CN112195243A - Kit for detecting polygene methylation and application thereof - Google Patents

Abstract

The invention provides a kit for detecting multi-gene methylation and application thereof. The kit for detecting multi-gene methylation comprises: a primer pair and a probe aiming at the methylation site of the SEPT9 gene promoter region; a primer pair and a probe aiming at the methylation site of the ALX4 gene promoter region; primer pairs and probes for methylation sites in the promoter region of the SDC2 gene. The invention adopts a multi-gene methylation joint detection method and simultaneously detects the methylation levels of three genes in plasma free DNA. Through screening optimization, the multiple probe method touchdown PCR initiated by the invention shows good sensitivity and specificity for early screening of colorectal cancer, the detection rate of adenoma in the colorectal progression stage is obviously improved, the detection result is stable, and the repeatability is good.

Description

Kit for detecting polygene methylation and application thereof

Technical Field

The invention belongs to the field of biology, and particularly relates to a kit for detecting multi-gene methylation and application thereof.

Background

Colorectal cancer is a common malignancy, including colon cancer and rectal cancer, originating in the colon, called colon cancer, and originating in the rectum, called rectal cancer; both organs are affected and are called colorectal cancer. The incidence of the large intestine cancer is rectum, sigmoid colon, caecum, ascending colon, descending colon and transverse colon from high to low. Its onset is closely related to life style, heredity, large intestine gland tumor, etc. Colorectal cancer is one of the most common lethal cancers, second to lung and prostate cancer in men and breast cancer in women, and the highest global mortality. In China, the incidence rate of colorectal cancer is increased at 4.71% per year, the incidence rate is far higher than the international level of 2%, more than 17 ten thousand new cases per year exist, and the colorectal cancer occupies 3 to 5 th malignant tumors.

The common early colorectal cancer screening means nowadays include stool detection, total colonoscopy, CT colonography, etc. The current noninvasive stool examination is not effective, and the sensitivity and specificity which are gradually improved do not overcome the resistance psychology which can be generated by a patient who needs to collect the stool for multiple times. Although effective, invasive tests such as colonoscopy and sigmoidoscopy require pre-patient examination bowel preparation, dietary restrictions, the use of spasmolytics and anesthetics, and inevitably invade patient privacy, thus failing to improve patient screening compliance. Therefore, there is a need to develop a new detection method based on molecular markers (biomarkers) in blood similar to cholesterol/blood lipids and Prostate Specific Antigen (PSA).

Tumors, including colorectal cancer, are closely related to abnormal methylation of DNA, and the methylation leads to the inhibition of gene transcription function, thereby affecting the normal expression of genes and inhibiting the loss of cancer functions. Free methylated DNA in blood of tumor patients is generally higher than that of normal people, and detection based on the free methylated DNA is therefore an important means for colorectal cancer screening. Cumulative studies over the past decades have incorporated the genes P16, ALX4, SEPT9, and TMEFF2 into candidate genes for early colorectal cancer screening, and more DNA methylation candidate genes have been mined.

Methods for detecting DNA methylation are numerous, the most commonly used being methylation specific pcr (msp). The most extensively studied DNA methylation assay at present is the SEPT9 gene, which is very effective in distinguishing between normal and colorectal cancer samples.

SEPT9(Septin9) gene methylation is a specific molecular marker in the early occurrence and development process of colorectal cancer, plasma SEPT9 DNA methylation detection is verified by domestic and foreign multi-center clinical tests, and a first generation detection method is applied to part of western countries^[1,2]. The second generation detection method is improved in technology, the sensitivity of the detected colorectal cancer is higher than that of the first generation technology (79.3% -95.6%), and the specificity is 84.8% -99%^[3]. Recently, a large-scale clinical test in China finds that the sensitivity and the specificity of the diagnosis of the colorectal cancer are 74.8 percent and 87.4 percent respectively^[4]All higher than the FOBT assay performed at the same time. Three products which are approved by the national food and drug administration (CFDA) are related to the methylation detection of the Septin9 gene. The product of Epigenomics AG, "SEPT 9 DNA methylation detection kit" (sensitivity is 74.8%, specificity is 97.5%), is used for auxiliary detection of early clinical diagnosis of colorectal cancer. The human Septin9 gene methylation DNA detection kit of Shanghai Jingjing Life technologies, Inc. and the human Septin9 gene methylation detection kit of Suzhou Zhenzhijing Biomedicine technologies, Inc., the former is used for the auxiliary diagnosis of colorectal cancer and is not suitable for screening, and the latter is only used as the qualitative standard of Septin9 methylation.

The methylation detection technology of single marker gene has its limitations, and the most widely known example is the possible species difference in marker gene detection by using SEPT9 single gene methylation detection kit^[17]And insufficient sensitivity to detection of advanced adenomas^[1,4,5,6]. The scholars improve the SYBR GREEN qPCR technology, and add a Touchdown PCR stage (named TqPCR) of 4 cycles before the quantitative PCR stage, and find that the TqPCR shows better amplification efficiency and higher sensitivity compared with the traditional qPCR, and improves the detection capability of low-expression genes^[14]. TqpCR is based on the SYBR GREEN dye method, SYBR GREEN is a dye with a GREEN excitation wavelength that binds to all double-stranded DNA helical minor groove regions. In the free state, SYBR GREEN emits weak fluorescence, but once bound to double-stranded DNA, fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of SYBR GREEN correlates with the amount of double-stranded DNA, and the amount of double-stranded DNA present in the PCR system can be detected from the fluorescence signal. Therefore, TqPCR has limitations, cannot well distinguish a specifically amplified target fragment from a non-specifically bound primer dimer, and cannot accurately distinguish the content of a single gene when a plurality of genes are detected simultaneously. The simultaneous detection of multiple genes can only be analyzed by a dissolution curve, and the resolving power is very limited.

In a word, the single gene methylation detection has the defects of ethnic difference and obvious insufficient detection in early canceration stage; TqPCR can improve the detection of low-trace genes, but the content of a single gene cannot be distinguished when multiple genes are detected simultaneously. Attempts to use a combination of methylation assays for multiple marker gene promoters may remedy these deficiencies^[18]。

The SDC2 gene has a promoter methylation rate of 89.4% in free plasma DNA of colorectal cancer patients and 81.1% in adenoma stage cancer patients^[8]. A recent study showed that SDC2 was 100% methylated in early-onset tumor tissue, 90.6% methylated in adenoma stage tissue, 90.9% methylated in hyperplastic polyp tissue, and no methylation was detected in normal tissue. Clinical stool DNA comparative detection of 6550 staged (I to IV) colorectal cancer patients, 6521 precancerous patientsThe feces of patients with pathological changes and 6522 normal patients show that the detection sensitivity of colorectal cancer and small polyp is 90.0 percent and 33.3 percent respectively, and the specificity is 90.9 percent^[9]. Serum detection also shows that the sensitivity of clinical screening of colorectal cancer by SDC2 methylation is 87.0 percent, the specificity is 95.2 percent, and the sensitivity reaches even 92.3 percent in the stage I^[10]。

Research shows that the sensitivity and specificity of detecting colorectal cancer by ALX4 gene promoter methylation are 68% and 88% respectively, and the detection can be used as a molecular marker for early screening of colorectal cancer^[11]. Another combined test using SEPT9 and ALX4 showed that the detection of precancerous lesions was increased from 29% to 37% compared to SEPT9 alone^[5]。

Disclosure of Invention

The drawbacks of the prior art as set forth above. The invention adopts a multi-gene methylation joint detection method and simultaneously detects the methylation levels of three genes in plasma free DNA. Through the screening of the marker gene to be detected and the optimization of the detection technology, the multiple fluorescence TaqMan probe method shows good sensitivity and specificity for early screening of colorectal cancer, the detection rate of adenoma in the colorectal progression stage is obviously improved, the detection result is stable, and the repeatability is good.

The invention aims to provide a kit for detecting multi-gene methylation, which can sensitively and efficiently detect the methylation of marker genes related to colorectal cancer. By using the kit, the methylation segments of a plurality of intestinal cancer related marker genes can be specifically and efficiently amplified synchronously in the same reaction system.

It is another object of the present invention to provide a method for detecting methylation of multiple genes. The invention innovatively combines the PCR of the multiple probe method and the touchdown PCR, and focuses on simultaneously carrying out fluorescence quantitative detection on a plurality of complex specific templates. The method is particularly suitable for multi-gene detection which does not know the homology degree of a primer and a target template and cannot optimize the annealing temperature uniformly, such as AFLP DNA fingerprint analysis and multiple plasma free marker methylation PCR.

The method for detecting the multi-gene methylation can be called as a multi-probe touchdown PCR, is a novel TaqMan probe multi-PCR, combines the advantages and the characteristics of touchdown PCR, and is used for solving the problems of inconsistent annealing temperature, low amplification efficiency and non-specific amplification among a plurality of pairs of primers caused by the specific template or limited length. Compared with the traditional multiple probe method, the multiple probe method landing PCR of the invention has better linear correlation, sensitivity, precision and lower detection limit, and can be used for detecting multiple specific nucleotide sequence probes or homologous sequence probes of different species.

Specifically, the innovative technology adopted by the invention is improved on the basis of the original multiple TaqMan PCR technology, and the improved multiple TaqMan PCR technology is combined with Touchdown PCR, so that the improved multiple TaqMan PCR technology is suitable for multiple TaqMan qPCR detection or TaqMan qPCR detection of homologous sequences of different species, and is named as multiple probe Touchdown PCR. Specificity is improved by using stringent annealing conditions in the first few cycles of PCR, where the cycles are initiated at an annealing temperature of about 15 ℃ above the estimated Tm and then reduced by 1 degree per cycle until the annealing temperature is 5 ℃ below Tm. The objective of this strategy is to ensure that the first primer and template hybridization event occurs between the most complementary reactants, i.e., specifically amplified reactants, when the annealing temperature is reduced to a level where non-specific amplification occurs, the specific product has already a geometric preponderance of the initial specific product at that time, and in the remaining reactions, the specific product competes with the non-specific product and preferentially amplifies the specific product all the time, thereby producing a single dominant amplification product.

The multiple probe touchdown PCR of the present invention is mainly used to avoid the occurrence of non-specific PCR products, especially when complex genomic DNA templates are used and non-specific annealing is likely to occur. The method is particularly effective for multiple gene detection which does not know the homology degree of the primer and the target template and cannot optimize the consistency at the annealing temperature, such as AFLP DNA fingerprint analysis and multiple plasma free marker methylation PCR.

In one aspect, the present invention provides a kit for detecting multiple gene methylation, comprising:

a primer pair and a probe aiming at the methylation site of the SEPT9 gene promoter region;

a primer pair and a probe aiming at the methylation site of the ALX4 gene promoter region;

primer pairs and probes for methylation sites in the promoter region of the SDC2 gene.

In one embodiment, the kit further comprises a primer pair and a probe for an internal reference gene (e.g., ACTB gene). Preferably, the ACTB gene is used as an internal reference gene.

In one embodiment, in the kit, the primer pair and probe for the methylation site of the promoter region of SEPT9 gene are selected from the group consisting of: SEQ ID NO 3 (forward primer) and SEQ ID NO 4 (reverse primer), SEQ ID NO 46 (probe); SEQ ID NO 5 (forward primer) and SEQ ID NO 6 (reverse primer), SEQ ID NO 47 (probe); SEQ ID NO 9 (forward primer) and SEQ ID NO 10 (reverse primer), SEQ ID NO 49 (probe).

In one embodiment, in the kit, the primer pair and probe for the methylation site of the promoter region of the ALX4 gene are selected from the group consisting of: 13 (forward primer) and 14 (reverse primer) SEQ ID NO, 51 (probe); SEQ ID NO 15 (forward primer) and SEQ ID NO 16 (reverse primer), SEQ ID NO 52 (probe); SEQ ID NO:29 (forward primer) and SEQ ID NO:30 (reverse primer), SEQ ID NO:59 (probe).

In a specific embodiment, in the kit, the primer pair and probe for the methylation site of the promoter region of the SDC2 gene are selected from the group consisting of: SEQ ID NO 31 (forward primer) and SEQ ID NO 32 (reverse primer), SEQ ID NO 60 (probe); SEQ ID NO 33 (forward primer) and SEQ ID NO 34 (reverse primer), SEQ ID NO 61 (probe); SEQ ID NO:39 (forward primer) and SEQ ID NO:40 (reverse primer), SEQ ID NO:64 (probe).

In one embodiment, in the kit, the primer pair and the probe for the reference gene (ACTB) are: SEQ ID NO:1 (forward primer) and SEQ ID NO:2 (reverse primer), SEQ ID NO:45 (probe).

In one embodiment, in the kit, the primer pair aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO. 3 (forward primer) and SEQ ID NO. 4 (reverse primer), and the probe aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO. 46; the primer pair aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO 13 (forward primer) and SEQ ID NO 14 (reverse primer), and the probe aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO 51; primer pairs for methylation sites of the promoter region of the SDC2 gene are: 31 (forward primer) and 32 (reverse primer), and the probe aiming at the methylation site of the promoter region of the SDC2 gene is SEQ ID NO: 60.

In one embodiment, in the kit, the primer pair aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO:5 (forward primer) and SEQ ID NO:6 (reverse primer), and the probe aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO: 47; the primer pair aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO. 15 (forward primer) and SEQ ID NO. 16 (reverse primer), and the probe aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO. 52; primer pairs for methylation sites of the promoter region of the SDC2 gene are: 33 (forward primer) and 34 (reverse primer), and the probe aiming at the methylation site of the promoter region of the SDC2 gene is SEQ ID NO 61.

In one embodiment, in the kit, the primer pair aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO:9 (forward primer) and SEQ ID NO:10 (reverse primer), and the probe aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO: 49; the primer pair aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO:29 (forward primer) and SEQ ID NO:30 (reverse primer), and the probe aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO: 59; primer pairs for methylation sites of the promoter region of the SDC2 gene are: 39 (forward primer) and 40 (reverse primer), and the probe aiming at the methylation site of the promoter region of the SDC2 gene is SEQ ID NO 64.

In one embodiment, in the kit, each of the probes is modified so that the detection can be performed simultaneously in one reaction system. For example, the probe may include the above modification, and any other modification combination may be used. Wherein, gene 1 is any one of the above-mentioned 4 genes (ACTB, SEPT9, SDC2, and ALX4), gene 2 is any one other than gene 1, gene 3 is any one other than gene 1 and gene 2, and gene 4 is the remaining one other than gene 1, gene 2, and gene 3:

name of probe	5' modification	3' modification
			Gene 1	FAM	BHQ1
Gene 2	JOE/VIC	BHQ1
			Gene 3	ROX/TEXAS RED	BHQ2
Gene 4	CY5	BHQ3

In one embodiment, the kit further comprises a DNA hot start polymerase Taq enzyme. Preferably, the DNA hot start polymerase Taq enzyme may be selected from FastFire qPCR PreMix (probe) and High Affinity hot start Taq of radix angelicae, hot start High Taq DNA polymerase of philippic, or the DNA hot start polymerase Taq enzyme may be the self-made DNA hot start polymerase Taq enzyme of the present invention, which may be prepared by the following method: a genomic DNA fragment of T.aquaticus YT1 (NCBI, Gene ID: 41352821, Location: NC-014974.1 (889676..892168)) containing a DNA polymerase (Taq-polymerase) Gene was cloned into an expression vector and expressed. Specifically, the self-prepared DNA hot start polymerization Taq enzyme can be prepared by the following method: 1) cloning; 2) inserting the gene fragment into a prokaryotic expression vector by an enzyme cutting method and carrying out sequencing verification; 3) transformation experiment, transferring into Escherichia coli; 4) marking and coating a plate; 5) screening blue white spots, selecting a single clone, putting the selected single clone into 10mL LB culture medium containing 80 mug/mL ampicillin resistance, and incubating overnight in a shaking table at the temperature of 37 ℃ and the speed of 250 rpm; 6) 500 μ L of overnight culture was added to 1L of LB medium containing ampicillin and incubated at 37 ℃ for 11 hours until OD600 was close to 0.8; 7) adding IPTG to a final concentration of 125 mg/L; 8) proliferating at 37 ℃ for 12 hours; 9) centrifuging at 2500rpm for 10 minutes at room temperature; 10) washing with 100mL of buffer solution A; 11) centrifuging at room temperature and 2500rpm for 10min, and discarding the liquid; 12) adding 50mL of pre-lysate for re-suspension; 13) incubation for 15 minutes at room temperature; 14) adding 50mL of lysate, and incubating for 1 hour at 75 ℃; 15) transferring the lysis mixture into a centrifuge tube, and centrifuging at 15,000rpm for 10 minutes at 4 ℃; 16) transferring the clarified supernatant into a new centrifuge tube; 17) adding 30g of ammonium sulfate powder into every 100mL of the solution, and rapidly stirring the solution at room temperature; 18) centrifugation at 15,000rpm for 10 minutes at room temperature; 19) resuspending the protein particles in 20mL of buffer A; 20) dialyzing the above-mentioned resuspended protein twice at 4 ℃ with 240mL of stock solution; 21) after dialysis, diluting with a sterilized stock solution in a ratio of 1: 1; 22) stored at-70 ℃ for use, at 20-fold dilution on start (about 5U/μ L), wherein lysate a: 50mM Tris-HCl pH7.9, 50mM dextran, 1mM EDTA, pre-lysate: adding 4mg/mL lysozyme into lysate A, and adding lysate: 10mM Tris-HCl pH7.9, 50mM KCl, 1mM EDTA, 1mM PMSF, 0.5% Tween-20, 0.5% NP-40, stock solutions: 50mM Tris-HCl pH7.9, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.5mM PMSF, 50% glycerol.

Further, in one embodiment, the kit comprises a gene methylation detection reagent. In one embodiment, the gene methylation detection reagent comprises the following components:

(1) CRC detection reagent 1 comprising DNA Hot Start polymerase Taq enzyme (preferably 87.5U), PCR bufferLiquid (preferably containing 0.02M Tris-HCl, 0.1M KCl, 3mM MgCl)₂) dNTP (preferably 400. mu.M).

(2) A CRC detection reagent 2 comprising the above-mentioned primer pair (preferably 5 to 10nM each) and probe (preferably 4 to 8nM each) for each gene to be detected (genes ACTB, SEPT9, SDC2 and ALX4), and a TE buffer for dissolving the above-mentioned primer pair and probe;

(3) a positive control comprising human cell genomic hypermethylated DNA (preferably 4.0. + -. 0.5 ng/. mu.L), and TE buffer (for dissolving the above human cell genomic hypermethylated DNA);

(4) a negative control comprising human leukocyte genomic hypomethylated DNA (preferably 4.0. + -. 0.5 ng/. mu.L), and TE buffer (for dissolving the above human leukocyte genomic hypomethylated DNA).

In one embodiment, the gene methylation detection reagent further comprises nuclease-free water.

In another aspect, the present invention provides a method for detecting methylation of multiple genes, which comprises detecting the methylation of multiple genes using the above-described kit.

In one embodiment, the method is multiplex probe touchdown PCR, wherein a touchdown PCR phase is provided prior to a conventional PCR phase. In the present invention, the conventional PCR is a PCR method comprising three stages of "denaturation, annealing and extension" which is generally used in the art.

Further, in one embodiment, the method comprises the steps of:

1) the polymerase is activated. Preferably, it is carried out at 95 ℃ for 10 minutes.

2) A touchdown PCR phase (e.g., 5 cycles) in which the estimated Tm is obtained by adding the melting temperature of the primer up to 15 ℃, the cycles starting at an annealing temperature about 15 ℃ above the estimated Tm, the annealing temperature decreasing by 0.5-2 ℃ per cycle until 5 ℃ below Tm. In this case, only the highly specific target template will be amplified initially, and these products will continue to be amplified in subsequent conventional quantitative PCR cycles, and will be expelled quantitatively competitively as amplification non-specific products.

3) A conventional PCR phase (e.g., 40 cycles).

In one embodiment, the above method, wherein the detection of genes (i) (SEPT9), (ALX4), and (SDC2) and the reference gene (ACTB) is performed in the same system; the annealing temperature of each primer pair is respectively calculated by design software or a primer order; calculating the average annealing temperature (Tm) ((Tm) + Tm ++ Tm + (ACTB))/4+10, taking the average annealing temperature plus 10-15 ℃ as the starting temperature and the average annealing temperature minus 5 ℃ as the ending temperature, calculating the Touchdown PCR temperature interval, and if the primers have the primer pairs which are not in the interval, properly adjusting the concentration of the pair of primers until the annealing temperature of the pair of primers is in the interval. Preferably, the primer and probe annealing stages are separated in the conventional PCR stage.

In one embodiment, in the above method, each probe concentration may be 200 nM.

In one embodiment, in the above method, each primer concentration may be 300 nM.

In one embodiment, the reaction conditions are as follows.

The method is mainly characterized in that a touchdown PCR stage (stage I) is added, and is one of the core points of the method, and specific fragments are effectively enriched by adding the touchdown PCR stage. The touchdown PCR can be used for condition selection without a preliminary experiment, and only one formal experiment is needed, so that the amplification test is carried out under ideal conditions even if the amplification test is not optimal, and ideal amplification effect can be obtained under most conditions. The basic operation is as follows: based on the calculation of the Tm of the primers, a large range of annealing temperatures (not one point, but a range that can span 10 to 20 degrees) is determined, and the calculated Tm should fall in the middle of this range. During the programming of the amplification cycle, the annealing temperature is gradually decreased from the highest temperature of the selected range, once every time, and finally ends at the lowest temperature of the selected range. Cycling twice at each dwell temperature. Thus, the primer hybridizes to the template at a higher temperature and initiates synthesis of an extension product, typically a specific amplification product, since it initiates synthesis at the earliest time, i.e., at a higher Tm, and the extension synthesis continues as the annealing temperature continues to decrease. It is conceivable that during the annealing temperature decrease, it is highly likely that the primer will also find a site on the template where second renaturation and primer extension will occur, but this site is usually "false positive amplification" because it will renaturate the primer at a lower Tm. These two products differ greatly in content. If the Tm values of the specific amplification and the false positive amplification differ by 3 degrees, since the synthesis of the false positive product is started after 6 cycles of the synthesis product with higher specificity since two cycles are performed at each temperature, the amount of the specific product is 64 times that of the false positive product, and the difference between the amounts of the specific product and the false positive product is significant.

Still further, in one embodiment, the method comprises the steps of:

(1) plasma free DNA extraction.

In one embodiment, the plasma-free DNA extraction comprises: the whole blood was separated to obtain plasma, and extracted with QIAamp Circulating Nucleic Acid Kit (QIAGEN Co.) or Magpure Circulating DNA Maxi Kit (MAGEN Co.).

(2) DNA sulfite transformation.

In one embodiment, the DNA sulfite conversion comprises: 2.1 extracting free DNA of blood plasma by adopting EZ DNA Methylation-Gold Kit of ZYMO company, and carrying out DNA conversion experiment strictly according to the specification; 2.2 after the concentration of the DNA is measured by a Qubit or an ultraviolet spectrophotometer, the DNA is stored for no more than 24 hours at the temperature of 2-8 ℃ and no more than 72 hours at the temperature of-25 to-15 ℃.

(3) A gene methylation assay comprising:

3.1 taking out the CRC detection reagent 1 and the CRC detection reagent 2 for thawing, thawing the DNA (the DNA after the transformation in 2.2) of the sample to be detected, the negative reference substance and the positive reference substance, and performing vortex mixing for 10-15 seconds, and performing instant separation;

3.2 calculate the reaction system as follows: the volume of the CRC detection reagent 1 is 12.5 × (2n +6) μ l, the volume of the CRC detection reagent 2 is 0.5 × (2n +6) μ l, the volume of water is 7.0 × (2n +6) μ l, and n represents the number of samples to be examined;

3.3 adding CRC detection reagent 1, CRC detection reagent 2 and water with corresponding volumes into the centrifuge tube in sequence, mixing uniformly for 10-15 seconds in a vortex manner, and separating instantly;

3.4 adding 20 mul of the mixed PCR reaction solution into the plate hole of the corresponding 96-hole PCR plate, adding 5 mul of DNA to be detected or a negative control product and a positive control product, and setting two adjacent holes as multiple holes;

3.5 sealing with a high-permeability sealing plate membrane, and centrifuging for 1 minute at 1000 +/-100 rcf;

3.6 the computer model is divided into an Applied Biosystem 7500PCR instrument, and the corresponding Software version is 7500Software v 2.3;

3.7 settings for the Applied Biosystem 7500PCR Instrument are as follows:

3.7.1 the Pasive Reference is set as 'none', ACTB selects FAM fluorescence channel, SEPT9 selects VIC channel, ALX4 selects ROX channel, SDC2 selects CY5 channel;

3.7.2 reaction procedure:

3.7.3 analysis conditions settings: setting a baseline value to be 3-15 cycles, setting the threshold of ACTB to be 80,000, setting the threshold of SEPT9 to be 120,000, setting the threshold of ALX4 to be 30,000, and setting the threshold of SDC2 to be 20,000;

3.7.4 PCR validation: the control sample should meet the following criteria

Ct value of control sample	ACTB	SEPT9	ALX4	SDC2
					Positive control	≤30	≤30	≤30	≤30
Negative control	≤30	Uncertainty	＞38	＞40

3.7.5 when the validity condition is satisfied, the sample is judged to be positive or negative: sample decision criteria

Ct value for methylation detection	ACTB	SEPT9	ALX4	SDC2
					Positive for	≤40	≤45	≤38	≤40
Negative of	≤40	Uncertainty	＞38	＞40
					Invalidation	＞40

In the method of touchdown PCR combined with conventional PCR, the main work is as follows:

considering that the annealing temperature difference of each pair of primers is huge, touchdown PCR is increased, a specific target gene is preferentially amplified, and fragments to be detected are enriched, so that the amplification efficiency of the original PCR is increased from ACTB 70.21%, SE 75.02%, AL 75.63% and SD 72.83% to ideal ACTB 108.49%, SE 104.65%, AL 102.37% and SDC2 91.88%, and the detection sensitivity is effectively improved;

increasing a touchdown PCR stage, reducing the inter-batch and intra-batch difference of an amplification result, reducing the CV between multiple wells from 0.04-4.39% to 0.04% -1.70%, and increasing R2 from 0.9344-0.9991 to 0.9875-0.9993;

r: the linear correlation coefficient is approximately close to 1, which indicates that the amplification result is more credible, and usually the R is more than or equal to 0.99 and is best;

and thirdly, the using amount of the probe and the template is optimized, the cost is reduced, and the requirement of blood plasma is reduced.

The detection method specifically comprises the following steps as described in the patent application: an enzyme activation phase, a touchdown PCR phase (5 cycles) and a conventional PCR phase (40 cycles).

The technical method comprises the screening of the marker gene to be detected and the optimization of the detection technology, and combines the multiple probe method PCR and the touchdown PCR so as to realize the specific and efficient synchronous amplification of a plurality of methylated fragments in the same reaction system. The invention has the following beneficial effects:

1. the detection of a plurality of methylated free fragment DNAs is simultaneously completed in the same system (Table a);

2. the overall sensitivity of colorectal cancer detection and the detection rate of advanced adenomas are improved (Table b).

TABLE a comparison of Gene and Performance for different colorectal cancer assays

TABLE b comparison of different colorectal cancer detection methods

Drawings

FIG. 1 is a flow chart of the detection carried out using the kit of the present invention.

FIG. 2 is a graph showing that the recruited population of the present invention is concentrated between 40-80 and substantially matches the distribution of the age structure of the Chinese population.

FIG. 3 is a graph showing the judgment of the experimental results in example 1. The single curve in each figure shows a single result in two multiple wells.

Detailed Description

Hereinafter, the present invention will be described in detail by way of examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: screening of candidate primer pairs and probes

The 4 genes adopted in the embodiment are ACTB, SEPT9, ALX4 and SDC2 respectively, the genes 1 to 4 correspond to any one of the genes respectively, the modification modes of the probes among the 4 genes are shown as follows, and the modification modes of the probes among the 4 genes can be interchanged:

name of probe	5' modification	3' modification	Purification mode
				Gene 1	FAM	BHQ1	HPL
Gene 2	JOE/VIC	BHQ1	HPL
				Gene 3	ROX/TEXAS RED	BHQ2	HPL
Gene 4	CY5	BHQ3	HPL

Candidate primer pairs are shown below:

candidate probes are shown below:

the screening process comprises the following steps:

1. the sample used for the detection comprises: a positive control substance, containing human leukocyte genome highly methylated DNA4.0 +/-0.5 ng/mu L, and dissolved in TE buffer solution; ② a negative reference substance, 4.0 plus or minus 0.5 ng/mu L of hypomethylated DNA containing human leukocyte genome, and TE buffer solution is dissolved; ③ blank control and TE buffer solution.

2. The hot-start enzyme used for detection was purchased from Shenzhen, China, Philor bio-Ltd, cat # MD 026; dNTPs used were purchased from ENZYMETICS under the cat number N2050-25; nuclease-free water was purchased from PROMEGA, cat No. P1195.

3. The instrument consumables used are as follows:

4. the experimental process comprises the following steps:

A. the detection sample adopts EZ DNA Methylation-Gold Kit (cat number D5006) of ZYMO company, and DNA transformation experiments are carried out strictly according to the requirements of the specification;

B. after the concentration of the converted ssDNA is measured by the Qubit 3.0, the ssDNA is stored at the temperature of 2-8 ℃ for no more than 24 hours and at the temperature of-25-15 ℃ for no more than 72 hours.

C. Thawing the reagent and the DNA of the sample to be detected, mixing uniformly for 10-15 seconds in a vortex mode, and separating instantly;

D. the reaction was prepared as follows:

component (A)	μL	Final concentration	Recommended concentration
				HotStart HiTaq DNA Polymerase	0.25
5×HotStart Buffer(Mg2+Plus)	5	1×
				Solution 1(10×)	2.5	1×
dNTPs(25mM)	0.2	200μM
				Forward primer (10. mu.M)	0.5	200nM	50～900nM
Reverse primer (10. mu.M)	0.5	200nM	50～900nM
				Probe (10 μ M)	0.5	200nM	50～250nM*
Stencil (10ng)	2	10ng	5～50ng
				NF H2O	13.55
Total of	25

E, uniformly mixing PCR reaction liquid, adding 23 mu l of the mixed solution into a plate hole of a corresponding 96-hole PCR plate, adding 2 mu l of DNA to be detected (namely a negative control product, a positive control product and a blank control product), and setting two adjacent holes as multiple holes;

F. sealing with a high-permeability sealing plate membrane, and centrifuging for 1 minute at 1000 +/-100 rcf;

G. the computer type is an Applied Biosystem 7500PCR instrument, and the corresponding Software version is 7500Software v 2.3;

H. settings for the Applied Biosystem 7500PCR Instrument: the Passive Reference is set as 'none', ACTB selects an FAM fluorescent channel, SEPT9 selects a VIC channel, ALX4 selects an ROX channel, and SDC2 selects a CY5 channel;

I. the reaction procedure was as follows:

5. the experimental results are as follows:

the experimental results were judged as follows: the positive reference should have a typical amplification curve, the negative reference and the blank non-amplified probes are qualified primer probe combinations (e.g., fig. 3).

The primer pairs and the probe pairs meeting the screening conditions are respectively as follows: SE-MF2, SE-MR2 and SE-Probe 2; AL-MF2, AL-MR2, and AL-Probe 2; SD-MF2, SD-MR2 and SD-Probe 2; SE-MF4, SE-MR4 and SE-Probe 4; AL-MF9, AL-MR9, and AL-Probe 9; SD-MF5, SD-MR5 and SD-Probe 5; SE-MF1, SE-MR1 and SE-Probe 1; AL-MF1, AL-MR1, and AL-Probe 1; SD-MF1, SD-MR1 and SD-Probe 1.

Example 2: effect of different Probe concentrations on amplification efficiency

Selecting a primer probe combination: SE-MF2, SE-MR2 and SE-Probe 2; AL-MF2, AL-MR2, and AL-Probe 2; SD-MF2, SD-MR2 and SD-Probe2 are used for optimizing the experimental system, and the influence of different Probe concentrations on the experimental result is respectively researched.

The test samples were the same as in item (one) 1 of example 1.

The detection reagent is the same as the item (one) 2 in example 1.

The detection apparatus and consumables are the same as those in item (one) 3 of example 1.

The threshold values are set as: ACTB 10000, (AXL 4) 25000, (SDC2) 50000, (SEPT9) 50000, and the rest are the same as (one) 4 items in example 1. The assay system was as follows, with the probe increasing at a 50 μ M concentration gradient:

component (A)	μL	Final concentration	Recommended concentration
				HotStart HiTaq DNA Polymerase	0.25
5×HotStart Buffer(Mg2+Plus)	5	1×
				Solution 1(10×)	2.5	1×
dNTPs(25mM)	0.2	200μM
				Forward primer (10. mu.M)	0.5	200nM	50～900nM
Reverse primer (10. mu.M)	0.5	200nM	50～900nM
				Probe (10 μ M)		0～500nM*	50～250nM*
Stencil (10ng)	2	10ng	5～50ng
				NF H2O
Total of	25

The experimental results are as follows:

when the concentration of the probe is too low, the Ct value of the detected target gene is large or even can not be detected; when the concentration of the probe is higher (600 mu M and 400 mu M), the detection effect on the target gene is not obviously improved. Therefore, subsequent experiments were performed with a probe concentration of 200 nM.

Example 3: effect of different primer concentrations on amplification efficiency

Selecting a primer probe combination: SE-MF2, SE-MR2 and SE-Probe 2; AL-MF2, AL-MR2, and AL-Probe 2; SD-MF2, SD-MR2 and SD-Probe2 are used for optimizing the experimental system, and the influence of different primer concentrations on the experimental result is respectively researched.

The test samples were the same as in item (one) 1 of example 1.

The detection reagent is the same as the item (one) 2 in example 1.

The detection apparatus and consumables are the same as those in item (one) 3 of example 1.

The threshold values are set as: ACTB 10000, AXL4 25000, SDC2 50000, SEPT9 50000, and the rest are item (one) 4 in example 1. The assay system was as follows, with primers increasing at a concentration gradient of 150 μ M:

component (A)	μL	Final concentration	Recommended concentration
				HotStart HiTaq DNA Polymerase	0.25
5×HotStart Buffer(Mg2+Plus)	5	1×
				Solution 1(10×)	2.5	1×
dNTPs(25mM)	0.2	200μM
				Forward primer (10. mu.M)		150～900nM	50～900nM
Reverse primer (10. mu.M)		150～900nM	50～900nM
				Probe (10 μ M)	0.5×4	200nM	50～250nM
Stencil (10ng)	2	10ng	5～50ng
				NF H2O
Total of	25

And (3) taking the positive control as a sample to be detected, setting a template gradient, and sequentially decreasing as follows: 8ng, 4ng and 2ng, standard curves were prepared.

4. The experimental results are as follows:

ACTB

SEPT9

AXL4

SDC2

wherein R represents a linear correlation coefficient, and the closer the R value is to 1, the more credible the experimental result is; slope is the Slope of the standard curve; e is amplification efficiency, and is a relatively excellent amplification result at 90% to 110%. As a result, it was found that the amplification efficiency was good at the primer concentration of 300nM for all genes except ALX 4.

Example 4: effect of different PCR plates on amplification efficiency

Selecting a primer probe combination: SE-MF2, SE-MR2 and SE-Probe 2; AL-MF2, AL-MR2, and AL-Probe 2; the SD-MF2, the SD-MR2 and the SD-Probe2 are used for optimizing an experimental system and researching the influence of different PCR plates on experimental results

The test samples were the same as in item (one) 1 of example 1.

The detection reagent is the same as the item (one) 2 in example 1.

The detection instrument is the same as the item (one) 3 in the embodiment 1, and the consumables are as follows:

instrument/consumable	Manufacturer of the product	Producing area	Model number
				96-hole PCR plate (frosted transparent plate)	Axygen	United states of America	PCR-96-LP-AB-C
96-well PCR plate (white plate)	Axygen	United states of America	PCR-96-LP-AB-W

The threshold values are set as: ACTB 10000, (AXL 4) 25000, (SDC2) 50000, (SEPT9) 50000, and the rest are the same as (one) 4 items in example 1. The detection system is as follows:

component (A)	μL	Final concentration	Recommended concentration
				HotStart HiTaq DNA Polymerase	0.25
5×HotStart Buffer(Mg2+Plus)	5	1×
				Solution 1(10×)	2.5	1×
dNTPs(25mM)	0.2	200μM
				Forward primer (10. mu.M)	0.75×4	300nM	50～900nM
Reverse primer (10. mu.M)	0.75×4	300nM	50～900nM
				Probe (10 μ M)	0.5×4	200nM	50～250nM
Stencil (10ng)	2	10ng	5～50ng
				NF H2O	7.05
Total of	25

The experimental results are as follows:

a transparent plate:

white board:

as can be seen from the above table, the use of a whiteboard affects the fluorescence measurement due to its opaque nature. This results in a significant decrease in PCR linearity and efficiency.

Example 5: effect of the touchdown PCR stage on the results

Selecting a primer probe combination: SE-MF2, SE-MR2 and SE-Probe 2; AL-MF2, AL-MR2, and AL-Probe 2; SD-MF2, SD-MR2 and SD-Probe2, with primer concentrations of 300nM and Probe concentrations of 200nM, respectively.

Axygen 96-AB-LP-C is selected as a detection plate, and ABI 7500 is selected as a detection instrument.

The results of the conventional PCR procedure (i.e. denaturation-annealing-extension) show good amplification efficiency, but the linear correlation coefficient R is not ideal, possibly affecting the reliability of the results, and the repeated sample testing of the conventional PCR shows a relatively large variability. Therefore, it is contemplated to add a touchdown PCR stage to reduce non-specific product amplification, reducing the variability of the replicate experiment.

The test samples were the same as in item (one) 1 of example 1.

The detection reagent is the same as the item (one) 2 in example 1.

The detection apparatus was as described in example 4.

The threshold values are set as:

ACTB 10000, AXL4 25000, SDC2 50000, SEPT9 50000, the reaction program is as follows, and the rest are as in item (one) 4 of example 1.

Conventional PCR procedure, i.e., the PCR procedure shown in item (one) 4. of example 1;

the multi-probe touchdown PCR, touchdown PCR stage (stage I), stage II is the conventional PCR stage. The whole program setting is as follows:

the experimental results are as follows:

conventional PCR:

group 1:

SEPT9

SDC2

ALX4

ACTB

group 2:

SEPT9

SDC2

ALX4

ACTB

group 3:

SEPT9

SDC2

ALX4

ACTB

general assembly

Group 1:

group 2:

group 3:

multiplex probe descent method PCR:

group 1:

SEPT9

SDC2

ALX4

ACTB

group 2:

SEPT9

SDC2

ALX4

ACTB

group 3:

SEPT9

SDC2

ALX4

ACTB

general assembly

Group 1:

group 2:

group 3:

template: sample, unit ng; rep: composite well Ct value; mean: composite pore mean value; SD: standard deviation among multiple holes; CV: the coefficient of variation between compound pores; r: a linear correlation coefficient; slope: a slope; e: efficiency of amplification

As shown in the table above, the results of the multiple touchdown PCR show smaller fluctuation CV (0.04-1.70%, conventional 0.04-4.39%); more stable R2 (0.9875-0.9993, conventional 0.9344-0.9991).

Example 6: effect of primer Probe Split annealing on Experimental results

Selecting a primer probe combination: SE-MF2, SE-MR2 and SE-Probe 2; AL-MF2, AL-MR2, and AL-Probe 2; SD-MF2, SD-MR2 and SD-Probe2, with primer concentrations of 300nM and Probe concentrations of 200nM, respectively.

Axygen 96-AB-LP-C is selected as a detection plate, and ABI 7500 is selected as a detection instrument.

Although the addition of touchdown PCR makes the results more stable and reliable, the above method still has the problem of insufficient PCR efficiency, probably because the stage II annealing probe primer is less efficient in binding to the template, considering separate annealing.

The test samples were the same as in item (one) 1 of example 1.

The detection reagent is the same as the item (one) 2 in example 1.

The detection apparatus was as described in example 4.

The threshold values are set as: ACTB 10000, AXL4 25000, SDC2 50000, SEPT9 50000, the reaction program is as follows, and the rest are as in item (one) 4 of example 1.

(a) The multi-probe falling method PCR comprises a falling PCR stage (stage I), a conventional PCR stage II and a primer annealing stage together. The whole program is set as follows;

(b) the multi-probe falling method PCR comprises a falling PCR stage (stage I), a stage II is a conventional PCR stage, and a primer annealing stage and a probe annealing stage are separated. The whole program setting is as follows:

the experimental results are as follows:

(a) stage II primers, probes anneal together at 50 ℃, 45 s:

(b) stage II primer, probe separate annealing:

it can be seen that separate annealing produces better amplification results.

Example 7: effect of different detection reagents on amplification efficiency

Selecting a primer probe combination: SE-MF2, SE-MR2 and SE-Probe 2; AL-MF2, AL-MR2, and AL-Probe 2; and the SD-MF2, the SD-MR2 and the SD-Probe2 are used for optimizing an experiment system and researching the influence of different detection reagents on an experiment result.

1. The test samples were the same as in item (one) 1 of example 1.

2. The detection reagent is as in item (one) 2 of example 1, and the hot start enzyme includes the following:

the self-developed hot start enzyme described in the above table was prepared as follows:

1) cloning of a genomic DNA fragment of t.aquaticus YT1 containing the DNA polymerase (Taq-polymerase) Gene (NCBI, Gene ID: 41352821, Location: NC _014974.1(889676.. 892168));

2) the gene fragment was inserted into a prokaryotic expression vector (pBV221-EGFP, biovector, cat #: 8600179) and sequencing for verification;

3) transformation experiments, transformation into e.coli (seimei fly, cat #: 18258012);

4) marking and coating a plate;

5) screening blue white spots, selecting a single clone, putting the selected single clone into 10mL LB culture medium containing 80 mug/mL ampicillin resistance, and incubating overnight in a shaking table at the temperature of 37 ℃ and the speed of 250 rpm;

6) 500 μ L of overnight culture was added to 1L of LB medium containing ampicillin and incubated at 37 ℃ for 11 hours until OD600 was close to 0.8;

7) adding IPTG to a final concentration of 125 mg/L;

8) proliferating at 37 ℃ for 12 hours;

9) centrifuging at 2500rpm for 10 minutes at room temperature;

10) washing with 100mL of buffer solution A;

11) centrifuging at room temperature and 2500rpm for 10min, and discarding the liquid;

12) adding 50mL of pre-lysate for re-suspension;

13) incubation for 15 minutes at room temperature;

14) adding 50mL of lysate, and incubating for 1 hour at 75 ℃;

15) transferring the lysis mixture into a centrifuge tube, and centrifuging at 15,000rpm for 10 minutes at 4 ℃;

16) transferring the clarified supernatant into a new centrifuge tube;

17) adding 30g of ammonium sulfate powder into every 100mL of the solution, and rapidly stirring the solution at room temperature;

18) centrifugation at 15,000rpm for 10 minutes at room temperature;

19) resuspending the protein particles in 20mL of buffer A;

20) dialyzing the above-mentioned resuspended protein twice at 4 ℃ with 240mL of stock solution;

21) after dialysis, diluting with a sterilized stock solution in a ratio of 1: 1;

22) storage at-70 ℃ for future use, 20-fold dilution on start (approximately 5U/. mu.L);

23) the enzyme activity was determined in comparison with commercial Taq enzyme.

Lysate a: 50mM Tris-HCl pH7.9, 50mM dextran, 1mM EDTA

Pre-lysis solution: lysate A plus 4mg/mL lysozyme

Lysis solution: 10mM Tris-HCl pH7.9, 50mM KCl, 1mM EDTA, 1mM PMSF, 0.5% Tween-20, 0.5% NP-40

Storage liquid: 50mM Tris-HCl pH7.9, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.5mM PMSF, 50% glycerol.

The detection apparatus is the same as that in item (one) 3 of example 1.

The threshold values are set as: ACTB 10000, AXL4 25000, SDC2 50000, SEPT9 50000. The detection system is strictly executed according to the kit use requirements of various manufacturers, and the program is set as follows:

the experimental results are as follows:

①

the amplification efficiency of the Probe qPCR Master Mix is not ideal;

secondly, the amplification efficiency of the THUNDERBIRD Probe qPCR Mix is not ideal;

③ Premix Ex Taq TM (Probe qPCR), the amplification effect is not ideal;

(iv) Fastfire qPCR Premix (probe) with good amplification result;

fifthly, High Affinity HotStart Taq has good amplification result;

sixthly, the HotStart HiTaq DNA polymerase has good amplification result;

seventhly, the hot start enzyme is ground, and the amplification result is excellent

The self-grinding hot start enzyme is self-made DNA hot start polymerization Taq enzyme, and the self-grinding buffer solution comprises (containing 0.02M Tris-HCl, 0.1M KCl and 3mM MgCl)₂)，dNTP 400μM；

FastFire qPCR Premix (probe) and High Affinity HotStart Taq of Tiangen, HotStart HiTaq DNA polymerase of Fic and hot start enzyme of Huada Gibby self-research all showed good results, and Huada self-research enzyme was used in consideration of cost, purchase, quality inspection and other factors.

Example 8: influence of salt ion concentration on the results of the experiment

Selecting a primer probe combination: SE-MF2, SE-MR2 and SE-Probe 2; AL-MF2, AL-MR2, and AL-Probe 2; SD-MF2, SD-MR2 and SD-Probe2, with primer concentrations of 300nM and Probe concentrations of 200nM, respectively.

Axygen 96-AB-LP-C is selected as a detection plate, and ABI 7500 is selected as a detection instrument.

The test samples were the same as in item (one) 1 of example 1.

The detection reagent is a self-grinding hot start enzyme of Aibia hua

The detection apparatus was as described in example 4.

The threshold values are set as: ACTB 10000, AXL4 25000, SDC2 50000, SEPT9 50000, the reaction program is as follows, and the rest are as in item (one) 4 of example 1.

The detection system is as follows:

component (A)	μL	Final concentration
			Huada autogenous grinding hot start enzyme (example 7)	2.5	1×
Huada Buiyuan enzyme buffer (2.5X)	8/10/12	0.8×/1.0×/1.2×
			ACTB Primer F(10μM)	0.75	300nM
ACTB Primer R(10μM)	0.75	300nM
			ALX4 Primer F(10μM)	0.75	300nM
AXL4 Primer R(10μM)	0.75	300nM
			SDC2 Primer F(10μM)	0.75	300nM
SDC2 Primer R(10μM)	0.75	300nM
			SEPT9 Primer F(10μM)	0.75	300nM
SEPT9Primer R(10μM)	0.75	300nM
			ACTB Probe(10μM)	0.5	200nM
AXL4 Probe(10μM)	0.5	200nM
			SDC2 Probe(10μM)	0.5	200nM
SEPT9 Probe(10μM)	0.5	200nM
			Stencil (10ng)	2
NF H2O
			Total of	25

Note: washington's enzyme buffer (2.5X) contains 0.025M Tris-HCl, 0.125M KCl, 3.75mM MgCl₂And dNTP 500. mu.M.

The experimental results are as follows:

group 1: 0.8 × Waals George enzyme buffer; group 2: 1.0 × Huada Boseki enzyme buffer; group 3: 1.2 × Huada Bushy enzyme buffer:

group 1:

group 2:

group 3:

group 1: 1.0 × Huada Boseki enzyme buffer; group 2: 1.2 × Huada Boseki enzyme buffer;

group 1:

group 2:

groups 1 and 2 are duplicate independent experiments.

The concentration of salt ions can affect the result of PCR detection, and the high salt concentration inhibits the detection efficiency. Suitable salt particle concentrations range from 0.025M Tris-HCl, 0.125M KCl, 3.75mM MgCl₂And dNTP 500. mu.M.

Example 9: the influence of the detection method on the detection limit

Selecting a primer probe combination: SE-MF2, SE-MR2 and SE-Probe 2; AL-MF2, AL-MR2, and AL-Probe 2; SD-MF2, SD-MR2 and SD-Probe2, with primer concentrations of 300nM and Probe concentrations of 200nM, respectively.

Axygen 96-AB-LP-C is selected as a detection plate, and ABI 7500 is selected as a detection instrument.

The test sample was subjected to 1/2-fold ultimate gradient dilution as in item (one) 1 in example 1, and the test result was examined.

The detection reagent was a self-immolative hot start enzyme (example 7).

The instrumentation and consumables were as described in example 4.

The threshold values are set as: ACTB 10000, AXL4 25000, SDC2 50000, SEPT9 50000, the reaction program is as follows, and the rest are as in item (one) 4 of example 1.

The detection system is as follows:

component (A)	μL	Final concentration
			Huada autogenous grinding hot start enzyme (example 7)	2.5	1×
Huada Buiyuan enzyme buffer (2.5X)	10	1×
			ACTB Primer F(10μM)	0.75	300nM
ACTB Primer R(10μM)	0.75	300nM
			ALX4 Primer F(10μM)	0.75	300nM
AXL4 Primer R(10μM)	0.75	300nM
			SDC2 Primer F(10μM)	0.75	300nM
SDC2 Primer R(10μM)	0.75	300nM
			SEPT9 Primer F(10μM)	0.75	300nM
SEPT9 Primer R(10μM)	0.75	300nM
			ACTB Probe(10μM)	0.5	200nM
AXL4 Probe(10μM)	0.5	200nM
			SDC2 Probe(10μM)	0.5	200nM
SEPT9 Probe(10μM)	0.5	200nM
			Stencil (10ng)	2
NF H2O	2.5
			Total of	25

Note: washington's enzyme buffer (2.5X) contains 0.025M Tris-HCl, 0.125M KCl, 3.75mM MgCl₂And dNTP 500. mu.M.

The experimental results are as follows:

SEPT9

SDC2

ALX4

ACTB

as shown in the table above, the multiplex-probe-drop-method PCR can reduce the detection limit from 132pg of the conventional PCR to 25pg, and the detection scheme has good linear correlation and amplification efficiency, stable and reliable results and improved detection rate. Therefore, the detection method of the invention innovatively combines the PCR and touchdown PCR of a multiple probe method and is suitable for simultaneously carrying out fluorescence quantitative detection on a plurality of complex specific templates. Compared with the conventional qPCR, the method has better amplification efficiency and higher sensitivity, improves the detection capability of low-expression genes, and reduces the detection limit.

Example 10: PCR optimization of primer probe combination clinical test in kit

(I) PCR detection reagent

The enzyme for PCR detection is Huada self-developed hot start enzyme, and its preparation method is described in example 7. Washington's enzyme buffer (2.5X) contains 0.025M Tris-HCl, 0.125M KCl, 3.75mM MgCl₂And dNTP 500. mu.M.

The specific implementation flow is as follows:

1. plasma free DNA extraction (upper left of FIG. 1)

1.1 separating 10ml of whole blood, and keeping the blood plasma for later use;

1.2 the plasma is recommended to be extracted by QIAamp Circulating Nucleic Acid Kit product of QIAGEN company or MagPure Circulating DNA Maxi Kit of MAGEN company;

DNA sulfite conversion (right in FIG. 1)

2.1 extracting free DNA of blood plasma by adopting EZ DNA Methylation-Gold Kit of ZYMO company, and carrying out DNA conversion experiment strictly according to the specification;

2.2 after the concentration of the DNA is measured by the Qubit 3.0, the DNA is preserved for no more than 24 hours at the temperature of 2-8 ℃ and no more than 72 hours at the temperature of-25 to-15 ℃.

3. Gene methylation assay (FIG. 3 bottom left)

3.1 taking out the CRC detection reagent 1 and the CRC detection reagent 2 for thawing, thawing the DNA (the DNA after the transformation in 2.2) of the sample to be detected, the negative reference substance and the positive reference substance, and performing vortex mixing for 10-15 seconds, and performing instant separation;

3.2 calculate the reaction system as follows: from the study of hot-start enzyme 2.5 × (2n +6) μ l and hot-start enzyme buffer 10 × (2n +6) μ l, the primer and probe combination of each test gene was added to a final concentration of 300nM for primer and 200nM for probe, and water was added to a total volume of 20 × (2n +6) μ l. n represents the number of samples to be examined. Wherein, the combination of the adopted probe primers is as follows;

combination I: primer List

Primer name	Sequence of	Purification mode
			ACTB-MF	5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′(SEQ ID NO:1)	PAG
ACTB-MR	5′-AACCAATAAAACCTACTCCTCCCTTAA-3′(SEQ ID NO:2)	PAG
			SE-MF	5'-CGCGGCGTTTTAGTTAGCGCG-3′(SEQ ID NO:5)	PAG
SE-MR	5'-CGTTAACCGCGAAATCCGAC-3′(SEQ ID NO:6)	PAG
			AL-MF	5′-GTTTATTATAGTTCGGTGTCG-3′(SEQ ID NO:15)	PAG
AL-MR	5′-TCCCTATACTTTAACGACGACCG-3′(SEQ ID NO:16)	PAG
			SD-MF	5′-AGAAAAGGATTGAGAAAACG-3′(SEQ ID NO:33)	PAG
SD-MR	5′-CGAAAACCAATAAACGCCGCG-3′(SEQ ID NO:34)	PAG

Combination I: probe list

And (2) combination II: primer List

Primer name	Sequence of	Purification mode
			ACTB-MF	5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′(SEQ ID NO:1)	PAG
ACTB-MR	5′-AACCAATAAAACCTACTCCTCCCTTAA-3′(SEQ ID NO:2)	PAG
			SE-MF	5'-TTTCGTCGTTGTTTTTCGC-3′(SEQ ID NO:9)	PAG
SE-MR	5'-ATCCAACTACGCGTTAACCG-3′(SEQ ID NO:10)	PAG
			AL-MF	5’-GATTTGCGTTTTTTATTGC-3′(SEQ ID NO:29)	PAG
AL-MR	5’-CGACCGACAAAAAAATTATAC-3′(SEQ ID NO:30)	PAG
			SD-MF	5′-GGATTTTTAGTTTTGCGGC-3′(SEQ ID NO:39)	PAG
SD-MR	5′-CCTACTTACGACACTCCCGTA-3′(SEQ ID NO:40)	PAG

And (2) combination II: probe list

Combination III: primer List

Primer name	Sequence of	Purification mode
			ACTB-MF	5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′(SEQ ID NO:1)	PAG
ACTB-MR	5′-AACCAATAAAACCTACTCCTCCCTTAA-3′(SEQ ID NO:2)	PAG
			SE-MF	5'-GCGTAGGGTTCGGGTTTCGTC-3′(SEQ ID NO:3)	PAG
SE-MR	5'-CAACTACGCGTTAACCGCG-3′(SEQ ID NO:4)	PAG
			AL-MF	5′-ATAGTTCGGTGTCGTAGAGTC-3′(SEQ ID NO:13)	PAG
AL-MR	5′-CCCCGAATCCCTATACTTTA-3′(SEQ ID NO:14)	PAG
			SD-MF	5′-TGGTTTTCGGAGTTGTTAATCG-3′(SEQ ID NO:31)	PAG
SD-MR	5′-TCTCCGAACTCCCCTAAACG-3′(SEQ ID NO:32)	PAG

Combination III: probe list

3.3 adding the corresponding volume of hot start enzyme, buffer solution, primer, probe and water into the centrifuge tube in turn. Vortex mixing is carried out for 10-15 seconds, and instant separation is carried out;

3.4 adding 20 mul of the mixed PCR reaction solution into the plate hole of the corresponding 96-hole PCR plate, adding 5 mul of DNA to be detected or a negative control product and a positive control product, and setting two adjacent holes as multiple holes;

3.5 sealing with a high-permeability sealing plate membrane, and centrifuging for 1 minute at 1000 +/-100 rcf;

3.6 the computer model is divided into an Applied Biosystem 7500PCR instrument, and the corresponding Software version is 7500Software v 2.3;

3.7 settings for the Applied Biosystem 7500PCR Instrument are as follows:

3.7.1 the Pasive Reference is set as 'none', ACTB selects FAM fluorescence channel, SEPT9 selects VIC channel, ALX4 selects ROX channel, SDC2 selects CY5 channel;

3.7.2 reaction procedure:

3.7.3 analysis conditions settings: setting a baseline value to be 3-15 cycles, setting the threshold of ACTB to be 80,000, setting the threshold of SEPT9 to be 120,000, setting the threshold of ALX4 to be 30,000, and setting the threshold of SDC2 to be 20,000;

3.7.4 PCR validation: the control sample should meet the following criteria

Ct value of control sample	ACTB	SEPT9	ALX4	SDC2
					Positive control	≤30	≤30	≤30	≤30
Negative control	≤30	Not determined	＞38	＞40

3.7.5 when the validity condition is satisfied, the sample is judged to be positive or negative: sample decision criteria

Ct value for methylation detection	ACTB	SEPT9	ALX4	SDC2
					Positive for	≤40	≤45	≤38	≤40
Negative of	≤40	Not determined	＞38	＞40
					Invalidation	＞40

(III) specific implementation:

this example is a cooperative project with Zhongshan tumor hospital to complete the analysis of 414 effective detection samples and evaluate the clinical detection results of primer probe combinations. The embodiment is as follows:

recruitment of volunteers and colorectal cancer patients:

1. the volunteers are subjected to excrement multi-target DNA methylation detection, the excrement detection is positive, then the excrement detection is confirmed by the enteroscopy, and only the person who is negative in the excrement detection and the person who is negative in the enteroscopy are taken as an asymptomatic person;

2. confirming the colorectal cancer patient through enteroscopy, and confirming the stage of pathological biopsy;

3. finally 164 colorectal cancer patients and 250 asymptomatic volunteers were recruited.

II, clinical process:

1. informing volunteers/patients to inform study information at least one day before sample collection, collecting information of the examinees and signing an informed consent;

2. collecting 10mL of peripheral venous blood from the elbow of the examinee in EDTA-K₂Centrifuging at 4 deg.C for 10min at 1,600g in an anticoagulation blood collection tube to collect supernatant;

3.16,000g,10min, re-centrifuging at 4 ℃, subpackaging 2mL of supernatant and freezing at-80 ℃;

4. stage information is confirmed by pathological biopsy of colorectal cancer patients.

Thirdly, an experimental process:

1. patients were grouped, 414 subjects (164 colorectal cancer patients and 250 symptomless volunteers) were participating in the trial study; (ii) a

2. Taking one tube of the examinees according to grouping results, subpackaging the blood plasma (about 2mL) in the tube, and thawing at 4 ℃;

3. extracting blood plasma by adopting MagPure Circulating DNA Maxi Kit of MAGEN company;

DNA sulfite transformation Using EZ DNA Methylation-Gold Kit from ZYMO;

5. gene methylation assays were as described previously;

6. and carrying out positive and negative discrimination analysis on the detection result.

Fourthly, information of instruments, reagents and primer sequences used for detecting the gene methylation:

1.

2.

reagent	Manufacturer of the product	Producing area
			cf-DNA extraction kit	MAGEN	China Guangzhou
Sulfite conversion reagent	ZYMO	California of America
			Gene methylation detection reagent	Example 11
Primer and method for producing the same	Invitrogen Synthesis
			Probe needle	Invitrogen Synthesis

The experimental results are as follows:

combination I:

the detection results are compared with clinical pathology results and enteroscopy results, and for 194 clinical samples, the detection results are as follows:

in the combination, the sensitivity is 48/68-70.6%, and the specificity is 104/126-82.5%

And (2) combination II:

the detection results are compared with clinical pathology results and enteroscopy results, and for 194 clinical samples, the detection results are as follows:

in the combination, the sensitivity is 48/68-70.6%, and the specificity is 71/72-98.6%

Combination III:

the detection results are compared with clinical pathology results and enteroscopy results, and for 194 clinical samples, the detection results are as follows:

in the combination, the sensitivity was 24/28-85.7%, and the specificity was 48/52-92.3%

Thus, the combination III has the highest accuracy, and can be selected as a primer probe combination for subsequent kit packaging. The results of clinical verification experiments prove that: the intestinal cancer of people over 40 years old is relatively high, and the recruited people are concentrated between 40 and 80 and basically accord with the age structure distribution of Chinese people (figure 2).

Example 11: reagent box package

The kit comprises the following components:

the PCR reaction solution I contains Taq DNA polymerase, dNTPs and buffer solution; the PCR reaction solution II contains primers and probes of each gene to be detected; the negative control substance contains peripheral leukocyte DNA and TE buffer solution of 1% BSA; the positive control contained whole genome methylated DNA, 1% BSA in TE buffer.

Preparation of CRC detection reagent 1

Taking out dNTPs and Tris-HCl, KCl and MgCl₂The following concentrations were formulated: PCR Buffer (0.025M Tris-HCl, 0.125M KCl, 3.75mM MgCl)₂) And dNTP 500. mu.M; wherein dNTPs and PCR Buffer are fully and uniformly mixed by vortex, and the tube wall liquid is dropped on the bottom of the tube by low-speed instantaneous separation;

example 7 the Chinese Dayan self-developed hot start enzyme finger flicks and mixes evenly, and the low speed instantaneous separation makes the tube wall liquid drop on the tube bottom;

the following components are sequentially added into a centrifugal tube marked with a CRC detection reagent 1:

component (A)	Volume (μ L)
		Huada autogenous grinding hot start enzyme (example 7)	175
Hot Start enzyme Buffer (2.5X) (PCR Buffer)	700

Wherein the enzyme activity is 5U/mu L. 2.5 × buffer contains: 0.025M Tris-HCl, 0.125M KCl, 3.75mM MgCl₂And dNTP 500. mu.M. Covering the tube cover, sealing and freezing at-20 ℃.

Preparation of CRC detection reagent 2

Taking out the probe and the primer dry powder, precipitating the dry powder at the bottom of the tube by low-speed instantaneous separation, and calculating the water volume required by preparing the 250 mu M primer/probe, wherein the calculation formula is as follows: v-nmol number × 4(μ L); adding corresponding volume of water, fully and uniformly mixing by vortex, and dropping the liquid on the tube wall at the tube bottom by low-speed instantaneous separation;

standing at room temperature for 5-10 min for full dissolution, sufficiently and uniformly mixing by vortex, and dropping the liquid on the tube wall at the tube bottom by low-speed instantaneous separation;

the prepared primers/probes were added sequentially to the brown centrifuge tube labeled "CRC detection reagent 2":

composition (I)	Volume (μ L)
		SE-MF(250μM)	2.1
SE-MR(250μM)	2.1
		SD-MF(250μM)	2.1
SD-MR(250μM)	2.1
		AL-MF(250μM)	2.1
AL-MR(250μM)	2.1
		ACTB-MF(250μM)	2.1
ACTB-MR(250μM)	2.1
		SE-Probe(250μM)	1.4
SD-Probe(250μM)	1.4
		AL-Probe(250μM)	1.4
ACTB-Probe(250μM)	1.4
		Nuclease-free water	12.6

Vortex, mix well, the low speed instantaneous separation makes the pipe wall liquid drop on the bottom of the pipe. Covering the tube cover, sealing and freezing at-20 ℃.

Preparation of negative control

The hypomethylated leukocyte genomes were diluted to 4.0. + -. 0.5 ng/. mu.L with 1% BSA in TE (i.e., 1g BSA in 100mL TE).

Fourth, preparation of negative control

The hypermethylated leukocyte genomes were diluted to 4.0. + -. 0.5 ng/. mu.L with TE solution containing 1% BSA.

Example 12: kit stability detection

PCR detection reagent:

the enzyme for PCR detection is Huada self-developed hot start enzyme, and its preparation method is described in example 7. Washington's enzyme buffer (2.5X) contains 0.025M Tris-HCl, 0.125M KCl, 3.75mM MgCl₂And dNTP 500. mu.M. And detecting the detection result of the prepared reagent and the reagent which is repeatedly frozen and thawed for 3 times on the same sample.

2. Experiment implementation:

the same samples were tested by repeated freeze-thawing 3 times using the freshly prepared self-research reagents or the kit prepared as in example 11. The detection method was the same as in example 10.

The experimental results are as follows:

the prepared reagent and the reagent which is repeatedly frozen and thawed for 3 times are used for detecting different concentrations of the same sample, and a standard curve is made. The Ct value detected by the kit and the amplification efficiency detected by the kit are almost unchanged, which indicates that the kit is stable.

Example 13: clinical sample detection of kit

PCR detection reagent

The specific implementation flow is as follows:

1. plasma free DNA extraction (upper left of FIG. 1)

1.1 separating 10ml of whole blood, and keeping the blood plasma for later use;

1.2 the plasma is recommended to be extracted by QIAamp Circulating Nucleic Acid Kit product of QIAGEN company or MagPure Circulating DNA Maxi Kit of MAGEN company;

DNA sulfite conversion (right in FIG. 1)

2.1 extracting free DNA of blood plasma by adopting EZ DNA Methylation-Gold Kit of ZYMO company, and carrying out DNA conversion experiment strictly according to the specification;

2.2 after the concentration of the DNA is measured by the Qubit 3.0, the DNA is preserved for no more than 24 hours at the temperature of 2-8 ℃ and no more than 72 hours at the temperature of-25 to-15 ℃.

3. Gene methylation assay (FIG. 1 bottom left)

3.1 taking out the CRC detection reagent 1 and the CRC detection reagent 2 for thawing, thawing the DNA (the DNA after the transformation in 2.2) of the sample to be detected, the negative reference substance and the positive reference substance, and performing vortex mixing for 10-15 seconds, and performing instant separation;

3.2 calculate the reaction system as follows: the volume of the CRC detection reagent 1 is 12.5 × (2n +6) μ l, the volume of the CRC detection reagent 2 is 0.5 × (2n +6) μ l, the volume of water is 7.0 × (2n +6) μ l, and n represents the number of samples to be examined;

3.3 adding CRC detection reagent 1, CRC detection reagent 2 and water with corresponding volumes into the centrifuge tube in sequence, mixing uniformly for 10-15 seconds in a vortex manner, and separating instantly;

3.4 adding 20 mul of the mixed PCR reaction solution into the plate hole of the corresponding 96-hole PCR plate, adding 5 mul of DNA to be detected or a negative control product and a positive control product, and setting two adjacent holes as multiple holes;

3.5 sealing with a high-permeability sealing plate membrane, and centrifuging for 1 minute at 1000 +/-100 rcf;

3.6 the computer model is divided into an Applied Biosystem 7500PCR instrument, and the corresponding Software version is 7500Software v 2.3;

3.7 settings for the Applied Biosystem 7500PCR Instrument are as follows:

3.7.1 the Pasive Reference is set as 'none', ACTB selects FAM fluorescence channel, SEPT9 selects VIC channel, ALX4 selects ROX channel, SDC2 selects CY5 channel;

3.7.2 reaction procedure:

3.7.3 analysis conditions settings: setting a baseline value to be 3-15 cycles, setting the threshold of ACTB to be 80,000, setting the threshold of SEPT9 to be 120,000, setting the threshold of ALX4 to be 30,000, and setting the threshold of SDC2 to be 20,000;

3.7.4 PCR validation: the control sample should meet the following criteria

Ct value of control sample	ACTB	SEPT9	ALX4	SDC2
					Positive control	≤30	≤30	≤30	≤30
Negative control	≤30	Uncertainty	＞38	＞40

3.7.5 when the validity condition is satisfied, the sample is judged to be positive or negative: sample decision criteria

Ct value for methylation detection	ACTB	SEPT9	ALX4	SDC2
					Positive for	≤40	≤45	≤38	≤40
Negative of	≤40	Uncertainty	＞38	＞40
					Invalidation	＞40

Reference documents:

[1]R.Grutzmann,B.Molnar,C.Pilarsky,J.K.Habermann,P.M.Schlag,H.D.Saeger,S.Miehlke,T.Stolz,F.Model,U.J.Roblick,H.P.Bruch,R.Koch,V.Liebenberg,T.Devos,X.Song,R.H.Day,A.Z.Sledziewski,C.Lofton-Day,Sensitive detection of colorectal cancer in peripheral blood by septin 9DNA methylation assay,PloS one 3(11)(2008)e3759.

[2]T.deVos,R.Tetzner,F.Model,G.Weiss,M.Schuster,J.Distler,K.V.Steiger,R.Grutzmann,C.Pilarsky,J.K.Habermann,P.R.Fleshner,B.M.Oubre,R.Day,A.Z.Sledziewski,C.Lofton-Day,Circulating methylated SEPT9DNA in plasma is a biomarker for colorectal cancer,Clinical chemistry 55(7)(2009)1337-46.

[3]K.Toth,F.Sipos,A.Kalmar,A.V.Patai,B.Wichmann,R.Stoehr,H.Golcher,V.Schellerer,Z.Tulassay,B.Molnar,Detection of methylated SEPT9 in plasma is a reliable screening method for both left-and right-sided colon cancers,PloS one 7(9)(2012)e46000.

[4]P.Jin,Q.Kang,X.Wang,L.Yang,Y.Yu,N.Li,Y.Q.He,X.Han,J.Hang,J.Zhang,L.Song,Y.Han,J.Q.Sheng,Performance of a second-generation methylated SEPT9 test in detecting colorectal neoplasm,Journal of gastroenterology and hepatology 30(5)(2015)830-3.

[5]M.Tanzer,B.Balluff,J.Distler,K.Hale,A.Leodolter,C.Rocken,B.Molnar,R.Schmid,C.Lofton-Day,T.Schuster,M.P.Ebert,Performance of epigenetic markers SEPT9 and ALX4 in plasma for detection of colorectal precancerous lesions,PloS one 5(2)(2010)e9061.

[6]T.R.Church,M.Wandell,C.Lofton-Day,S.J.Mongin,M.Burger,S.R.Payne,E.Castanos-Velez,B.A.Blumenstein,T.Rosch,N.Osborn,D.Snover,R.W.Day,D.F.Ransohoff,I.Presept Clinical Study Steering Committee,T.Study,Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer,Gut 63(2)(2014)317-25.

[7]C.Lofton-Day,F.Model,T.Devos,R.Tetzner,J.Distler,M.Schuster,X.Song,R.Lesche,V.Liebenberg,M.Ebert,B.Molnar,R.Grutzmann,C.Pilarsky,A.Sledziewski,DNA methylation biomarkers for blood-based colorectal cancer screening,Clinical chemistry 54(2)(2008)414-23.

[8]B.K.Bartak,A.Kalmar,B.Peterfia,A.V.Patai,O.Galamb,G.Valcz,S.Spisak,B.Wichmann,Z.B.Nagy,K.Toth,Z.Tulassay,P.Igaz,B.Molnar,Colorectal adenoma and cancer detection based on altered methylation pattern of SFRP1,SFRP2,SDC2,and PRIMA1 in plasma samples,Epigenetics 12(9)(2017)751-763.

[9]T.J.Oh,H.I.Oh,Y.Y.Seo,D.Jeong,C.Kim,H.W.Kang,Y.D.Han,H.C.Chung,N.K.Kim,S.An,Feasibility of quantifying SDC2 methylation in stool DNA for early detection of colorectal cancer,Clinical epigenetics 9(2017)126.

[10]T.Oh,N.Kim,Y.Moon,M.S.Kim,B.D.Hoehn,C.H.Park,T.S.Kim,N.K.Kim,H.C.Chung,S.An,Genome-wide identification and validation of a novel methylation biomarker,SDC2,for blood-based detection of colorectal cancer,The Journal of molecular diagnostics:JMD 15(4)(2013)498-507.

[11]R.Salehi,N.Atapour,N.Vatandoust,N.Farahani,F.Ahangari,A.R.Salehi,Methylation pattern of ALX4gene promoter as a potential biomarker for blood-based early detection of colorectal cancer,Adv Biomed Res 4(2015)252.

[12]T.F.Imperiale,D.F.Ransohoff,S.H.Itzkowitz,B.A.Turnbull,M.E.Ross,G.Colorectal Cancer Study,Fecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population,The New England journal of medicine 351(26)(2004)2704-14.

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[15]B.Fu,P.Yan,S.Zhang,Y.Lu,L.Pan,W.Tang,S.Chen,S.Chen,A.Zhang,W.Liu,Cell-Free Circulating Methylated SEPT9 for Noninvasive Diagnosis and Monitoring of Colorectal Cancer,Disease markers 2018(2018)6437104.

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SEQUENCE LISTING

<110> Beijing Hua Dajibiai Biotech Co., Ltd

<120> kit for detecting multi-gene methylation and application thereof

<130> DI20-0046-XC37

<160> 66

<170> PatentIn version 3.5

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agtcggcgta gttatagcgc 20

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tagttaaggc gcgcggtggc gt 22

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gacgttaaga gtcgggttcg t 21

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tagtcgttta ggggagttcg ga 22

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tatagtagag taagaagagt 20

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cgttgagggg tggaggttgt a 21

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gtttattggt tttcggagtt gt 22

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<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> SD-Probe7

<400> 66

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Claims (10)

1. A kit for detecting multiple gene methylation comprising:

a primer pair and a probe aiming at the methylation site of the SEPT9 gene promoter region;

a primer pair and a probe aiming at the methylation site of the ALX4 gene promoter region;

primer pairs and probes for methylation sites in the promoter region of the SDC2 gene.

2. The kit of claim 1, wherein the kit further comprises a primer pair and a probe for an internal reference gene (preferably the ACTB gene).

3. The kit according to claim 1 or 2,

wherein, the primer pair and the probe aiming at the methylation site of the SEPT9 gene promoter region are selected from the following: SEQ ID NO 3 (forward primer) and SEQ ID NO 4 (reverse primer), SEQ ID NO 46 (probe); SEQ ID NO 5 (forward primer) and SEQ ID NO 6 (reverse primer), SEQ ID NO 47 (probe); SEQ ID NO 9 (forward primer) and SEQ ID NO 10 (reverse primer), SEQ ID NO 49 (Probe), andor

Wherein, the primer pair and the probe aiming at the methylation site of the ALX4 gene promoter region are selected from the following: 13 (forward primer) and 14 (reverse primer) SEQ ID NO, 51 (probe); SEQ ID NO 15 (forward primer) and SEQ ID NO 16 (reverse primer), SEQ ID NO 52 (probe); SEQ ID NO:29 (forward primer) and SEQ ID NO:30 (reverse primer), SEQ ID NO:59 (probe), andor

Wherein, the primer pair and the probe aiming at the methylation site of the promoter region of the SDC2 gene are selected from the following: SEQ ID NO 31 (forward primer) and SEQ ID NO 32 (reverse primer), SEQ ID NO 60 (probe); SEQ ID NO 33 (forward primer) and SEQ ID NO 34 (reverse primer), SEQ ID NO 61 (probe); SEQ ID NO:39 (forward primer) and SEQ ID NO:40 (reverse primer), SEQ ID NO:64 (probe).

4. The kit according to claim 1 or 2, wherein,

the primer pair aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO. 3 (forward primer) and SEQ ID NO. 4 (reverse primer), and the probe aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO. 46; the primer pair aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO 13 (forward primer) and SEQ ID NO 14 (reverse primer), and the probe aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO 51; primer pairs for methylation sites of the promoter region of the SDC2 gene are: 31 (forward primer) and 32 (reverse primer), and the probe aiming at the methylation site of the promoter region of the SDC2 gene is SEQ ID NO 60, or

The primer pair aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO. 5 (forward primer) and SEQ ID NO. 6 (reverse primer), and the probe aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO. 47; the primer pair aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO. 15 (forward primer) and SEQ ID NO. 16 (reverse primer), and the probe aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO. 52; primer pairs for methylation sites of the promoter region of the SDC2 gene are: 33 (forward primer) and 34 (reverse primer), and the probe aiming at the methylation site of the promoter region of the SDC2 gene is SEQ ID NO 61, or

The primer pair aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO. 9 (forward primer) and SEQ ID NO. 10 (reverse primer), and the probe aiming at the methylation site of the SEPT9 gene promoter region is SEQ ID NO. 49; the primer pair aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO:29 (forward primer) and SEQ ID NO:30 (reverse primer), and the probe aiming at the methylation site of the ALX4 gene promoter region is SEQ ID NO: 59; primer pairs for methylation sites of the promoter region of the SDC2 gene are: 39 (forward primer) and 40 (reverse primer), and the probe aiming at the methylation site of the promoter region of the SDC2 gene is SEQ ID NO 64.

5. The kit according to claim 1 or 2, wherein the kit further comprises a DNA hot start polymerase Taq enzyme, preferably selected from FastFire qPCR PreMix (probe) and High Affinity hot start Taq of heaven, hot start High Taq DNA polymerase; or the DNA hot start polymerization Taq enzyme is prepared by the following method: a genomic DNA fragment of t.aquaticus YT1 containing a DNA polymerase (Taq-polymerase) gene was cloned into an expression vector and expressed.

6. The kit according to claim 1 or 2, wherein the kit comprises a gene methylation detection reagent comprising the following components:

(1) CRC detection reagent 1 comprising DNA Hot Start polymerase Taq enzyme (preferably 87.5U), PCR buffer (preferably containing 0.02M Tris-HCl, 0.1M KCl, 3mM MgCl₂) dNTP (preferably 400. mu.M);

(2) a CRC detection reagent 2 comprising the primer pair (preferably 5 to 10nM each) and the probe (preferably 4 to 8nM each) described in any one of claims 1 to 4, dissolved in TE buffer;

(3) a positive control comprising hypermethylated DNA of the genome of a human cell (preferably 4.0. + -. 0.5 ng/. mu.L), and TE buffer;

(4) negative control comprising human leukocyte genomic hypomethylated DNA (preferably 4.0. + -. 0.5 ng/. mu.L), and TE buffer.

7. A method for detecting multiple gene methylation, characterized in that detection is carried out using a kit according to any one of claims 1 to 6.

8. The method of claim 7, wherein the touchdown PCR is performed using a multiplex probe touchdown PCR that sets a touchdown PCR phase prior to a conventional PCR phase, the multiplex probe touchdown PCR comprising the steps of:

1) activating a polymerase;

2) a touchdown PCR phase wherein the estimated Tm is obtained by adding the melting temperature of the primer up to 15 ℃, the cycles are started at an annealing temperature about 15 ℃ above the estimated Tm, and the annealing temperature is lowered by 0.5-2 ℃ per cycle until 5 ℃ below Tm;

3) the traditional PCR stage is preferably a stage in which the primer and probe annealing stages are separated,

preferably, the reaction conditions of the process are as follows:

9. the method according to claim 7 or 8,

wherein each probe concentration is 200nM, and/or

Wherein, the concentration of each primer can be 300 nM.

10. The method according to claim 7 or 8, comprising the steps of:

(1) extracting free DNA of blood plasma;

(2) DNA sulfite conversion;

(3) a gene methylation assay comprising:

3.1 taking out the CRC detection reagent 1 and the CRC detection reagent 2 of claim 6 for thawing, thawing the sample DNA (the DNA after transformation in 2.2) to be detected, the negative control and the positive control, mixing uniformly for 10-15 seconds in a vortex mode, and separating instantly;

3.2 calculate the reaction system as follows: the volume of the CRC detection reagent 1 is 12.5 × (2n +6) μ l, the volume of the CRC detection reagent 2 is 0.5 × (2n +6) μ l, the volume of water is 7.0 × (2n +6) μ l, and n represents the number of samples to be examined;

3.3 adding CRC detection reagent 1, CRC detection reagent 2 and water with corresponding volumes into the centrifuge tube in sequence, mixing uniformly for 10-15 seconds in a vortex manner, and separating instantly;

3.4 adding 20 mul of the mixed PCR reaction solution into the plate hole of the corresponding 96-hole PCR plate, adding 5 mul of DNA to be detected or a negative control product and a positive control product, and setting two adjacent holes as multiple holes;

3.5 sealing with a high-permeability sealing plate membrane, and centrifuging for 1 minute at 1000 +/-100 rcf;

3.6 the computer model is divided into an Applied Biosystem 7500PCR instrument, and the corresponding Software version is 7500Software v 2.3;

3.7 settings for the Applied Biosystem 7500PCR Instrument are as follows:

3.7.1 the Pasive Reference is set as 'none', ACTB selects FAM fluorescence channel, SEPT9 selects VIC channel, ALX4 selects ROX channel, SDC2 selects CY5 channel;

3.7.2 reaction procedure:

3.7.3 analysis conditions settings: setting a baseline value to be 3-15 cycles, setting the threshold of ACTB to be 80,000, setting the threshold of SEPT9 to be 120,000, setting the threshold of ALX4 to be 30,000, and setting the threshold of SDC2 to be 20,000;

3.7.4 PCR validation: the control sample should meet the following criteria

Ct value of control sample ACTB SEPT9 ALX4 SDC2 Positive control ≤30 ≤30 ≤30 ≤30 Negative control ≤30 Uncertainty ＞38 ＞40

3.7.5 when the validity condition is satisfied, the sample is judged to be positive or negative: sample decision criteria

Ct value for methylation detection ACTB SEPT9 ALX4 SDC2 Positive for ≤40 ≤45 ≤38 ≤40 Negative of ≤40 Uncertainty ＞38 ＞40 Invalidation ＞40

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