CN106893777B - Multi-site methylation kit for detecting colorectal cancer related genes and application thereof - Google Patents

Multi-site methylation kit for detecting colorectal cancer related genes and application thereof Download PDF

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CN106893777B
CN106893777B CN201710121118.3A CN201710121118A CN106893777B CN 106893777 B CN106893777 B CN 106893777B CN 201710121118 A CN201710121118 A CN 201710121118A CN 106893777 B CN106893777 B CN 106893777B
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张良禄
董兰兰
姚希辉
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Wuhan Aimisen Life Technology Co ltd
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Abstract

The invention discloses a kit for detecting methylation of multiple sites of genes related to colorectal cancer and application thereof. The invention also provides a primer combination for detecting methylation, application of the probe in colorectal cancer and precancerous polyp screening and a method thereof. The method comprises the following steps: adding cell lysate into cell sediment, fully and uniformly mixing, then adding PVPP (polyvinyl pyrrolidone) for crude extraction, adding a small amount of protein and RNA in a cell sediment sample after primary treatment, then adding proteinase K and RNase to completely remove the protein and RNA, adding the cell lysate into a centrifugal column with an adsorption membrane, washing impurities by using DNA rinsing liquid, then adding DNA eluent to elute DNA from the adsorption membrane, and finally centrifuging and collecting to obtain DNA solution. The high-efficiency tube combination detection is carried out aiming at a plurality of gene loci, the sensitivity and specificity are improved, and the requirement of early screening of tumors can be met.

Description

Multi-site methylation kit for detecting colorectal cancer related genes and application thereof
Technical Field
The invention relates to the technical field of medical treatment, in particular to a multi-site methylation kit for detecting colorectal cancer related genes and application thereof.
Background
Colorectal cancer is the third most malignant tumor in the world. In China, the incidence rate of colorectal cancer rises year by year, about 40 million cases are estimated to occur every year, and the second case is listed in China's digestive system malignant tumor. The 5-year survival rate of early colorectal cancer patients can reach more than 90 percent after operation, the 5-year survival rate of middle and late stage patients is only about 10 percent, and about 80 percent of clinically diagnosed colorectal cancer is in the middle and late stage, which is one of the key factors causing the death rate to be high. The incidence rate of early colorectal cancer patients effectively treated is reduced by 60 percent, and the fatality rate is reduced by 80 percent, so that early discovery, early diagnosis and early treatment of colorectal cancer are particularly important.
Current studies indicate that early onset of colorectal cancer is strongly associated with methylation of the promoter region of genes associated with colorectal cancer. Methylation is an epigenetic modification, and abnormal changes thereof can cause changes in DNA conformation, interaction mode of DNA and protein, and the like, so as to control gene expression, and thus, the methylation is closely related to early occurrence of colorectal cancer. In the prior patent CN 105543354A, magnetic beads are used for capturing human excrement DNA, then the human excrement DNA is subjected to bisulfite treatment, and finally PCR is used for detecting the methylation condition of the promoter region of the SDC2 gene.
Problems and disadvantages with this approach:
1. the gene detected by this method is single, and although the sensitivity is relatively high, the specificity is insufficient. According to the invention, the methylation of a plurality of gene promoters is related to the occurrence of colorectal cancer, and the joint detection is carried out aiming at a plurality of gene loci, so that the sensitivity and specificity are improved, and the requirement of early screening of tumors can be met.
2. The method has limitation on the capture of target gene DNA fragments, can only capture the sequence of the magnetic bead specific fragments, and cannot detect a plurality of gene loci. The inventive fecal DNA extraction technology and kit can obtain high-purity and high-quality genomic DNA, and facilitate subsequent detection.
Therefore, a multi-site methylation kit for detecting colorectal cancer related genes and application thereof are provided.
Disclosure of Invention
The invention provides a multi-site methylation kit for detecting colorectal cancer related genes and application thereof, and aims to solve the problems in the background technology.
The kit is used for detecting the multi-site methylation of genes related to colorectal cancer and application, and a fecal sample is placed in a genome extraction kit for processing, and the steps are as follows:
a1, adding the sample into the nucleic acid lysis solution, mixing well, adding PVPP to perform crude extraction, and removing a large amount of impurities such as pigment and protein;
a2, adding a small amount of protein and RNA in the nucleic acid sample obtained after the primary treatment, and then adding proteinase K and RNase to completely remove the protein and RNA;
a3, adding the nucleic acid sample into a centrifugal column with an adsorption film, repeatedly rinsing and centrifuging with a DNA rinsing solution, adding a DNA eluent to elute the DNA from the adsorption film, and centrifuging to collect a DNA solution.
And carrying out CT conversion on the purified DNA by using the bisulfite conversion kit disclosed by the invention, wherein the kit comprises a dissolving buffer solution, a methylation conversion reagent, a binding buffer solution and a rinsing buffer solution.
Specific primer pairs and probes aiming at the methylation of promoter regions of colorectal cancer related genes NDRG4, SDC2, TFPI2 and BMP3 carry out high-throughput tube combination detection aiming at the methylation sites of a plurality of gene promoters.
The invention provides a multi-site methylation kit for detecting colorectal cancer related genes and application thereof, wherein the kit comprises a primer combination, the primer combination comprises a first primer pair, a second primer pair, a third primer pair, a fourth primer pair and a fifth primer pair, the base sequence of the first primer pair is shown as SEQ ID NO.1-2, the base sequence of the second primer pair is shown as SEQ ID NO.3-4, the base sequence of the third primer pair is shown as SEQ ID NO.5-6, the base sequence of the fourth primer pair is shown as SEQ ID NO.7-8, and the base sequence of the fifth primer pair is shown as SEQ ID NO. 9-10; the kit also comprises probes, wherein the probes comprise a first probe, a second probe, a third probe, a fourth probe and a fifth probe, the base sequence of the first probe is shown as SEQ ID NO.11, the sequence of the second probe is shown as SEQ ID NO.12, the sequence of the third probe is shown as SEQ ID NO.13, the sequence of the fourth probe is shown as SEQ ID NO.14, and the sequence of the fifth probe is shown as SEQ ID NO. 15; the probe is characterized in that a fluorescent group is marked at the 5 'end of the probe, a quenching group is marked at the 3' end of the probe, the fluorescent group is one of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 and NED, and the quenching group is one of 6-TAMRA, BHQ-1-3 and a non-fluorescence quenching agent combined with a molecular groove.
Further, the first primer pair is an NDRG4 detection primer, the upstream detection primer comprises a nucleotide sequence shown by SEQ ID NO.1, and the nucleotide sequence shown by SEQ ID NO.1 is 5'-TGTTTTTTAGGTTTC-3'.
Further, the NDRG4 downstream detection primer comprises a nucleotide sequence shown by SEQ ID NO.2, and the nucleotide sequence shown by SEQ ID NO.2 is 5'-GCGTAACTTCCGCCTTCTA-3'.
Further, the NDRG4 probe comprises a nucleotide sequence shown in SEQ ID NO.11, and the nucleotide sequence shown in SEQ ID NO.11 is 5'-CGTTCGTTTTTTCGTTCGTTTATCGG-3'.
Furthermore, the 5 'end of the detection probe is marked with a fluorescent group, and the 3' end of the detection probe is marked with a quenching group.
Further, the probe may be labeled at its 5' end with any one of the following fluorescent groups: FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670, NED, any of the following quenching groups is selected to be marked at the 3' end of the compound: 6-TAMRA, BHQ-1-3, and a non-fluorescence quencher (MGB NFQ) combined with a molecular Groove. Preferably, the fluorescent label is selected from FAM and the quencher is selected from BHQ.
Further, the second primer pair and the second probe are a primer pair SDC2 and a probe SDC 2.
Further, the SDC2 upstream primer comprises a nucleotide sequence shown in SEQ ID NO.3, and the nucleotide sequence shown in SEQ ID NO.3 is 5'-TTAATAAGTGAGAGGGCGTCGC-3'.
Further, the SDC2 downstream primer comprises a nucleotide sequence shown in SEQ ID NO.4, and the nucleotide sequence shown in SEQ ID NO.4 is 5'-CGACTCAAACTCGAAAACTC-3'.
Further, the SDC2 probe comprises a nucleotide sequence shown in SEQ ID NO.12, and the nucleotide sequence shown in SEQ ID NO.12 is 5'-CGTAGTTGCGGGCGGCGGGAGTAGGC-3'.
Further, the probe may be labeled at its 5' end with any one of the following fluorescent groups: FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670, NED, any of the following quenching groups is selected to be marked at the 3' end of the compound: 6-TAMRA, BHQ-1-3, and a non-fluorescence quencher (MGB NFQ) combined with a molecular Groove. Preferably, the fluorescent marker is VIC and the quencher is MGB.
Further, the third primer pair and the third probe are a TFPI2 primer pair and a TFPI2 probe.
Further, the TFPI2 upstream primer comprises a nucleotide sequence shown in SEQ ID NO.5, and the nucleotide sequence shown in SEQ ID NO.5 is 5'-GGCGAAGTTGTTATTAGTCGTC-3'.
Further, the TFPI2 downstream primer comprises a nucleotide sequence shown by SEQ ID NO.6, and the nucleotide sequence shown by SEQ ID NO.6 is 5'-ATAAATACCCTACGCGCA-3'.
Further, the TFPI2 probe comprises a nucleotide sequence shown in SEQ ID NO.13, and the nucleotide sequence shown in SEQ ID NO.13 is 5'-AGCGCGAGAGTTTTGGGTGCGCG-3'.
The probe can be marked at the 5' end by any one of the following fluorescent groups: FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670, NED, any of the following quenching groups is selected to be marked at the 3' end of the compound: 6-TAMRA, BHQ-1-3, and a non-fluorescence quencher (MGB NFQ) combined with a molecular Groove. Preferably, the fluorescent label is selected from FAM and the quencher is selected from BHQ.
Further, the fourth primer pair and the fourth probe are the primer pair of BMP3 and the probe of BMP 3.
The BMP3 upstream primer comprises a nucleotide sequence shown by SEQ ID NO.7, and the nucleotide sequence shown by SEQ ID NO.7 is 5'-TTATTTCGTTGTATTCGGTC-3'.
The BMP3 downstream primer comprises a nucleotide sequence shown by SEQ ID NO.8, and the nucleotide sequence shown by SEQ ID NO.8 is 5'-ACCGAAAATTAAACTCCAA-3'.
Further, the BMP3 probe comprises a nucleotide sequence shown in SEQ ID NO.14, and the nucleotide sequence shown in SEQ ID NO.14 is 5'-CGGGTTTCGTGCGTTTTCGTT-3'.
The probe can be marked at the 5' end by any one of the following fluorescent groups: FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670, NED, any of the following quenching groups is selected to be marked at the 3' end of the compound: 6-TAMRA, BHQ-1-3, and a non-fluorescence quencher (MGB NFQ) combined with a molecular Groove. Preferably, the fluorescent label is selected for VIC and the quencher is selected for BHQ.
Further, the fifth primer pair and the fifth probe are an Internal Reference (IR) primer pair and an Internal reference probe.
The internal reference primer pair comprises an internal reference upstream primer and an internal reference downstream primer and is used for amplifying actin genes of human.
The IR upstream primer comprises a nucleotide sequence shown by SEQ ID NO.9, and the nucleotide sequence shown by SEQ ID NO.9 is 5'-TGGGATGGGGAGTTTGTTTAGATT-3'.
The IR downstream primer comprises a nucleotide sequence shown by SEQ ID NO.10, and the nucleotide sequence shown by SEQ ID NO.10 is 5'-AATACTAACACTAACTCATATAAC-3'.
Further, the IR probe comprises a nucleotide sequence shown in SEQ ID NO.15, and the nucleotide sequence shown in SEQ ID NO.15 is 5'-TGTATTTATTTAATATATATTTTAAGGTTG-3'.
The probe can be marked at the 5' end by any one of the following fluorescent groups: FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670, NED, any of the following quenching groups is selected to be marked at the 3' end of the compound: 6-TAMRA, BHQ-1-3, and a non-fluorescence quencher (MGB NFQ) combined with a molecular Groove. Preferably, the fluorescent label is NED and the quencher is BHQ-2.
The invention also provides a kit for detecting the multi-site methylation of genes related to colorectal cancer and application thereof, wherein the kit comprises the primer combination and the probe for detecting NDRG4, SDC2, TFPI2, BMP3 and actin, and one or more of a sample preservation solution, a nucleic acid extracting solution, a PCR reaction solution, a positive control substance, a negative control substance, a process control sample, a standard substance, a dissolving buffer solution, a methylation conversion reagent, a binding buffer solution and a rinsing buffer solution.
The kit for detecting the multi-site methylation of the colorectal cancer related gene and the application have the advantages that:
1. according to the invention, the methylation of a plurality of gene promoters is related to the occurrence of colorectal cancer, and high-throughput convergent tube detection is carried out aiming at the methylation sites of the plurality of gene promoters, so that the sensitivity and specificity are improved, and the requirement of early screening of tumors can be met;
2. the inventive fecal DNA extraction technology and kit can obtain high-purity and high-quality genomic DNA, and facilitate subsequent detection.
Drawings
FIG. 1 is a linear plot of NDRG 4;
FIG. 2 is a line graph of BMP 3;
figure 3 is a linear plot of SDC 2;
FIG. 4 is a linear plot of TFPI 2;
FIG. 5 is a linear relationship diagram of Actin;
FIG. 6 is a graph showing the results of the detection of NDRG 4;
FIG. 7 is a graph showing the results of assaying a sample of BMP 3;
figure 8 is a sample detection diagram of SDC 2;
fig. 9 is a sample detection diagram of TFPI 2;
FIG. 10 is a sample detection map of actin.
Detailed Description
The invention is further illustrated by the following examples.
Example 1
The invention provides a multi-site methylation kit for detecting genes related to colorectal cancer and application thereof, wherein a fecal genome extraction kit is used for DNA separation and extraction to obtain a fecal genome, and the steps are as follows:
1. the excrement of a hospital patient is collected by a special excrement collecting box, wherein the excrement contains excrement genome preservation solution, the preservation solution contains 0.1mol/L EDTA and 0.5mol/L TRIS, and the function of the preservation solution is to protect genome DNA from being degraded by nuclease in excrement.
2. In this example, after the fecal sample was fully shaken and mixed in the sample preservation solution, 1.5ml of the mixed solution was put into a 2.0ml EP tube, one ninth of the sample of 40% SDS lysate was added, 8. mu.L of protease K was added, and after shaking and mixing, the mixture was lysed at 70 ℃ for 10 min.
3.12000 rpm for 8min, 900. mu.L of the supernatant was transferred to a new EP tube.
4. Adding a piece of PVPP tablet, fully shaking and uniformly mixing, and then centrifuging at 12000rpm for 5 min.
5. Transfer supernatant to a new 2.0ml EP tube.
6. According to 8M guanidine hydrochloride: isopropyl alcohol: adding 8M guanidine hydrochloride and isopropanol into the supernatant at a mass-to-volume ratio of 1:1:1, respectively, properly mixing the supernatant and the guanidine hydrochloride before adding the isopropanol, and reversing and mixing the mixture after adding the isopropanol. .
7. Purifying with adsorption column or magnetic bead
The column purification procedure was as follows:
a1, taking 750uL of the liquid obtained in the step 11 and putting the liquid into an adsorption column with a corresponding number. The column was centrifuged at 12000rpm for 30sec to discard the waste liquid from the column. And (4) putting the adsorption column into the recovery collecting column again. This step was repeated until all of the above liquid passed through the adsorption column. A2, W1500 uL. mu.L was put on an adsorption column placed on a collection column, centrifuged at 12,000rpm for 30sec, the waste liquid in the collection column was discarded, and the adsorption column was replaced with the collection column. A3, taking W2600 uL. mu.L into an adsorption column, centrifuging at 12,000rpm for 30sec, pouring the waste liquid from the collection column, and placing the adsorption column into the collection column again. A4, repeating the step A3 once
Centrifuging at 12,000rpm for 2min under A5, pouring off waste liquid, placing the adsorption column in the collection column, opening the cover of the adsorption column, standing at room temperature for 5-10min, and allowing ethanol to volatilize. A6, transfer the adsorption column to a new 1.5mL collection tube, suspend the adsorption column, add 50uL of μ LTB (without contact with adsorption membrane), cover with lid, and let stand for 5 min.
A7, centrifugation at 12,000rpm for 2min, and collection of DNA solution.
Step of extracting fecal genome DNA by paramagnetic particle method
And B1, adding 20 mu L of magnetic beads into a small amount of specimen supernatant or feces homogenizing solution into which guanidine percarbonate and isopropanol are added in equal volume, fully oscillating and uniformly mixing, standing and combining for 10 minutes, uniformly mixing for several times during the period, and avoiding magnetic bead sedimentation.
B2, throwing the EP tube for a few seconds to enable the liquid drops attached to the tube cover and the wall to fall to the bottom of the EP tube, putting the EP tube into a magnetic frame, fully attaching the magnetic beads to one side of the inner wall of the EP tube after 1 minute, sucking the liquid by a pipette and abandoning the liquid.
B3, adding 1mL of rinsing liquid W1, immersing the magnetic beads in the washing liquid, fully oscillating and uniformly mixing, separating the EP tube for a few seconds, allowing the liquid drops attached to the tube cover and the wall to fall to the bottom of the EP tube, placing the EP tube into a magnetic frame, fully attaching the magnetic beads to one side of the inner wall of the EP tube after 1 minute, and sucking out the liquid by a pipette and discarding the liquid.
B4, adding 1mL of rinsing liquid W2, immersing magnetic beads in the washing liquid, fully shaking and uniformly mixing, shortening the EP tube for a few seconds to enable the liquid drops attached to the tube cover and the wall to fall to the bottom of the EP tube, placing the EP tube into a magnetic frame, fully attaching the magnetic beads to one side of the inner wall of the EP tube after 1 minute, sucking out the redundant W2 liquid by a pipette, and discarding the liquid.
And B5, repeating the step 4.
And B6, drying in the air, and volatilizing the ethanol in the tube.
And B7, adding 50 mu L of TB for elution, fully shaking and uniformly mixing, separating the EP tube for a few seconds to enable the liquid drops attached to the tube cover and the wall to fall to the bottom of the EP tube, putting the EP tube into a magnetic frame, fully attaching magnetic beads to one side of the inner wall of the EP tube after 1 minute, and sucking out the DNA solution to a new 1.5mL centrifuge tube by using a pipette for later use.
Example 2
The embodiment provides a multi-site methylation kit for detecting colorectal cancer related genes and application thereof, wherein the primer combination comprises a first primer pair, a second primer pair, a third primer pair, a fourth primer pair and a fifth primer pair, the base sequence of the first primer pair is shown as SEQ ID NO.1-2, the base sequence of the second primer pair is shown as SEQ ID NO.3-4, the base sequence of the third primer pair is shown as SEQ ID NO.5-6, the base sequence of the fourth primer pair is shown as SEQ ID NO.7-8, and the base sequence of the fifth primer pair is shown as SEQ ID NO. 9-10; the probe comprises a first probe, a second probe, a third probe, a fourth probe and a fifth probe, wherein the base sequence of the first probe is shown as SEQ ID NO.11, the sequence of the second probe is shown as SEQ ID NO.12, the sequence of the third probe is shown as SEQ ID NO.13, the sequence of the fourth probe is shown as SEQ ID NO.14, and the sequence of the fifth probe is shown as SEQ ID NO. 15;
the primer combination comprises five primer pairs, the base sequences of the primer pairs are respectively shown as SEQ ID NO.1-10, and the base sequences of the probes are respectively shown as SEQ ID NO. 11-15.
The NDRG4 upstream primer is:
SEQ ID NO.1：5’-TGTTTTTTAGGTTTCGCGTC-3’，
the downstream primer of the NDRG4 is:
SEQ ID NO.2:5’-GCGTAACTTCCGCCTTCTA-3’，
the SDC2 upstream primer is:
SEQ ID NO.3：5’-CGGGAGTAGGCGTAGGAG-3’，
the SDC2 downstream primers are as follows:
SEQ ID NO.4：5’-AAATACCGCAACGATTACGAC-3’，
the TFPI2 upstream primer is as follows:
SEQ ID NO.5：5’-GGCGAAGTTGTTATTAGTCGTC-3’，
the TFPI2 downstream primer is:
SEQ ID NO.6：5’-AAATAAATACCCTACGCGCAC-3’，
the upstream primer of BMP3 is:
SEQ ID NO.7：5’-TTATTTCGTTGTATTCGGTC-3’，
the downstream primer of BMP3 is:
SEQ ID NO.8：5’-ACCGAAAATTAAACTCCAA-3’，
the IR upstream primer is:
SEQ ID NO.9：TGGGATGGGGAGTTTGTTTAGATT
the IR downstream primer is:
SEQ ID NO.10：AATACTAACACTAACTCATATAAC
the probe is as follows:
SEQ ID NO.11：CGTTCGTTTTTTCGTTCGTTTATCGG
SEQ ID NO.12：TCGCGTTTTCGGGGCG
SEQ ID NO.13：AGCGCGAGAGTTTTGGGTGCGCG
SEQ ID NO.14：CGGGTTTCGTGCGTTTTCGTT
SEQ ID NO.15：TGTATTTATTTAATATATATTTTAAGGTTG
carrying out amplification treatment on the treated primer, wherein the reaction program comprises 50 ℃ UNG enzyme digestion for 10 min; pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 10s, annealing and extension at 58-65 ℃ for 30s, and collection of fluorescence signals for 40 cycles.
Cell lysate, PVPP, protease K, RNA enzyme, DNA rinsing liquid, DNA eluent and DNA preserving liquid are all preserved in the genome extraction kit.
Method of use of the kit reference example 2.
Example 3
The invention provides a multi-site methylation kit for detecting colorectal cancer related genes and application thereof, wherein the kit further comprises one or more of a sample preservation solution, a nucleic acid extracting solution, a PCR reaction solution, a positive quality control substance, a negative quality control substance, an internal standard control and a standard substance reagent.
The kit also comprises one or more of a dissolving buffer solution, a methylation conversion reagent, a binding buffer solution and a rinsing buffer solution, and the purified DNA can be subjected to CT conversion.
The dissolving buffer solution comprises 1-5mol/L NaOH and 5-20mmol/L hydroquinone, and the preferable effect is the best effect when the contents are 3mol/L NaOH and 10mmol/L hydroquinone.
The methylation conversion reagent comprises, but is not limited to, salts containing sulfitation groups, such as sodium bisulfite, sodium sulfite, ammonium bisulfite, ammonium sulfite and the like, and the concentration is 1-10mol/L, preferably ammonium bisulfite, and the concentration is 9 mol/L.
The binding buffer includes, but is not limited to guanidine salts such as guanidine hydrochloride and guanidine isothiocyanate, and the concentration is 1-10mol/L, preferably guanidine hydrochloride, and the concentration is 6 mol/L.
The rinsing buffer solution comprises Tris-HCl with the concentration of 20-100mol/L and ethanol or isopropanol with the concentration of 50% -80%, and the preferable concentration is 80%.
Example 4
The kit is used for detecting colorectal cancer related gene multi-site methylation and application, the detection primer combination and the probe are subjected to linear range and sensitivity detection, and the internal standard IR is actin. Respectively diluting plasmids of NDRG4, SDC2, TFPI2 and BMP3 according to a gradient of 10 times to obtain concentrations of 106、105、104、103、102And (3) carrying out amplification reaction according to the reaction system and the reaction program by using the copy of concentration gradient solution as a template.
The gene recombinant plasmids of NDRG4, SDC2, TFPI2, BMP3 and actin were quantified to 1010Copy/. mu.L, and dilute in a gradient to give 1X 106Copy/. mu.L, 1X 105Copy/. mu.L, 1X 104Copy/. mu.L, 1X 103Copy/. mu.L, 1X 102Copies/. mu.L of different concentration gradient dilutions were used as standard DNA.
Reaction System configuration referring specifically to example 2, 1. mu.L of a plasmid template diluted in a gradient was added to each reaction tube.
Result judgment
The results are shown in FIGS. 1-5, which represent the plasmid amplification curves for NDRG4, SDC2, TFPI2, BMP3 and actin, respectively, and the 5 curves in each figure represent the corresponding plasmid at a ten-fold dilution of 106、105、104、103、102The amplification results of the individual copies have obvious S-shaped amplification curves, and the reaction system is 106-102The distribution is linear, and R2 is more than or equal to 0.99. According to the Ct value of the amplification curve, the concentration and the Ct result of the amplification cycle number have good linear relation.
As shown in FIG. 2, the IR plasmid was sequentially diluted to 106、105、104、103、102The amplification results of the concentration gradient solutions of the copies have obvious S-shaped amplification curves, the reaction system is linearly distributed between 106 and 102, and the template with the lowest detection rate of 100 copies/. mu.L is detected. The results show that the primer and the probe have higher amplification sensitivity, and the lowest detection limit experiment shows that the template with the lowest copy/muL of 100 copies is detected in the reaction system.
Description of stability experiments, 20 replicates, CV < 5%.
Example 5
This example uses the first, second, third, fourth and fifth primer pairs provided in example 2 to detect methylated DNA in stool samples from colorectal cancer patients. In this example, 85 colorectal cancer patient samples were set, including 27 cases of differentiated adenocarcinoma, 21 cases of highly differentiated adenocarcinoma, 19 cases of high grade intraepithelial neoplasia of partial glands, 18 cases of poorly differentiated adenocarcinoma, and 55 other tumor samples, including 16 cases of gastric tubular adenocarcinoma, 22 cases of gastric adenosquamous carcinoma, 11 cases of gastric ulcer neoplasia, 6 cases of pancreatic small cell carcinoma, 25 cases of healthy controls, one positive quality control sample, one negative quality control sample, and internal reference IR as actin, and were subjected to PCR amplification, and the PCR amplification system and reaction program were referred to in example 2.
The results showed that the sensitivity of detection was 92.6% and the specificity was 88.2% for the intermediate adenocarcinoma.
For the highly differentiated adenocarcinoma, the detection sensitivity is 90.4% and the specificity is 85.3%.
For glandular high-grade intraepithelial neoplasia, the detection sensitivity is 84.2% and the specificity is 89.3%.
For the poorly differentiated adenocarcinoma, the detection sensitivity was 88.9% and the specificity was 87.7%.
Partial results are shown in fig. 6-9, where the Ct values of the colorectal cancer patient samples are within 35 amplification cycles, the Ct values of other tumor samples, normal human samples and blank controls are 39 cycles except for 2 pancreatic small cell carcinomas and 1 gastric tubular adenocarcinoma, and the Ct values of 1 normal human sample are 39 cycles, and the Ct values of the other samples do not reach the Ct threshold value within the given amplification cycle range. Therefore, the primer combination and the probe have strong specificity and high sensitivity.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (8)

1. The kit for detecting the multi-site methylation of the colorectal cancer related genes is characterized by comprising a primer combination, wherein the primer combination comprises a first primer pair, a second primer pair, a third primer pair, a fourth primer pair and a fifth primer pair; the base sequence of the first primer pair is shown as SEQ ID NO. 1-2; the base sequence of the second primer pair is shown as SEQ ID NO. 3-4; the base sequence of the third primer pair is shown as SEQ ID NO. 5-6; the base sequence of the fourth primer pair is shown as SEQ ID NO.7-8, the fifth primer pair is an internal reference primer pair, and the base sequence of the fifth primer pair is shown as SEQ ID NO. 9-10; the kit further comprises probes, wherein the probes comprise a first probe, a second probe, a third probe, a fourth probe and a fifth probe; the base sequence of the first probe is shown as SEQ ID NO. 11; the sequence of the second probe is shown as SEQ ID NO. 12; the sequence of the third probe is shown as SEQ ID NO. 13; the sequence of the fourth probe is shown as SEQ ID NO. 14; the fifth probe is an internal reference probe, and the sequence of the fifth probe is shown in SEQ ID NO. 15; the probe is characterized in that a fluorescent group is marked at the 5 'end of the probe, a quenching group is marked at the 3' end of the probe, the fluorescent group is one of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670 and NED, and the quenching group is one of 6-TAMRA, BHQ-1-3 and a non-fluorescence quenching agent combined with a molecular groove.
2. The kit of claim 1, further comprising a sample preservation solution, a nucleic acid extraction solution, a PCR reaction solution, a positive quality control product, a negative quality control product, an internal standard control and a standard reagent.
3. The kit of claim 2, further comprising a lysis buffer, a methylation conversion reagent, a binding buffer, and a rinsing buffer, wherein the purified DNA can be subjected to CT conversion.
4. The kit of claim 3, wherein the dissolution buffer comprises 1-5mol/L NaOH and 5-20mmol/L hydroquinone.
5. The kit of claim 3, wherein the methylation conversion reagent comprises a salt comprising a sulfite group at a concentration of 1 to 10 mol/L.
6. The kit of claim 3, wherein the binding buffer comprises a guanidinium salt at a concentration of 1 to 10 mol/L.
7. The kit of claim 3, wherein the rinse buffer comprises a Tris-HCl concentration of 20-100mol/L and an ethanol or isopropanol concentration of 50% -80%.
8. The use of the kit of claim 1 in the preparation of a product for high-throughput tube-closing detection of promoter methylation region of colorectal cancer-related genes, wherein the colorectal cancer-related genes are NDRG4, SDC2, TFPI2 and BMP 3.
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