CN111748636A

CN111748636A - Composition and kit for auxiliary diagnosis of colorectal cancer and application of composition and kit

Info

Publication number: CN111748636A
Application number: CN202010892075.0A
Authority: CN
Inventors: 戴立忠; 周镕; 纪博知; 刘佳
Original assignee: Sansure Biotech Inc
Current assignee: Sanway Gene Tech Inc
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2020-10-09
Anticipated expiration: 2040-08-31
Also published as: CN111748636B

Abstract

The invention relates to the field of molecular biological detection, in particular to methylation detection of three genes including Septin9, NDRG4 and SDC 2. The invention provides a composition for auxiliary diagnosis of colorectal cancer; also provided are kits containing the compositions, and uses of the compositions. The composition can be used for screening early colorectal cancer with high sensitivity and specificity, and the three-gene combined detection reduces the sample loading amount and operation, avoids the sample from being excessively diluted, and has low cost and convenient operation.

Description

Composition and kit for auxiliary diagnosis of colorectal cancer and application of composition and kit

Technical Field

The invention relates to the field of molecular biological detection, in particular to the field of auxiliary diagnosis of colorectal cancer, and more particularly relates to methylation detection of three genes including Septin9, NDRG4 and SDC 2.

Background

Colorectal cancer, a relatively high incidence of digestive tract tumors at present, is of great importance for the treatment of colorectal cancer because most people are already in the middle and advanced stages at the time of diagnosis due to its occult nature of onset.

There are two main techniques currently used in colorectal cancer screening. One is fecal occult blood test. The method comprises detecting blood components (hemoglobin) in stool, and if multiple and continuous positive reactions indicate gastrointestinal hemorrhage, further checking to alert intestinal tumor. The method has the advantages of fast and clear result display and semi-quantitative result. But the method has the defects of poor specificity and poor interference resistance, and the accurate judgment of the method as colorectal cancer diagnosis is influenced by factors such as poor sensitivity, more primary limiting conditions and the like. Another method is enteroscopy. At present, the enteroscopy is the most effective and reliable diagnosis method for diagnosing intestinal lesions, and is also the main means which cannot be replaced by other diagnosis and treatment means at present. However, it also has a series of disadvantages: the method comprises two aspects of unfriendliness of preparation work in the early stage of examination and pain of patients in the examination process, and greatly influences the practical application and popularization of the technology.

In addition to the above two methods, molecular diagnostic methods are cancer diagnostic methods that have been rapidly developed in recent years. At present, a large number of molecular markers for colorectal cancer have been studied and reported in tissue and blood samples, for example:

the Septin9 gene is one of Septin gene family members found by Hartwell et al, and has guanosine triphosphatase (GTPase) activity, Septin9 gene is located in human chromosome 17q25.3, contains 17 exons, and has length of about 2.40 × 10⁵bp, Septin9 protein. However, since the transcript contains multiple translation origins and variant splicing, Septin9 is mutated to generate 18 variant spliceosomes, encoding 15 polypeptides. The Septin9 gene and its expression product are widely involved in various physiological processes of human body, such as cell polarization, cell proliferation and apoptosis, substance transport inside and outside cells, cytoskeleton regulation, etc. Research shows that Septin9 gene plays the role of inhibiting cancer gene in colorectal cancer, and methylation of Septin9 gene can inhibit normal expression of Septin gene and make Septin9 gene have cancer inhibiting functionLoss and eventually cell division and carcinogenesis.

NDRG4 (N-myc downy-regulated gene 4) is a member of the cancer suppressor gene NDRG gene family, and the gene is 32kb in length and consists of 17 exons and 16 introns. Numerous studies have shown that the NDRG4 gene is involved in the development, development and metastasis of tumors, and its 5' regulatory region contains CpG islands, which are frequently methylated during the development of colorectal cancer, and are considered to be an important biological feature of colorectal cancer. Therefore, the detection of the methylation level of the promoter region of the NDRG4 gene provides an effective way for noninvasive colorectal cancer screening.

The SDC2 gene is a known protein involved in cell division, migration, and expression in mesenchymal cells of the colon. It was found that the methylation level of the SDC2 target region was significantly higher in tumor tissue than the SDC2 target region in paired adjacent non-tumor tissue. By analyzing the methylation level of SDC2 in 133 CRC patients' primary tumors and their paired adjacent non-tumor tissue samples, researchers found that tumor tissue samples showed significantly higher methylation levels than control adjacent non-tumor tissue samples in the transcriptional regulatory region of the SDC2 gene.

At present, according to different pretreatment modes of samples, the existing DNA methylation detection technologies mainly have three main types:

1) methylation detection techniques based on restriction enzyme digestion pretreatment: the method is characterized in that DNA is digested into fragments with different sizes by utilizing the characteristic that methylation sensitive restriction endonuclease does not cut a methylation region, and then products are separated through PCR amplification so as to clarify the methylation state of a target fragment;

2) methylation detection technology based on sulfite modification pretreatment: in this method, unmethylated cytosine (C) is converted to uracil (U) after sulfite treatment of DNA, and methylated cytosine remains unchanged. Sequencing the treated DNA or using a specific primer PCR to obtain very accurate DNA sequence methylation site information;

3) methylation detection technology based on affinity enrichment pretreatment: the affinity enrichment-based DNA methylation detection technologies mainly include a methylated DNA immunoprecipitation technology (MeDIP) and a methylated CpG binding protein affinity capture technology (MBDCap): the MeDIP method is characterized in that after genome DNA is broken and denatured by ultrasonic waves, a methylated fragment is enriched by using a 5' -methylcytosine specific antibody, then the methylated DNA fragment is obtained by separation and purification, and then the methylated DNA fragment is analyzed by methods such as sequencing and the like; the MBDCap technique is similar to the MeDIP method in that methylated DNA binding proteins are used to immunoprecipitate methylated DNA because, unlike the enrichment proteins used, MeD IP generally enriches methylated regions of low CpG density, whereas MBDCap generally enriches methylated regions of high CpG density.

However, the conventional marker combinations have problems such as poor sensitivity and specificity. Therefore, there is a need in the art for a related product that enables early screening for colorectal cancer with high sensitivity and specificity.

Disclosure of Invention

In light of the above needs, the inventors have surprisingly found that marker combinations of Septin9, NDRG4 and SDC2 have high sensitivity and specificity for early screening of colorectal cancer.

In a first aspect, the present invention provides a composition for aiding in the diagnosis of colorectal cancer, the composition comprising:

primer for detecting methylation of Septin9 gene:

a forward primer: 5'-GCGGGAACCTGATCCGCCCGGGAGGCGGGG-3' (SEQ ID NO: 1);

reverse primer: 5'-CCTGGCTCAGCTGAATGAATGGGGGAGG-3' (SEQ ID NO: 2);

fluorescent probe for detecting methylation of Septin9 gene:

a fluorescent probe: 5'-AGGGGCCGGCGCCCGCCTTC-3' (SEQ ID NO: 3);

primers for detecting methylation of NDRG4 gene:

a forward primer: 5'-GGATATTTTTGTTAGGGGATGTTTT-3' (SEQ ID NO: 4);

reverse primer: 5'-CTCCCAAAAAAAACCTTAAACCTAC-3' (SEQ ID NO: 5);

fluorescent probe for detecting methylation of NDRG4 gene:

a fluorescent probe: 5'-TAAGGATATTTTCGGG-3' (SEQ ID NO: 6);

primers for detecting methylation of SDC2 gene:

a forward primer: 5'-GGGAGTGTAGAAATTAATAAGTGAGAG-3' (SEQ ID NO: 7);

reverse primer: 5'-ACCAAACCCAAAATAAACAAAATC-3' (SEQ ID NO: 8); and

fluorescent probe for detecting methylation of SDC2 gene:

a fluorescent probe: 5'-CCCCGAGCCCGAGTCCCCGAGC-3' (SEQ ID NO: 9).

Further, the fluorescent reporter groups of the fluorescent probes in the composition do not interfere with each other.

As used herein, "non-interfering" means that the fluorescent reporter groups used in each fluorescent probe in the composition are not identical and do not interfere with each other's detection, i.e., they can be detected using different channels. For example, FAM, HEX, ROX, VIC and CY5 can be used, which do not have close absorbance values and can select different channels so that they do not interfere with each other.

The composition can be used for screening early colorectal cancer with high sensitivity and specificity, and the three-gene joint detection reduces the sample loading amount and operation, avoids the sample from being excessively diluted, and has low cost and convenient operation.

The specific promoter regions of the Septin9 targeted by the composition of the invention are: human chr.17: 75369186-75369304;

the specific promoter regions of NDGR4 to which the compositions of the invention are directed are: human chr.16: 58497133. 58497310;

the specific promoter regions of SDC2 for which the compositions of the invention are directed are: human chr.8: 97506600-97506800.

Further, the composition may further comprise a blocking probe.

In a specific embodiment, the blocking probe of the Septin9 gene is: 5'-TGGTGCCTGCCTTCCTCCCC-3' (SEQ ID NO: 10).

By using the blocking probe, under the condition that the blocking probe does not influence the whole multiplex PCR detection system, the unmethylated fragments can not be amplified by PCR, and the detection accuracy is further improved.

Further, the composition comprises: an internal standard upstream primer, an internal standard downstream primer and an internal standard probe for monitoring.

In a specific embodiment, the internal standard is a β -acting gene.

In a particular embodiment, the composition further comprises: an internal standard upstream primer shown as SEQ ID NO. 11, an internal standard downstream primer shown as SEQ ID NO. 12, and an internal standard probe shown as SEQ ID NO. 13.

In the present invention, the fluorescent reporter group may be selected from FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3 and JOE, but is not limited thereto.

In a specific embodiment, the fluorescent reporter group of the Septin9 gene fluorescent probe shown in SEQ ID NO. 3 is FAM; the fluorescent reporter group of the NDRG4 gene fluorescent probe shown as SEQ ID NO. 6 is VIC; the fluorescent reporter group of the SDC2 gene fluorescent probe shown as SEQ ID NO. 9 is ROX.

In a specific embodiment, the fluorescent reporter of the internal standard fluorescent probe shown in SEQ ID NO. 13 is CY 5.

In a particular embodiment, the ingredients of the composition of the invention are present in separate packages.

Further, the ingredients of the composition of the present invention are present in a mixed form.

In a second aspect, the present invention provides the use of the above-mentioned composition of the present invention in the preparation of a kit for aiding in the diagnosis of colorectal cancer.

In a third aspect, the present invention provides a kit for aiding in the diagnosis of colorectal cancer, the kit comprising the above-described composition.

Further, the kit further comprises a nucleic acid releasing reagent, dNTP, DNA polymerase, UDG enzyme, PCR buffer solution and Mg²⁺At least one of (1).

Common PCR buffers are Tris-HCl, MgCl₂、KCl、TritonX-100 and the like. The total volume of a single PCR reaction tube is 20-200 mu l.

Further, the amount of the primer in the composition is 20-100 nM; the dosage of the fluorescent probe in the composition is 10-100 nM; the amount of probe blocking probe in the composition is 20-100 nM.

More preferably, the amounts of the components of the kit are as follows:

。

drawings

FIG. 1 shows the results of examining a sample of intestinal cancer with the composition of the present invention;

FIG. 2 shows the results of examining a sample of intestinal cancer with the composition of the present invention;

FIG. 3 shows the results of examining a sample of intestinal cancer with the composition of the present invention;

FIG. 4 shows the results of a sensitive assay of a composition of the present invention;

FIG. 5 shows the result of the specificity test of the composition of the present invention;

FIG. 6A is a graph showing the results of a test in which the composition of the present invention does not have blocking probes;

FIG. 6B shows the detection of the composition of the present invention with a blocking probe;

FIG. 7 shows the result of detecting a colon cancer sample alone with BMP3 gene;

FIG. 8 shows the detection result of the Septin9 gene alone for detecting intestinal cancer samples;

FIG. 9 shows the results of examining intestinal cancer samples with the SDC2 gene alone;

FIG. 10 shows the detection result of NDGR4 gene alone in intestinal cancer samples;

FIG. 11 shows the result of detection of an intestinal cancer sample by the VIM gene alone;

FIG. 12 shows the results of examination of intestinal cancer samples with the SFRP2 gene alone;

FIG. 13 shows the result of detecting intestinal cancer sample alone with Septin9 gene of comparative example composition of the present invention;

FIG. 14 shows the results of testing intestinal cancer samples with SDC2 gene alone according to comparative example composition of the present invention;

FIG. 15 shows the result of examination of intestinal cancer samples alone using NDGR4 gene in comparative example composition of the present invention.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Example 1 primers and probes used in the present invention

Primer for detecting methylation of Septin9 gene:

a forward primer: 5'-GCGGGAACCTGATCCGCCCGGGAGGCGGGG-3' (SEQ ID NO: 1);

reverse primer: 5'-CCTGGCTCAGCTGAATGAATGGGGGAGG-3' (SEQ ID NO: 2);

fluorescent probe for detecting methylation of Septin9 gene:

a fluorescent probe: 5'-AGGGGCCGGCGCCCGCCTTC-3' (SEQ ID NO: 3);

primers for detecting methylation of NDRG4 gene:

a forward primer: 5'-GGATATTTTTGTTAGGGGATGTTTT-3' (SEQ ID NO: 4);

reverse primer: 5'-CTCCCAAAAAAAACCTTAAACCTAC-3' (SEQ ID NO: 5);

fluorescent probe for detecting methylation of NDRG4 gene:

a fluorescent probe: 5'-TAAGGATATTTTCGGG-3' (SEQ ID NO: 6);

primers for detecting methylation of SDC2 gene:

a forward primer: 5'-GGGAGTGTAGAAATTAATAAGTGAGAG-3' (SEQ ID NO: 7);

reverse primer: 5'-ACCAAACCCAAAATAAACAAAATC-3' (SEQ ID NO: 8);

fluorescent probe for detecting methylation of SDC2 gene:

a fluorescent probe: 5'-CCCCGAGCCCGAGTCCCCGAGC-3' (SEQ ID NO: 9);

blocking probe of Septin9 gene:

5’-TGGTGCCTGCCTTCCTCCCC-3’ （SEQ ID NO:10）；

primers for detecting beta-actin:

a forward primer: 5'-TTTGATTTAGGATTTAAAAATTGGAA-3' (SEQ ID NO: 11);

reverse primer: 5'-AACTAAACCATTCTCCTTAAAAAAAA-3' (SEQ ID NO: 12);

fluorescent probe for detecting beta-actin:

and (3) probe: 5'-GTGACAGCAGTCGGTTGGA-3' (SEQ ID NO: 13).

Wherein, the fluorescence reporter group of the Septin9 gene fluorescence probe shown in SEQ ID NO. 3 is FAM; the fluorescent reporter group of the NDRG4 gene fluorescent probe shown as SEQ ID NO. 6 is VIC; the fluorescent reporter group of the SDC2 gene fluorescent probe shown as SEQ ID NO. 9 is ROX; the fluorescent reporter group of the internal standard fluorescent probe shown as SEQ ID NO. 13 is CY 5. The 3' end of the probe also has a BHQ1 or BHQ2 quenching group.

Example 2 method for aiding in the diagnosis of colorectal cancer

1. Extraction of genomic DNA

Taking out the sample from a refrigerator with the temperature of-80 ℃, taking 10-20 mg of tissues, and putting the sample tissues and the grinding beads into a centrifuge tube or a grinding tank;

placing the centrifuge tube or grinding pot into an adapter;

loading the adapter into a grinder, setting working parameters and operating equipment;

the equipment is finished running and taken outCentrifuging the sample for 1 min;

adding 200 mu L of homogenate sample into a 1.5mL centrifuge tube, adding 50 mu L of nucleic acid extraction and purification reagent pre-cracking liquid PL, adding 10 mu L of protease K, and shaking for 10 s;

adding 450 μ L of nucleic acid extraction and purification reagent extract 1, shaking for 1min, centrifuging instantly, and standing at 60 deg.C for 10 min;

adding 150 μ L of nucleic acid extraction and purification reagent extractive solution 2, shaking for 1min, centrifuging instantly, and standing at room temperature for 5 min;

placing the centrifugal tube on a magnetic separator, and slowly absorbing waste liquid after 2min (taking care not to touch magnetic beads absorbed on the inner side of the tube wall);

adding 800 μ L of nucleic acid extraction and purification reagent washing solution 1, shaking for 30s, placing the centrifuge tube on a magnetic separator, and slowly absorbing waste liquid after 2min (taking care not to touch magnetic beads adsorbed on the inner side of the tube wall);

adding 800 μ L of nucleic acid extraction and purification reagent washing solution 2, shaking for 30s, placing the centrifuge tube on a magnetic separator, and slowly absorbing waste liquid after 2min (taking care not to touch magnetic beads adsorbed on the inner side of the tube wall); adding 400 mu L of nucleic acid extraction and purification reagent washing solution 2, shaking for 30s, placing the centrifugal tube on a magnetic separator, and slowly absorbing waste liquid after 2min (taking care not to touch magnetic beads adsorbed on the inner side of the tube wall); instantaneous moment of actionCentrifuging, placing the centrifugal tube on a magnetic separator, carefully absorbing and discarding the liquid at the bottom of the tube, and standing at room temperature for 5 min; adding 50-100 mu L of eluent S, shaking and uniformly mixing for 30S, eluting the magnetic beads on the wall of the centrifugal tube to the bottom of the centrifugal tube, standing for 10min at room temperature (or standing for 10min at 56 ℃ can be selected, so that the nucleic acid elution efficiency of part of complex samples can be improved), placing the centrifugal tube on a magnetic separator again for magnetic absorption for 2min, and then transferring the eluted nucleic acid into a new 1.5mL centrifugal tube.

2. Sulfite conversion

Sequentially taking 20 mu L of extracted DNA samples (diluted according to the concentration, the total amount of DNA is not more than 500 ng) and adding into an eight-connecting tube, then adding 130 mu L of Lighting Conversion Reagent, mixing uniformly and centrifuging;

putting the mixed eight connecting tubes into a PCR instrument, and setting a program:

8mins at 98 ℃; 60mins at 54 ℃; 20mins at 4 ℃ (4 ℃ being an optional choice);

taking out the eight-connected tube after the operation of the instrument is finished, sucking out the solution in the tube, adding the solution into a new purifying tube, adding 600 mu L M-Binding Buffer, putting the purifying tube into a collecting tube, and covering a tube cover;

turning the tube upside down for several times, and mixing;

centrifuging at 12000rpm for 30s, and discarding the waste liquid in the collecting tube;

adding 100 mu L M-Wash Buffer, centrifuging at 12000rpm for 30s, and removing waste liquid in the collection tube;

adding 200 mu L L-depletion Buffer, standing at room temperature (20-30 ℃) for 15-20 mins, centrifuging at 12000rpm for 30s, and removing waste liquid in a collection tube;

adding 200 mu L M-Wash Buffer, centrifuging at 12000rpm for 30s, and removing waste liquid in a collection tube;

repeating the step 8;

the purified tube is placed into a new EP tube with the volume of 1.5mL, 20 mu L M-precipitation Buffer is added, the tube is centrifuged at 12000rpm for 30s, and the centrifuged EP tube is the purified DNA sample which can be directly used for experiments.

3. PCR procedure

Preparing the designed primer probe and other components into PCR reaction solution according to a proportion, and adding 2 mu L of the converted template;

performing amplification detection by using Shanghai macrolith SLAN-96P real-time fluorescent quantitative PCR, wherein the amplification program comprises the following steps: denaturation at 95 deg.C for 10 min; 45 cycles of 95 ℃ for 15s and 60 ℃ for 30s (fluorescence acquisition).

4. Quality control and result analysis

1) The target detection signals are FAM, VIC and ROX, and the internal reference detection signal is CY 5;

2) setting Baseline: baseline is generally set to be 3-15 cycles, and can be adjusted according to actual conditions. The adjustment principle is as follows: selecting a region with stable fluorescence signal before exponential amplification, wherein the starting point (Start) avoids the signal fluctuation in the initial stage of fluorescence acquisition, and the End point (End) is reduced by 1-2 cycles compared with the sample Ct with the earliest exponential amplification. Setting Threshold: setting a principle that a threshold line just exceeds the highest point of a normal negative control product;

3) according to the test result, the Ct value of the internal standard (CY 5 channel) is Ct less than or equal to 36, which indicates that the detection is effective, and the subsequent experiment is continued;

A) if the FAM channel detects a typical S-type amplification curve and Ct is less than 40, the result of the methylation detection of the Septin9 gene is positive;

B) if the VIC channel detects a typical S-type amplification curve and Ct is less than 40, the NDGR4 gene methylation detection result is positive;

C) if the ROX channel detects a typical S-type amplification curve and Ct is less than 40, the methylation detection result of the SDC2 gene is positive;

interpretation criteria: judging that the sample is positive if any one target in the three genes is drawn and Ct is less than 40;

4) if the internal standard does not detect Ct or Ct is more than 36 in the CY5 channel, the result indicates that the concentration of the detected sample is too low or the interfering substances inhibit the reaction, and the experiment needs to be prepared again.

Example 3 results of testing clinical specimens with the composition of the invention

40 cancer tissue samples were tested using the composition described in example 1 according to the method described in example 2, and exemplary test results for 3 samples are shown in FIGS. 1-3. As can be seen from the figure, the composition of the present invention is well able to amplify samples of colorectal cancer. And the positive detection rate of the composition of the present invention was 100% (40/40) in all 40 cancer tissue samples.

Example 4 detection results of sensitivity of the composition of the present invention

Nucleic acid was extracted from a tissue sample determined to be intestinal cancer, and then sulfite conversion was performed, and the sample was diluted to a concentration of 0.05 ng/. mu.L, 0.025 ng/. mu.L, 0.01 ng/. mu.L, 0.005 ng/. mu.L, 0.0025 ng/. mu.L, 0.001 ng/. mu.L, or 0.0005 ng/. mu.L, and then subjected to multiplex PCR to verify the sensitivity threshold. The detection results are shown in fig. 4 and table 1.

According to the detection result, the sample can still be normally detected when the concentration of the sample is 0.001 ng/. mu.L, namely, the sensitivity of the composition of the invention is 0.001 ng/. mu.L.

Example 5 detection results of the specificity of the composition of the invention

In order to ensure the detection accuracy of the one-tube method, the primers cannot interfere with each other and cannot generate the condition of non-specific amplification, primer probes of septin9, NDRG4 and SDC2 are mixed to prepare PCR reaction liquid, 40 cases of normal human blood samples are used for extracting nucleic acid, and detection is carried out after methylation transformation. The results of the detection are shown in FIG. 5. As can be seen from the figure, the detection results of all the 40 negative samples are negative and the internal standard amplification is normal, and the experiment proves that no crosstalk exists between the primers, the specificity is good, and the non-specific amplification cannot occur to the negative samples.

Example 6 Effect of blocking Probe on test results

Furthermore, in order to ensure the amplification accuracy and avoid the occurrence of false positive results, a PNA blocking probe is added into the reaction system, and the PNA blocking probe can be specifically combined with the unmethylated single-chain fragment and cannot be hydrolyzed and enzyme-digested, so that the reaction system can specifically amplify the methylated fragment, and the detection accuracy is improved. The detection result is shown in fig. 6, and it can be seen from the figure that, after the blocking probe is added, the amplification of the Septin9 is more accurate, and the result of false positive is avoided.

Example 7 results of the combination of multiple Gene targets for colorectal cancer detection

To further illustrate the superiority of the present composition, in addition to the targets to which the present invention relates, the inventors have also investigated the remaining colorectal cancer-related targets: BMP3 gene, VIM gene and SFRP2 gene.

First, the detection rate of a single gene target was investigated. Methylation of the gene was detected in 10 samples identified as colorectal cancer, and the results are shown in FIGS. 7 to 12. As can be seen from the figure, the individual detection rates of the 6 genes are respectively: septin9 (8/10), SDC2 (7/10), NDRG4 (6/10), BMP3 (4/10), VIM (6/10) and SFRP2 (7/10) were 80% (8/10), 70% (7/10), 60% (6/10) and 70% (6/10).

Secondly, the detection rate of different gene target compositions is researched, 40 cancer tissue samples are detected, and the results of different three-gene joint detection are shown as follows:

septin9+ NDRG4+ SDC2 is 100% (40/40);

septin9+ VIM + SFRP2 was 90% (36/40);

septin9+ NDRG4+ VIM 92% (37/40);

92% (37/40) of Septin9+ NDRG4+ SFRP 2;

NDRG4+ SDC2+ VIM is 95% (38/40);

NDRG4+ SDC2+ SFRP2 is 95% (38/40);

90% (36/40) for SDC2+ VIM + SFRP 2;

NDRG4+ VIM + SFRP2 was 85% (34/40);

90% (36/40) of BMP3+ Septin9+ NDRG 4;

95% (38/40) of BMP3+ Septin9+ SDC 2;

90% of BMP3+ Septin9+ VIM (36/40);

90% (36/40) of BMP3+ Septin9+ SFRP 2;

95% (38/40) for BMP3+ NDRG4+ SDC 2;

85% of BMP3+ NDRG4+ VIM (34/40);

85% (34/40) for BMP3+ NDRG4+ SFRP 2;

72% for BMP3+ SDC2+ VIM (29/40);

72% (29/40) for BMP3+ SDC2+ SFRP 2;

90% of BMP3+ VIM + Septin9 (36/40);

72% of BMP3+ VIM + SFRP2 (29/40).

As can be seen from the results, although the detection rates of the VIM and SFRP2 genes, in particular, were consistent with those of the genes used in the composition of the present invention when detected individually, the detection rates were clearly different when they were combined together for detection. Further illustrating the advantages of the composition of the invention, the composition of the invention enables to achieve a complete coverage of the sample of colorectal cancer avoiding the risk of missed detection.

Example 8 comparison of the results of detection of the compositions of the invention with the Single PCR

In order to show that the multiplex PCR adopted by the invention does not have great difference of detection effect compared with single PCR of each gene, the invention further verifies whether the multiplex PCR and the single amplification have difference or not, and 40 cases of intestinal cancer clinical samples are verified by adopting the multiplex PCR and the single PCR for comparison experiments. The results of the experiments are shown in tables 2-3 below.

According to the comparison result, the detection rate of the multiplex PCR is the same as 100 percent (40/40) compared with that of the single PCR, the Ct value of the multiplex PCR amplification is slightly deduced, but does not exceed 1 Ct value, and no obvious difference exists, which indicates that the composition has the advantages of sensitivity and accuracy of the single PCR.

Example 9 comparative example test results of inventive composition

To further illustrate the superiority of the compositions of the invention, the inventors also designed the remaining plurality of primers and probes. The inventor finds another region in the promoter region of Septin9, and the specific promoter region is as follows: human Chr.16:75277963 and 75278145; another region was found in the promoter region of NDRG4, which was specifically the promoter region: human chr.16: 58498500-58498753; another region was found in the promoter region of SDC2, with specific promoter regions: human chr.8: 97507512-97507773. However, primers and probes were designed for the above-mentioned genes, and then methylation of the genes was detected in 10 cases of the samples identified as colorectal cancer, and the results of the detection are shown in FIGS. 13 to 15. As can be seen from the figure, the individual detection rates of the 3 genes are respectively: septin9 was 50% (5/10), SDC2 was 40% (4/10), NDRG4 was 40% (4/10).

Secondly, the detection rate of different gene target compositions is researched, 40 cancer tissue samples are detected, and the result of the three-gene joint detection is as follows: septin9+ NDRG4+ SDC2 was 65% (26/40).

Therefore, it can be seen that the detection effect of the comparative example composition of the present invention is much lower than that of the composition of the present invention, demonstrating the superiority of the composition of the present invention in the detection effect.

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Claims

1. A composition for aiding in the diagnosis of colorectal cancer, the composition comprising:

primer for detecting methylation of Septin9 gene:

a forward primer: 5'-GCGGGAACCTGATCCGCCCGGGAGGCGGGG-3' (SEQ ID NO: 1);

reverse primer: 5'-CCTGGCTCAGCTGAATGAATGGGGGAGG-3' (SEQ ID NO: 2);

fluorescent probe for detecting methylation of Septin9 gene:

a fluorescent probe: 5'-AGGGGCCGGCGCCCGCCTTC-3' (SEQ ID NO: 3);

primers for detecting methylation of NDRG4 gene:

a forward primer: 5'-GGATATTTTTGTTAGGGGATGTTTT-3' (SEQ ID NO: 4);

reverse primer: 5'-CTCCCAAAAAAAACCTTAAACCTAC-3' (SEQ ID NO: 5);

fluorescent probe for detecting methylation of NDRG4 gene:

a fluorescent probe: 5'-TAAGGATATTTTCGGG-3' (SEQ ID NO: 6);

primers for detecting methylation of SDC2 gene:

a forward primer: 5'-GGGAGTGTAGAAATTAATAAGTGAGAG-3' (SEQ ID NO: 7);

reverse primer: 5'-ACCAAACCCAAAATAAACAAAATC-3' (SEQ ID NO: 8); and

fluorescent probe for detecting methylation of SDC2 gene:

a fluorescent probe: 5'-CCCCGAGCCCGAGTCCCCGAGC-3' (SEQ ID NO: 9).

2. The composition of claim 1, wherein the fluorescent reporter groups of the fluorescent probes do not interfere with each other.

3. The composition of claim 2, wherein the composition further comprises a blocking probe.

4. The composition of claim 3, wherein the blocking probe is 5'-TGGTGCCTGCCTTCCTCCCC-3' (SEQ ID NO: 10).

5. The composition of any one of claims 1 to 4, wherein the composition further comprises an internal standard upstream primer, an internal standard downstream primer and an internal standard probe for monitoring.

6. The composition of claim 5, wherein the composition comprises an internal standard upstream primer shown as SEQ ID NO. 11, an internal standard downstream primer shown as SEQ ID NO. 12, and an internal standard probe shown as SEQ ID NO. 13.

7. The composition of claim 6, wherein the fluorescent reporter of the Septin9 gene fluorescent probe shown in SEQ ID NO. 3 is FAM; the fluorescent reporter group of the NDRG4 gene fluorescent probe shown as SEQ ID NO. 6 is VIC; the fluorescent reporter group of the SDC2 gene fluorescent probe shown as SEQ ID NO. 9 is ROX; the fluorescent reporter group of the internal standard fluorescent probe shown as SEQ ID NO. 13 is CY 5.

8. Use of a composition according to any one of claims 1 to 7 in the manufacture of a kit for aiding in the diagnosis of colorectal cancer.

9. A kit for aiding in the diagnosis of colorectal cancer, the kit comprising the composition of any one of claims 1 to 7.

10. The kit according to claim 9, wherein the kit further comprises the remaining reagents required for PCR, such as nucleic acid releasing reagents, dntps, DNA polymerase, UDG enzyme, PCR buffer and Mg²⁺At least one of (1).