CN112501305B - Primer probe combination for quantitative methylation detection of TFPI2 gene, application, kit and method thereof - Google Patents

Primer probe combination for quantitative methylation detection of TFPI2 gene, application, kit and method thereof Download PDF

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CN112501305B
CN112501305B CN202011508431.0A CN202011508431A CN112501305B CN 112501305 B CN112501305 B CN 112501305B CN 202011508431 A CN202011508431 A CN 202011508431A CN 112501305 B CN112501305 B CN 112501305B
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tfpi2
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CN112501305A (en
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罗光华
甘圣春
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Jiangsu Mojin Bioengineering Technology Co ltd
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/154—Methylation markers
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Abstract

The invention provides a primer probe combination for quantitative detection of TFPI2 gene methylation, application, a kit and a method thereof, belonging to the technical field of genetic engineering, wherein the primer probe combination comprises a primer probe for amplifying TFPI2 genes and ACTB genes; the primer probe of the TFPI2 gene comprises T2-F, T2-R and T2-P, and the nucleotide sequence is shown as SEQ ID No. 1-3 in sequence; the primer probe of the ACTB gene comprises AB-F, AB-R and AB-P, and the nucleotide sequence is shown in SEQ ID No. 4-6 in sequence. The primer probe combination for quantitative detection of the TFPI2 gene methylation can realize quantitative detection of the TFPI2 gene methylation.

Description

Primer probe combination for quantitative methylation detection of TFPI2 gene, application, kit and method thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a primer probe combination for quantitative detection of TFPI2 gene methylation, and application, a kit and a method thereof.
Background
Colorectal cancer is one of the most common malignant tumors worldwide, and according to the report by the national cancer center in 2018, colorectal cancer is the third most common malignancy in men and the second most common malignancy in women, with death cases being ranked fourth and third in men and women, respectively, worldwide. Colorectal cancer incidence is significantly higher in developed countries than in developing countries, which is related to factors such as higher obesity rates, unhealthy eating habits, etc.
Large-scale crowd screening is an important way for reducing the incidence rate and the death rate of colorectal cancer, and the traditional colorectal cancer screening has the problems of poor sensitivity, low patient acceptance and the like; the new screening methods (such as blood, fecal DNA detection) have the problems of high price, low patient acceptance, etc.
DNA methylation (DNA methylation) is a form of chemical modification of DNA that can alter genetic manifestations without altering the DNA sequence. By DNA methylation is meant covalent bonding of a methyl group at the 5' carbon position of cytosine of a genomic CpG dinucleotide under the action of a DNA methyltransferase. Numerous studies have shown that DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby controlling gene expression. In recent years, a great deal of research shows that abnormal methylation of DNA has a close relation with the occurrence, development and canceration of tumors. The biological effect of DNA methylation is used as an early marker for the development of tumor, and the tissue canceration degree at the current stage can be better marked and the tissue canceration risk can be predicted at the tissue pathological level. However, the methylation detection in the prior art is limited to qualitative detection, and no quantitative detection of the methylation of the TFPI2 gene is realized by designing a primer probe.
Disclosure of Invention
In view of the above, the invention aims to provide a primer probe combination for quantitative detection of TFPI2 gene methylation, and application, a kit and a method thereof, and the primer probe combination for quantitative detection of TFPI2 gene methylation can be used for realizing quantitative detection of TFPI2 gene methylation.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer probe combination for quantitative detection of TFPI2 gene methylation, which comprises a primer probe for amplifying TFPI2 genes and ACTB genes;
the primer probe of the TFPI2 gene comprises T2-F, T2-R and T2-P, wherein the nucleotide sequence of the T2-F is shown as SEQ ID No.1, the nucleotide sequence of the T2-R is shown as SEQ ID No.2, and the nucleotide sequence of the T2-P is shown as SEQ ID No. 3;
the primer probe of the ACTB gene comprises AB-F, AB-R and AB-P, wherein the nucleotide sequence of the AB-F is shown as SEQ ID No.4, the nucleotide sequence of the AB-R is shown as SEQ ID No.5, and the nucleotide sequence of the AB-P is shown as SEQ ID No. 6.
Preferably, 6-FAM is added to the 5 '-end of the T2-P, and BHQ1 is added to the 3' -end;
ROX is added to the 5 '-end of the AB-P, and BHQ2 is added to the 3' -end.
The invention also provides a kit for quantitative detection of TFPI2 gene methylation, which comprises quantitative PCR reaction liquid, taq enzyme, positive calibrator, positive control and negative control; the quantitative PCR reaction liquid contains the primer probe combination in the technical scheme.
Preferably, the quantitative PCR reaction solution comprises per 22.5. Mu.L: 10 XBuffer 2.5-5. Mu.L, 100. Mu.M concentration of T2-F0.02-0.1. Mu.L, 100. Mu.M concentration of T2-R0.02-0.1. Mu.L, 100. Mu.M concentrationAB-F0.02-0.1. Mu.L, AB-R0.02-0.1. Mu.L at 100. Mu.M, T2-P0.02-0.1. Mu.L at 100. Mu.M, AB-P0.02-0.1. Mu.L at 100. Mu.M, dNTPs 0.1-1. Mu.L at 10mM, mg 2+ 0.5-2. Mu.L of 50mM solution, ddH 2 O18.25μL。
Preferably, the positive calibrator is 4 solutions containing T2 plasmid, and the concentration of T2 plasmid in the 4 solutions is 1×10 respectively 5 copies/μL、1×10 4 copies/μL、1×10 3 COPIES/. Mu.L and 1X 10 2 copies/μL。
Preferably, the enzyme activity of the Taq enzyme is 50U/. Mu.L; the positive control is DNA positive for TFPI2 gene methylation, and the negative control is DNA negative for TFPI2 gene methylation.
The invention also provides application of the primer probe combination in preparation of a reagent for predicting colorectal cancer recurrence risk.
The invention also provides a quantitative detection method for TFPI2 gene methylation based on the kit based on the technical scheme, which comprises the following steps:
1) Performing sulfite conversion on the sample DNA to obtain converted DNA;
2) Performing fluorescent quantitative PCR reaction on the converted DNA obtained in the step 1) by using the kit, and taking a positive calibrator as a quantitative calibrator to obtain the copy number of the TFPI2 gene and the copy number of the ACTB gene in the sample; dividing the copy number of the TFPI2 gene by the copy number of the ACTB gene to obtain the methylation rate of the TFPI2 gene in the sample, and realizing quantitative detection of the methylation of the TFPI2 gene.
Preferably, the conditions of the fluorescent quantitative PCR reaction of step 2) include: 95 ℃ for 10min; 15s at 95℃and 30s at 60℃for 40 cycles.
The invention provides a primer probe combination for quantitative detection of TFPI2 gene methylation. The invention designs specific primers aiming at CpG island and ACTB sequence of TFPI2 gene respectively, uses probe to carry out real-time quantitative PCR, uses T2 plasmid as quantitative calibrator to obtain copy number of TFPI2 and ACTB in sample, obtains methylation rate of TFPI2 of each sample by multiplying TFPI2 copy number/ACTB copy number by 100, and realizes quantitative detection of TFPI2 gene methylation.
Drawings
FIG. 1 is a scatter plot of the statistical distribution of TFPI2 methylation rates (ordinate) versus different colorectal tissue types (abscissa), at pathological levels, according to the progression of colorectal development, into six major classes of colorectal tissue, each group measuring TFPI2 methylation rates for several tens of samples, resulting in a group TFPI2 methylation rate; cancer tissue: 32, paracancerous tissue: 32, high-grade intraepithelial neoplasia: 50, low grade intraepithelial neoplasia: 52, polyp tissue: 49, normal tissue: 49, total number of samples: 264.
Detailed Description
The invention provides a primer probe combination for quantitative detection of TFPI2 gene methylation, which comprises a primer probe for amplifying TFPI2 genes and ACTB genes; the primer probe of the TFPI2 gene comprises T2-F, T2-R and T2-P, wherein the nucleotide sequence of the T2-F is shown as SEQ ID No.1, the nucleotide sequence of the T2-R is shown as SEQ ID No.2, and the nucleotide sequence of the T2-P is shown as SEQ ID No. 3; the primer probe of the ACTB gene comprises AB-F, AB-R and AB-P, wherein the nucleotide sequence of the AB-F is shown as SEQ ID No.4, the nucleotide sequence of the AB-R is shown as SEQ ID No.5, and the nucleotide sequence of the AB-P is shown as SEQ ID No. 6.
Specific primers are designed aiming at CpG island and ACTB sequences of TFPI2 genes respectively. In the present invention, the TFPI2 gene has an accession number of ng_032914 and the ACTB gene has an accession number of ng_007992. In the invention, the CpG island of the TFPI2 gene is positioned in a promoter region and is a key region for regulating gene transcription, the C methylation of a specific site in the CpG island has a great influence on the expression activity of the gene, and the specific site methylation condition can be detected by designing a specific primer probe sequence, so that the positive expression risk is high, and the negative expression risk is low.
ACTB is a housekeeping gene commonly used in cells, and the sequence of a detection primer probe is designed at the junction of an exon and an intron of the gene, so that the cell number can be specifically calibrated, and the detection primer probe is necessary for accurate methylation quantification.
In the invention, the nucleotide sequence of the T2-F is shown as SEQ ID No.1, and is specifically as follows:
GATCGTAGTGTAAGACGTTC；
the nucleotide sequence of the T2-R is shown as SEQ ID No.2, and is specifically as follows:
ATAACGAACACACGATCCG；
the nucleotide sequence of the T2-P is shown as SEQ ID No.3, and is specifically as follows:
AAAGAGCGTATGGCGAGATG, the invention preferably adds 6-FAM at the 5 '-end of T2-P and BHQ1 at the 3' -end.
In the invention, the nucleotide sequence of the AB-F is shown as SEQ ID No.4, and is specifically as follows:
TTATTGTGGGGTGTTTTAGGTATTAG；
the nucleotide sequence of the AB-R is shown as SEQ ID No.5, and is specifically as follows:
AAACACCCTTACCTCCCACC；
the nucleotide sequence of the AB-P is shown as SEQ ID No.6, and is specifically as follows:
agttggttgggtggggtagttttggg in the invention, ROX is preferably added at the 5 '-end of AB-P, and BHQ2 is preferably added at the 3' -end.
The invention also provides a kit for quantitative detection of TFPI2 gene methylation, which comprises quantitative PCR reaction liquid, taq enzyme, positive calibrator, positive control and negative control; the quantitative PCR reaction liquid contains the primer probe according to the technical scheme.
In the present invention, the quantitative PCR reaction solution preferably comprises, per 22.5. Mu.L: 10 XBuffer 2.5-5. Mu.L, 100. Mu.M T2-F0.02-0.1. Mu.L, 100. Mu.M T2-R0.02-0.1. Mu.L, 100. Mu.M AB-F0.02-0.1. Mu.L, 100. Mu.M AB-R0.02-0.1. Mu.L, 100. Mu.M T2-P0.02-0.1. Mu.L, 100. Mu.M AB-P0.02-0.1. Mu.L, 10mM dNTPs 0.1-1. Mu.L, mg 2+ 0.5-2. Mu.L of 50mM solution, ddH 2 O18.25. Mu.L. The source of the above-mentioned reagent is not particularly limited, and a conventional commercially available product may be used. The invention has no special limitation on the amount of the quantitative PCR reaction liquid placed in the kit, and the quantitative PCR reaction liquid can be placed in the conventional kit。
In the present invention, the positive calibrator is preferably 4 solutions containing T2 plasmids, wherein the concentrations of T2 plasmids in the 4 solutions are respectively 1X 10 5 copies/μL、1×10 4 copies/μL、1×10 3 COPIES/. Mu.L and 1X 10 2 COPies/. Mu.L. The invention takes T2 plasmid as quantitative calibrator. The invention has no special limitation on the amount of the positive calibrator placed in the kit, and the positive calibrator can be obtained by adopting the conventional method.
In the invention, the nucleotide sequence of the T2 plasmid is shown as SEQ ID No.7, and is specifically as follows: wherein, the bold part is a TFPI2 amplified fragment, and the italic part is an ACTB amplified fragment. GTTCGTTGGGTAAGGCGTTCGAGAAAGCGTTTGGCGGGAGGAGGTGCGCGGTTTTTTGTTTTAGGCGGTTCGGGTGTTCGTTTTCTAAACCCTAAACCCTAAACCCTAAACCCTAAACCCTAAACCCTAAACCCTAAACCCTAACCCTAAACCCTAACCCTAAACCCTAAACCCTAAACCCTAAACCCTAAACCCTAAACAGCTGACAGTACGATAGATCCACGCGAGAGGAACCGGAGAGACAACGGGATCCAGGCGCCAGCGACGGATCCGGGCGAGAGGGGAGTGGAGATCATGGATCCGTGCGGGAGGGGAAGAAGTCGCCGAATCCGACCCTCCCATCGCCATCGACAGTAGGTCTGCGCGAGAGGGGCACCGGCGCTGGCTCTGAGCGAGATGCAACGCCGGCCGGCTTGGAGAGTAACTCAAGAGAGACAGAATGGAAGATAGAGAACAAGAGAGTGAGAGGATAAGGATATAGACCAGACCACACAATTTTCTCTTCTTTTTAACTTTGTATTAAGATCTTTTATGGAACATCTTTTATTGTTGATATCAAAATAACTGAAACTTATACTTTAATATTTTTTGAGACAAAAAGTAACAATCGTAAAAAAAAGTTCCACGAGAGTTACCCCACCACCCATGTCTCGAGCCGACCAGATCTGCCGCCCACGACCCGAGTCATCGTTGGATCCACCACCCACAACCCGCGACCGAGCTACGCCTCCTGTGGATTGATGCCACTGCCCTGAGCTTCAC
TGCCGGTACTGTCGCTCGCGACCCGAGCTCCGCCGCTGGTACCGTCGC
CCACAACTCAAGCTGTGTTGCCACTAGAGAAAAAGGAGGCGAGAGCG
AGGGCAAAACGAGGCAAGGCGTGGCGTGACATGGTGTGACTGGTTGG
TCAATTAGCCGACAATGGATCCACCGCCCATGAACCGAGCCACCGTTG
GATCCGCCACCCGCAGATTCGGCTGTCCCCTCTCCCTCAACTTATTCTT
CCTCTCTTGATTCAACGGGAGGAGTGGCAACTGACGGCAACAGAGCT
CGCCTCATGGGCGATGGAACCAGCGGCGAAGCTCGGAGGAGGAAGCG
AGCGTTTTCGAGTTTCGAGTTCGAGTTTTCGAGTTTGAGTCGTAATCGT
TGCGGTATTTTGTTTCGGATTCGTGGCGGAGGGGTTGAGCCACATGAG
GCGATGGCGACAATGAGGCGAGACATGGCGTGGCTGGCTGTTACATTT
TGTTTTGATGAAAAGCATAACCATGCGGATGATATTTTTATTATAGACTA
GAGATGATTATTGAATAGACATGCTCTTAACCATTTTTAACTCTAATTCC
ACCTACACTATGAATATTAGAAAAAAAATACAATGTATACTCTTAAGAA
GTTTGCGCTATCATAGCCATCATACTCATTAAAACATTTTTCTTCACATG
TTGCAACGCATGGGCATTTTGCTAGTTAGAGAATAAAAATAAATTTGTA
GGTAAAACTTTTATATATGTGTTCTCGGTGACTTAAAAGCCAATGCTTA
AAAATAAACTACGTTAAAAATATCTTAAAATCAATATTAAAATTAAGTTT
GAAAATTTAAATTTTGGCTTTTTCTTTGACTGAATAGGCCATCTGATGG
AAGCTGAATAAGCCATCCGATGGGAGCTGTGACAGTGATGAAAAGGA
GAGCAGTAAGAGAGCAGTAAGAGCATCCCAGCAGCTCCTCTATCTAGG
CATCCATCCGATATTTGGAGTATGGAGGAGAAAAACAGTGCTCCAGCA
GAGTCTCCATCACATGCTTCATTTTTGGAGGTCCTCCAAATCTAGCCCT
TCCCAAGCCAAATATGGAGGTTCTCTCTCCTCACTCCATCCTCAACTAC
CCACTTCCCAAGATTTTTTTTGAGTGGTGAAAGGTGAGAGGAATAGGA
GTAAAAATGTGTGGTAGTTGAAAAATATTACTAAAATATAGGATGTTGG
TATATGTAGGATGTAATATGAATGATCTGTTGGAGTGAAAAGTTATATAG
AGTGGAAATCTTTTTAAATGGCCATCCAAATGAGAGTATGAAATTTAGA
TGAGCTGGTGGGGATGCTCTAAGAGAACGAGAGAAGCACAGAGCAGA
TAAACCACACCCACAGGCACCACCGTCCTTGTTGGTAATGAAGAAGAC
TTATTGTGGGGTGTTTTAGGTATTAGGTAGGGGAGTTGGTTGGGTGGGGTAGTTTTGGGAGTGGGTGGGAGGTAAGGGTGTTT。
In the present invention, the enzyme activity of the Taq enzyme is preferably 50U/. Mu.L, and the amount of the Taq enzyme placed in the kit is not particularly limited, and the amount of the enzyme placed in the kit is conventionally used.
In the present invention, the positive control is preferably a DNA positive for TFPI2 gene methylation, and the negative control is preferably a DNA negative for TFPI2 gene methylation. In the invention, the positive control is DNA extracted from a tumor cell strain positive for TFPI2 gene methylation, and the negative control is DNA extracted from a cell strain negative for TFPI2 gene methylation. The specific nucleotide sequence is as follows:
TFPI2 gene methylation positive control (SEQ ID No. 8):
GTTCGTTGGGTAAGGCGTTCGAGAAAGCGTTTGGCGGGAGGAGGTGCGCGGTTTTTTGTTTTAGGCGGTTCGGGTGTTCGTTTT；
TFPI2 gene methylation negative control (SEQ ID No. 9):
GTTTGTTGGGTAAGGTGTTTGAGAAAGTGTTTGGTGGGAGGAGGTGTGTGGTTTTTTGTTTTAGGTGGTTTGGGTGTTTGTTTT。
the invention also provides application of the primer probe combination in preparation of a reagent for predicting colorectal cancer recurrence risk.
The invention also provides a quantitative detection method for TFPI2 gene methylation based on the kit based on the technical scheme, which comprises the following steps:
1) Performing sulfite conversion on the sample DNA to obtain converted DNA;
2) Performing fluorescent quantitative PCR reaction on the converted DNA obtained in the step 1) by using the kit, and taking a positive calibrator as a quantitative calibrator to obtain the copy number of the TFPI2 gene and the copy number of the ACTB gene in the sample; dividing the copy number of the TFPI2 gene by the copy number of the ACTB gene to obtain the methylation rate of the TFPI2 gene in the sample, and realizing quantitative detection of the methylation of the TFPI2 gene.
In the present invention, the sample is preferably colorectal cancer tissue. The method for extracting DNA of colorectal cancer is not particularly limited, and a conventional method for extracting DNA in tissues is adopted. The method of sulfite conversion of sample DNA according to the present invention is not particularly limited, and is preferably carried out according to the instructions in the Qiagen59104EpiTect Bisulfite Kit kit. In the present invention, the principle of sulfite conversion is: the methylated C is not converted in the sulfite environment, the unmethylated C is converted into T in the sulfite environment, and after the sulfite conversion, the PCR fluorescent probe method is used for distinguishing whether the C at a specific site is converted into T or not, so that whether the C at the site has methylation or not is judged.
In the present invention, the conditions for the fluorescent quantitative PCR reaction preferably include: 95 ℃ for 10min; 15s at 95℃and 30s at 60℃for 40 cycles. In the present invention, the system of the fluorescent quantitative PCR reaction is shown in Table 1:
TABLE 1 fluorescent quantitative PCR reaction System
Composition of the components Volume of
10×Buffer 2.5μL
T2-F(10μM) 0.4μL
T2-R(10μM) 0.4μL
AB-F(10μM) 0.4μL
AB-R(10μM) 0.4μL
T2-P(10μM) 0.6μL
AB-P(10μM) 0.3μL
dNTPs(10mM) 0.5μL
Mg 2+ (50mM) 1μL
Water and its preparation method 16μL
Taq enzyme (5U/. Mu.L) 0.5μL
Post-transformation DNA (DNA concentration 10 ng/. Mu.L) 2μL
Total volume of 25μL
The present invention preferably uses ABI7500 PCR apparatus to perform fluorescence quantitative PCR. And taking the positive calibrator as a quantitative calibrator to obtain the copy number of the TFPI2 gene and the copy number of the ACTB gene in the sample. In the invention, four kinds of calibrator synchronously perform PCR reaction and calibrate corresponding concentrations (TFPI 2 and ACTB copy number) when detecting samples, sample concentrations (TFPI 2 and ACTB copy number) are calculated through analysis of software of a PCR instrument according to the relation between a Ct value and the concentrations (TFPI 2 and ACTB copy number), and methylation rate is calculated through the formula: the methylation rate of each sample was calculated as TFPI2 copy number/ACTB copy number x 100 (so this methylation rate can be understood as the number of TFPI2 methylation positive cells out of 100 cells).
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Reagent and instrument sources: qiagen EpiTect Bisulfite Kit (48) kit was purchased from Shanghai Youning vitamin technologies Co., ltd; omega FFPE DNA Kit D3399 paraffin-embedded tissue DNA extraction kit is purchased from Nanjing Youlbo biotechnology Co., ltd; primer probe commission biological engineering (Shanghai) Co., ltd; taq enzyme was purchased from Bioline (Meridian LifeScience, inc.); dNTPs are purchased from Norwegian biotechnology Co., ltd; the calibrator (T2 plasmid) was synthesized by gold Style biotechnology Co., ltd; positive control is whole genome DNA extracted from HT-29 cell strain; the negative control was whole genomic DNA extracted from 293T cell line.
Example 1
1. Preparing TFPI2 methylation positive and negative control substances by using HT-29 cell lines and 293T cell lines; synthesis of plasmid (T2) containing fragments of TFPI2 and ACTB Gene, dilution to 1X 10 5 copies/mL、1×10 4 copies/mL、1×10 3 copies/mL、1×10 2 The concentration gradient of the copies/mL4 was used for quantification. Taking six samples of paraffin sections of colorectal cancer tissues and corresponding paracancerous tissues as an example, TFPI2 gene methylation was quantitatively detected.
Paraffin tissue DNA extraction
1. Taking 3-8 paraffin slice samples with the diameter of 10-20 mu m to a 1.5mL centrifuge tube, adding 1mL of dimethylbenzene, and mixing evenly by intense vortex for 10 s. Centrifuge at 10,000g for 2min, carefully pipette off the supernatant (not to the sections).
3. 1mL of absolute ethanol is added and mixed by vortex.
Centrifuge at 4.10,000g for 2min, carefully pipette off the supernatant (not to pellet); uncapped air drying at 37deg.C for 5min until ethanol is completely volatilized (e.g. wax cake is not removed and dewaxing can be repeated).
5. 200. Mu.L of BufferFTL and 20. Mu.L of OB protease were added and vortexed.
Water bath at 6.55 deg.c for 3 hr or until tissue digestion, and mixing once every 20-30 min. If necessary, can also be digested overnight.
Water bath at 7.90 deg.c for 60min.
8. Standing at room temperature for 5min.
9. 220 μl of BufferBL was added and vortexed.
10. Add 250 μl of absolute ethanol, vortex at high speed for 15s and mix well.
11. The mixture was transferred to a HiBind DNA column fitted with a collection tube, centrifuged at 10,000g for 1min at room temperature, and the filtrate was discarded.
12. The column was packed in a collection tube, 500. Mu.L HB Buffer was added, and the filtrate was discarded by centrifugation at 10,000g for 1min at room temperature.
13. The column was packed in a fresh collection tube, 500. Mu. L DNA Wash Buffer was added and centrifuged at 10,000g for 1min at room temperature.
14. The filtrate was discarded and step 13 was repeated once. After the filtrate was discarded, the column was returned to the collection tube and centrifuged at 10,000g for 3min.
15. The column was placed in a clean 1.5mL centrifuge tube, 80. Mu.L of EluthonBuffer preheated at 70℃was added to the column matrix, and after standing at room temperature for 3min, the column was centrifuged at 10,000g for 1min at room temperature to elute the DNA, and the eluate containing the DNA was retained.
(II) determination of DNA concentration
DNA concentration was measured using a Shanghai Yuan-Xie visible light photometer UV-5500PC and DNA was diluted to 100 ng/. Mu.L.
(III) sulfite conversion
Transformation was performed according to Qiagen59104EpiTectBisulfite Kit, with the specific steps of:
1. preparing a conversion reaction system:
TABLE 2 conversion reaction System
Composition of the components Volume (mu L)
DNA 5
RNase-freewater 15
BisulfiteMix 85
DNAProtectBuffer 35
Total volume of 140
2. After the preparation is completed, the components are evenly mixed and placed on a PCR instrument, and the following PCR program is set:
TABLE 3PCR procedure
Step (a) Time Temperature (temperature)
A 5min 95℃
B 25min 60℃
C 5min 95℃
D 85min 60℃
E 5min 95℃
F 175min 60℃
G 1min 30℃
3. The transformation procedure was run and after transformation was completed, the transformed DNA was recovered.
4. The transformed DNA was aspirated into a fresh 1.5ml centrifuge tube.
5. 310. Mu.L of BufferBL containing 10. Mu.L/ml of carrier rRNA was added and mixed by shaking for 5s.
6. 250 mu L of absolute ethyl alcohol is added, and the mixture is stirred and mixed for 5s.
7. The liquid after the mixing in step 6 was added to EpiTect spin column and centrifuged at 10,000g for 1min.
8. The filtrate was discarded, 500. Mu.L of BufferBW was added to the recovery column and centrifuged at 10,000g for 1min.
9. The filtrate was discarded, 500. Mu.L Buffer BD was added to the recovery column, and the mixture was allowed to stand at room temperature for 15min and centrifuged at 10,000g for 1min.
10. The filtrate was discarded, 500. Mu.L of BufferBW was added to the recovery column and centrifuged at 10,000g for 1min.
11. The step 10 is repeated once.
12. The recovery column was placed in a new 1.5mL centrifuge tube and allowed to stand at room temperature for 5min.
13. 50. Mu.L of BuffereB (preheated at 56 ℃) was added to the column and allowed to stand at room temperature for 5min.
Centrifuging at 14.10,000g for 2min, and preserving the eluted converted DNA for later use.
Six colorectal cancer tissues and corresponding paracancerous tissues were selected for methylation quantitative analysis.
The transformed DNA is extracted according to the above procedure, and a PCR reaction system is prepared according to the following components:
TABLE 1PCR reaction System
The ABI7500 PCR instrument was set up to perform a fluorescent PCR reaction with the following procedure:
TABLE 4 procedure for fluorescent PCR reactions
Analysis of results:
the results of methylation quantitative detection of six colorectal cancer tissues and corresponding paracancerous tissues are shown in Table 5, wherein 13-1 to 18-1 are the cancer tissues of six different patients and the corresponding paracancerous tissues:
1. quantitative calibrators for the 4 concentrations described above were set as S in the ABI7500 PCR instrument software sample set-up and the corresponding concentrations were indicated.
2. The baseline, threshold line, is adjusted.
3. Standard curve judgment: checking whether the Curve is accurate in Standard Curve, R 2 And is more than or equal to 0.98 percent.
4. Sample copy number calculation: after the standard curve is checked well, clicking the Export menu bar to generate the copy numbers of the samples TFPI2 and ACTB.
5. The methylation rate of each sample was calculated in the generated Excel table: each sample corresponds to two fluorescent channels, FAM (TFPI 2) and ROX (ACTB). The formula of methylation rate is: TFPI2 copy number/ACTB copy number 100, 13-1 to 18-1 and 13-3 to 18-3 methylation rates were calculated according to this formula.
TABLE 5 quantitative methylation detection results of six colorectal cancer tissues and corresponding paracancerous tissues
Cancer tissue Methylation Rate Tissue beside cancer Methylation Rate
13-1 26.00 13-3 1.99
14-1 14.01 14-3 0.60
15-1 7.01 15-3 0.48
16-1 31.99 16-3 0.51
17-1 10.11 17-3 0.00
18-1 11.41 18-3 2.90
As can be seen from Table 5, colorectal cancer tissue is highly methylated and there may be a small amount of methylation in the paracancerous tissue.
FIG. 1 shows statistical analysis of samples taken from different stages of colorectal cancer development using the primer probe combinations and methods described above, initially setting a TFPI2 methylation rate greater than 5 at high risk (susceptibility to recurrence, residual cancer cells, etc.).
The detection limit experiment is carried out according to the following steps:
1.500ng of methylation positive cell DNA was transformed, the recovery volume was 50. Mu.l, and the concentration of DNA after transformation was about 10 ng/. Mu.l.
2.500ng of methylation negative cell DNA was transformed, the recovery volume was 50. Mu.l, and the concentration of DNA after transformation was about 10 ng/. Mu.l.
3. And accurately quantifying ACTB of the positive and negative DNA after transformation by using a standard curve, and adjusting the copy numbers of the ACTB and the ACTB to the same level.
4. According to methylation positive DNA: methylation negative DNA = 1:99 ratio 1% methylated DNA was formulated.
And (3) carrying out 25 times of PCR repeated detection by taking 1% methylated DNA as a template, wherein the detection times of TFPI2 are not less than 23 times.
The results were: the minimum limit of detection was 1% methylation (10 ng/. Mu.l).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
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Claims (12)

1. The primer probe combination for quantitative detection of TFPI2 gene methylation is characterized by comprising a primer probe for amplifying the TFPI2 gene and the ACTB gene;
the primer probe of the TFPI2 gene comprises T2-F, T2-R and T2-P, wherein the nucleotide sequence of the T2-F is shown as SEQ ID No.1, the nucleotide sequence of the T2-R is shown as SEQ ID No.2, and the nucleotide sequence of the T2-P is shown as SEQ ID No. 3;
the primer probe of the ACTB gene comprises AB-F, AB-R and AB-P, wherein the nucleotide sequence of the AB-F is shown as SEQ ID No.4, the nucleotide sequence of the AB-R is shown as SEQ ID No.5, and the nucleotide sequence of the AB-P is shown as SEQ ID No. 6.
2. The primer probe combination according to claim 1, wherein 6-FAM is added to the 5 'end of T2-P and BHQ1 is added to the 3' end;
ROX is added to the 5 '-end of the AB-P, and BHQ2 is added to the 3' -end.
3. The kit for quantitative detection of TFPI2 gene methylation is characterized by comprising quantitative PCR reaction liquid, taq enzyme, a positive calibrator, a positive control and a negative control; the quantitative PCR reaction solution contains the primer probe combination according to any one of claims 1 to 2.
4. The kit of claim 3, wherein the quantitative PCR reaction solution comprises: 10 XBuffer 2.5-5. Mu.L, 100. Mu.M T2-F0.02-0.1. Mu.L, 100. Mu.M T2-R0.02-0.1. Mu.L, 100. Mu.M AB-F0.02-0.1. Mu.L, 100. Mu.M AB-R0.02-0.1. Mu.L, 100. Mu.M T2-P0.02-0.1. Mu.L, 100. Mu.M AB-P0.02-0.1. Mu.L, 10mM dNTPs 0.1-1. Mu.L, mg 2+ 0.5-2. Mu.L of 50mM solution, ddH 2 O18.25μL。
5. The kit according to claim 3, wherein the positive calibrator is 4 solutions containing T2 plasmid, and the concentration of T2 plasmid in the 4 solutions is 1X 10 respectively 5 copies/μL、1×10 4 copies/μL、1×10 3 COPIES/. Mu.L and 1X 10 2 cobies/. Mu.L; the T2 plasmid is a plasmid containing a TFPI2 gene methylation positive sequence and an ACTB gene fragment sequence, and the nucleotide sequence is shown as SEQ ID No. 7.
6. The kit according to claim 3, wherein the enzyme activity of the Taq enzyme is 50U/. Mu.L; the positive control is DNA positive for TFPI2 gene methylation, and the negative control is DNA negative for TFPI2 gene methylation.
7. The kit of claim 6 wherein the DNA sequence that is methylation positive for the TFPI2 gene is shown in SEQ ID No.8 and the DNA sequence that is methylation negative for the TFPI2 gene is shown in SEQ ID No. 9.
8. Use of a primer probe combination according to any one of claims 1-2 for the preparation of a reagent for detecting the onset and progression of colorectal cancer.
9. A method for quantitative detection of TFPI2 gene methylation for non-diagnostic purposes based on the kit of any one of claims 3 to 7, comprising the steps of:
1) Performing sulfite conversion on the sample DNA to obtain converted DNA;
2) Performing fluorescent quantitative PCR reaction on the converted DNA obtained in the step 1) by using the kit, and taking a positive calibrator as a quantitative calibrator to obtain the copy number of the TFPI2 gene and the copy number of the ACTB gene in the sample; dividing the copy number of the TFPI2 gene by the copy number of the ACTB gene to obtain the methylation rate of the TFPI2 gene in the sample, and realizing quantitative detection of the methylation of the TFPI2 gene.
10. The method according to claim 9, wherein the sample in step 1) is colorectal cancer tissue; the sulfite conversion of the sample DNA was performed using the Qiagen59104EpiTectBisulfite Kit kit.
11. The method of claim 9, wherein the conditions for the fluorescent quantitative PCR reaction of step 2) include: 95 ℃ for 10min; 15s at 95℃and 30s at 60℃for 40 cycles.
12. The method of claim 9, wherein said step 2) fluorescent quantitative PCR reaction uses an ABI7500 PCR instrument for fluorescent quantitative PCR.
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