CN109207592B - Kit for colorectal cancer detection and application thereof - Google Patents

Abstract

The invention discloses a kit for colorectal cancer detection and application thereof; wherein the kit comprises a primer composition and a probe composition; the primer composition comprises BS-S9-F, BS-S9-R, BS-N4-F, BS-N4-R, BS-SDC2-F, BS-SDC2-R, BS-ACT-F and BS-ACT-R; the probe composition included BS-S9-P, BS-N4-P, BS-SDC2-P and BS-ACT-P. The kit can be applied to colorectal cancer detection and has the advantages of high sensitivity, high specificity and the like.

Description

Kit for colorectal cancer detection and application thereof

Technical Field

The invention relates to the field of biotechnology and medicine, in particular to a kit for detecting colorectal cancer and application thereof.

Background

Cancer has become the biggest killer threatening human health, and statistics of chinese cancer data in 2018 show that: the number of new cancer patients in the country in 2014 is 380.4 ten thousands, 12 thousands of cancer patients are increased compared with 2013, and the increase is 3.2%. According to the prediction of Wanqing Chen in Cancer Statistics in China,2015, the number of newly added Cancer cases in China reaches 429.2 ten thousand and the number of newly added Cancer death cases reaches 281.4 ten thousand in 2015.

Colorectal cancer is a common malignancy of the digestive tract, with no significant symptoms at an early stage and no significant abnormal changes in the patient, thus leading to many patients missing the best diagnosis and being in the middle and late stages of cancer once diagnosed.

According to the statistics of Chinese cancer data in 2018, the colorectal cancer incidence rate in China is the fourth and the third of all cancer incidence rates of men and women. The incidence rate of colorectal cancer in the United states is higher than that in China, but the number of colorectal cancer patients in China is unprecedented and almost accounts for 1/4 worldwide in consideration of population base. According to WHO statistics, the method comprises the following steps: the early detection rate of colorectal cancer in the United states reaches 39%, and the 5-year survival rate of patients reaches 65%; however, in China, the early colorectal cancer discovery rate is only 10%, and the 5-year survival rate of patients is only 32%.

The discovery rate is low, the death rate is high, and the reasons for the discovery are that the prevention consciousness is weak, the screening technology is slow to develop, the coverage rate of screening people is low, and the like. Therefore, the early colorectal cancer screening technology in China is improved, the self-prevention consciousness of the people is popularized, and the urban and rural screening coverage is enlarged.

DNA methylation refers to the selective addition of methyl to cytosine at two nucleotides of DNA CG under the catalysis of methyltransferase to form 5-methylcytosine, which is commonly found in 5'-CG-3' sequences of genes. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.

A great deal of research shows that the methylation of the promoter region of the cancer suppressor gene has close correlation with the occurrence of colorectal cancer, which provides a theoretical basis for developing a new colorectal cancer screening technology. The FDA has approved the product Cologuard for colorectal cancer screening in 2014, but this product is mainly targeted to populations in north america. CFDA has also approved the methylation level of SEPT9 in blood for use in the auxiliary diagnosis of colorectal cancer. The blood SEPT9 methylation detection product is on the market at present, but the sensitivity of the product is still to be improved.

Clinically, the gold standard for colorectal cancer diagnosis is confirmation of enteroscopy plus histological pathological section. Prior to performing an enteroscopy, subjects require fasting and bowel cleansing, while the procedure is invasive and risks infection and trauma. In China, due to the fact that cancer screening consciousness of people is not high enough and the enteroscopy has large resistance psychology, people who obviously suffer from intestinal discomfort are mostly subjected to active enteroscopy, and the intestinal discomfort is caused to be caused to the fact that most intestinal cancer patients enter the middle and late stages of cancer once diagnosis is confirmed, and the optimal treatment time is missed.

Other types of colorectal cancer detection means also include fecal occult blood detection, protein marker detection and the like, but are limited by sensitivity, and the methods cannot effectively and accurately screen the colorectal cancer.

Feces are formed from the intestine and pass through the hepatic portal where large numbers of exfoliated cells of intestinal tissue are present. Human nucleic acid in the feces is detected, and cancer risk assessment is carried out on the examined person according to whether the relevant gene has methylation or not, and the confirmed diagnosis is carried out through a gold standard. The nucleic acid detected by the fecal sample is directly from the intestinal tract, and compared with blood detection, the performance is more sensitive and the reaction is more intuitive; compared with the complexity and the complexity of enteroscopy, the stool sampling is noninvasive and convenient, and is more easily accepted by the examinee, thereby being beneficial to the large-scale development of colorectal cancer screening.

At present, the methylation detection products aiming at colorectal cancer related genes in China only have the methylation detection of SEPT9 based on plasma samples, and although the specificity is high, the sensitivity is far from reaching the clinical requirement.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a kit for detecting colorectal cancer, which has the advantages of high sensitivity, high specificity and the like. The application of the kit in detecting colorectal cancer is further provided, the stool sample is used as the detection sample, the sampling is convenient, and the discovery rate of the advanced adenoma and the early colorectal cancer can be effectively improved.

A kit for detecting colorectal cancer comprises a primer composition and a probe composition;

the primer composition comprises a primer pair BS-S9-F and BS-S9-R for detecting SEPT9 gene, a primer pair BS-N4-F and BS-N4-R for detecting NDRG4 gene, a primer pair BS-SDC2-F and BS-SDC2-R for detecting SDC2 gene, and a primer pair BS-ACT-F and BS-ACT-R for detecting ACTB gene,

the DNA sequence of the BS-S9-F is shown in SEQ ID NO.1,

the DNA sequence of the BS-S9-R is shown in SEQ ID NO.2,

the DNA sequence of the BS-N4-F is shown in SEQ ID NO.3,

the DNA sequence of the BS-N4-R is shown in SEQ ID NO.4,

the DNA sequence of the BS-SDC2-F is shown in SEQ ID NO.5,

the DNA sequence of the BS-SDC2-R is shown in SEQ ID NO.6,

the DNA sequence of the BS-ACT-F is shown in SEQ ID NO.7,

the DNA sequence of the BS-ACT-R is shown in SEQ ID NO. 8;

the probe composition includes BS-S9-P, BS-N4-P, BS-SDC2-P and BS-ACT-P,

the DNA sequence of the BS-S9-P is shown in SEQ ID NO.9,

the DNA sequence of the BS-N4-P is shown in SEQ ID NO.10,

the DNA sequence of the BS-SDC2-P is shown in SEQ ID NO.11,

the DNA sequence of the BS-ACT-P is shown in SEQ ID NO. 12.

The kit preferably further comprises a blocking sequence composition comprising BS-S9-C, BS-N4-C and BS-SDC2-C,

the DNA sequence of the BS-S9-C is shown in SEQ ID NO. 13;

the DNA sequence of the BS-N4-C is shown in SEQ ID NO. 14;

the DNA sequence of the BS-SDC2-C is shown in SEQ ID NO. 15.

Preferably, the concentration of each blocking sequence in the blocking sequence composition is 0.1. mu.M.

In the kit, the concentration of each primer in the primer composition is preferably 0.2. mu.M.

In the kit, the concentration of each probe in the probe composition is preferably 0.15. mu.M.

The kit as described above, preferably, in the probe composition,

the BS-S9-P is marked by FAM and BHQ 1;

and/or, the BS-N4-P is marked by ROX and BHQ 2;

and/or, the BS-SDC2-P is marked by CY5 and BHQ 2;

and/or, the BS-ACT-P is labeled with HEX and BHQ 1.

In the kit, preferably, in the closed sequence composition, the BS-S9-C, the BS-N4-C and the BS-SDC2-C are all subjected to phosphorylation modification.

Preferably, the kit further comprises 2x 360Master Mix, glycerol, tetramethylammonium chloride (TMAC) and deionized water.

In the kit, the concentration of the tetramethylammonium chloride is preferably 10 to 40 mM.

Preferably, the volume concentration of the glycerol in the kit is 8-20%.

As a general technical concept, the invention also provides an application of the kit in detecting colorectal cancer.

In the above application, preferably, the application method comprises the following steps:

(1) dissolving the excrement sample in excrement sample protective solution, and extracting excrement DNA;

(2) performing bisulfite conversion on the extracted fecal DNA to obtain methylated fecal DNA;

(3) performing fluorescent quantitative PCR detection on the methylated fecal DNA by using a kit, and calculating a P value according to a logistic regression formula by using the amplified Ct values of SEPT9, NDRG4, SDC2 and ACTB, wherein the logistic regression formula is as follows: p ═ e^Score/(1+e^Score)，

Score＝w1×Ct1+w2×Ct2+w3×Ct3+w4×Ct4+B；

Wherein P is the colorectal cancer risk index,

e is a natural constant, and the natural constant is,

w1 is-0.14867, w2 is-0.12201, w3 is-0.21562, w4 is 0.20329, B is 13.68188;

ct1 is the Ct value of SEPT9 amplification,

ct2 is the NDRG4 amplification Ct value,

ct3 is the Ct value for SDC2 amplification,

ct4 is the ACTB amplification Ct value;

when the P value is greater than or equal to 0.362, the result is judged to be positive; when the average molecular weight is less than 0.362, the test result is judged to be negative.

In the above application, preferably, the reaction procedure of the fluorescence quantitative PCR detection is: 2min at 50 ℃; pre-denaturation at 95 ℃ for 10 min; then, taking the denaturation at 95 ℃ for 15s and the annealing at 60 ℃ for 30s as a cycle, and carrying out 45 times; finally, the temperature is kept at 20 ℃ for 2 min.

In the above application, preferably, the fecal sample protective solution comprises the following components: 0.25M-0.8M tris-HCl, 1.0M-4.0M guanidinium isothiocyanate, 5 mM-20 mM EDTA, 0.1M/v% -0.5M/v% Sodium Dodecyl Sulfate (SDS), 0.5 v/v% -3 v/v% Triton X-100, 0.5 v/v% -3 v/v% Tween 20, 3 v/v% -10 v/v% glycerol.

Further, the fecal sample protective solution comprises the following components: 0.5M tris-HCl, 1.5M guanidinium isothiocyanate, 10mM EDTA, 0.2M/v% Sodium Dodecyl Sulfate (SDS), 1 v/v% Triton X-100, 1 v/v% Tween 20, 5 v/v% glycerol.

The detection principle of the invention is as follows: unmethylated cytosine (C) in a nucleic acid sequence is converted to uracil (U) after bisulfite treatment, and methylated cytosine is not converted, and a primer probe is designed for a sequence converted from a methylated gene, the primer probe can only amplify a nucleic acid converted from a methylated gene, and a nucleic acid converted from a non-methylated gene is not amplified. In order to further avoid false positive results caused by non-specific amplification, the invention adds a 3' end phosphorylation modified oligonucleotide sequence which is complementarily matched with a sequence after non-methylated gene conversion, and the sequence can block the combination of an amplification primer and a non-methylated nucleic acid conversion sequence, thereby playing a role in improving the detection specificity.

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

(1) the invention provides a kit for colorectal cancer detection, which adopts SEPT9, NDRG4 and SDC2 for combined detection, adopts ACTB housekeeping genes as internal standards, and monitors the content of human-derived nucleic acid in a sample; the housekeeping gene amplification fragment is a section of non-methylated sequence, and the amplification fragment is used as an internal standard, so that the total amount of human nucleic acid contained in a sample added during detection can be fully monitored, and false positive or false negative of a detection result can be prevented. Methylation of three genes SEPT9, NDRG4 and SDC2 is the most common gene methylation in colorectal cancer patients, but the three genes are not necessarily methylated at the same time, so that the positive detection rate can be effectively improved by adopting a joint detection mode, false negative is reduced, and the detection sensitivity is improved. The kit is high in sensitivity and specificity, and can effectively improve the discovery rate of advanced adenomas and early colorectal cancer, so that colorectal cancer is prevented from occurring or radically cured, and the 5-year survival rate of patients is improved.

(2) The invention provides a kit for detecting colorectal cancer, provides a closed sequence for shielding an unmethylated sequence, and can improve the specificity of a detection reagent.

(3) The invention provides a kit for detecting colorectal cancer, aiming at the problem that in the conversion process, unmethylated cytosine (C) is converted into uracil (U), so that the ratio of guanine (G) to cytosine (C) in an amplification area is greatly reduced, and the amplification efficiency is reduced, tetramethylammonium chloride (TMAC) and glycerol (glycerol) are added into a reaction system to improve the specific amplification efficiency.

(4) The invention provides a kit for detecting colorectal cancer, wherein a detection system is a 4-joint detection reagent, a real-time fluorescent quantitative PCR technology is adopted, and four detection genes adopt different fluorescent markers, so that the kit is convenient to distinguish and the detection accuracy is improved.

(5) The invention provides application of a kit for detecting colorectal cancer in colorectal cancer detection, wherein a stool sample is used as a detection sample for auxiliary diagnosis or screening of colorectal cancer, the sampling is convenient, the kit belongs to noninvasive detection, the acceptance degree of an examinee can be improved, the coverage of colorectal cancer screening can be expanded, the incidence of colorectal cancer in China is reduced, and the guarantee is provided for the health of the whole people.

Drawings

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.

FIG. 1 is a schematic diagram of the kit of example 1.

FIG. 2 is a graph showing the results of analysis of the model ROC curve in example 2.

FIG. 3 is a graph showing the internal standard curve for sample detection in example 3.

FIG. 4 is a graph showing the detection curve of a positive sample in example 3.

FIG. 5 is a wild-type amplification curve in example 4 without addition of a blocking sequence.

FIG. 6 is a wild-type amplification curve with the addition of blocking sequences and additives as in example 4.

FIG. 7 shows the ACTB assay curve of the internal standard in example 5, with a DNA content of 10 ng/reaction.

FIG. 8 is a graph of the SEPT9 sensitivity detection curve (10 ng/reaction, with 1% SEPT9 methylation) in example 5.

FIG. 9 is a graph of the sensitivity detection of NDRG4 in example 5 (10 ng/reaction, containing 1% NDRG4 methylation).

FIG. 10 is a graph of the sensitivity detection curve for SDC2 in example 5 (10 ng/reaction with 1% SDC2 methylation).

Detailed Description

The invention is further described below with reference to the drawings and specific preferred embodiments of the description, without thereby limiting the scope of protection of the invention.

Examples

The materials and equipment used in the following examples are commercially available.

Example 1:

the kit for detecting colorectal cancer comprises a primer composition, a probe composition, a blocking sequence composition, a 2x 360Master Mix, tetramethylammonium chloride (TMAC), glycerol and deionized water.

Wherein the components of the primer composition are listed in table 1.

Table 1: primer composition of example 1

BS-S9-F	5’-TTTGTATTGTAGGAGCGCGGGC-3’(SEQ ID NO.1)
		BS-S9-R	5’-CAACGACGAAAAAACGCCCCCG-3’(SEQ ID NO.2)
BS-N4-F	5’-GTTTCGCGTCGCGGTTTTCGTTC-3’(SEQ ID NO.3)
		BS-N4-R	5’-CGTAACTTCCGCCTTCTACGCG-3’(SEQ ID NO.4)
BS-SDC2-F	5’-AGAAATTAATAAGTGAGAGGGCGTCGC-3’(SEQ ID NO.5)
		BS-SDC2-R	5’-AACGCTCGCTTCCTCCTCCTACG-3’(SEQ ID NO.6)
BS-ACT-F	5’-GAAAGGGTGTAGTTTTGGGAGGTTAG-3’(SEQ ID NO.7)
		BS-ACT-R	5’-CCCAAAACCAACCACAAAAAAAT-3’(SEQ ID NO.8)

In Table 1, the primer pair BS-S9-F and BS-S9-R was used to amplify the SEPT9 gene; the primer pair BS-N4-F and BS-N4-R is used for amplifying the NDRG4 gene; primer pairs BS-SDC2-F and BS-SDC2-R were used to amplify the SDC2 gene, and primer pairs BS-ACT-F and BS-ACT-R were used to amplify the ACTB gene.

The components of the probe composition are listed in table 2.

Table 2: probe composition of example 1

In Table 2, the BS-S9-P probe was used to identify SEPT9 gene, and BS-N4-P was used to identify NDRG4 gene; BS-SDC2-P is used to identify SDC2 gene, BS-ACT-P is used to identify ACTB gene.

The components of the blocking sequence compositions are listed in table 3.

Table 3: blocking sequence composition of example 1

BS-S9-C	5'-TTTGTATTGTAGGAGTGTGGGT-3' phosphorylation modification (SEQ ID NO.13)
		BS-N4-C	5'-GTTTTGTGTTGTGGTTTTTGTTC-3' phosphorylation modification (SEQ ID NO.14)
BS-SDC2-C	5'-AGAAATTAATAAGTGAGAGGGCGTCGC-3' phosphorylation modification (SEQ ID NO.15)

In Table 3, BS-S9-C was used to block SEPT9 gene and BS-N4-C was used to block NDRG4 gene; BS-SDC2-C is used to block the SDC2 gene.

FIG. 1 is the principle of operation of the kit of example 1: when the gene of interest is unmethylated, the blocking sequence is preferred over the binding of the primer to its transformed sequence, thereby blocking the binding of the primer to the wild-type transformed sequence. When methylation exists in a target gene, a primer and a probe are combined with a sequence after conversion, and a fluorescent reporter group (FAM, ROX, CY5 and HEX) at the 5 'end of the probe is separated from a quenching group (BHQ1 and BHQ2) at the 3' end under the action of exonuclease activity from the 5 'end to the 3' end of DNA polymerase in the PCR process to generate a fluorescent signal; if the target gene in the sample is not methylated, the primer and the probe cannot be combined with the template, and no amplification reaction exists, so that a fluorescent signal cannot be generated.

Example 2:

the kit of example 1 for detecting colorectal cancer, the method of use comprising the steps of:

sampling: 5-8 g of the fecal sample is collected and placed in 15mL of fecal sample protective solution, and the components of the fecal sample protective solution are listed in Table 4.

Table 4: excrement sample protective liquid ingredient table

Components	Concentration of
		tris-HCl	0.5M
Guanidine isothiocyanate	1.5M
		EDTA	10mM
Sodium dodecyl sulfate SDS	0.2％(m/v)
		Triton X-100	1％(v/v)
Tween 20	1％(v/v)
		Glycerol	5％(v/v)

(2) Extraction: the extraction is carried out by adopting a Jinmaige excrement genome extraction kit (magnetic bead method) of the human and future biotechnology (Changsha) limited company, and the specific steps are as follows:

2.1, fully shaking and mixing the fecal sample (about 20mL) to be in a homogenized state, centrifuging for 15min at 3000g, transferring 10mL of supernatant to another 50mL centrifuge tube, and storing the rest sample for later use.

2.2, 10mL lysine Buffer is added into the centrifuge tube, the mixture is inverted and mixed evenly, and the mixture is incubated in a water bath at 55 ℃ for 20 min.

2.3, 30. mu.L of Acryl Carrier, 240. mu.L of proteinase K, 60. mu.L of magnetic beads were added and mixed in a vertical mixer for 30 min.

2.4, 3000g for 3min, and the supernatant was poured into a waste tank, leaving about 1.8mL of supernatant.

2.5, after fully and uniformly mixing the residual solution and the magnetic beads, transferring the mixture into another 2mL centrifuge tube, placing the centrifuge tube on a magnetic frame for adsorption until the mixture is clarified, and absorbing and discarding the supernatant.

2.6, adding 800 mu L of wash buffer 1, fully oscillating and uniformly mixing, standing for 1min, placing on a magnetic frame for adsorption until the mixture is clear, and absorbing and discarding the supernatant.

2.7, adding 500 mu L of wash buffer 2, fully oscillating and uniformly mixing, standing for 1min, placing on a magnetic frame for adsorption until the mixture is clear, and absorbing and discarding the supernatant.

2.8, repeating the step 2.7 and sucking and discarding residual liquid.

2.9, adding 800. mu.L of wash buffer 3, standing for 1min, and then removing the supernatant by suction.

2.10, 60. mu.L of precipitation buffer was added, and the mixture was subjected to 55 ℃ water bath for 15 min.

2.11, performing high-speed instantaneous centrifugation, placing the solution on a magnetic frame for adsorption until the solution is clarified, and adsorbing the supernatant into a new centrifugal tube to obtain the extracted DNA.

(3) And (3) transformation: the extracted fecal DNA was transformed with bisulfite and purified using a transformation kit EZ-96DNA Methylation-Gold MagPrep (cat # D5042) from ZYMO. The conversion treatment is carried out by converting unmethylated C to U so that the methylated sequence and unmethylated sequence are different in sequence after conversion and thus detected, and the sequences of the methylated sequence and unmethylated sequence are the same when conversion treatment is not carried out.

SEPT9 wild type pre-transformation sequence (SEQ ID NO. 16):

TTCATTCAGCTGAGCCAGGGGGCCTAGGGGCTCCTCCGGCGGCTAGCTCTGCACTGCAGGAGCGCGGGCGCGGCGCCCCAGCCAGCGCGCAGGGCCCGGGCCCCGCCGGGGGCGCTTCCTCGCCGCTGCCCTCCGCGCGACCCGCTGCCCACCAGCCATCATGTCGGACCCCGCGGTCAACGCGCAGCTGGATGGGATCATTTCGGACTTCGAAGGTGGGTGCTGGGCTGGCTGCTGCGGCCGCGGACGTGCTGGAGAGGACCCTGCGGGTGGGCCTGGCGCGGGACGGGGGTGCGCTGAGGGGAGACGGGAGTGCGCTGAGGGGAGACGGGAC。

SEPT9 methylated Gene post-transformation sequence (SEQ ID NO. 17):

TTTATTTAGTTGAGTTAGGGGGTTTAGGGGTTTTTTCGGCGGTTAGTTTTGTATTGTAGGAGCGCGGGCGCGGCGTTTTAGTTAGCGCGTAGGGTTCGGGTTTCGTCGGGGGCGTTTTTTCGTCGTTGTTTTTCGCGCGATTCGTTGTTTATTAGTTATTATGTCGGATTTCGCGGTTAACGCGTAGTTGGATGGGATTATTTCGGATTTCGAAGGTGGGTGTTGGGTTGGTTGTTGCGGTCGCGGACGTGTTGGAGAGGATTTTGCGGGTGGGTTTGGCGCGGGACGGGGGTGCGTTGAGGGGAGACGGGAGTGCGTTGAGGGGAGACGGGAT。

NDRG4 methylated Gene transformation Pre-sequence (SEQ ID NO. 18):

GGGCCTCGCAGCGCACCCAGCACAGTCCGCGCGGCGGAGCGGGTGAGAAGTCGGCGGGGGCGCGGATCGACCGGGGTGTCCCCCAGGCTCCGCGTCGCGGTCCCCGCTCGCCCTCCCGCCCGCCCACCGGGCACCCCAGCCGCGCAGAAGGCGGAAGCCACGCGCGAGGGACCGCGGTCCGTCCGGGACTAGCCCCAGGCCCGGCACCGCCCCGCGGGCCGAGCG。

NDRG4 methylated gene post-transformation sequence (SEQ ID NO. 19):

GGGTTTCGTAGCGTATTTAGTATAGTTCGCGCGGCGGAGCGGGCGAGAAGTCGGCGGGGGCGCGGATCGATCGGGGTGTTTTTTAGGTTTCGCGTCGCGGTTTTCGTTCGTTTTTTCGTTCGTTTATCGGGTATTTTAGTCGCGTAGAAGGCGGAAGTTACGCGCGAGGGATCGCGGTTCGTTCGGGATTAGTTTTAGGTTCGGTATCGTTTCGCGGGTCGAGCG。

SDC2 methylated Gene transformation Pre-sequence (SEQ ID NO. 20):

TCCGCGGAGGAGCAAAACCACAGCAGAGCAAGAAGAGCTTCAGAGAGCAGCCTTCCCGGAGCACCAACTCCGTGTCGGGAGTGCAGAAACCAACAAGTGAGAGGGCGCCGCGTTCCCGGGGCGCAGCTGCGGGCGGCGGGAGCAGGCGCAGGAGGAGGAAGCGAGCGCCCCCGAGCCCCGAGCCCGAGTCCCCGAGCCTGAGCCGCAATCGCTGCGGTACTCTGCTCCGGATTCGTGTGCGCGGGCTGCGCCGAGCGCTGGGCAGGAGGCTTCGTTTTGCCCTGGTTGCAAGCAGCGGCTGGGAGCAGCCGGTCCCTGGGGAATATGCGGCGC。

SDC2 methylated Gene post-transformation sequence (SEQ ID NO. 21):

TTCGCGGAGGAGTAAAATTATAGTAGAGTAAGAAGAGTTTTAGAGAGTAGTTTTTTCGGAGTATTAATTTCGTGTCGGGAGTGTAGAAATTAATAAGTGAGAGGGCGTCGCGTTTTCGGGGCGTAGTTGCGGGCGGCGGGAGTAGGCGTAGGAGGAGGAAGCGAGCGTTTTCGAGTTTCGAGTTCGAGTTTTCGAGTTTGAGTCGTAATCGTTGCGGTATTTTGTTTCGGATTCGTGTGCGCGGGTTGCGTCGAGCGTTGGGTAGGAGGTTTCGTTTTGTTTTGGTTGTAAGTAGCGGTTGGGAGTAGTCGGTTTTTGGGGAATATGCGGCGT。

(4) and (3) detection: and (3) carrying out fluorescent quantitative PCR detection on 5 mu L of the nucleic acid transformed in the step (3), wherein the total volume of the detection system is 30 mu L, and the following components are listed in the following table 5.

Table 5: component and dosage table of detection system

Components	Dosage of
		2x 360Master Mix	15μL
Each primer	0.2μM
		Each probe	0.15μM
Tetramethyl ammonium chloride	20mM
		Glycerol
	10％(v/v)
		Each closed sequence	0.1μM
Deionized water	4.2μL
		Form panel	5μL

The amplification procedure was: pre-denaturation at 50 deg.C for 2 min; denaturation at 95 deg.C for 10 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, fluorescence collection (from the first cycle), and 45 cycles; 20 ℃ for 2 min.

(5) And (4) analyzing results:

the test is negative when the enteroscopy is approximately normal, polyps are detected by the enteroscopy and are verified to be positive by pathological detection, and the test is used as a gold standard control. 100 stool samples were selected, 50 positive samples and 50 negative samples. Logistic regression fitting was performed with the amplified Ct values of SEPT9, NDRG4, SDC2, ACTB as inputs.

The logistic regression formula used is as follows:

P＝e^Score/(1+e^Score)，

Score＝w1×Ct1+w2×Ct2+w3×Ct3+w4×Ct4+B。

wherein P is the colorectal cancer risk index,

e is a natural constant, and the natural constant is,

w1, w2, w3, w4, B is a constant,

ct1 is the Ct value of SEPT9 amplification,

ct2 is the NDRG4 amplification Ct value,

ct3 is the Ct value for SDC2 amplification,

ct4 is the ACTB amplification Ct value.

Specific values of w1, w2, w3, w4 and B in 100 clinical samples are determined, and a P value with the highest overall coincidence rate is selected as a threshold value for judging whether colorectal cancer risk exists through ROC curve analysis to obtain a prediction model. Table 6 below shows the specific values of w1, w2, w3, w4 and w B, P after the determination of 100 clinical samples.

Table 6: tables of values for w1, w2, w3, w4 and w B, P

w1	w2	w3	w4	B
					-0.14867	-0.12201	-0.21562	0.20329	13.68188

FIG. 2 shows the results of the model ROC curve analysis. As can be seen from fig. 2: when the P value is selected to be 0.362, the highest accuracy is obtained, wherein the threshold is set to 0.362 since the sensitivity is 0.962 and the specificity is 0.732.

When the P value obtained by sample detection calculation is greater than or equal to 0.362, the sample is judged to be positive; when the average molecular weight is less than 0.362, the test result is judged to be negative.

Example 3:

the sensitivity, specificity and accuracy of the method of example 2 were examined. Based on the prediction model in embodiment 2, prediction is performed on 90 newly added detection samples, and the prediction is compared with an actual value to obtain a sample confusion matrix. Table 7 below is a sample matrix.

Table 7: sample matrix table

The sensitivity, specificity and accuracy of the prediction model of the present application were calculated from the predicted values and actual values of table 7.

Wherein, the sensitivity is true positive/(true positive + false negative) × 100%;

specificity ═ true negative/(true negative + false positive) × 100%;

the accuracy rate is (true positive + true negative)/total number of tests x 100%.

From the results in table 7, the prediction model of example 2 showed a sensitivity of 0.8857; specificity is 0.8909; accuracy is 0.8889; kappa is 0.7686.

FIG. 3 is a schematic diagram of an internal standard curve for sample detection; FIG. 4 is a schematic diagram of a positive sample detection curve.

Example 4: specificity detection

Since wild-type nucleic acids still have a very high degree of similarity to methylated nucleic acids after transformation, primer probes can still bind to wild-type templates, possibly resulting in non-specific amplification. This example examines the specificity of the two kits without and with the addition of blocking sequences.

FIG. 5 is a wild-type amplification curve without the addition of blocking sequences; FIG. 6 is a wild-type amplification curve with addition of blocking sequences and additives. As can be seen from fig. 5: amplification of pure wild-type sequence results in weak non-specific amplification when no blocking sequence is added. As can be seen in fig. 6: after addition of blocking sequences and additives, non-specific amplification is eliminated and the minimum detection limit of the present invention is not affected.

Example 5: sensitivity detection

According to the method of example 2, DNA from a wild-type tissue sample (negative) and DNA from a CRC cell line (positive) were diluted to 2.5 ng/. mu.l, respectively, before transformation. Mixing the diluted wild type tissue sample DNA and the CRC cell line DNA according to the volume ratio of 99: 1 to obtain a mixed solution. Then, 50ng of the mixed solution was transformed by the method described in example 2, 25. mu.l of the solution was eluted, and 5. mu.l (corresponding to 10ng of DNA before transformation) of the solution was subjected to fluorescent quantitative PCR. The results of the tests are shown in FIGS. 7 to 10.

Fig. 7 is a graph of ACTB detection in internal standard, and fig. 8-10 are sensitivity amplification graphs of SEPT9, NDRG4, and SDC2, respectively. As can be seen from the figure, under the condition that the DNA detection content is 10 ng/reaction and the methylation ratio of the target gene is 1%, the reagent provided by the invention can well detect the target gene, so that the sensitivity of the detection reagent provided by the invention can be as low as 10 ng/reaction containing 1% methylation of the target gene, namely 2 ng/. mu.L of nucleic acid sample containing 1% methylation of the target gene.

Example 6: gene screening research

5 genes of KRAS, BMP3, SEPT9, SDC2 and NDRG4 are selected as candidate colorectal cancer screening markers and detected according to the method of example 2. In the study of 100 clinical samples, all genes were detected by fluorescent quantitative PCR to obtain the Ct value detected by each gene. Characteristic value selection was performed by LASSO regression, and the results are shown in table 8.

Table 8: examination table of different colorectal cancer screening markers

Gene	Coefficient (coefficient)
		KRAS	0
SEPT9	-0.30444915
		BMP3	0
NDRG4	-0.12050771
		SDC2	-0.07247555
ACTB	0.14267033

As can be seen from table 8, KRAS and BMP3 contributed 0 to the model, and therefore both markers KRAS and BMP3 were discarded.

Example 7: marker combination study

And combining different markers, inspecting models and ROC curves of different combinations, calculating to obtain corresponding AUC values, and determining the optimal marker combination. See table 9 below for specific combinations and findings.

Table 9: model AUC values for different marker combinations

Combination of	AUC
		KRAS+BMP3+NDRG4+SEPT9+SDC2	0.912
BMP3+NDRG4+SEPT9+SDC2	0.862
		NDRG4+SETP9+SDC2	0.909
SETP9+SDC2	0.701
		NDRG4+SETP9	0.735

From table 9, it can be seen that: the combined model of KRAS + BMP3+ NDRG4+ SEPT9+ SDC2 and the combined model of NDRG4+ SETP9+ SDC2 have the highest detection accuracy, and the combined detection of SEPT9, NDRG4 and SDC2 is determined for the balance of model simplification and detection accuracy guarantee.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.

Sequence listing

<110> human and future Biotechnology (Changsha) Ltd

<120> kit for colorectal cancer detection and application thereof

<160>21

<170>SIPOSequenceListing 1.0

<210>1

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

tttgtattgt aggagcgcgg gc 22

<210>2

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

caacgacgaa aaaacgcccc cg 22

<210>3

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

gtttcgcgtc gcggttttcg ttc 23

<210>4

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

cgtaacttcc gccttctacg cg 22

<210>5

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

agaaattaat aagtgagagg gcgtcgc 27

<210>6

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

aacgctcgct tcctcctcct acg 23

<210>7

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gaaagggtgt agttttggga ggttag 26

<210>8

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

cccaaaacca accacaaaaa aat 23

<210>9

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

cgttttagtt agcgcgtagg gttcggg 27

<210>10

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gttttttcgt tcgtttatcg ggtattttag tcg 33

<210>11

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

ttttcggggc gtagttgcgg gcgg 24

<210>12

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

cctcttctaa taaccacctc cctccttcct aac 33

<210>13

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

tttgtattgt aggagtgtgg gt 22

<210>14

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

gttttgtgtt gtggtttttg ttc 23

<210>15

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

agaaattaat aagtgagagg gcgtcgc 27

<210>16

<211>334

<212>DNA

<213> human (human)

<400>16

ttcattcagc tgagccaggg ggcctagggg ctcctccggc ggctagctct gcactgcagg 60

agcgcgggcg cggcgcccca gccagcgcgc agggcccggg ccccgccggg ggcgcttcct 120

cgccgctgcc ctccgcgcga cccgctgccc accagccatc atgtcggacc ccgcggtcaa 180

cgcgcagctg gatgggatca tttcggactt cgaaggtggg tgctgggctg gctgctgcgg 240

ccgcggacgt gctggagagg accctgcggg tgggcctggc gcgggacggg ggtgcgctga 300

ggggagacgg gagtgcgctg aggggagacg ggac 334

<210>17

<211>334

<212>DNA

<213> human (human)

<400>17

tttatttagt tgagttaggg ggtttagggg ttttttcggc ggttagtttt gtattgtagg 60

agcgcgggcg cggcgtttta gttagcgcgt agggttcggg tttcgtcggg ggcgtttttt 120

cgtcgttgtt tttcgcgcga ttcgttgttt attagttatt atgtcggatt tcgcggttaa 180

cgcgtagttg gatgggatta tttcggattt cgaaggtggg tgttgggttg gttgttgcgg 240

tcgcggacgt gttggagagg attttgcggg tgggtttggc gcgggacggg ggtgcgttga 300

ggggagacgg gagtgcgttg aggggagacg ggat 334

<210>18

<211>225

<212>DNA

<213> human (human)

<400>18

gggcctcgca gcgcacccag cacagtccgc gcggcggagc gggtgagaag tcggcggggg 60

cgcggatcga ccggggtgtc ccccaggctc cgcgtcgcgg tccccgctcg ccctcccgcc 120

cgcccaccgg gcaccccagc cgcgcagaag gcggaagcca cgcgcgaggg accgcggtcc 180

gtccgggact agccccaggc ccggcaccgc cccgcgggcc gagcg 225

<210>19

<211>225

<212>DNA

<213> human (human)

<400>19

gggtttcgta gcgtatttag tatagttcgc gcggcggagc gggcgagaag tcggcggggg 60

cgcggatcga tcggggtgtt ttttaggttt cgcgtcgcgg ttttcgttcg ttttttcgtt 120

cgtttatcgg gtattttagt cgcgtagaag gcggaagtta cgcgcgaggg atcgcggttc 180

gttcgggatt agttttaggt tcggtatcgt ttcgcgggtc gagcg 225

<210>20

<211>333

<212>DNA

<213> human (human)

<400>20

tccgcggagg agcaaaacca cagcagagca agaagagctt cagagagcag ccttcccgga 60

gcaccaactc cgtgtcggga gtgcagaaac caacaagtga gagggcgccg cgttcccggg 120

gcgcagctgc gggcggcggg agcaggcgca ggaggaggaa gcgagcgccc ccgagccccg 180

agcccgagtc cccgagcctg agccgcaatc gctgcggtac tctgctccgg attcgtgtgc 240

gcgggctgcg ccgagcgctg ggcaggaggc ttcgttttgc cctggttgca agcagcggct 300

gggagcagcc ggtccctggg gaatatgcgg cgc 333

<210>21

<211>333

<212>DNA

<213> human (human)

<400>21

ttcgcggagg agtaaaatta tagtagagta agaagagttt tagagagtag ttttttcgga 60

gtattaattt cgtgtcggga gtgtagaaat taataagtga gagggcgtcg cgttttcggg 120

gcgtagttgc gggcggcggg agtaggcgta ggaggaggaa gcgagcgttt tcgagtttcg 180

agttcgagtt ttcgagtttg agtcgtaatc gttgcggtat tttgtttcgg attcgtgtgc 240

gcgggttgcg tcgagcgttg ggtaggaggt ttcgttttgt tttggttgta agtagcggtt 300

gggagtagtc ggtttttggg gaatatgcgg cgt 333

Claims (5)

1. A kit for detecting colorectal cancer is characterized by comprising a primer composition, a probe composition and a closed sequence composition; the primer combination consists of a primer pair BS-S9-F and BS-S9-R for detecting SEPT9 gene, a primer pair BS-N4-F and BS-N4-R for detecting NDRG4 gene, a primer pair BS-SDC2-F and BS-SDC2-R for detecting SDC2 gene, and a primer pair BS-ACT-F and BS-ACT-R for detecting ACTB gene,

the DNA sequence of the BS-S9-F is shown in SEQ ID NO.1,

the DNA sequence of the BS-S9-R is shown in SEQ ID NO.2,

the DNA sequence of the BS-N4-F is shown in SEQ ID NO.3,

the DNA sequence of the BS-N4-R is shown in SEQ ID NO.4,

the DNA sequence of the BS-SDC2-F is shown in SEQ ID NO.5,

the DNA sequence of the BS-SDC2-R is shown in SEQ ID NO.6,

the DNA sequence of the BS-ACT-F is shown in SEQ ID NO.7,

the DNA sequence of the BS-ACT-R is shown in SEQ ID NO. 8;

the probe composition consists of BS-S9-P, BS-N4-P, BS-SDC2-P and BS-ACT-P,

the DNA sequence of the BS-S9-P is shown in SEQ ID NO.9,

the DNA sequence of the BS-N4-P is shown in SEQ ID NO.10,

the DNA sequence of the BS-SDC2-P is shown in SEQ ID NO.11,

the DNA sequence of the BS-ACT-P is shown in SEQ ID NO. 12;

the closed sequence composition consists of BS-S9-C, BS-N4-C and BS-SDC2-C,

the DNA sequence of the BS-S9-C is shown in SEQ ID NO. 13;

the DNA sequence of the BS-N4-C is shown in SEQ ID NO. 14;

the DNA sequence of the BS-SDC2-C is shown in SEQ ID NO. 15;

the kit also comprises 2x 360Master Mix, tetramethylammonium chloride, glycerol and deionized water; the concentration of the tetramethylammonium chloride is 10-40 mM; the volume concentration of the glycerol is 8-20%;

when the kit is used for detecting colorectal cancer, the method comprises the following steps:

(1) dissolving the excrement sample in excrement sample protective solution, and extracting excrement DNA;

(2) carrying out bisulfite conversion on the extracted excrement DNA to obtain converted excrement DNA;

(3) and (3) performing fluorescent quantitative PCR detection on the transformed fecal DNA by using the kit, and calculating a P value according to a logistic regression formula by using the amplified Ct values of SEPT9, NDRG4, SDC2 and ACTB, wherein the logistic regression formula is as follows: p = e^Score/ （1+e^Score），Score = w1×Ct1 + w2×Ct2 + w3×Ct3 + w4×Ct4 + B；

Wherein P is the colorectal cancer risk index,

e is a natural constant, and the natural constant is,

w1 is-0.14867, w2 is-0.12201, w3 is-0.21562, w4 is 0.20329, B is 13.68188;

ct1 is the Ct value of SEPT9 amplification,

ct2 is the NDRG4 amplification Ct value,

ct3 is the Ct value for SDC2 amplification,

ct4 is the ACTB amplification Ct value;

when the P value is greater than or equal to 0.362, the result is judged to be positive; when the average molecular weight is less than 0.362, the test result is judged to be negative.

2. The kit of claim 1, wherein the blocking sequence composition comprises phosphorylation modifications at the 3' end of each of the BS-S9-C, BS-N4-C, and BS-SDC 2-C.

3. The kit according to claim 1, wherein in the probe composition,

the BS-S9-P is marked by FAM and BHQ 1;

and/or, the BS-N4-P is marked by ROX and BHQ 2;

and/or, the BS-SDC2-P is marked by CY5 and BHQ 2;

and/or, the BS-ACT-P is labeled with HEX and BHQ 1.

4. The kit according to any one of claims 1 to 3, wherein the concentration of each primer in the primer composition is 0.2 μ M;

and/or, the concentration of each probe in the probe composition is 0.15 μ M;

and/or, the concentration of each blocking sequence in the blocking sequence composition is 0.1. mu.M.

5. Use of a kit according to any one of claims 1 to 4 in the manufacture of a kit for the detection of colorectal cancer.

Images