CN108642177A - A kind of method, kit and the application of detection excrement LAD1 gene methylations - Google Patents

Abstract

The present invention provides a kind of method, kit and the applications of detection excrement LAD1 gene methylations.By the DNA fragmentation extracted from human faecal mass with bisulf iotate-treated, using specific primer pair of the invention and/or it can carry out real-time quantitative PCR with the probe of site to be measured complementation, methylate and be detected to the LAD1 of sample.The detection method of the present invention has high pass flow characteristic and hypersensitivity, and without carrying out the operations such as electrophoresis and molecule hybridization after PCR, pollution and operating error in experimentation can be reduced, the time can also be saved and save reagent consumption, testing cost can not only be reduced, detection efficiency is improved, will be early diagnosed for colorectal cancer and noninvasive rapid screening provides effective means, and had a good application prospect.

Description

Method and kit for detecting methylation of LAD1 gene in feces and application of kit

Technical Field

The invention belongs to the field of biological detection, and particularly relates to a method for detecting the methylation of a fecal LAD1 gene, a kit and application.

Background

Although methylation detection such as SPG20, FBN1 and VIM is used for early diagnosis of colorectal cancer, the characteristics of poor specificity or sensitivity are often appeared. Methods commonly used to detect gene methylation include methylation-specific PCR (MS-PCR), bisulfite treatment, and sequencing.

The MS-PCR method is economical and practical, does not need special instruments, and is the most widely applied method at present. After bisulfite treatment, MS-PCR can be performed. In conventional MSP methods, two primer pairs are typically designed, one MSP primer pair amplifying bisulfite-treated DNA template and the other pair amplifying unmethylated fragments. If the first primer pair is capable of amplifying a fragment, methylation at the detection site is indicated, and if the second primer pair is capable of amplifying a fragment, methylation at the detection site is not indicated. The method has high sensitivity, can be used for paraffin embedding samples, and is not limited by endonuclease. However, there is a certain disadvantage that it is important to know the DNA sequence of the fragment to be tested in advance and design good primers. In addition, if there is a case where the bisulfite treatment is incomplete, it may lead to false positives.

Bisulfite treatment and sequencing were once considered the gold standard for DNA methylation analysis. The process is as follows: after bisulfite treatment, the target fragment is amplified by PCR, the PCR product is sequenced, and the sequence is compared with the untreated sequence to judge whether the CpG sites are methylated. The method is reliable and high in accuracy, can determine the methylation state of each CpG site in a target fragment, but needs a large amount of clone sequencing, and is complex and expensive in process.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, improve the problems of low specificity or sensitivity and the like of methylation detection and provide a method for detecting the methylation of the LAD1 gene in feces.

Another objective of the invention is to provide a kit for detecting methylation of LAD1 gene in feces.

Still another object of the present invention is to provide the use of the method or the kit for detecting methylation of LAD1 gene in feces.

The purpose of the invention is realized by the following technical scheme:

a method for detecting fecal LAD1 gene methylation comprises the steps of extracting DNA from a fecal sample, carrying out real-time quantitative PCR on LAD1-mF and LAD1-mR by using specific primers, and detecting the methylation degrees of 26 th and 27 th sites of a LAD1 gene promoter region (SEQ ID NO.7) in a fecal sample to be detected (figure 1).

Wherein the sequence of LAD1-mF is: GATTATCGGGTGTCGTAGTTC, (shown in SEQ ID NO. 1);

the sequence of LAD1-mR is: CCCTAACGCAACGTACGCG are provided. (shown as SEQ ID NO. 2).

The real-time quantitative PCR can be a probe method or a dye method.

When the real-time quantitative PCR is a probe method, the fluorescent probe matched with the specific primer pair comprises the following arbitrary sequences:

(1) TGAGTAGAATAAGGAGAGATTAT, (shown as SEQ ID NO. 3)

(2) ACAACTAACACTTAACATTATAAC are provided. (as shown in SEQ ID NO. 4)

When real-time fluorescent PCR is performed using a fluorescent dye, the optimal reaction conditions are: pre-denaturation at 95 deg.C for 10 min; at 95 ℃, 15s, 60 ℃, 60s, 40 cycles; the dissolution curve was obtained by increasing the temperature from 75 ℃ to 95 ℃ at a rate of 1 ℃ per 20 s.

When the fluorescent probe is used for real-time fluorescent PCR, the optimal reaction conditions are as follows: at 95 ℃ for 20 min; 50 PCR cycles, wherein in each cycle, the temperature is firstly increased to 62 ℃ for 5s, then is reduced to 56 ℃ for reaction for 35s, and finally is increased to 94 ℃ for reaction for 20 s; after 50 cycles of reaction, the temperature is reduced to 40 ℃ and the reaction lasts for 30 s.

The method for detecting the methylation of the LAD1 gene in the excrement can further confirm the result of the non-methylation of the LAD1 gene by carrying out real-time quantitative PCR on the primer pair shown in SEQ ID NO. 5-6 so as to further improve the accuracy of the result. If only the methylated primer is amplified, the methylation of the 26 th position and the 27 th position is shown; if both the methylated and unmethylated primers are amplified, it indicates that only position 26 or 27 of the site is methylated; if only the unmethylated primers are amplified, it is indicated that both sites are completely unmethylated.

A kit for detecting methylation of a fecal LAD1 gene comprises the specific primer pair.

The kit for detecting methylation of the fecal LAD1 gene can also comprise the following primer pairs for further confirming the non-methylation result:

LAD 1-uF: AGATTATTGGGTGTTGTAGTTT, (shown as SEQ ID NO. 5)

LAD 1-uR: AACCCTAACACAACATACACA are provided. (as shown in SEQ ID NO. 6)

The kit for detecting the methylation of the LAD1 gene in the excrement also comprises the probe or the fluorescent dye.

The kit for detecting the methylation of the LAD1 gene in the excrement can also comprise at least one of a positive standard substance, a negative quality control substance, a PCR reaction solution and a PCR enzyme.

The positive standard substance is a vector plasmid inserted with a nucleotide sequence shown as SEQ ID NO. 7.

The method for detecting the methylation of the LAD1 gene in the excrement or the kit for detecting the methylation of the LAD1 gene in the excrement is applied to colorectal cancer detection.

The invention aims to detect the methylation of the LAD1 gene in the DNA of a sample to be detected by a methylation fluorescence method. And performing real time PCR detection on the specific methylation sites by adopting a probe method and a dyeing method. The signal intensity is proportional to the amount of PCR product, from which the degree of methylation of the sample can be calculated.

The invention utilizes methylation fluorescence method to detect LAD1 gene methylation in feces DNA as an auxiliary means for early diagnosis of colorectal cancer. LAD1 is a colorectal cancer suppressor gene newly discovered by the applicant, and the immunohistochemical result indicates that the gene is negatively related to the TNM stage and lymph node metastasis of colorectal cancer, and theoretical research indicates that the gene is related to anoikis of colorectal cancer cells. However, no research report exists on the detection method of LAD1 alone or in combination with other genes as early diagnosis of colorectal cancer. In view of the biological function of LAD1 and the research results of the applicant, the fecal LAD1 gene methylation is successfully utilized to assist the early stage of colorectal cancerAnd (6) diagnosis. The invention adopts a methylation fluorescence method to detect the methylation of LAD1 gene in fecal DNA, and utilizes PCR primer combinationProbes or fluorescent dyes to distinguish between methylated and unmethylated DNA. Firstly, DNA fragments extracted from human feces are treated by bisulfite, and the LAD1 methylation of a sample is detected by utilizing the specific primer pair and/or the probe or the fluorescent dye which can be complementary with a site to be detected and then carrying out real-time quantitative PCR. Applicants found that DNA from a test sample (e.g., blood sample) from a normal human is not methylated at LAD1, whereas DNA from a test sample (e.g., blood sample) from a colorectal cancer patient is significantly methylated at LAD 1. Therefore, the future patient is advised to firstly detect the DNA of a sample (such as a blood sample) to be detected, and if the methylation degree of the LAD1 is obviously increased, the patient is advised to carry out routine detection such as endoscopy and the like so as to be convenient for the accurate diagnosis of the disease condition. Therefore, the present invention can calculate the methylation degree of the sample according to the fluorescence signal released by the hybridization of the probe and the DNA.

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

1. the detection method used by the invention has high flux characteristic and high sensitivity, and provides a certain reference for noninvasive and rapid colorectal cancer screening.

2. The invention does not need operations such as electrophoresis and molecular hybridization after PCR, not only can reduce pollution and operation errors in the experimental process, but also can save time and reagent consumption, so the invention can not reduce the detection cost and improve the detection efficiency.

3. The detection method used by the invention has the characteristics of high sensitivity and the like, and methylated LAD1 appears in early colon cancer, and the detection method provides reference for early diagnosis of colorectal cancer.

4. The invention provides a basis for confirming the reliability of the detection method and the accuracy of the detection result by double detection of the results of non-methylation and non-methylation of the specific site of the LAD1 gene by using real-time quantitative PCR.

5. According to the invention, two real-time quantitative PCR methods are used for research, so that the LAD1 gene specific site methylation detection is proved to have experimental feasibility, and an important basis is provided for clinical application.

Drawings

FIG. 1 is a sequence diagram of the promoter region of LAD1 gene.

FIG. 2 is a graph of the fluorescence quantitative PCR lysis and amplification of primers LAD1-mF and LAD1-mR in example 1 (colorectal cancer patient).

FIG. 3 is a fluorescent quantitative PCR lysis curve and amplification curve of the reference gene ATCB in example 1.

FIG. 4 is a graph showing the fluorescent quantitative PCR lysis and amplification profiles (for normal persons) for the primers LAD1-uF and LAD1-uR in example 1.

FIG. 5 is a graph showing the specificity of LAD1 in example 2, wherein the horizontal axis represents 1-specificity and the vertical axis represents sensitivity.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1 fluorescent dye method

DNA extraction

The fecal DNA extraction kit (QiaGen fecal DNA extraction kit) is used for extracting the fecal DNA of normal people and colorectal cancer patients, and the specific operation steps are as follows:

(1) collecting 200-220 mg of feces to a 2mL centrifuge tube, and placing on ice. (in the case of frozen specimens, the DNA cannot be thawed until buffer ASL is added, otherwise the DNA is easily degraded.)

(2) Add 1.4mL buffer ASL and continue vortexing for 1 minute or until uniform mixing is achieved, taking care to vortex completely, otherwise severely reduce yield.

(3) The resuspend was incubated at 70 ℃ for 5 minutes. This heating step can increase the DNA yield by 3-5 fold and help lyse bacteria and parasites. For some cells that are difficult to lyse (e.g., gram positive bacteria), this can be increased to 95 ℃.

(4) Vortex for 15 seconds and let stand at room temperature for 1 minute. The fecal pellets were pelleted by centrifugation at highest speed for 1 minute.

(5) Transfer 900. mu.L of the supernatant to a 1.5mL centrifuge tube, add 100. mu.L of impurity scavenger AB, vortex immediately for 1 minute or until uniform and stand at room temperature for 1 minute. The impurities were removed by centrifugation at maximum speed for 3 minutes.

(6) Transfer all supernatants to a 1.5mL centrifuge tube and centrifuge at top speed for 3 minutes.

(7) Transfer 210. mu.L of the supernatant to a 1.5mL centrifuge tube, add 20. mu.L of proteinase K (20mg/mL) solution, mix well, add 200. mu.L of binding solution CB, vortex for 15 seconds, mix well. Incubate at 70 ℃ for 10 minutes. (if the yield is lower, more supernatant can be transferred and the corresponding proportion of proteinase K and conjugate and subsequent isopropanol usage increased.)

(8) After cooling, 100. mu.L of isopropanol was added and vortexed to mix well.

(9) The solution obtained in the previous step and any precipitate that may have appeared are added to an adsorption column AC, which is placed in a collection tube and centrifuged at 13,000rpm for 30 seconds, and the collection tube is discarded.

(10) 500. mu.L of inhibitor-removing solution IR was added thereto, and the mixture was centrifuged at 12,000rpm for 30 seconds, and the waste liquid was discarded.

(11) Add 600. mu.L of the WB rinse (please first check if absolute ethanol has been added), centrifuge at 12,000rpm for 30 seconds, and discard the waste.

(12) 600. mu.L of the rinse WB was added, centrifuged at 12,000rpm for 30 seconds, and the waste was discarded.

(13) The adsorption column AC was returned to the empty collection tube and centrifuged at 13,000rpm for 2 minutes to remove the rinse as much as possible so as not to inhibit downstream reactions by residual ethanol in the rinse.

(14) Taking out the adsorption column AC, putting into a clean centrifuge tube, adding 100-150 μ L of elution buffer EB (the elution buffer can be preheated in water bath at 65-70 deg.C in advance) in the middle of the adsorption membrane, standing at room temperature for 2 min, and centrifuging at 12,000rpm for 1 min. The resulting solution was again introduced into the centrifugal adsorption column, and left at room temperature for 2 minutes, followed by centrifugation at 12,000rpm for 1 minute. The larger the elution volume, the higher the elution efficiency, and if the DNA concentration is required to be high, the elution volume can be reduced appropriately, but the minimum volume should not be less than 50. mu.L, and the too small volume reduces the DNA elution efficiency and the DNA yield.

(15) The DNA can be stored at 2-8 ℃ and-20 ℃ if the DNA is stored for a long time.

2. Bisulfite treatment of DNA

The genomic DNA was Bisulfite treated with a methylation Kit EpiTect bisufite Kit (48) from Qiagen, Germany, and the procedure was as described in the specification.

The bisulfite mixture was dissolved in RNase-free water, and the corresponding substances were added to a 200. mu.L PCR tube (130. mu.L system) in accordance with the reaction system shown in Table 1:

TABLE 1 PCR reaction System

Fully and uniformly mixing, standing at room temperature for 15-25 minutes, and then carrying out the following PCR program reaction on a PCR instrument: 95 ℃ for 5 minutes, 60 ℃ for 25 minutes, 95 ℃ for 5 minutes, 60 ℃ for 85 minutes, 95 ℃ for 5 minutes, 60 ℃ for 175 minutes, and finally into 20 ℃ hold.

Taking out the PCR tube, slightly centrifuging, transferring the content to a 1.5mL Eppendorf tube, adding 560 μ L of fresh BL buffer solution, adding carrier RNA into BL according to the concentration of 10 μ g/mL, fully mixing, slightly centrifuging, transferring the content to a centrifugal column at room temperature, centrifuging for 1 minute at the maximum speed, discarding the liquid after centrifugation, putting the centrifugal column back to the collection tube again, washing once with 500 μ L of BW solution, adding 500 μ L of BD solution, standing for 15 minutes at room temperature, centrifuging for 1 minute at room temperature and the maximum speed, washing twice (500 μ L each time) with BW solution, then emptying for 1 minute to remove the residual liquid as much as possible, opening the cover of the centrifugal column, putting a new 1.5mLeppendorf tube, standing for 5 minutes at 56 ℃, dropwise adding 20 μ L of EB solution to the membrane of the centrifugal column, and centrifuging for 1 minute at room temperature and obtaining the treated DNA to be detected.

3. And (3) taking the DNA to be detected obtained in the step (2) as a template, and respectively carrying out quantitative PCR by using corresponding primer pairs.

(1) 0.2mL of the PCR tube was used to prepare the reaction system shown in Table 2, and 3 tubes were prepared for each reverse transcription product.

TABLE 2 m-LAD1 primer PCR reaction System

TABLE 3 u-LAD1 primer PCR reaction System

(2) 0.2mL of the PCR tube was used to prepare reaction systems shown in Table 4, and 3 tubes were prepared for each reaction system.

TABLE 4 reference gene ATCB PCR reaction system

The ACTB gene and probe primers are as follows:

ACTB-F GTGATGGAGGTTTAGTAAGTT

ACTB-R CCAATAAAACCTACTCCTCCCTTAA

probe primer ACCACCACCCAACACACAATAACAAACACA

(3) PCR amplification

Pre-denaturation at 95 deg.C for 10 min;

cycles (40 times) 95 ℃, 15s → 60 ℃, 60 s;

the dissolution curve 75 ℃ → 95 ℃ and the temperature rises 1 ℃ every 20 s.

(4) Analysis of melting curves

Running SDS software, and setting a melting curve program: 15s at 95 ℃; 15s at 60 ℃; 95 ℃ for 15 s.

4. Analysis of results

And (3) analyzing a real-time fluorescent quantitative PCR result: and after amplification is finished, entering a result analysis interface, and looking at the CT value, the initial copy number, the standard deviation, the dissolution curve and the like. Relative quantitative values (RQ values) of the expression of the target genes were obtained by comparing the ATCB as an internal reference gene with a control group, and the RQ values were used for statistical analysis. If methylation occurs at the detection site, the corresponding fluorescence intensity occurs in Q-PCR; whereas no fluorescence was present over 40 cycles, indicating that the site was not methylated.

Setting Ct value and calculating relative expression: the threshold is a critical value at which fluorescence exceeds background or baseline during amplification, and may indirectly reflect the amount of starting cDNA template. The essential requirement for the threshold selection is that the probability of the measured background fluorescence signal being greater than the threshold due to incidental errors in the measurement is less than 10^-5. When the fluorescence signal is greater than the threshold, then it can be determined that the detected fluorescence signal is due to PCR amplification. The Threshold cycle (Ct) is the Threshold crossing-line and the PCR amplification curveThe number of cycles corresponding to the intersection of the lines. The threshold and threshold cycle number (Ct) are typically automatically generated by software. In some cases, the amplification curve may be obtained with the number of cycles as the abscissa and the intensity of the fluorescence signal as the ordinate. The study mainly adopts the automatic analysis result of SDS software and adopts a relative quantitative method to analyze data.

FIG. 2 is an amplification curve and a lysis curve of DNA extracted from a stool sample of a colon cancer patient, the lysis curve having a single peak and a lysis temperature of about 85.8 ℃. FIG. 3 shows an amplification curve and a melting curve of the reference gene ACTB.

Further, the primers (LAD1-uF and LAD1-uR) of u-LAD1 are used for carrying out reverse verification on a normal human fecal sample, and if the corresponding fluorescence intensity appears in 40 cycles in Q-PCR, the site is in a non-methylation state; no fluorescence was present over 40 cycles, indicating a site change to methylated state. The results are shown in FIG. 4, which shows that the method for detecting LAD1 gene methylation established by the invention is accurate and reliable.

EXAMPLE 2 Probe method

DNA extraction

The procedure is as in example 1.

2. Bisulfite treatment of DNA

The procedure is as in example 1.

3. Real-time quantitative PCR

And (3) taking the DNA to be detected obtained in the step (2) as a template, and respectively carrying out quantitative PCR by using corresponding primer pairs and probes.

(1) PCR reaction system for sample to be detected

The total volume of the PCR reaction system was 30. mu.L.

The procedure of PCR reaction is 95 ℃ for 20 min; 50 PCR cycles, wherein in each cycle, the temperature is firstly increased to 62 ℃ for 5s, then is reduced to 56 ℃ for reaction for 35s, and finally is increased to 94 ℃ for reaction for 20 s; and after 50 cycles of reaction, cooling to 40 ℃, reacting for 30s, and finishing the PCR reaction.

30 mu L of each PCR reaction system, and the final reaction system concentration reaches: magnesium chloride (MgCl)₂)2.5mM, dNTP0.25mM, 350nM of LAD1 primers (LAD1-mF, LAD1-mR), 100nM of blocker primer (shown in SEQ ID NO. 11), 70nM of LAD1 probe (shown in SEQ ID NO.3, 5 'end of the sequence is marked with FAM, 3' end is marked with TAMRA), 170nM of ACTB gene primer (upstream and downstream), 70nM of ACTB gene probe, 1.5U of DNA polymerase, 2-20 ng/uL of DNA template, and 10 XPCR buffer is supplemented to make the total volume 30 uL.

The primers and probes for the upstream and downstream ACTB gene were the same as those in example 1.

(2) Analysis of results

And (3) analyzing a real-time fluorescent quantitative PCR result: and after amplification is finished, entering a result analysis interface, and viewing the CT value, the initial copy number, the standard deviation and the like. Relative quantitative values (RQ values) of the expression of the target genes were obtained by comparing the ATCB as an internal reference gene with a control group, and the RQ values were used for statistical analysis. If methylation occurs at the detection site, the corresponding fluorescence intensity occurs in Q-PCR; whereas no fluorescence was present over 40 cycles, indicating that the site was not methylated.

EXAMPLE 3 Probe method

DNA extraction

The procedure is as in example 1.

2. Bisulfite treatment of DNA

The procedure is as in example 1.

3. Real-time quantitative PCR

And (3) taking the DNA to be detected obtained in the step (2) as a template, and respectively carrying out quantitative PCR by using corresponding primer pairs and probes.

(1) PCR reaction system for sample to be detected

The total volume of the PCR reaction system was 30. mu.L.

The procedure of PCR reaction is 95 ℃ for 20 min; 50 PCR cycles, wherein in each cycle, the temperature is firstly increased to 62 ℃ for 5s, then is reduced to 56 ℃ for reaction for 35s, and finally is increased to 94 ℃ for reaction for 20 s; and after 50 cycles of reaction, cooling to 40 ℃, reacting for 30s, and finishing the PCR reaction.

30 mu L of each PCR reaction system, and the final reaction system concentration reaches: magnesium chloride (MgCl)₂)2.5mM, dNTP0.25mM, 350nM of LAD1 primers (LAD1-mF, LAD1-mR), 100nM of blocker primer (shown as SEQ ID NO. 11), 70nM of LAD1 probe (shown as SEQ ID NO.4, FAM labeled at the 5 'end of the sequence and TAMRA labeled at the 3' end of the sequence), 170nM of ACTB gene primer (upstream and downstream), 70nM of ACTB gene probe, 1.5U of DNA polymerase, 2-20 ng/uL of DNA template, and 10 XPCR buffer supplemented to total volume of 30 uL.

The primers and probes for the upstream and downstream ACTB gene were the same as those in example 1.

(2) Analysis of results

And (3) analyzing a real-time fluorescent quantitative PCR result: and after amplification is finished, entering a result analysis interface, and viewing the CT value, the initial copy number, the standard deviation and the like. Relative quantitative values (RQ values) of the expression of the target genes were obtained by comparing the ATCB as an internal reference gene with a control group, and the RQ values were used for statistical analysis. If methylation occurs at the detection site, the corresponding fluorescence intensity occurs in Q-PCR; whereas no fluorescence was present over 40 cycles, indicating that the site was not methylated.

Further carrying out reverse validation on a feces sample of a normal person by using primers (LAD1-uF and LAD1-uR) of u-LAD1, wherein if corresponding fluorescence intensity appears in 40 cycles in Q-PCR, the site is in a non-methylation state; no fluorescence was present over 40 cycles, indicating a site change to methylated state.

EXAMPLE 4 clinical Effect example

DNA was extracted from 50 cases of colorectal cancer surgically excised cancer tissues, paracancerous tissues and preoperative stools thereof, and the methylation level of LAD1 gene in colorectal cancer stools was detected using the fluorescence quantification method of example 1; the sensitivity and specificity of the LAD1 methylation detection in colorectal cancer tissue and stool was compared in the diagnosis of colorectal cancer, using post-operative pathological diagnosis as a standard.

The results are shown in fig. 1, in 126 cases of colorectal cancer, the detection rate of LAD1 in cancer tissues is 83.9%, the detection rate of paracarcinoma tissues is 8.1%, the two types of colorectal cancer are statistically different (P <0.01), and the specificity is 93.1%; the methylation detection result of the colorectal cancer feces LAD1 gene is consistent with the detection result of cancer tissues. Conclusion the colorectal cancer tissue LAD1 gene methylation abnormality has high incidence; abnormal methylation of fecal LAD1 can be used as a tumor marker for early diagnosis of colorectal cancer.

The combined detection can not only discover the colorectal cancer patients at the late stage, but also avoid the missed diagnosis of the early stage patients, which has important significance for the early discovery, early diagnosis and early treatment of tumor diseases.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

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Claims (10)

1. A method for detecting methylation of LAD1 gene in feces, which is characterized by comprising the following steps:

extracting DNA from a fecal sample, and carrying out real-time quantitative PCR on LAD1-mF and LAD1-mR by using specific primers to detect the methylation degree of the LAD1 gene in the fecal sample to be detected; wherein,

the sequence of LAD1-mF is: GATTATCGGGTGTCGTAGTTC the flow of the air in the air conditioner,

the sequence of LAD1-mR is: CCCTAACGCAACGTACGCG are provided.

2. The method of detecting methylation of the fecal LAD1 gene according to claim 1, wherein:

the real-time quantitative PCR can be a probe method or a dye method.

3. The method of detecting methylation of the fecal LAD1 gene according to claim 2, wherein:

when the real-time quantitative PCR is a probe method, a fluorescent probe matched with the specific primer pair is shown as any sequence of SEQ ID NO. 3-4.

4. The method for detecting methylation of the LAD1 gene in feces according to any one of claims 1 to 3, wherein the methylation of the LAD1 gene is as follows:

the non-methylation result of the LAD1 gene can be further confirmed by carrying out real-time quantitative PCR on the primer pair shown in SEQ ID No. 5-6.

5. A colorectal cancer kit taking feces as a detection sample is characterized in that:

comprises the specific primer pair LAD1-mF and LAD 1-mR.

6. The colorectal cancer kit using feces as a detection sample according to claim 5, wherein:

primer pairs shown as SEQ ID NO. 5-6 for further confirming the non-methylation result can also be included.

7. Colorectal cancer kit using feces as a detection sample according to claim 5 or 6, characterized in that:

also comprises a probe or fluorescent dye shown as any sequence of SEQ ID NO. 3-4.

8. The colorectal cancer kit using feces as a detection sample according to any one of claims 5 to 7, wherein:

and at least one of a positive standard substance, a negative quality control substance, a PCR reaction solution and a PCR enzyme can be included.

9. The colorectal cancer kit using feces as a detection sample according to claim 8, wherein:

the positive standard substance is a vector plasmid inserted with a nucleotide sequence shown as SEQ ID NO. 7.

10. The method for detecting methylation of LAD1 gene in feces according to claims 1 to 4 or the use of the kit according to claims 5 to 9 in colorectal cancer detection.