GENE MARKER COMBINATION AND USE THEREOF

Abstract

The present invention relates to a tumor marker, a methylation detection reagent, a kit and use thereof. By detecting the methylation level of SDC2, COL4A1/COL4A2 and ITGA4 genes, colorectal cancer specimens can be well distinguished from fecal specimens, and the detection sensitivity and specificity for the colorectal cancer can be 90% or above.

Claims

1-12. (canceled)

13. A detection reagent or kit, comprising: at least one reagent for methylation detection of SDC2 gene; at least one reagent for methylation detection of COL4A1/COL4A2; and at least one reagent for methylation detection of ITGA4 gene.

14. The reagent or kit of claim 13, wherein: the at least one reagent for methylation detection of SDC2 gene comprises: a capture sequence for the SDC2 gene; a primer pair for the SDC2 gene; and a probe for the SDC2 gene; the at least one reagent for methylation detection of COL4A1/COL4A2 comprises: a capture sequence for the COL4A1/COL4A2 gene; a primer pair for the COL4A1/COL4A2 gene; and a probe for the COL4A1/COL4A2 gene; and the at least one reagent for methylation detection of ITGA4 gene comprises: a capture sequence for the ITGA4 gene; a primer pair for the ITGA4 gene; and a probe for the ITGA4 gene.

15. A method for diagnosing a colorectal tumor in a subject, comprising: detecting a sum of a methylation level of SDC2 gene, a methylation level of COL4A1/COL4A2 gene, and a methylation level of ITGA4 gene, of a sample derived from the subject; obtaining a deviation between the sum of the methylation level of the SDC2 gene, the methylation level of the COL4A1/COL4A2 gene, and the methylation level of the ITGA4 gene, of the sample, and a sum of a normal methylation level of SDC2 gene, a normal methylation level of COL4A1/COL4A2 gene, and a normal methylation level of the ITGA4 gene; and diagnosing a colorectal tumor in the subject based on the deviation.

16. A method for treating a colorectal tumor in a patient comprising administering to the subject a drug for treating the colorectal tumor, wherein the patient is diagnosed as a patient with the colorectal tumor by the method of claim 15.

17. The method of claim 15, wherein the colorectal tumor is colorectal cancer or adenoma.

18. The method of claim 15, wherein the sample is selected from a group consisting of tissue, body fluid and excrement.

19. The method of claim 15, wherein the sample is feces.

20. The method of claim 15, wherein said detecting of the sum of the methylation level of the SDC2 gene, the methylation level of the COL4A1/COL4A2 gene, and the methylation level of ITGA4 gene, is by methylation-specific quantitative PCR (qMSP).

21. The method of claim 15, wherein said detecting of the sum of the methylation level of the SDC2 gene, the methylation level of the COL4A1/COL4A2 gene, and the methylation level of ITGA4 gene, comprises: contacting the sample with at least one reagent for methylation detection of SDC2 gene; contacting the sample with at least one reagent for methylation detection of COL4A1/COL4A2; and contacting the sample with at least one reagent for methylation detection of ITGA4 gene.

22. The method of claim 21, wherein the at least one reagent for methylation detection of SDC2 gene comprises: a capture sequence for the SDC2 gene; a primer pair for the SDC2 gene; and a probe for the SDC2 gene; the at least one reagent for methylation detection of COL4A1/COL4A2 comprises: a capture sequence for the COL4A1/COL4A2 gene; a primer pair for the COL4A1/COL4A2 gene; and a probe for the COL4A1/COL4A2 gene; and the at least one reagent for methylation detection of ITGA4 gene comprises: a capture sequence for the ITGA4 gene; a primer pair for the ITGA4 gene; and a probe for the ITGA4 gene.

23. The method of claim 22, wherein the capture sequence for the SDC2 gene comprises a first nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in any one of SEQ ID NOs: 1 and 2, and a first complementary nucleotide sequence complementary to the first nucleotide sequence; and/or the primer pair for the SDC2 gene comprises a second nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 3, and a second complementary nucleotide sequence complementary to the second nucleotide sequence; and/or the primer pair for the SDC2 gene comprises a third nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 4, and a third complementary nucleotide sequence complementary to the third nucleotide sequence; and/or the probe for the SDC2 gene comprises a fourth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 5, and a fourth complementary nucleotide sequence complementary to the fourth nucleotide sequence; and/or the capture sequence for the COL4A1/COL4A2 gene comprises a fifth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 6, and a fifth complementary nucleotide sequence complementary to the fifth nucleotide sequence; and/or the primer pair for the COL4A1/COL4A2 gene comprises a sixth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in any one of SEQ ID NOs: 7 and 14, and a sixth complementary nucleotide sequence complementary to the sixth nucleotide sequence; and/or the primer pair for the COL4A1/COL4A2 gene comprises a seventh nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in any one of SEQ ID NOs: 8 and 15, and a seventh complementary nucleotide sequence complementary to the seventh nucleotide sequence; and/or the probe for the COL4A1/COL4A2 gene comprises an eighth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 9, and an eighth complementary nucleotide sequence complementary to the eighth nucleotide sequence; and/or the capture sequence for the ITGA4 gene comprises a ninth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 10, and a ninth complementary nucleotide sequence complementary to the ninth nucleotide sequence; and/or the primer pair for the ITGA4 gene comprises a tenth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in at least one of SEQ ID NOs: 11 and 16, and a tenth complementary nucleotide sequence complementary to the tenth nucleotide sequence; and/or the primer pair for the ITGA4 gene comprises an eleventh nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in at least one of SEQ ID NOs: 12 and 17, and an eleventh complementary nucleotide sequence complementary to the eleventh nucleotide sequence; and/or the probe for the ITGA4 gene comprises a twelfth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 13, and a twelfth complementary nucleotide sequence complementary to the twelfth nucleotide sequence.

24. The reagent or kit of claim 14, wherein the capture sequence for the SDC2 gene comprises a first nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in any one of SEQ ID NOs: 1 and 2, and a first complementary nucleotide sequence complementary to the first nucleotide sequence.

25. The reagent or kit of claim 14, wherein the primer pair for the SDC2 gene comprises a second nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 3, and a second complementary nucleotide sequence complementary to the second nucleotide sequence; or the primer pair for the SDC2 gene comprises a third nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 4, and a third complementary nucleotide sequence complementary to the third nucleotide sequence.

26. The reagent or kit of claim 14, wherein the probe for the SDC2 gene comprises a fourth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 5, and a fourth complementary nucleotide sequence complementary to the fourth nucleotide sequence.

27. The reagent or kit of claim 14, wherein the capture sequence for the COL4A1/COL4A2 gene comprises a fifth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 6, and a fifth complementary nucleotide sequence complementary to the fifth nucleotide sequence.

28. The reagent or kit of claim 14, wherein the primer pair for the COL4A1/COL4A2 gene comprises a sixth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in any one of SEQ ID NOs: 7 and 14, and a sixth complementary nucleotide sequence complementary to the sixth nucleotide sequence; or the primer pair for the COL4A1/COL4A2 gene comprises a seventh nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in any one of SEQ ID NOs: 8 and 15, and a seventh complementary nucleotide sequence complementary to the seventh nucleotide sequence.

29. The reagent or kit of claim 14, wherein the probe for the COL4A1/COL4A2 gene comprises an eighth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 9, and an eighth complementary nucleotide sequence complementary to the eighth nucleotide sequence.

30. The reagent or kit of claim 14, wherein the capture sequence for the ITGA4 gene comprises a ninth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 10, and a ninth complementary nucleotide sequence complementary to the ninth nucleotide sequence.

31. The reagent or kit of claim 14, wherein the primer pair for the ITGA4 gene comprises a tenth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in at least one of SEQ ID NOs: 11 and 16, and a tenth complementary nucleotide sequence complementary to the tenth nucleotide sequence; or the primer pair for the ITGA4 gene comprises an eleventh nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in at least one of SEQ ID NOs: 12 and 17, and an eleventh complementary nucleotide sequence complementary to the eleventh nucleotide sequence.

32. The reagent or kit of claim 14, wherein the probe for the ITGA4 gene comprises a twelfth nucleotide sequence having at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identity to the sequences as set forth in SEQ ID NO: 13, and a twelfth complementary nucleotide sequence complementary to the twelfth nucleotide sequence.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0160] FIG. 1 shows a ROC curve of SDC2, COL4A1/COL4A2 and ITG4 gene combined detection of colorectal cancer and adenoma (l cm) of 935 fecal specimens in Example 1.

[0161] FIG. 2 shows detection results of fecal specimens before and after operation of 23 colorectal cancer patients detected by a method of SDC2, COL4A1/COL4A2 and ITG4 gene combined detection in Example 2.

[0162] FIG. 3 shows a ROC curve of SOX21 gene for colorectal cancer and adenoma detection of 240 fecal specimens in Comparative Example 1.

[0163] FIG. 4 shows a ROC curve of ITGA4, COL4A1/COL4A2 gene combined detection of colorectal cancer of 109 fecal specimens in Comparative Example 2.

DETAILED DESCRIPTION OF THE INVENTION

[0164] The technical solutions of the present disclosure are further described by specific examples below, and the specific examples do not pose a limitation on the protection scope of the present disclosure. Some non-essential modifications and adjustments made by others based on the concept of the present disclosure still fall within the protection scope of the present disclosure.

[0165] The “capture sequence”, “primer” or “probe” in the present disclosure refers to an oligonucleotide that contains a region complementary to a sequence of at least 6 consecutive nucleotides of a target nucleic acid molecule (e.g., a target gene). In some embodiments, at least part of the sequence of the primer or probe is not complementary to an amplified sequence. In some embodiments, the primer or probe contains a region complementary to a sequence of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of a target molecule. When a primer or probe contains a region “complementary to at least x consecutive nucleotides of a target molecule”, the primer or probe is at least 95% complementary to at least x consecutive or discontinuous block nucleotides of the target molecule. In some embodiments, the primer or probe is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target molecule.

[0166] In the present disclosure, “normal” samples refer to samples of the same type isolated from individuals who are known to be free of the cancer or tumor.

[0167] In the present disclosure, the “subject” is a mammal, such as a human.

[0168] The samples for methylation detection in the present disclosure include but are not limited to DNAs, or RNAs, or DNA and RNA samples containing mRNAs, or DNA-RNA hybrids. The DNA or RNA may be single-stranded or double-stranded.

[0169] In the present disclosure, the “methylation level” is equivalent to “methylation degree”, which can usually be expressed as the percentage of methylated cytosine, which is obtained through dividing the number of methylated cytosine by the sum of the number of methylated cytosine and the number of unmethylated cytosine; be expressed with the generally used method of dividing the number of methylation target genes by the number of internal reference genes; and be expressed with other methods of expressing the methylation level in the prior at.

[0170] In the present disclosure, the “sample” is equivalent to “specimen”.

[0171] As used herein, the term “and/or” refers to and encompasses any and all possible combinations of one or more of associated listed items. When used in a list of two or more items, the term “and/or” means that any of the listed items can be used alone, or any combination of two or more of the listed items can be used. For example, if a composition, a combination, a structure, and the like are described as including (or comprising) components A, B, C and/or D, then the composition may include A alone; B alone; C alone; D alone; a combination of A and B; a combination of A and C; a combination of A and D; a combination of B and C; a combination of B and D; a combination of C and D; a combination of A. B and C; a combination of A. B and D; a combination of A, C and D; a combination of B, C and D; or a combination of A, B. C and D.

Example 1

[0172] 935 fecal specimens (359 cases of colorectal cancer, 67 cases of adenoma (≥1 cm), 509 cases of non-tumor individuals, all confirmed by colonoscopy or pathology) were selected for grinding and centrifugation, 50 ul of capture magnetic beads (containing capture sequences of SDC2, COL4A2. ITGA4 and reference gene ACTB) were added, and the operations were carried out according to the technical solution described below.

[0173] The technical solution was as follows:

1) Fecal specimens of normal people and colorectal tumor patients with colonoscopy pathology results were collected, mixed and ground according to 1 g of fences: 4 mL of protective solution, and then centrifuged at 5000 rpm for 10 min, a supernatant was taken and precipitates were discarded.
2) 10 mL of supernatant was taken and centrifuged again, and 3.2 mL of supernatant was taken, into which 2 mL of lysate and 50 ul of capture magnetic beads M1 were added, incubated at 95° C. for 15 min, and then placed at room temperature for 30 min.
3) The mixture was placed on a magnetic stand, pat of supernatant was discarded, the magnetic beads were washed off, the mixture was transferred to a 2 mL centrifuge tube, 800 ul of washing solution W1 was added, incubation was carried out at 1300 rpm for 1 min at room temperature, the mixture was placed on the magnetic stand to aspirate the supernatant, and this process was repeated 2 times;
4) 50 ul of eluent was added, incubated at room temperature at 1300 rpm for 5 minutes, and placed on a magnetic stand, and an eluent was transferred to a new EP tube in 3 minutes.
5) DNA fragments in the previous step was methylated by using an EZ DNA Methylation Kit (Zymo Research) according to the method in Reference [2], and 20 ul of final eluate was used for qMSP detection.

[0174] Finally, 20 ul of Bisulfite-transformed DNA was obtained. Then qMSP detection was carried out, and finally the methylation level of the SDC2, COL4A1/COL4A2 and ITG4 gene combination in the specimen was determined according to the CT value.

[0175] The qMSP reaction system of this example: 30 ul (nuclease-free water 2.98 ul, 5×Coloress GoTaq Flexi Buffer 6 ul, MgCl.sub.2 (25 mM) 5 ul, dNTPs (10 mM) 1 ul, GoTaq Hot Start polymerase 0.6 ul, ACTB-FP (100 uM) 0.08 ul, ACTB-RP (100 uM) 0.08 ul, ACTB-Probe (100 uM) 0.06 ul, SDC2-FP (100 uM) 0.12 ul, SDC2-RP (100 uM) 0.12 ul, SDC2-Probe (100 uM) 0.04 ul, COL4A2-FP (100 uM) 0.06 ul, COL4A2-RP (100 uM) 0.06 ul. COL4A2-Probe (100 uM) 0.04 ul, ITGA4-FP (100 uM) 0.06 ul, ITGA4-RP (100 uM) 0.06 ul, ITGA4-Probe (100 uM) 0.04 ul, DNA 10 ul). Reaction procedure: 95° C. 5 minutes, (95° C. 15 s, 58° C. 30 s, 72° C. 30 s)×48 Cycles, 40° C. 30 s.

[0176] The capture and PCR reaction uses ACTB as the reference gene, and finally the methylation level in the specimen is determined according to the CT value. The target gene is determined as positive with a CT value≤38, and as negative with a CT value>38.

[0177] Because the 5′ ends of the COL4A and COL4A2 genes are close to each other with a gap of 127 bp, they share a bidirectional promoter region. The methylation region detected in this example is the bidirectional promoter region of the COL4A1 and COL4A2 genes. Therefore, in this example, the gen direction of COL4A2 of the two genes is selected to label sequence information.

[0178] The methylation sites of the SDC2, COL4A1/COL4A2 and ITGA4 genes are mainly located in the promoter region or CpG island nearby.

[0179] In this example, the PCR probes of SDC2, COL4A2 and ITGA4 are labeled with a same fluorescent group, so the sum of methylation lewis of these genes can be easily detected through one fluorescent channel, without the need of detecting each gene by using different fluorescence channels, which reduces the complexity of test.

[0180] The capture sequences, primers and probes of this example are as follows:

TABLE-US-00001 SEQ ID NO. 1: capture sequence 1 of SDC2: 5′-AGCCCGCGCACACGAATCCGGAGCAGAGIACCG-3′ or SEQ ID NO. 2: capture sequence 2 of SDC2: 5′-CTCCTGCCCAGCGCTCGGCGCAGCCCGC-3′ qMSP primer and probe of SDC2: SEQ ID NO. 3: SDC2-FP: 5′-GAGGAAGCGAGCGTITTC-3′ SEQ ID NO. 4: SDC2-RP: 5′-AAAATACCGCAACGATTACGA-3′ SEQ ID NO. 5: SDC2-Probe:  5′-AGTTTCGAGTTCGAGTTTTCGAGTTTG-3′

[0181] The two capture sequences of SDC2 have the same capture effect, and either one can be selected.

TABLE-US-00002 SEQ ID NO. 6: capture sequence of COL4A1/COL4A2: 5′-GCTGCTGCCCGAACGCATTGGCCCTTCCAGAAGCA-3′ qMSP primer and probe of COL4A1/COL4A2: SEQ ID NO. 7: COL4A1/COL4A2-FP: 5′-AGAGAGTTTAGTAAGGTCGGGC-3′ SEQ ID NO. 8: COL4A1/COL4A2-RP: 5′-GACTTCAAAAACTACTACCCG-3′ or SEQ ID NO. 14: COL4A1/COL4A2-FP: 5′-AGAGAGTTTAGTAAGGTCGGAC-3′ SEQ ID NO. 15: COL4A1/COL4A2-RP: 5′-GACTTCAAAAACTACTACCCG-3′ SEQ ID NO. 9: COL4A1/COL4A2-Probe: 5′-TGTCGGTGTGTCGTCGGC-3′

[0182] The two pairs of primers of COL4A1/COL4A2 have the same amplification specificity and can distinguish positive and negative samples well, and either one can be selected.

TABLE-US-00003 SEQ ID NO. 10: capture sequence of ITGA4: 5′-CTACGCGCGGCTGCAGGGGGCGCTGGGGAACCT-3′ qMSP primer and probe of ITGA4: SEQ ID NO. 11: ITGA4-FP: 5′-ACGCGAGTTTTGCGTAGAC-3′-3′ SEQ ID NO. 12: ITGA4-RP: 5′-GCTAAATAAAATCCCGAACG-3′ or SEQ ID NO. 16: ITGA4-FP 5′-ACGCGAGTTTTGCGTAGTC-3′ SEQ ID NO. 17: ITGA4-RP: 5′-GCTAAATAAAATCCCGAACG-3′ SEQ ID NO. 13: ITGA4-Probe: 5′-ACGGAGTTCGGTTTTGCGTTTTC-3′

[0183] The two pairs of primers of ITGA4 have the same amplification specificity and can distinguish positive and negative samples well, and either one can be selected.

[0184] According to the results of these 935 focal specimens, the ROC curve of the marker combination for colorectal cancer and adenoma detection is drawn using IBMSPSS statistics 20 software, as shown in FIG. 1.

[0185] The results show that the SDC2, COL4A1/COL4A2 and ITG4 gene combined methylation detection has a sensitivity of 91.36% for colorectal cancer and a sensitivity of 50.75% for adenoma when the specificity is 95.28%; and with the SDC2, COL4A1/COL4A2 and ITG4 gene combined methylation detection, the area under the ROC curve for colorectal cancer detection is 0.979, 95% CI is 0.970 to 0.988, the area under the ROC curve for adenoma detection is 0.832, and 95% CI is 0.771 to 0.894.

Example 2

[0186] 23 patients diagnosed with colorectal cancer were selected, fecal specimens of the patients were collected pre- and 3-6 months post-operation, the marker combination (SDC2. COL4A1/COL4A2 and ITGA4) of the present disclosure was used to detect 46 fecal specimens pre- and post-operation, and the methylation levels of the marker combination in the feces before and after operation were compared.

[0187] The detection process and result determination standard of the marker combination are the same as in Example 1. Target gene is determined as positive with a CT value≤38, and as negative with a CT value>38. The results are as shown in FIG. 2.

[0188] The results show that the detection results of these 23 patients with colorectal cancer are all positive before the operation. The fecal specimens are tested again after the operation, and the results are all negative. It indicates that the methylation level of the marker combination is significantly reduced after the tumor is excised, suggesting that the marker combination can be used for postoperative follow-up monitoring of patients with colorectal cancer.

Comparative Example 1

[0189] The studies find that the detection of methylated SOX21 gene in feces can be used for auxiliary diagnosis of colorectal cancer, and the detection of colorectal cancer in feces with methylated SOX21 is now used as a comparative example. The following is the procedure and results of detecting SOX21 gone in feces.

[0190] 240 fecal specimens (80 cases of colorectal cancer, 77 cases of adenoma (≥1 cm), 83 cases of normal, all confirmed by colonoscopy or pathology) were selected for grinding and centrifugation. 50 ul of capture magnetic beads (containing capture sequences of SOX21 and reference gene ACTB) were added, and the operations were carried out according to the technical solution described in Example 1. Finally, 20 ul of Bisulfite-transformed DNA was obtained. Then qMSP detection was carried out, and finally the methylation level of SOX21 in the specimen was determined according to the CT value.

[0191] For the capture sequence, the primer and the probe contained in the reagent, and the qMSP reaction system and the procedures, see reference 121. Based on the results of 240 fecal specimens, the ROC curve of SOX21 gene for colorectal cancer and adenoma detection is drawn, as shown in FIG. 3.

[0192] The results show that when the specificity of methylated SOX21 is 97.6%, the sensitivity to colorectal cancer is 80% and the sensitivity to adenoma is 33.8%; and the area under the ROC curve for SOX21 colorectal cancer detection is 0.926, 95% CI is 0.885 to 0.968, the area under the ROC curve for adenoma detection is 0.667, and 95% CI is 0.583 to 0.751.

Comparative Example 2

[0193] 161 fecal specimens (79 cases of colorectal cancer, 82 cases of normal, all confirmed by colonoscopy or pathology) were selected for grinding and centrifugation, 50 ul of capture magnetic beads (containing capture sequences of SOX21, SDC2 and reference gene ACTB) were added and the operations were carried out according to the technical solution described in Example 1. Finally, 20 ul of Bisulfite-transformed DNA was obtained. Then qMSP detection was carried out, and finally the methylation levels of SOX21 and SDC2 in the specimen were determined according to the CT value.

[0194] The capture sequence, the primer and the probe of SOX21, and the qMSP reaction system and the procedures were the same as those in Comparative Example 1. The capture sequence, the primer and the probe of SDC2, and the qMSP reaction system and the procedures were the same as those in Example 1.

[0195] The detection results show that the methylated SDC2 gene detection has a sensitivity of 88.6%, and a specificity of 93.9%; and the area under the curve is 0.939. The methylated SOX21 gene has a sensitivity of 79.7% and a specificity of 86.6%; and the area under the curve is 0.924. The methylated SOX21 gene and methylated SDC2 gene combination has a detection sensitivity of 87.34% and a specificity of 93.9%. The detection sensitivity of the methylated SDC2 gene and methylated SOX21 gene combined detection is lower than that of the methylated SDC2 gene alone as a marker for colon cancer, and is also lower than that of the combined detection of SDC2, COL4A1/COL4A2 and ITG4 genes of the present disclosure.

Comparative Example 3

[0196] 109 fecal specimens (61 cases of colorectal cancer, and 48 cases of normal, all confirmed by colonoscopy or pathology) were selected for grinding and centrifugation, 50 ul of capture magnetic beads (containing capture sequences of ITGA4, COL4A1/COL4A2 and reference gene ACTB) were added.

[0197] The capture sequences, the primers and the probes of ITGA4, COL4A1/COL4A2, and the qMSP reaction system and the procedures were the same as in Example 1.

[0198] Based on the results of these 109 fecal specimens, the ROC curve of the marker combination for colorectal cancer detection is drawn, as shown in FIG. 4.

[0199] The results show that the area under the curve distinguishing colorectal cancer from normal human samples is 0.964. When the positive determination Ct value is 38, the methylated ITGA4+COL4A1/COL4A2 gene has a sensitivity of 86.89% and a specificity of 91.67% for colorectal cancer detection. Its sensitivity and specificity are both lower than the sensitivity and specificity of the SDC2, COL4A1/COL4A2 and ITG4 gene combined methylation detection of the present disclosure.

[0200] The descriptions above are only some implementations of the present disclosure. It should be pointed out that for a person of ordinary skill in the art, without departing from the principle of the present disclosure, several improvements and modifications can be made, and these improvements and modifications shall also fall within the protection scope of the present disclosure.

REFERENCES

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