CN112159846A - Composition, kit and application thereof - Google Patents
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Abstract
The invention provides compositions, kits and uses thereof. The composition comprises a first detection reagent for detecting at least two methylation target regions of a CpG island of the Septin9 gene and a second detection reagent for detecting at least two methylation target regions of a CpG island of the SDC2 gene. The composition of the embodiment of the invention has at least the following beneficial effects: the embodiment of the invention carries out combined detection on a plurality of target genes related to colorectal cancer, selects site combinations of different methylated target regions which are separated from each other by a certain distance for different methylated sites on the same gene, and respectively and independently sets detection reagents for detection, effectively increases detection target sites, judges according to superposed signals of different sites, effectively enhances detection signals, improves the utilization efficiency of limited samples, and thus improves the detection sensitivity.
Description
Technical Field
The invention relates to the technical field of cancer detection, in particular to a composition, a kit and application thereof.
Background
The related data show that the incidence and mortality of colorectal cancer are the third and fifth of the incidence and mortality of tumors in China, and the incidence and mortality of colorectal cancer rapidly increase with the aging population and the change of dietary structure in recent years. In clinical practice, early detection of colorectal cancer is very important for effective treatment of cancer patients. DNA methylation is an early event of tumor occurrence and is closely related to the occurrence and development of tumors, the high methylation of CpG islands of related genes is earlier than the occurrence of tumors, and meanwhile, the DNA methylation exists stably and can be detected through a PCR amplification effect. Therefore, the methylation state of the genes can be used as a potential tumor marker for early screening, auxiliary diagnosis and curative effect evaluation of tumors. Compared with other cancer species, the methylation detection of colorectal cancer is carried out more, and several products enter clinical application at home and abroad, but the clinical sensitivity and specificity are generally lower, and the gap exists between the clinical sensitivity and the clinical requirement. Therefore, it is necessary to provide a composition having higher detection sensitivity.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a composition with higher detection sensitivity and capable of being used for colorectal cancer detection, a kit and application thereof.
In a first aspect, one embodiment of the invention provides a composition comprising a first detection reagent that detects at least two methylation target regions of a CpG island of the Septin9 gene and a second detection reagent that detects at least two methylation target regions of a CpG island of the SDC2 gene.
The composition of the embodiment of the invention has at least the following beneficial effects:
the embodiment of the invention carries out combined detection on a plurality of target genes related to colorectal cancer, selects site combinations of different methylated target regions which are separated from each other by a certain distance for different methylated sites on the same gene, and respectively and independently sets detection reagents for detection, effectively increases detection target sites, judges according to superposed signals of different sites, effectively enhances detection signals, improves the utilization efficiency of limited samples, and thus improves the detection sensitivity.
According to the compositions of some embodiments of the invention, the methylation target region of the Septin9 gene includes a first target region of Septin9 and a second target region of Septin 9; the methylated target regions of the SDC2 gene include a SDC2 first target region and a SDC2 second target region.
A plurality of methylation sites are distributed in the first target region and the second target region of the Septin9 gene and the SDC2 gene, the sites have very obvious differential methylation in colorectal cancer patients and normal human bodies, and accumulated signals are obtained by respectively detecting the combination of different methylation sites in the target regions of any gene, so that the detection sensitivity is effectively improved.
According to the composition of some embodiments of the invention, the first detection reagent comprises a first probe set comprising a first probe (Septin9-1) that specifically detects a first target region of Septin9 and a second probe (Septin9-2) that specifically detects a second target region of Septin 9; the second detection reagent comprises a second set of probes comprising a third probe (SDC2-1) specific for detecting the first target region of SDC2 and a fourth probe (SDC2-2) specific for detecting the first target region of SDC 2;
wherein the nucleotide sequences of the first probe, the second probe, the third probe and the fourth probe are as follows:
first probe Septin 9-1: TAACCGCGAAATCCGAC (SEQ ID No. 3);
second probe Septin 9-2: CGTCCGCGACCGC (SEQ ID No. 7);
third probe SDC 2-1: GCGCGCGTGGATTTTGT (SEQ ID No. 11);
fourth probe SDC 2-2: GAGGGCGCCGCGTTCCCGGG (SEQ ID No. 15).
According to the composition of some embodiments of the invention, the 5 'end of the first probe, the second probe, the third probe and the fourth probe is labeled with a fluorescent reporter group, and the 3' end of the first probe, the second probe, the third probe and the fourth probe is labeled with a fluorescent quencher group; the emission spectra of the fluorescent reporter group and the fluorescent quencher group of the probes of different probe sets are different.
According to some embodiments of the invention, the fluorescent reporter group is selected from FAM, VIC, HEX, JOE, ROX, CY5 and the fluorescence quencher group is selected from BHQ1, BHQ2, TAMRA.
According to the composition of some embodiments of the invention, the fluorescent reporter group of the first probe and the fluorescent reporter group of the second probe are FAM, and the fluorescent quencher group is BHQ 1; the fluorescence reporter group of the third probe and the fourth probe is ROX, and the fluorescence quencher group is BHQ 2.
According to the composition of some embodiments of the invention, the first detection reagent further comprises a first specific primer set comprising a first primer pair (Septin9-1-F/R) that specifically amplifies a first target region of Septin9 and a second primer pair (Septin9-2-F/R) that specifically amplifies a second target region of Septin 9; the second detection reagent further comprises a second specific primer set comprising a third primer pair (SDC20-1-F/R) that specifically amplifies the first target region of SDC2 and a fourth primer pair (SDC20-2-F/R) that specifically amplifies the second target region of SDC 2;
wherein the nucleotide sequences of the first primer pair, the second primer pair, the third primer pair and the fourth primer pair are as follows:
the upstream primer Septin9-1-F of the first primer pair: GATTCGTTGTTTATTAGTTATTATG (SEQ ID No.1),
the downstream primer Septin9-1-R of the first primer pair: AAATAATCCCATCCAACT (SEQ ID No. 2);
the upstream primer Septin9-2-F of the second primer pair: GGTGTTGGGTTGGTTGT (SEQ ID No.5),
the downstream primer Septin9-2-R of the second primer pair: CACCCGCAAAATCCTCT (SEQ ID No. 6);
the upstream primer SDC2-1-F of the third primer pair: TAGTCGGTTTTTGGGGA (SEQ ID No.9),
the downstream primer SDC2-1-R of the third primer pair: AACCACCAAACCCAAAATA (SEQ ID No. 10);
the upstream primer SDC2-2-F of the fourth primer pair: GAGTGCAGAAACCAACAAGT (SEQ ID No.13),
the downstream primer SDC2-2-R of the fourth primer pair: CCTCCTCCTGCGCCTGCTC (SEQ ID No. 14).
In accordance with some embodiments of the invention, the first detection reagent further comprises a first blocking sequence (Blocker) set comprising a first blocking sequence (Blocker1) and a second blocking sequence (Blocker2), and the second detection reagent further comprises a second blocking sequence set comprising a third blocking sequence (Blocker3) and a fourth blocking sequence (Blocker 4); the Blockers 1-4 are respectively used for being combined with non-methylated sequences of a first target region of Septin9, a second target region of Septin9, a first target region of SDC2 and a second target region of SDC2, so that the non-methylated sequences are prevented from interfering with the nucleotide sequences of methylation detection.
Wherein the nucleotide sequences of the first blocking sequence, the second blocking sequence, the third blocking sequence and the fourth blocking sequence are respectively as follows:
first blocking sequence Blocker 1: TTATTATGTTGGATTTTGTGGTTAATGTGTA (SEQ ID No.4),
second blocking sequence Blocker 2: TTGTTGTGGTTGTGGATGTGTTG (SEQ ID No. 8);
third blocking sequence Blocker 3: TAAACAAAATCCACACACACCACATATT (SEQ ID No.12),
fourth blocking sequence Blocker 4: GTGAGAGGGCGCCGCGTTCCCGGGGCGCAGCTGCGGGCGGCGGG (SEQ ID No. 16).
According to the composition of some embodiments of the present invention, the 3' ends of the first blocking sequence, the second blocking sequence, the third blocking sequence and the fourth blocking sequence are modified by phosphorylation.
In a second aspect, one embodiment of the present invention provides a kit comprising the composition described above.
The kit according to some embodiments of the present invention, further comprising a detection reagent for the reference gene. The expression of internal reference genes such as ACTB is constant in different tissue cells, and the internal reference genes are used as reference substances to correct experimental errors during sample loading, so that the accuracy of results is ensured.
According to the kit of some embodiments of the present invention, the detection reagent for the reference gene includes a detection probe for the reference gene and upstream and downstream primers.
According to the kit of some embodiments of the present invention, the nucleotide sequence of the detection probe for the reference gene is as follows:
ACTB probe: CTTTACACCAACCTCATAACCTTATCAC (SEQ ID No. 19).
According to the kit of some embodiments of the present invention, the 5 'end of the detection probe of the reference gene is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group; the fluorescence reporter group is VIC, and the fluorescence quencher group is BHQ 1.
According to the kit of some embodiments of the present invention, the nucleotide sequences of the upstream and downstream primers of the reference gene are as follows:
ACTB-F：GTGTTTAAGATAGTGTTGTGG(SEQ ID No.17)，
ACTB-R：CTACTTAATACACACTCCAAAAC(SEQ ID No.18)。
a kit according to some embodiments of the invention further comprises a buffer, a polymerase, a divalent cation, and dNTPs.
A kit according to some embodiments of the invention further comprises a positive control and/or a negative control.
According to the kit of some embodiments of the present invention, the positive control comprises HeLa cells.
According to the kit of some embodiments of the present invention, the positive control is a mixture of HeLa cells, bovine serum albumin and TE buffer.
According to the kit of some embodiments of the invention, the negative control comprises Jurkat cells.
According to the kit of some embodiments of the present invention, the negative control is a mixture of Jurkat cells, bovine serum albumin, and TE buffer.
In a third aspect, an embodiment of the present invention provides a use of the above composition or the above kit for preparing a diagnostic reagent for colorectal cancer. The composition or the kit performs combined detection on a plurality of target genes related to colorectal cancer, so that more target sites are detected, the superposed signals of different sites are stronger, and the sensitivity of colorectal cancer detection is higher.
Drawings
FIG. 1 is a graph of amplification of FAM channels from the detection of early colorectal cancer samples in accordance with one embodiment of the present invention.
FIG. 2 is a graph showing the amplification of VIC channels from the detection of early colorectal cancer samples in accordance with one embodiment of the present invention.
FIG. 3 is a graph showing the amplification of the ROX channel in the detection of early colorectal cancer samples according to an embodiment of the present invention.
FIG. 4 is a graph showing the amplification of FAM channels from samples of different methylation ratios according to another embodiment of the present invention.
FIG. 5 is a graph showing the amplification of VIC channels from samples with different methylation ratios according to another embodiment of the present invention.
FIG. 6 is a graph showing the amplification of ROX channels for the detection of samples with different methylation ratios according to another embodiment of the present invention.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
Herein, the "Septin 9 gene" is one of a family of conserved genes having guanosine triphosphatase activity, and is related to many cell functions such as chromosome separation, DNA repair, migration and apoptosis. The Septin9 gene acts as a cancer suppressor gene in colorectal cancer, and methylation of the gene can inhibit normal expression of the gene, so that the cancer suppressor function is lost, and finally cell division and canceration are caused. The methylation abnormality is an important molecular characteristic of the colorectal cancer, and can be used for early diagnosis of the colorectal cancer.
Herein, the "SDC 2 gene" is the syndecan 2 (SDC 2) gene. The SDC2 gene shows high-level methylation in colorectal cancer and adenocarcinoma tissues, and the high-level methylation is a sign that the SDC2 gene has high value as a diagnostic marker of colorectal cancer.
Herein, "methylation specific PCR" is a method that utilizes differential PCR amplification to detect the presence or absence of methylated modifications of DNA. The method mainly utilizes NaOH and sodium bisulfite to carry out sulfur conversion pretreatment on a DNA template, unmethylated cytosine base (C) is converted into uracil (U), methylated cytosine is not converted, and then specific PCR primers and probes are used for identifying the methylated or unmethylated DNA template.
As used herein, "blocking sequence (Blocker)" refers to a nucleotide sequence that is used to bind to an unmethylated sequence to avoid interference with methylation detection by the unmethylated sequence.
The reagents used in the following examples include:
free nucleic acid extraction kit, Qiagen, germany;
bisulfite conversion kit, ZYMO RESEARCH, usa;
taq enzyme, TAKARA, Japan;
10 XPCR Buffer, TAKARA, Japan;
dNTPs, TAKARA, Japan;
MgCl2japan TAKARA.
The following examples used instruments including: ABI 7500 fluorescent quantitative PCR instrument, water bath, vortex mixer, high speed centrifuge, and vacuum pump.
Example 1
Design of primers and probes
Two methylation target regions are selected as detection targets for target genes Septin9 and SDC2 respectively, each methylation target region comprises 2-4 methylation sites, and an upstream amplification primer, a downstream amplification primer, a probe and a blocking sequence are designed according to the methylation target regions respectively. Wherein, the upstream and downstream amplification primers and the probe are designed based on the sequence after sulfur conversion, namely C of the non-methylation site in the original sequence is completely converted into a sequence of T; the blocking sequence is used for combining with the non-methylated sequence to avoid the non-methylated sequence from interfering the methylation detection, so the blocking sequence is designed after further converting C of the methylated site into T on the basis of the converted sequence. Converting a section of sequence of the reference gene ACTB into a sequence after sulfur conversion, and setting corresponding upstream and downstream amplification primers and probes. The final primers, probes, and blocking sequences are shown in Table 1, and the sequences were sent to the synthesis supplier for synthesis, and the synthesized sequences were dissolved and diluted to 10. mu.M for use.
TABLE 1 detection reagent sequences
Wherein, the 5 'end of the Septin9-1 probe and the Septin9-2 probe is marked with a fluorescence reporter group FAM, and the 3' end is marked with a fluorescence quenching group BHQ 1; the 5 'ends of the SDC2-1 probe and the SDC2-2 probe are marked with a fluorescence reporter group ROX, and the 3' ends are marked with a fluorescence quenching group BHQ 2; the ACTB probe is labeled with a fluorescent reporter group VIC at the 5 'end and a fluorescent quencher group BHQ1 at the 3' end.
Example 2
Detection experiment
Preparation of a reference substance: culturing HeLa cells, separating, adding bovine serum albumin and 1 XTE buffer solution to obtain final concentration of 4 × 103And (4) obtaining a positive control substance, wherein the total concentration of the bovine serum albumin is 50 g/mL. Culturing Jurkat cells, separating, adding bovine serum albumin and 1 XTE buffer solution to give final concentration of Jurkat cells of 4 × 103And (4) obtaining a negative control substance, wherein the final concentration of the bovine serum albumin is 50 g/L.
Plasma samples of two colorectal cancer patients were taken and detected by methylation-specific PCR using the detection reagent designed in example 1, and the specific steps were as follows:
(1) 10mL of peripheral blood of the patient was extracted by a Streck tube, centrifuged, and plasma was separated.
(2) And (4) taking the plasma of the patient sample, a positive control substance and a negative control substance, and performing parallel extraction by using a free nucleic acid extraction kit to obtain a free DNA sample.
(3) Free DNA was subjected to sulfur conversion and purification using a bisulfite conversion kit.
(4) The purified product was subjected to fluorescent quantitative PCR detection, the final PCR reaction system was 50. mu.L, and contained 24. mu.L of PCR reaction mixture, 1. mu.L of Taq enzyme, and 25. mu.L of purified product, the final concentrations of the reaction components are shown in Table 2, and the reaction procedures are shown in Table 3.
TABLE 2 PCR reaction System
TABLE 3 PCR reaction procedure
After the reaction is finished, the result is judged according to the Ct value, and the result is shown in figures 1-3. As can be seen from the figure, Ct values of the internal references of the 4 groups of reactions are all below 40, Ct values of two colorectal cancer samples and positive controls have obvious amplification curves in FAM and ROX channels, the Ct values are below 40, negative controls have no amplification curves in FAM and ROX channels, test results are in line with expectations, and the kit can be proved to be capable of effectively detecting plasma positive samples.
Example 3
Comparative experiment
Samples of early colorectal cancer patients were collected in 70 cases, and healthy human plasma was collected in 100 cases, and DNA was extracted and sulfur-transformed by the method of example 2. The PCR reaction systems of example 2 and comparative example 1 were used to test the samples after sulfur conversion. Comparative example 1 is a single target region reaction system, each target gene only has one target region to be included in the detection, see table 4.
TABLE 4 Single target region detection PCR reaction System
After the detection is finished, judging the result, judging the positive control FAM channel, the VIC channel and the ROX to be normal if the Ct values of the positive control FAM channel, the VIC channel and the ROX are less than or equal to 40, and judging the negative control VIC channel to be normal if the Ct value of the reference gene is less than 40 and no Ct value exists in both FAM and ROX; if the Ct value of the VIC channel of the detected sample is greater than 40, determining that the sample is unqualified; the Ct value of the VIC channel of the detection sample is less than or equal to 40, and at least one of the Ct values of FAM and ROX is less than or equal to 42, the detection sample is judged to be positive; and (3) judging the sample to be negative if the Ct value of the VIC channel is less than or equal to 40, and the Ct values of both the FAM channel and the ROX channel are greater than 42 or no amplification exists.
TABLE 5 comparative test results
The results are shown in Table 5, from which it can be seen that: for 70 early colorectal cancer samples, the reaction system of comparative example 1 detected 56 positive samples, and the reaction system of example 2 detected 62 positive samples; for 100 healthy human plasma samples, the reaction system of comparative example 1 detected 4 positives, the reaction system of example 2 detected 5 positives, and the detection sensitivities and the detection specificities of comparative example 1 and example 2 were 80% and 88.6%, respectively, and 96% and 95%, respectively. The result shows that the detection sensitivity of the multi-gene multi-target area detection system adopted by the embodiment of the invention is obviously improved compared with that of a multi-gene single-target area detection system, the specificity is slightly reduced but the difference is very small, and the detection sensitivity of the multi-gene multi-target area detection reagent provided by the invention can be effectively improved compared with the traditional detection.
Example 4
Methylation gradient experiment
DNA was extracted from Hela and Jurkat cells, and then quantified and diluted to 1 ng/. mu.L after sulfur conversion, mixed DNA templates (50%, 5%, 1%, 0.5% for methylated templates, respectively) were prepared, and the mixture was examined using the reaction systems shown in Table 2, wherein the ratios of methylated templates were 50%, 5%, 1%, and 0.5%, respectively. As shown in FIGS. 4 to 6, this system was able to detect positive results for all of the above templates. The existing detection reagent has a false negative result when the methylation proportion of the total template is low and the total amount of the methylated template is very low. The detection reagent provided by the embodiment of the invention respectively detects at least two target areas containing a plurality of methylation sites to obtain accumulated signals, can effectively detect samples with the methylation proportion as low as 0.5%, and increases the detection rate of the methylation sites and effectively improves the detection sensitivity compared with the traditional detection method.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> 1
SHENZHEN NUCLEAR GENE TECHNOLOGY Co.,Ltd.
<120> composition, kit and application thereof
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<170> PatentIn version 3.5
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ggtgttgggt tggttgt 17
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Claims (9)
1. A composition comprising a first detection reagent that detects at least two methylation target regions of the CpG island of the Septin9 gene and a second detection reagent that detects at least two methylation target regions of the CpG island of the SDC2 gene.
2. The composition of claim 1, wherein the methylation target region of the Septin9 gene comprises a first target region of Septin9 and a second target region of Septin 9; the methylated target regions of the SDC2 gene include a SDC2 first target region and a SDC2 second target region;
the first detection reagent comprises a first probe set comprising a first probe that specifically detects a first target region of the Septin9 and a second probe that specifically detects a second target region of the Septin 9; the second detection reagent comprises a second set of probes comprising a third probe that specifically detects the first target region of SDC2 and a fourth probe that specifically detects the second target region of SDC 2;
wherein the nucleotide sequences of the first probe, the second probe, the third probe and the fourth probe are respectively as follows:
first probe Septin 9-1: TAACCGCGAAATCCGAC (SEQ ID No. 3);
second probe Septin 9-2: CGTCCGCGACCGC (SEQ ID No. 7);
third probe SDC 2-1: GCGCGCGTGGATTTTGT (SEQ ID No. 11);
fourth probe SDC 2-2: GAGGGCGCCGCGTTCCCGGG (SEQ ID No. 15);
preferably, the 5 'ends of the first probe, the second probe, the third probe and the fourth probe are labeled with fluorescent reporter groups, and the 3' ends of the first probe, the second probe, the third probe and the fourth probe are labeled with fluorescent quencher groups; the emission spectra of the fluorescence reporter group and the fluorescence quencher group of the probes of different probe sets are different;
preferably, the fluorescence reporter group is selected from FAM, VIC, HEX, JOE, ROX, CY5, and the fluorescence quencher group is selected from BHQ1, BHQ2 and TAMRA.
3. The composition of claim 2, wherein the first detection reagent further comprises a first specific primer set comprising a first primer pair that specifically amplifies a first target region of Septin9 and a second primer pair that specifically amplifies a second target region of Septin 9; the second detection reagent further comprises a second specific primer set comprising a third primer pair that specifically amplifies the first target region of SDC2 and a fourth primer pair that specifically amplifies the second target region of SDC 2;
preferably, the nucleotide sequences of the first primer pair, the second primer pair, the third primer pair and the fourth primer pair are as follows:
the upstream primer Septin9-1-F of the first primer pair: GATTCGTTGTTTATTAGTTATTATG (SEQ ID No.1),
the downstream primer Septin9-1-R of the first primer pair: AAATAATCCCATCCAACT (SEQ ID No. 2);
the upstream primer Septin9-2-F of the second primer pair: GGTGTTGGGTTGGTTGT (SEQ ID No.5),
the downstream primer Septin9-2-R of the second primer pair: CACCCGCAAAATCCTCT (SEQ ID No. 6);
the upstream primer SDC2-1-F of the third primer pair: TAGTCGGTTTTTGGGGA (SEQ ID No.9),
the downstream primer SDC2-1-R of the third primer pair: AACCACCAAACCCAAAATA (SEQ ID No. 10);
the upstream primer SDC2-2-F of the fourth primer pair: GAGTGCAGAAACCAACAAGT (SEQ ID No.13),
the downstream primer SDC2-2-R of the fourth primer pair: CCTCCTCCTGCGCCTGCTC (SEQ ID No. 14).
4. The composition of claim 3, wherein the first detection reagent further comprises a first blocking sequence group comprising a first blocking sequence and a second blocking sequence; the second detection reagent further comprises a second blocking sequence group, wherein the second blocking sequence group comprises a third blocking sequence and a fourth blocking sequence;
preferably, the nucleotide sequences of the first blocking sequence, the second blocking sequence, the third blocking sequence and the fourth blocking sequence are respectively as follows:
first blocking sequence Blocker 1: TTATTATGTTGGATTTTGTGGTTAATGTGTA (SEQ ID No.4),
second blocking sequence Blocker 2: TTGTTGTGGTTGTGGATGTGTTG (SEQ ID No. 8);
third blocking sequence Blocker 3: TAAACAAAATCCACACACACCACATATT (SEQ ID No.12),
fourth blocking sequence Blocker 4: GTGAGAGGGCGCCGCGTTCCCGGGGCGCAGCTGCGGGCGGCGGG (SEQ ID No. 16);
preferably, the 3' ends of the first blocking sequence, the second blocking sequence, the third blocking sequence and the fourth blocking sequence are modified by phosphorylation.
5. Kit, characterized in that it comprises a composition according to any one of claims 1 to 4.
6. The kit according to claim 5, further comprising a detection reagent for an internal reference gene;
preferably, the detection reagent of the reference gene comprises a detection probe and upstream and downstream primers of the reference gene;
preferably, the nucleotide sequence of the detection probe of the reference gene is as follows:
ACTB probe: CTTTACACCAACCTCATAACCTTATCAC (SEQ ID No. 19);
preferably, the nucleotide sequences of the upstream and downstream primers of the reference gene are as follows:
ACTB-F：GTGTTTAAGATAGTGTTGTGG(SEQ ID No.17)，
ACTB-R：CTACTTAATACACACTCCAAAAC(SEQ ID No.18)。
7. the kit of claim 5, further comprising a buffer, a polymerase, a divalent cation, and dNTPs.
8. The kit of claim 5, further comprising a positive control and/or a negative control;
preferably, the positive control comprises HeLa cells;
preferably, the negative control comprises Jurkat cells.
9. Use of the composition of any one of claims 1 to 4, or the kit of any one of claims 5 to 8, for the preparation of a colorectal cancer detection reagent.
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