gtcg 24

<210> 9

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

caccaatatc tataaaccct acctcg 26

<210> 10

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

gggaaggttg ttattggtta gtcg 24

<210> 11

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tacaccaata tctataaacc ctacctcg 28

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gggaaggttg ttattggtta gtcg 24

<210> 13

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

ccaatatcta taaaccctac ctcgc 25

<210> 14

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

gggaaggttg ttattggtta gtcg 24

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

tttgtattta acgtgggttg ggaat 25

<210> 16

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

cgacctaact actacacctc gatc 24

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

gatttgtatt taacgtgggt tggga 25

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

cgacctaact actacacctc gatc 24

<210> 19

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

tgtatttaac gtgggttggg aatt 24

<210> 20

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

cgacctaact actacacctc gatc 24

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

tatttaacgt gggttgggaa ttagg 25

<210> 22

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

cgacctaact actacacctc gatc 24

<210> 23

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

gagggatttg tatttaacgt gggt 24

<210> 24

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

cgacctaact actacacctc gatc 24

<210> 25

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

tttgacgtta cggtatttat tatggt 26

<210> 26

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

cgaactcgcg cttcgatt 18

<210> 27

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

tcggatttaa ttttagttta ggggtat 27

<210> 28

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

cgaactcgcg cttcgatt 18

<210> 29

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

gacgttacgg tatttattat ggtgtt 26

<210> 30

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

cgaactcgcg cttcgatt 18

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

gaggttttcg agggcgtttt 20

<210> 32

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

cgaactcgcg cttcgatt 18

<210> 33

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

tgacgttacg gtatttatta tggtgt 26

<210> 34

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

cgaactcgcg cttcgatt 18

<210> 35

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

tacctccgaa tatttcctct ctcc 24

<210> 36

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

gttagagtag ttacgcgaag ttgg 24

<210> 37

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

ccacctcctc gccctcccct ctaa 24

<210> 38

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

ccacctcctc gccctcccct ctaaa 25

<210> 39

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

cacctcctcg ccctcccctc taaaac 26

<210> 40

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 40

ccctacctaa acgacttcca ccgcc 25

<210> 41

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

cccaaaccta ctcgcaacac cctact 26

<210> 42

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 42

ccccgcaccg cctaattccg aaaa 24

<210> 43

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 43

gtgatggagg aggtttagta a 21

<210> 44

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 44

ccaataaaac ctactcctcc ct 22

<210> 45

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 45

cacccaacac acaataacaa acaca 25

Claims (7)

1. Use of a reagent for the preparation of an early diagnostic product for colorectal advanced adenoma, characterized in that the reagent is a reagent for detecting the methylation state or level of a gene;

wherein the reagent comprises a reagent for detecting SLC32A1-MEGF10 composite markers; reagent for detecting SLC32A1-MEGF10-SYNPR combined marker, SLC32A1-MEGF10-LSM2 combined marker, SLC32A1-LSM2-SYNPR combined marker and SLC32A1-MEGF10-LSM2-SYNPR combined marker;

the specific methylation sites of the SYNPR gene are cg09462808, cg04785972 and cg06314761 methylation sites located in the transcription initiation region of chromosome 3, and the DNA base sequences of the sites and 200bp upstream and downstream thereof are shown in SEQ ID NO:1 is shown in the specification;

specific methylation sites of the MEGF10 gene are cg08874609, cg04609576 and cg23018092 methylation sites located in a chromosome 5 transcription initiation region, and DNA base sequences of the sites and 200bp upstream and downstream thereof are shown in SEQ ID NO:2 is shown in the figure;

specific methylation sites of the LSM2 gene are cg13806231, cg01937314, cg07935151 and cg07797068 methylation sites located in the exon region of chromosome 6, and DNA base sequences of the sites and 200bp upstream and downstream thereof are shown in SEQ ID NO:3 is shown in the figure;

the specific methylation sites of the SLC32A1 gene are cg07033372, cg25307168 and cg12180703 methylation sites of a 20 # chromosome transcription initiation region, and the DNA base sequences of the sites and 200bp upstream and downstream thereof are shown in SEQ ID NO: 4.

2. Use of an agent according to claim 1 for the preparation of an early diagnostic product for colorectal advanced adenoma, comprising: a specific primer pair and a specific probe for detecting methylation status or level of 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 number of the holes in the steel plate,

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 nucleic acid sequence of the third primer pair is shown as SEQ ID NO:9 and SEQ ID NO: as shown in the drawing 10,

the nucleic acid sequence of the fourth primer pair is shown as SEQ ID NO:11 and SEQ ID NO: as shown in the drawing 12,

the nucleic acid sequence of the fifth primer pair is shown as SEQ ID NO:13 and SEQ ID NO: as shown in the drawing 14,

the nucleic acid sequence of the probe is shown as SEQ ID NO: 37. SEQ ID NO:38 and SEQ ID NO: 39.

3. Use of an agent according to claim 1 for the preparation of an early diagnostic product for colorectal advanced adenoma, comprising: 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 indicated by the numeral 16,

the nucleic acid sequence of the seventh primer pair is shown as SEQ ID NO:17 and SEQ ID NO: as indicated at 18, the number of the cells,

the nucleic acid sequence of the eighth primer pair is shown as SEQ ID NO:19 and SEQ ID NO: as shown in the drawing 20,

the nucleic acid sequence of the ninth primer pair is shown as SEQ ID NO:21 and SEQ ID NO: as indicated by the reference numeral 22,

the nucleic acid sequence of the tenth primer pair is shown as SEQ ID NO:23 and SEQ ID NO: as indicated at 24, the number of the cells,

the nucleic acid sequence of the probe is shown as SEQ ID NO: 40.

4. Use of an agent according to claim 1 for the preparation of an early diagnostic product for colorectal advanced adenoma, comprising: specific primer pairs and probes for detecting the methylation status or level of the LSM2 gene;

the nucleic acid sequence of the eleventh primer pair is shown as SEQ ID NO:25 and SEQ ID NO: as indicated at 26, the number of the cells,

the nucleic acid sequence of the twelfth primer pair is shown as SEQ ID NO:27 and SEQ ID NO: as indicated at 28, the number of the cells in the cell,

the nucleic acid sequence of the thirteenth primer pair is shown as SEQ ID NO:29 and SEQ ID NO: as indicated by the numeral 30,

the nucleic acid sequence of the fourteenth primer pair is shown in SEQ ID NO:31 and SEQ ID NO: as indicated by the numeral 32,

the nucleic acid sequence of the fifteenth primer pair is shown in SEQ ID NO:33 and SEQ ID NO: as indicated by the numeral 34,

the nucleic acid sequence of the probe is shown as SEQ ID NO: shown at 41.

5. Use of an agent according to claim 1 for the preparation of an early diagnostic product for colorectal advanced adenoma, 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 indicated by the numeral 36,

the nucleic acid sequence of the probe is shown as SEQ ID NO: shown at 42.

6. Use of an agent according to any one of claims 2-5 for the preparation of an early diagnostic product of colorectal advanced adenoma, characterized in that cytosine in the specific methylation site is converted to 5' -methylcytosine by catalysis of DNA methyltransferase; the probe is marked with a fluorescence report group and a fluorescence quenching group.

7. The use of an agent according to claim 1 for the preparation of an early diagnostic product for colorectal advanced adenoma comprising an agent that simultaneously detects the methylation status or levels of the SLC32A1 gene and the MEGF10 gene.