CN114507739A

CN114507739A - Primer and probe for detecting colorectal cancer gene methylation level in human fecal sample

Info

Publication number: CN114507739A
Application number: CN202210339236.2A
Authority: CN
Inventors: 严林海; 唐卫中; 钟华戈; 莫显伟
Original assignee: Guangxi Medical University Affiliated Tumour Hospital
Current assignee: Guangxi Medical University Affiliated Tumour Hospital
Priority date: 2022-04-01
Filing date: 2022-04-01
Publication date: 2022-05-17

Abstract

The invention discloses a primer and a probe for detecting the methylation level of colorectal cancer genes in a human fecal sample, which comprise a primer and a probe for detecting the methylation level of SDC2 and/or a primer and a probe for detecting the methylation level of Septin9 and/or a primer and a probe for detecting the methylation level of MLH 1. The primer and the probe provided by the invention can be used for early diagnosis of colorectal tumors, and a simple, convenient and feasible solution is provided for early discovery of colorectal tumors.

Description

Primer and probe for detecting colorectal cancer gene methylation level in human fecal sample

Technical Field

The invention relates to the technical field of molecular biology, in particular to a primer, a probe and a kit for detecting colorectal cancer gene methylation level in a human fecal sample.

Background

Colorectal cancer (bowel cancer) is the third largest malignancy in the world. Early treatment effect of intestinal cancer is good, prognosis of middle and late stages is extremely poor, great burden is brought to families and society, development of the large health industry is seriously influenced, early diagnosis is the most important means for improving prognosis, and the active development of intestinal cancer screening and early prevention and treatment work is also important for reducing government-related medical cost.

At present, the following types of screening are mainly available: (1) the conventional blood tests have weak specificity sensitivity and are traumatic to the body; (2) imaging, such as enteroscopy, soft sigmoidoscope, CT colonography (virtual enteroscopy), Double Contrast Barium Enema (DCBE), etc., which requires treatment of intestinal cleansing, anesthesia, etc. before examination and is relatively serious to body trauma; (3) fecal occult blood tests such as Fecal Occult Blood Test (FOBT), Fecal Immunochemical Test (FIT) and the like are convenient and noninvasive, but have weak specificity and sensitivity. And the methods are implemented in hospitals, and the treatment period is long or the cost is high.

The intestinal cancer is a heterogeneous disease of intestinal epithelium and is characterized in that host gene mutation is accumulated, so that the detection of abnormal DNA segments of tumor cells can be used for screening and early diagnosis of colorectal cancer, the method does not cause physical trauma, and can be used for screening of people of all adult ages, but the fact that a kit with high specificity and sensitivity is screened is of great importance to the detection method.

Disclosure of Invention

The invention discloses a primer and a probe for detecting the methylation level of colorectal cancer genes in a human fecal sample, and the provided primer and probe can be used for early diagnosis of colorectal tumors, thereby providing a simple, convenient and feasible solution for early discovery of colorectal tumors.

In order to achieve the purpose, the technical scheme of the invention is as follows:

primers and probes for detecting the methylation level of colorectal cancer genes in human fecal samples comprise primers and probes for detecting the methylation level of SDC2 and/or primers and probes for detecting the methylation level of Septin9 and/or primers and probes for detecting the methylation level of MLH 1;

the primers used for detecting the methylation level of SDC2 were: the upstream primer is selected from a sequence shown by SEQ ID No. 1, the downstream primer is selected from a sequence shown by SEQ ID No. 2, and the probe is selected from a sequence shown by SEQ ID No. 3;

the primers for detecting the methylation level of Septin9 are as follows: the upstream primer is selected from a sequence shown by SEQ ID No. 4, the downstream primer is selected from a sequence shown by SEQ ID No. 5, and the probe is selected from a sequence shown by SEQ ID No. 6;

the primers for detecting the methylation level of MLH1 are as follows: the upstream primer is selected from a sequence shown by SEQ ID No. 7, the downstream primer is selected from a sequence shown by SEQ ID No. 8, and the probe is selected from a sequence shown by SEQ ID No. 9;

furthermore, HEX fluorescent groups are marked at the 5 'ends of the sequences shown in SEQ ID No. 3, SEQ ID No. 6 and SEQ ID No. 9, and MGB quenching groups are marked at the 3' ends of the probes.

The method for detecting the methylation level of the colorectal cancer gene in the human fecal sample comprises the following steps:

(1) preparation of a sample: extracting DNA and treating sulfite from the collected human excrement sample;

(2) and (3) PCR amplification: adding the primers and the probes, and carrying out fluorescent quantitative PCR detection on the DNA extracted in the step (1) and treated by sulfite;

(3) methylation level analysis and microbial level analysis: and calculating the difference value of the amplification Ct ratio of the target genes SDC2, Septin9, MLH1 and the reference gene ACTB to obtain the relative methylation level of the sample.

Wherein, when a Roche LightCycler 480 II instrument is adopted for result analysis, an AbsQuant/2nd Derivative Max analysis method is used for analyzing the amplification cycle number; ABI7500 Instrument results analysis the instrument default baseline was used. When the analysis is carried out by SDS V2.3.1 software, the value of FAM channel (for detecting SDC2 gene) is set to 65000, the threshold value of TexasRed channel (for detecting ACTB gene) is set to 95000, and the Ct value can be calculated only when the amplification curves of FAM channel and Texas Red channel show "S" type amplification.

Further, in the step (2), the primers used for amplifying the ACTB are:

the upstream primer is a sequence shown as SEQ ID No. 10, and the downstream primer is a sequence shown as SEQ ID No. 11;

the probe used for ACTB amplification is a sequence shown in SEQ ID No. 12, the 5 'end of the probe is marked with a HEX fluorescent group, and the 3' end of the probe is marked with a BHQ2 quenching group;

primers used for ACTB amplification after sulfite treatment were:

the upstream primer is a sequence shown as SEQ ID No. 13, and the downstream primer is a sequence shown as SEQ ID No. 14;

the probe used for ACTB amplification is a sequence shown in SEQ ID No. 15, the 5 'end of the probe is marked with a HEX fluorescent group, and the 3' end of the probe is marked with a BHQ2 quenching group.

Further, the reaction system of the PCR is as follows:

2×AceQ qPCR Probe Master Mix 10 μL

Primer Mix 4.6μL

50× ROX Reference Dye 2 0.4μL

Template DNA 5μL。

further, the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 2min, 10s at 95 ℃ and 30s at 60 ℃, amplification for 50 cycles, and final extension at 12 ℃.

SDC2, Septin9 and MLH1 have high methylation expression in colorectal cancer cells, a large number of clinical samples are used for verifying, designing and screening primer probes, and the primers and the probes have high sensitivity and high specificity when used for detection.

The detection method of the invention is based on a methylated DNA detection technology of quantitative PCR, and utilizes a probe and a PCR primer to distinguish methylated DNA from unmethylated DNA. Firstly, treating a DNA fragment by using bisulfite, designing a probe primer which can be complementary with a site to be detected, determining the CpG site which has the most relevance to colorectal tumor in the gene by real-time quantitative PCR determination, simultaneously determining an internal reference gene ACTB in a sample, and determining the methylation level of a target gene. The method has the advantages of high flux and high sensitivity, and does not need operations such as electrophoresis, hybridization and the like after PCR, thereby reducing pollution and operation errors. Can provide information for doctors for early diagnosis of colorectal tumors and provides a simple and easy solution for early discovery of colorectal tumors.

Millions of cells of ordinary people are shed from the colon wall to enter the excretory system including cells in the process of intestinal tumor formation every day, and the cells after entering the excretory system contain corresponding DNA, wherein the corresponding DNA contains genetic information closely related to tumors, and the process of colorectal cancer or tumor formation is reflected. Early intestinal cancer cells are a precursor of intestinal cancer due to methylation conversion of some genes, and these cells naturally fall into the intestinal tract and are excreted together with feces. By detecting these DNA markers, the presence of colorectal cancer, tumor, or the like on the intestinal wall can be found.

Therefore, the preferred detection method of the invention adopts human-derived DNA of excrement, is obtained from common excrement, is non-invasive, non-invasive and painless, is convenient for household sampling, does not need to go to a hospital, and has no time limit.

In conclusion, the primer and the probe provided by the invention can detect the methylation levels of SDC2, Septin9 and MLH1 in human fecal samples, have high sensitivity and specificity, are used for early diagnosis of colorectal tumors, greatly improve the sensitivity of colorectal tumor diagnosis, do not cause trauma to bodies, are suitable for screening of all adult age groups, and have wide application prospects.

Drawings

FIG. 1 is a fluorescent quantitative PCR amplification graph (positive) of the reference gene ACTB.

FIG. 2 is a graph of fluorescence quantitative PCR amplification of ACTB (Q-ACTB) after transformation (Positive).

Figure 3 is a graph of SDC2 fluorescent quantitative PCR amplification (positive).

FIG. 4 is the fluorescent quantitative PCR amplification curve (positive) for Septin 9.

FIG. 5 is a graph of MLH1 fluorescent quantitative PCR amplification (positive).

Detailed Description

Example 1

primers used to detect the methylation level of SDC2 were: the upstream primer is selected from a sequence shown by SEQ ID No. 1, the downstream primer is selected from a sequence shown by SEQ ID No. 2, and the probe is selected from a sequence shown by SEQ ID No. 3;

the primers used to detect the methylation level of Septin9 were: the upstream primer is selected from a sequence shown by SEQ ID No. 4, the downstream primer is selected from a sequence shown by SEQ ID No. 5, and the probe is selected from a sequence shown by SEQ ID No. 6;

primers used to detect the level of methylation of MLH1 were: the upstream primer is selected from a sequence shown by SEQ ID No. 7, the downstream primer is selected from a sequence shown by SEQ ID No. 8, and the probe is selected from a sequence shown by SEQ ID No. 9;

HEX fluorescent groups are marked at the 5 'ends of the sequences shown in SEQ ID No. 3, SEQ ID No. 6 and SEQ ID No. 9, and MGB quenching groups are marked at the 3' ends of the probes.

TABLE 1 primer and Probe sequence Listing

Example 2

(1) nucleic acid extraction: treatment for extracting host cell DNA and sulfite from 100-200 mg of excrement sample

The scheme is suitable for extracting host cell DNA from 100-200 mg of excrement sample

1.1 high temperature lysis method (conventional assay)

A1. Transferring 100-200 mg of excrement sample to a 2ml centrifuge tube, adding 1.0 ml of Buffer ATL/PVP-10 into the sample, and scattering the sample by maximum vortex for 1 minute. When liquid feces are sucked, a small part of the head of a 1ml pipette tip is cut off to facilitate sample transfer. Before use, the PVP-10 powder is poured into a Buffer ATL bottle, inverted and mixed evenly, and used after being completely dissolved. The method is used for treating fiber-rich animal waste samples (such as cattle and sheep) with the sample amount controlled at 70-140 mg and treating animal waste (such as mouse waste) with little water, wherein the sample amount is 50-70 mg.

A2.70 ℃ water bath for 10 minutes.

A3. Centrifuge at 13000 rpm for 3 minutes at room temperature.

A4. Transfer 0.6ml of supernatant to a new centrifuge tube, add 0.6ml Buffer PCI to the lysate, vortex and mix for 15 seconds, and stand for 3 minutes.

A5. Centrifuge at 13000 rpm for 3 minutes at room temperature.

1.2 transferring 500 mul of supernatant to a 2ml centrifuge tube, adding 4 mul of RNase Solution to the supernatant, and standing for 10 minutes at room temperature.

1.3 Add 20. mu.l Proteinase K and 500. mu.l Buffer AL to the supernatant. Mix by inversion and digest at 70 ℃ for 10 min.

1.4 adding 500 mul of absolute ethyl alcohol to the mixed solution, and uniformly mixing for 15 seconds by vortex.

1.5 HiPure DNA Mini Column II was placed in a 2ml collection tube and half the volume of the mixture was transferred to the Column. Centrifuging at 10000rpm for 30-60 seconds.

1.6 discard the filtrate and pack the column back into the collection tube. The remaining mixture was transferred to the column. Centrifuging at 10000rpm for 30-60 seconds.

1.7 pour the effluent and place the column back into the collection tube. 500 μ l of Buffer GW1 (diluted with absolute ethanol) was added to the column. Centrifuging at 10000rpm for 30-60 seconds. Buffer GW1 was diluted with absolute ethanol and was diluted as indicated in the bottle label or the instructions.

1.8 discard the filtrate and pack the column back into the collection tube. 650 μ l of Buffer GW2 (diluted with ethanol) was added to the column. Centrifuging at 10000rpm for 30-60 seconds. Buffer GW2 was diluted with absolute ethanol. Dilution is performed as indicated by the bottle label or instructions.

1.9 discard the filtrate and pack the column back into the collection tube. Centrifuge at 13000 rpm for 2 minutes and spin dry the column.

1.10 the columns were packed in 1.5ml centrifuge tubes. Adding 30-100 mul of Buffer AE or sterilized water preheated to 65 ℃ to the center of the membrane of the column, and standing for 2 minutes at room temperature. Centrifuge at 13000 rpm for 1 minute.

1.11. The DNA binding column was discarded. DNA is stored at 2-8 deg.C and-20 deg.C for long-term storage.

(2) DNA sulfuration (use kit II)

Preparation work: adding 30 ml of absolute ethyl alcohol into BW buffer, and storing at room temperature. ② adding 27 ml of absolute ethyl alcohol into the BD buffer, and storing at 2-8 ℃. ③ 310 ul of water is added into Carrier RNA, and the Carrier RNA is stored at the temperature of minus 20 ℃ after being subpackaged. And fourthly, the sample and the buffer need to be balanced to the room temperature. BL buffer 60 ℃ water bath.

2.1 thawing DNA: ensure complete dissolution of the sulphurising agent and if necessary, vortex at 60 ℃ until the ingredients are dissolved. Note: the sulfurizing agent cannot be placed on ice.

2.2 build-up of the vulcanization reaction, Table 2 below:

note: if the total volume is less than 140ul, the water is used for supplementing to 140.

And 2.3, covering the PCR tube cover, and fully and uniformly mixing to ensure that the sample is at normal temperature. Note: the DNA protection buffer turns blue, indicating that the pH of the sulfurization reaction is correct.

2.4 the PCR instrument was run according to Table 3. The PCR instrument was hot-capped to 105 ℃. Note: if the maximum volume of the PCR instrument is adjusted to be less than 140ul, the maximum volume is adjusted.

Note: an extension time of up to 20min at 60 ℃ is necessary to allow complete sulphurized transformation of the DNA. The transformed DNA can be used overnight on a PCR instrument, and the experimental results have no influence.

2.5 Place PCR tube in PCR instrument and start incubation.

2.6 sulfurized samples, briefly centrifuge, transfer the liquid to a new 1.5ml centrifuge tube.

2.7 Add 310 ul of currently prepared BL buffer, briefly vortex, centrifuge Mix. If the total amount of DNA is less than 100 ng, BL Buffer for dissolving carrier RNA is added, and only the required amount (Table 4) needs to be prepared, note: is a mix containing DNA.

TABLE 4 Carrier RNA and BL buffer volumes

2.8 an additional 250ul of absolute ethanol (96-100%) was added, pulsed for 15 seconds, and then briefly centrifuged to remove the droplets from the lid.

2.9 the mixture was incubated at 65 ℃ for 3min to see the state of crystallization, and if there was no crystallization, the mixture was immediately added to a preheated MinElute DNA spin column and immediately centrifuged. Centrifuge at full speed for 1 min, discard the liquid, place the column back into the centrifuge tube.

2.10 Add 500ul BW buffer, centrifuge at full speed for 1 min, discard the liquid, place the column back into the centrifuge tube.

2.11 Add 500ul BD buffer to avoid aspiration to pellet. The lid was closed and incubated at room temperature for 15 min. Note: when the BD buffer is used, the BD buffer can contact air in the shortest time to prevent acidification. Centrifuge at full speed for 1 min, discard the liquid, place the column back into the centrifuge tube.

2.12 Add 500ul BW buffer, centrifuge at full speed for 1 min, discard the liquid, place the column back into the centrifuge tube. And repeating the steps once.

2.13 Add 250ul absolute ethanol (96-100%), centrifuge at full speed for 1 min.

2.14 Place the column in a new 2ml collection tube and centrifuge at full speed for 1 min. The residual liquid is removed.

2.15 Place the column in a new 1.5ml centrifuge tube, add 60ul EB Buffer directly to the middle of the column membrane, close the lid. Incubate at room temperature for 1 min, and centrifuge for 1 min. Note: the minimum volume of eluent added is 10 ul.

(3) And (3) PCR amplification:

the modified sample DNA was subjected to fluorescent quantitative PCR using the following primers and probes according to the PCR reaction system of Table 5 and the PCR reaction program of Table 6.

The following reaction system was used to add the ingredients:

TABLE 5 PCR reaction System

After the sample is added, the mixture is fully and uniformly mixed and then centrifuged, and the PCR tube is placed into a PCR instrument.

The reaction conditions for PCR were set as follows:

TABLE 6 PCR reaction procedure

(4) Methylation level analysis: and calculating the difference value of the amplification Ct ratio of the target genes SDC2, Septin9, MLH1 and the reference gene ACTB to obtain the relative methylation level of the sample. Setting analysis conditions: analyzing the amplification cycle number by using an AbsQuant/2nd Derivative Max analysis method when analyzing the results of a Roche LightCycler 480 II instrument; ABI7500 Instrument results analysis Using the default baseline for the instrument, the value of the FAM channel (for detecting the SDC2 gene) was set to 65000 and the threshold of the TexaRed channel (for detecting the ACTB gene) was set to 95000 for the SDS V2.3.1 software analysis. The Ct values can be calculated only when the amplification curves of the FAM channel and the Texas Red channel show an "S" type amplification.

TABLE 7 Positive judgment values

Interpretation of test results:

1. and (3) after the test is finished, operating the Ct value calculation, and when the Ct value of the operating non-template control (NTC) is more than or equal to 40 or no amplification curve appears, indicating that the test has no pollution, and continuously analyzing the test condition.

2. Operating FAM channels of the positive quality control product and the negative quality control product, wherein when the Ct value of the positive quality control is less than or equal to 35, the Ct value of the negative quality control is more than or equal to 40 or no Ct value; when Ct values of the Texas Red channel negative quality control product and the positive quality control product are both less than or equal to 35, the experiment system is normal, and the analysis experiment can be continued.

3. In a Texas Red channel in a reaction hole for detecting a sample, the Ct value is generally less than or equal to 36, the analysis can be continued, and if the Ct value is greater than 36, the DNA amount of the sample is insufficient or the degradation is serious, so that the sample is not suitable for experimental analysis.

4. In the FAM channel of the detection sample reaction hole, the sample is negative when no amplification curve appears or the Ct value is more than 38. The sample with S-type amplification curve and Ct value less than or equal to 38 is positive.

5. The patients with positive samples are recommended to be subjected to enteroscopy for confirmation; patients judged negative do not exclude the possibility of disease; and judging that the sample is unqualified, and recommending the patient to perform resampling detection.

6. The interference experiment result shows that: the fecal samples contained the following materials: animal DNA (500 ng/ml), plant tissue (500 ng/ml), plant oil (10. mu.l/ml), laxative mannitol (75 mg/ml), laxative capsule (7.25 mg/ml), hemorrhoid ointment (24.75 mg/ml), moroxydine (0.14 mg/ml), tetracycline (10.65 mg/ml), penicillin (2.56 mg/ml), ibuprofen capsule 75. mu.g/ml, stomach drug stavudine (6.84 mg/ml), omeprazole (2.82 mg/ml), cefixime (20.5 mg/ml), levofloxacin hydrochloride (0.15 mg/ml), cimetidine (2 mg/ml), Ganmaoling capsule (0.27 mg/ml) had no effect on the test results.

And (3) specificity test: 138 intestinal cancer samples and 17 healthy people are selected; and (3) sensitivity test: 138 intestinal cancer samples were selected, and 17 healthy people were selected

。

Sequence listing

<110> tumor hospital affiliated to Guangxi medical university

<120> primers and probes for detecting colorectal cancer gene methylation level in human fecal samples

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<141> 2022-04-01

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<400> 1

tttgagtttt gagtttgagt tttt 24

<210> 2

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<212> DNA

<213> SDC2 downstream primer (SDC2)

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tcctacccaa cgctcgacg 19

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<213> SDC2 Probe (SDC2)

<400> 3

ttgtaattgt tgtggtatt 19

<210> 4

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<213> Septin9 upstream primer (Septin9)

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agtttgaaat gattttattt agttgtgt 28

<210> 5

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<213> Septin9 downstream primer (Septin9)

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cctcaccact accctccg 18

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<213> Septin9 Probe (Septin9)

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cactacccac caaccatcat a 21

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<213> MLH1 upstream primer (MLH1)

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cgatagatta ggtatagggt tttatcgttt ttc 33

<210> 8

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<213> MLH1 downstream primer (MLH1)

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gcccaaaaaa aacaaaataa aaatcgacg 29

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<213> MLH1 Probe (MLH1)

<400> 9

acgttgggtt tattcgggtc ggaa 24

<210> 10

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<213> ACTB amplification upstream primer (ACTB)

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gtgacgtgga catccgcaaa 20

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ggaaagacac ccaccttgat cttc 24

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Claims

1. Primers and probes for detecting the level of colorectal cancer gene methylation in human stool samples, characterized in that: comprises a primer and a probe for detecting the methylation level of SDC2 and/or a primer and a probe for detecting the methylation level of Septin9 and/or a primer and a probe for detecting the methylation level of MLH 1;

the primers for detecting the methylation level of MLH1 are as follows: the upstream primer is selected from a sequence shown in SEQ ID No. 7, the downstream primer is selected from a sequence shown in SEQ ID No. 8, and the probe is selected from a sequence shown in SEQ ID No. 9.

2. The primers and probes according to claim 1, characterized in that:

3. The method for detecting the methylation level of the colorectal cancer gene in the human fecal sample is characterized by comprising the following steps:

(2) and (3) PCR amplification: adding the primer and the probe of claim 1, and carrying out fluorescent quantitative PCR detection on the DNA extracted in the step (1) and treated by sulfite;

(3) methylation level analysis: and calculating the difference value of the amplification Ct ratio of the target genes SDC2, Septin9, MLH1 and the reference gene ACTB to obtain the relative methylation level of the sample.

4. The detection method according to claim 3, characterized in that:

in the step (2), the primers used for ACTB amplification are as follows:

primers used for amplification of ACTB after sulfite treatment were:

5. The detection method according to claim 3, characterized in that:

the reaction system of the PCR is as follows:

2×AceQ qPCR Probe Master Mix 10 μL

Primer Mix 4.6μL

50× ROX Reference Dye 2 0.4μL

Template DNA 5μL。

6. the detection method according to claim 3 or 5, characterized in that:

and (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 2min, 10s at 95 ℃ and 30s at 60 ℃, amplification for 50 cycles, and final extension at 12 ℃.

7. Use of the primers and probes of claim 1 or 2 for the preparation of a reagent for early diagnosis of colorectal cancer.