CN114561464A

CN114561464A - Primer probe set and kit for diagnosing, detecting or screening advanced adenoma

Info

Publication number: CN114561464A
Application number: CN202111565525.6A
Authority: CN
Inventors: 刘晶; 吴春燕; 陈煜庶; 王云霞; 王美丛
Original assignee: Shanghai Realbio Technology Co ltd
Current assignee: Qingdao Ruiyi Precision Medical Test Co ltd; Shanghai Realbio Technology Co ltd
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2022-05-31

Abstract

The invention discloses a methylation marker for early screening of advanced adenoma, a primer probe combination and application. Specific DNA methylation markers related to early diagnosis of advanced adenomas mainly screened by public databases comprise RXRG, ISLR2, EVC2, ZNF793 and NKAIN4 gene methylation. The DNA methylation molecular marker disclosed by the invention can be used for identifying advanced adenomas, and the obtained model has better differential diagnosis efficiency, wherein the AUC is 0.926-0.970. The optimal marker combination is ISLR2-RXRG, the AUC is 0.970, and the marker has the characteristics of high sensitivity and high specificity.

Description

Primer probe set and kit for diagnosing, detecting or screening advanced adenoma

Technical Field

The invention relates to the technical field of early screening of advanced adenomas, in particular to a methylation marker combination and a primer probe combination for early screening of advanced adenomas and a kit for early screening of advanced adenomas.

Background

Colorectal cancer is one of the most common digestive tract tumors in China, and the incidence rate and the fatality rate of the colorectal cancer tend to rise year by year. Therefore, the morbidity and mortality of colorectal cancer in China are reduced, and the important public health problem to be solved urgently is formed.

According to the multi-stage theory of the process of carcinogenesis, colorectal cancer occurs morphologically as normal mucosal hyperplasia, adenomatous formation, staged evolution of adenoma canceration to invasive metastasis, with the majority of colorectal cancers originating from colorectal adenomas, particularly advanced adenomas >10mm in diameter, being the most common precancerous lesion. The development of adenoma into colorectal cancer needs 10-15 years, the colorectal adenoma removed in time is found to reduce the incidence rate of colon cancer by more than 75%, and the cure rate of colorectal cancer found in the early stage can reach more than 90%.

Intervention by screening is an effective measure to reduce the incidence and mortality of CRC. Colorectal cancer screening methods commonly used in China include colonoscopy, immunochemical fecal occult blood test (FIT), fecal DNA test (sDNA) and the like. Colonoscope is the golden standard for colorectal cancer diagnosis, but because of its strong invasiveness and tedious intestinal tract preparation, the compliance of Chinese colonoscope screening is low, and at the present stage, medical resources of everyone in China are extremely lacking, and large-scale application of colonoscope as screening causes great resource waste. 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. Fecal DNA testing has been developed in many ways, but has high sensitivity and specificity for colorectal cancer and poor diagnostic performance for advanced adenomas.

Disclosure of Invention

The invention aims to screen out a specific DNA methylation marker for early diagnosis of advanced adenoma by analyzing methylation chip data in a public database, and provides a methylation marker for early screening of advanced adenoma and a primer probe combination thereof. The provided composition can be used for detecting and diagnosing advanced adenomas, has high specificity and sensitivity, and can be used for auxiliary clinical detection.

In a first aspect of the invention, there is provided a methylation marker combination that can be used for early screening of advanced adenomas.

The invention provides cg17885806, cg13901501, cg22675486, cg11717564, cg01913568 and cg08623787 (the CpG site DNA base sequence is shown in SEQ ID NO: 1) positioned in an RXRG gene region;

the invention provides cg08703540, cg06041677, cg12017911, cg27200833, cg25705558 and cg02709205(CpG locus DNA base sequence is shown in SEQ ID NO: 2) positioned in ISLR2 gene region;

the invention provides cg14654886, cg27434509, cg17255450, cg06426416, cg05542338 and cg01834022(CpG site DNA base sequence is shown in SEQ ID NO: 3) positioned in EVC2 gene region;

the invention provides cg24078985, cg07482795, cg 2458375, cg02711801, cg23296010, cg14732998 and cg15139588 (the CpG site DNA base sequence is shown in SEQ ID NO: 4) positioned in a ZNF793 gene region;

the invention provides cg06763829, cg15571277, cg21258057, cg09196068, cg07785314 and cg18412834(CpG site DNA base sequence is shown as SEQ ID NO: 5) positioned in NKAIN4 gene region.

The invention provides an application of a target gene DNA methylation combination as a molecular marker in early screening of adenoma in a progressive stage, wherein the target gene DNA methylation combination is composed of at least one of RXRG, ISLR2, EVC2, ZNF793 and NKAIN4 genes; the nucleotide sequence is shown as SEQ ID NO: 1-5.

In the second part of the invention, a method for screening a methylation marker specific to an adenoma at a progressive stage by using a database is provided, which comprises the following steps:

1) downloading 450k methylated chip data related to advanced adenomas from a GEO database;

2) dividing the sample into healthy people and advanced adenoma patients by utilizing the GEO database data;

3) analyzing a cancer sample and a healthy human sample of a patient with advanced adenoma in GEO, screening methylation sites which are different in adenoma and a normal sample, meeting the requirement that 6 different methylation sites exist within 150bp, and then verifying by utilizing the data of GEO;

4) and finally screening the optimal site combination from the specific methylation sites of the advanced adenoma by using a random forest modeling method, namely the methylation marker combination for screening the advanced adenoma.

In a third aspect of the invention, there is provided a primer combination and a probe for amplifying a DNA methylation marker according to the first aspect of the invention.

The nucleic acid sequence of the primer probe of the specific methylation site of the RXRG gene is as follows:

the forward primer is SEQ ID NO: any one of the compounds shown in the formulas 6-9,

the reverse primer is shown as SEQ ID NO: as shown in the figure 10 of the drawings,

the probe is SEQ ID NO: 11-12.

The primer probe nucleic acid sequence of the specific methylation site of the ISLR2 gene is as follows:

the forward primer is SEQ ID NO: any one of the ones shown in figures 13-17,

the reverse primer is shown as SEQ ID NO: as shown at 18, the flow of air is,

the probe is shown as SEQ ID NO: 19, respectively.

Primer probe nucleic acid sequences of specific methylation sites of EVC2 gene are as follows:

the forward primer is SEQ ID NO: any one of the elements shown at 20-22,

the reverse primer is SEQ ID NO: 23-24 of the plurality of first and second members,

the probe is SEQ ID NO: 25-26.

Primer probe nucleic acid sequences of specific methylation sites of ZNF793 gene are as follows:

the forward primer is SEQ ID NO: any one of the ones shown at 27-29,

the reverse primer is SEQ ID NO: any one of the ones shown at 30-31,

the probe is SEQ ID NO: 32-33.

Primer probe nucleic acid sequences of specific methylation sites of the NKAIN4 gene are as follows:

the forward primer is SEQ ID NO: any one of the components shown at 34-36,

the reverse primer is SEQ ID NO: any one of the ones shown in 37-38,

the probe is SEQ ID NO: 39-40.

In a fourth aspect of the present invention, there is provided a method for detecting a DNA methylation marker according to the first aspect of the present invention, comprising the steps of:

I) extracting genomic DNA from a sample;

II) carrying out conversion treatment on the DNA obtained in the step I) by using sulfite to obtain a DNA conversion product;

III) carrying out methylation quantitative PCR detection on the obtained DNA conversion product by using the primer probe of the third aspect of the invention;

IV) detecting the methylation status of the amplification product obtained in step III).

Preferably, the sample is selected from feces.

Preferably, the probe is labeled with a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end.

Preferably, the 5 'end of the probe is labeled with a fluorescence reporter group of FAM and the 3' end is labeled with a fluorescence quencher group of MGB.

Compared with the prior art, the invention has the following advantages:

1. the invention screens the unreported specific marker combination of the advanced adenoma with the clinical application potential by utilizing the data of the advanced adenoma of the public database and combining with a modeling means.

2. The DNA methylation molecular marker discovered for the first time can be used for judging the progress of the adenoma in the colorectal progression stage, and the obtained differential diagnosis model has better diagnosis efficiency. The AUC is 0.926-0.970; the optimal marker combination is ISLR2-RXRG, AUC 0.970. 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.

3. The invention can realize early diagnosis and screening of the adenoma in the advanced stage by utilizing a methylation detection method, and effectively reduces the incidence rate and the fatality rate of the colorectal cancer.

Drawings

FIG. 1 is a flow chart of an analysis of methylation markers specific for an advanced adenoma using public databases.

FIG. 2 is a ROC plot of RXRG, ISLR2, EVC2, ZNF793 and NKAIN4 genes from example 3 alone or in combination to detect advanced adenomas.

FIG. 3 is a ROC graph of any two genes selected from RXRG, ISLR2, EVC2, ZNF793 and NKAIN4 in example 3 in combination to detect colorectal adenoma samples.

FIG. 4 is a ROC graph of any three of the genes RXRG, ISLR2, EVC2, ZNF793 and NKAIN4 in example 3 in combination to detect colorectal adenoma samples.

FIG. 5 is a ROC graph of any four combinations of genes RXRG, ISLR2, EVC2, ZNF793 and NKAIN4 in example 3 to detect colorectal adenoma samples.

FIG. 6 is a ROC graph of the combination of the five genes RXRG, ISLR2, EVC2, ZNF793 and NKAIN4 in example 3 to detect colorectal adenoma samples.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples.

The invention provides a marker combination for detecting advanced adenomas, which comprises a primer and a probe combination for detecting the methylation level of RXRG gene, ISLR2 gene, EVC2 gene, ZNF793 gene and NKAIN4 gene.

The methylation modification of the gene mentioned herein is closely related to the occurrence and development of cancer, and shows a significant difference between normal tissues and cancer-affected tissues.

For example, the RXRG gene is an oncosuppressor gene, the retinoic acid receptor, also known as NR2B3 (nuclear receptor subfamily 2, group B, member 3) is a human nuclear receptor, and high levels of methylation are associated with a variety of cancers. The hypermethylation of RXRG leads to a decrease in the level of gene expression, and the low level of RXRG expression in lung cancer cells correlates with low patient survival (Lee SM et al, 2010).

The ISLR2 gene is a leucine rich repeat immunoglobulin superfamily 2, and ISLR2 promoter methylation can be used as a glioblastoma multiforme prognostic marker (Mock A et al, 2016).

The EVC2 gene encodes Ellis van Creveld syndrome 2 (limb protein), and plays a role in early embryonic development. Researchers have found mutations in EVC2 in metastatic breast cancer, a gene that may play a role in the development of cancer (Matt RP et al, 2020).

The ZNF793 gene encodes a zinc finger protein, and has DNA binding transcription activity. ZNF793 methylation can be used as a marker of gastric cancer (G Lou mez A et al, 2021).

The NKAIN4 gene encodes potassium-sodium channel protein interacting protein 4.

Example 1: screening process for DNA methylation markers

The inventor carries out methylation degree difference analysis according to the data of tissues of patients with adenomas and normal tissues based on an Illumina450K chip of adenomas in the advanced stage of GEO database, and identifies and obtains sites with obvious methylation difference degrees.

Example 2: detection method and experimental process of DNA methylation marker

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 into a new centrifuge tube, sucking 600 mu L of supernatant of the previous step into 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.

What is described here is: extraction of human DNA from feces was performed using a commercial Kit QIAamp Fast DNA StoolMini Kit, cat # 51604. Thus, the PCR inhibitor in the fecal sample can be efficiently removed while ensuring high yield of DNA from the fecal sample, and is suitable for downstream detection.

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 of CTconversion Reagent (ready for use) into the extracted 20 μ L DNA sample, mixing uniformly by vortex, and performing conversion reaction under the conditions of (98 deg.C, 10 min; 64 deg.C, 2.5 h)); 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 removing the lower layer solution by suction; repeating the above steps; the 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, it was centrifuged at 10000rpm for 1 min. DNA after sulfite conversion is obtained for subsequent detection.

What is described here is: host DNA extracted from feces is methylated according to the principle that bisulfite converts all unmethylated cytosines to uracil, while methylated cytosines remain unchanged. The commercialized kit for DNA methylation is EZ DNA methylation kit manufactured by ZYMO Research company, cat number D5005. Therefore, the loss rate of the host DNA in the methylation transformation process is further reduced, and the detection sensitivity of the methylation of the low-copy host DNA is improved.

Tri, qMSP detection

And carrying out methylation quantitative PCR on the DNA converted by the sulfite by using the primers and the probes in the kit.

In this example, the same sample gene was separately tested, that is, multiplex testing was not performed.

Specifically, for RXRG gene detection, a PCR probe primer premix for detecting RXRG gene may be prepared, where the PCR probe primer premix includes: 0.1 muM RXRG gene detection probe, 0.3 muM RXRG gene forward primer, 0.3 muM RXRG gene reverse primer, and deionized water to make up to 2 muL. The qMSP system for detecting RXRG gene is shown in the table 1-1:

TABLE 1-1

For beta-actin detection, a PCR probe primer premix for detecting beta-actin can be prepared, and the PCR probe primer premix comprises: 0.1 mu M beta-actin detection probe, 0.3 mu M beta-actin forward primer, 0.3 mu M beta-actin reverse primer, and deionized water to make up to 2 mu L.

The qMSP system for detecting beta-actin is shown in the table 1-2:

tables 1 to 2

Composition (I)	Addition amount (μ L)
		PCR probe primer premix for detecting beta-actin gene	2
Self-prepared 2 XTaqman Taq mix buffer	15
		DNA template after sulfite conversion	13
Total up to	30

The qMSP program for each gene detection is shown in tables 1-3:

tables 1 to 3

Temperature (. degree.C.)	Time	Number of cycles
			95	15min	1
95	15s	45
			60	1min	45

Example 3: advanced adenoma patient sample

First, DNA extraction of fecal samples

Stool samples from 151 enteroscopies and pathological examinations confirmed advanced adenoma patients and 60 enteroscopies confirmed normal patients were collected clinically. And (3) carrying out DNA extraction on the obtained excrement sample.

The specific extraction method can comprise the following steps:

firstly, according to the standard operation flow of a QIAamp Fast DNASoolMini Kit: adding 1mL of inhibitor Buffer into the sample, swirling for 1min until the sample is fully 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; 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 was continued at 16000g 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 manipulation.

Conversion of di-and sulphite

And performing 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 of CTconversion 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 removing the lower layer solution by suction; repeating the above steps; the 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, it was centrifuged at 10000rpm for 1 min. Obtaining DNA after sulfite conversion for detecting sensitivity of subsequent DNA methylation.

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 more 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, the genes are respectively assigned with different weights, and result judgment is carried out through a logical operation formula, wherein a, b, c, d and e are determined through clinical test data distribution.

The logical operation formula is as follows:

Score＝e^p/(1+e^p)

p＝aΔCt(RXRG)+bΔCt(ISLR2)+cΔCt(EVC2)+dΔCt(ZNF793)+eΔCt(NKAIN4)+F

then obtaining the Score value of the risk Score of the sample, determining a threshold value according to clinical distribution, and determining that the sample is positive when the Score is greater than or equal to the threshold value Score; a Score value less than the threshold is considered negative.

In this example, the RXRG gene, ISLR2 gene, EVC2 gene, ZNF793 gene, NKAIN4 gene and β -actin gene of the same sample were detected separately, that is, multiplex detection was not performed.

Fourth, result analysis

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, five gene combination and the like, inspecting models and ROC curves of different combinations, calculating to obtain corresponding AUC values, and determining the optimal marker combination. The specific combinations and the results of the investigation are as follows.

(1) Single gene methylation assay results

Marker substance	Sensitivity of the probe	Specificity of	Area under subject curve
				RXRG	91.39％	90.00％	0.961
ISLR2	89.40％	86.67％	0.951
				EVC2	90.07％	83.33％	0.939
ZNF793	89.40％	75.00％	0.926
				NKAIN4	88.74％	81.67％	0.929

(2) Two genes joint methylation detection results (10 combinations in total)

(3) Three genes joint methylation detection results (total 10 combinations)

Marker substance	Sensitivity of the probe	Specificity of	Area under subject curve
				EVC2-ISLR2-RXRG	92.05％	88.33％	0.963
ZNF793-ISLR2-RXRG	91.39％	90.00％	0.969
				NKAIN4-ISLR2-RXRG	92.72％	90.00％	0.966
ZNF793-EVC2-RXRG	92.05％	88.33％	0.968
				NKAIN4-EVC2-RXRG	91.39％	93.33％	0.963
NKAIN4-ZNF793-RXRG	93.38％	90.00％	0.968
				ZNF793-EVC2-ISLR2	90.73％	81.67％	0.957
NKAIN4-EVC2-ISLR2	90.07％	88.33％	0.950
				NKAIN4-ZNF793-ISLR2	92.72％	90.00％	0.956
NKAIN4-ZNF793-EVC2	90.73％	85.00％	0.951

(4) Results of four-gene joint methylation detection (5 combinations in total)

(5) Five-gene combined methylation detection result

When NKAIN4-ZNF793-EVC2-ISLR2-RXRG five genes are combined to be used as biomarkers, the sensitivity of detecting colorectal advanced adenomas is 92.72%, the specificity is 90.00%, and the area under the curve of a subject is 0.967.

Note:

sensitivity (true positive rate), true positive number/(true positive number + false negative number) × 100%.

Refers to the degree to which a patient is correctly judged, i.e., the percentage of patients that are actually ill and correctly diagnosed.

Specificity (true negative rate, specificity) is true negative number/(true negative number + false positive number) × 100%.

Refers to the degree of non-patient judgment, i.e., the percentage of patients that are actually disease free and correctly diagnosed as disease free.

The results show that the invention can obtain better detection performance than single gene detection by joint detection of a plurality of genes. The optimal genome combination with high sensitivity and specificity and lower detection cost is ISLR2-RXRG, the sensitivity is 92.05%, the specificity is 90.00% and the AUC is 0.970.

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 as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aaaatcttcc atcccctacc gt 22

<210> 11

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tctcctaacc tacaccgaac gatcatacat 30

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cgcctctctc ctaacctaca ccgaacg 27

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ttagggttaa cgtgggtagc g 21

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggcggaaatc gggttttagt taa 23

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ttttcgtcgt tgaaagaaat gtcg 24

<210> 16

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cggaaatcgg gttttagtta aatgtt 26

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

agggttaacg tgggtagcgt a 21

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tcacacaaca accgacttcg a 21

<210> 19

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

aactcgctat tcccgactat cgctccc 27

<210> 20

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ggattttagg ttcgtttcgt cgt 23

<210> 21

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tttggaataa atattcgtat ttggggttt 29

<210> 22

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gagtatgttt agtgcgagtc gtc 23

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

cgctctaatt cgatttaaac gcaaa 25

<210> 24

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

cgctctaatt cgatttaaac gca 23

<210> 25

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

aaacctaacc gccgaacgcc taaacc 26

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ccaaacctaa ccgccgaacg cctaa 25

<210> 27

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tttggtttag taaagggttt aattattcgt 30

<210> 28

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gttttaaata tggtcgtttt aggtaggag 29

<210> 29

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ggagttaagt gggcgtcgtt a 21

<210> 30

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

taaccgatac gcaaactcaa ttacg 25

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

cgatacgcaa actcaattac gac 23

<210> 32

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

aaactatcaa ccgccacccg acaacc 26

<210> 33

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

caaccgccac ccgacaacct tcca 24

<210> 34

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

aaatttggtc gagggagttt cg 22

<210> 35

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gggtagaatg gaaatttggt cga 23

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

cgagggagtt tcgattaatt tcg 23

<210> 37

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

acgtaaaact acgcgaaaac ga 22

<210> 38

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gtaaaactac gcgaaaacga aaa 23

<210> 39

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

aaaccgatac ccgaaacgca acgc 24

<210> 40

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

ctaaaaccga tacccgaaac gcaacgc 27

Claims

1. A primer probe set for diagnosing, detecting or screening advanced adenomas is characterized by comprising a primer probe of a specific methylation site of at least one of RXRG gene, ISLR2 gene, EVC2 gene, ZNF793 gene and NKAIN4 gene.

2. The primer probe set for diagnosing, detecting or screening advanced adenomas according to claim 1, wherein the specific methylation site of RXRG gene is cg17885806, cg13901501, cg22675486, cg11717564, cg01913568 and/or cg08623787, and the primer probe nucleic acid sequence of the specific methylation site of RXRG gene is as follows:

the forward primer is SEQ ID NO: any one of those shown in the numbers 6-9,

the reverse primer is shown as SEQ ID NO: as shown in figure 10 of the drawings,

the probe is SEQ ID NO: 11-12.

3. The primer probe set for diagnosing, detecting or screening advanced adenomas according to claim 1, wherein the specific methylation site of ISLR2 gene is cg08703540, cg06041677, cg12017911, cg27200833, cg25705558 and/or cg02709205, and the primer probe nucleic acid sequence of the specific methylation site of ISLR2 gene is as follows:

the forward primer is SEQ ID NO: any one of the ones shown in figures 13-17,

the reverse primer is shown as SEQ ID NO: as shown at 18, the flow of air is,

the probe is shown as SEQ ID NO: 19, respectively.

4. The primer probe set for diagnosing, detecting or screening advanced adenomas according to claim 1, wherein the specific methylation site of EVC2 gene is cg14654886, cg27434509, cg17255450, cg06426416, cg05542338 and/or cg01834022, and the primer probe nucleic acid sequence of the specific methylation site of EVC2 gene is as follows:

the forward primer is SEQ ID NO: any one of the elements shown at 20-22,

the reverse primer is SEQ ID NO: any one of the ones shown in figures 23-24,

the probe is SEQ ID NO: 25-26.

5. The primer probe set for diagnosing, detecting or screening advanced adenomas according to claim 1, wherein the specific methylation sites of ZNF793 gene are cg24078985, cg07482795, cg 2488375, cg02711801, cg23296010, cg14732998 and/or cg15139588, and the primer probe nucleic acid sequences of the specific methylation sites of ZNF793 gene are as follows:

the forward primer is SEQ ID NO: any one of the ones shown at 27-29,

the reverse primer is SEQ ID NO: any one of the ones shown at 30-31,

the probe is SEQ ID NO: 32-33.

6. The primer-probe set for diagnosing, detecting or screening advanced adenomas according to claim 1, wherein the primer-probe nucleic acid sequence of the NKAIN4 gene with specific methylation sites is cg06763829, cg15571277, cg21258057, cg09196068, cg07785314 and/or cg18412834, and the specific methylation sites of NKAIN4 gene is as follows:

the forward primer is SEQ ID NO: any one of the components shown at 34-36,

the reverse primer is SEQ ID NO: any one of the ones shown in 37-38,

the probe is SEQ ID NO: 39-40.

7. The primer probe set for diagnosing, detecting or screening advanced adenoma according to claim 1, wherein the object to be detected is a DNA sample obtained by extracting genomic DNA from human feces, and then performing bisulfite conversion and purification.

8. The primer probe set for diagnosing, detecting or screening advanced adenomas as in claim 1, 2 or 3, comprising: primer probes for specific methylation sites of ISLR2 gene and RXRG gene.

9. Use of a primer probe set for diagnosing, detecting or screening advanced adenomas according to any one of claims 1 to 8 in the preparation of a product for detecting advanced adenomas.

10. A kit for diagnosing, detecting or screening advanced adenomas, comprising the primer probe set of any one of claims 1 to 8 for diagnosing, detecting or screening advanced adenomas.