CN109943638A - A kind of primer combination of probe detecting NDRG4, SDC2 and BMP3 gene methylation and its application and kit - Google Patents
A kind of primer combination of probe detecting NDRG4, SDC2 and BMP3 gene methylation and its application and kit Download PDFInfo
- Publication number
- CN109943638A CN109943638A CN201910379603.XA CN201910379603A CN109943638A CN 109943638 A CN109943638 A CN 109943638A CN 201910379603 A CN201910379603 A CN 201910379603A CN 109943638 A CN109943638 A CN 109943638A
- Authority
- CN
- China
- Prior art keywords
- probe
- primer
- actin
- sdc2
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000523 sample Substances 0.000 title claims abstract description 134
- 238000007069 methylation reaction Methods 0.000 title claims abstract description 99
- 230000011987 methylation Effects 0.000 title claims abstract description 98
- 101000995332 Homo sapiens Protein NDRG4 Proteins 0.000 title claims abstract description 63
- 102100026087 Syndecan-2 Human genes 0.000 title claims abstract description 46
- 102100034432 Protein NDRG4 Human genes 0.000 title claims abstract description 44
- 101150118392 sdc-2 gene Proteins 0.000 title claims abstract description 44
- 101150006966 bmp3 gene Proteins 0.000 title claims abstract description 42
- 108010085238 Actins Proteins 0.000 claims abstract description 60
- 102000007469 Actins Human genes 0.000 claims abstract description 60
- 238000001514 detection method Methods 0.000 claims abstract description 43
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 37
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims abstract description 36
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims description 71
- 101000692109 Homo sapiens Syndecan-2 Proteins 0.000 claims description 24
- 102100024504 Bone morphogenetic protein 3 Human genes 0.000 claims description 23
- 101000762375 Homo sapiens Bone morphogenetic protein 3 Proteins 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 201000010989 colorectal carcinoma Diseases 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 7
- 239000013642 negative control Substances 0.000 claims description 7
- 239000013641 positive control Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 5
- 230000002093 peripheral effect Effects 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 101150010855 cma gene Proteins 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 60
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 17
- 239000006228 supernatant Substances 0.000 description 13
- 239000006210 lotion Substances 0.000 description 12
- 238000005406 washing Methods 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- 239000000284 extract Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 239000011324 bead Substances 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 7
- 206010028980 Neoplasm Diseases 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 5
- 230000002550 fecal effect Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 108010067770 Endopeptidase K Proteins 0.000 description 4
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 4
- 239000007984 Tris EDTA buffer Substances 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 208000003200 Adenoma Diseases 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 210000001072 colon Anatomy 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 210000000664 rectum Anatomy 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 229960004198 guanidine Drugs 0.000 description 2
- 229960000789 guanidine hydrochloride Drugs 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 206010001233 Adenoma benign Diseases 0.000 description 1
- 230000007067 DNA methylation Effects 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 208000037062 Polyps Diseases 0.000 description 1
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 235000013809 polyvinylpolypyrrolidone Nutrition 0.000 description 1
- 229920000523 polyvinylpolypyrrolidone Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of primer combination of probe for detecting NDRG4, SDC2 and BMP3 gene methylation and its application and kits, belong to cma gene detection technique field, and primed probe includes detection target gene methylated primers probe and β-actin primed probe.Sensitivity to colorectal cancer is improved using the primer combination of probe of detection NDRG4, SDC2 and BMP3 gene methylation provided by the invention, sensitivity 88.1% is just capable of detecting when in colorectal cancer early stage, convenient without invasive.
Description
Technical field
The invention belongs to cma gene detection technique field more particularly to a kind of detection NDRG4, SDC2 and BMP3 gene first
The primer combination of probe of base and its application and kit.
Background technique
Colorectal cancer (Colorectal Cancer, CRC) is common one of malignant tumour, is studied according to international cancer
Statistics in 2012 is organized, colorectal cancer incidence rate is in cancer and composes the 3rd in world wide, about 1,360,000 people of new cases.It accounts for
The 10% of all cancers: disease leads to dead 690,000 people, occupies cancer mortality and composes the 2nd.As reform and opening-up and modernization are built
If gradually in-depth, rural and urban population life style increasingly westernization.Past 10 years China's colorectal cancer incidence rates and case fatality rate
Overall present gradually rises trend.China's colorectal cancer incidence rate in 2013 is 17.2/10 ten thousand, case fatality rate is 7.76/10 ten thousand, compared with
14.6/10 ten thousand in 2008 and 6.18/10 ten thousand have obvious rising.The 2 months 2018 newest numbers of National Cancer Center Nattonal Cancer
According to display, colorectal cancer arranges the 3rd in national disease incidence, and the death rate arranges the 5th.
Colorectal Cancer concealment, but mostly according to classical adenoma-atypical hyperplasia, gland cancer pattern progression.From benign breath
Meat development is that advanced tumors usually require 5~15 years.Colorectal cancer prognosis and clinicopathologia are by stages closely related.Early stage ties
5 years survival rates of the carcinoma of the rectum are up to 90%, and advanced colorectal cancer is less than 10%.Just because of colorectal cancer have onset concealment,
The characteristics of course of disease is long and early diagnoses good prognosis, so that screening has very important status in colorectal cancer totality prevention and control.
To mitigate personal and society Disease Spectrum, various countries increase the investment of colorectal cancer screening project year by year.At present
The main method of colorectal cancer conventional detection has a fecal occult blood detection (FOBT) and Fecal Immunochemical method (FIT), but this method
Sensitivity it is low, the sensitivity to progressive stage polyp is only 20~25%.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of primers for detecting NDRG4, SDC2 and BMP3 gene methylation
Probe combinations and its application and kit improve detection Human colorectal carcinoma using primer combination of probe provided by the invention
Sensitivity.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
The present invention provides a kind of primer combination of probe for detecting NDRG4, SDC2 and BMP3 gene methylation, the primers
Probe includes detection target gene methylated primers probe and β-actin primed probe；
The sequence of the primer of NDRG4 gene methylation is detected as shown in No.1~2 SEQ ID, detects NDRG4 gene methyl
The sequence of the probe of change is as shown in SEQ ID No.3；
The sequence of the primer of SDC2 gene methylation is detected as shown in No.4~5 SEQ ID, detects SDC2 gene methylation
Probe sequence as shown in SEQ ID No.6；
The sequence of the primer of BMP3 gene methylation is detected as shown in No.7~8 SEQ ID, detects BMP3 gene methylation
Probe sequence as shown in SEQ ID No.9；
The sequence of β-actin primer is as shown in No.10~11 SEQ ID, the sequence of β-actin probe such as SEQ ID
Shown in No.12.
Preferably, in the end the 5` addition FAM label of the probe of the detection target gene methylation, MGB is added at the end 3`
Label.
Preferably, in the end 5` of β-actin probe addition VIC label, in the end 3` addition MGB label.
The present invention also provides the primer combination of probe described in above-mentioned technical proposal to detect Human colorectal carcinoma reagent in preparation
Application in box.
The present invention also provides a kind of kits, including NDRG4 reaction solution, SDC2 reaction solution and BMP3 reaction solution；
The NDRG4 reaction solution includes nuclease-free water, 10 × PCRbuffer, dNTP, inspection described in above-mentioned technical proposal
Survey the probe of detection NDRG4 gene methylation, above-mentioned technology described in the primer of NDRG4 gene methylation, above-mentioned technical proposal
β-actin probe described in β-actin primer and above-mentioned technical proposal described in scheme；
The SDC2 reaction solution includes nuclease-free water, 10 × PCRbuffer, dNTP, inspection described in above-mentioned technical proposal
Survey the probe of detection SDC2 gene methylation, above-mentioned technical side described in the primer of SDC2 gene methylation, above-mentioned technical proposal
β-actin probe described in β-actin primer and above-mentioned technical proposal described in case；
The BMP3 reaction solution includes nuclease-free water, 10 × PCRbuffer, dNTP, inspection described in above-mentioned technical proposal
Survey the probe of detection BMP3 gene methylation, above-mentioned technical side described in the primer of BMP3 gene methylation, above-mentioned technical proposal
β-actin probe described in β-actin primer and above-mentioned technical proposal described in case.
Preferably, in the NDRG4 reaction solution, the concentration of 10 × PCRbuffer is 1 × PCRbuffer, the concentration of dNTP
For 0.25mM, the concentration for detecting the primer of SDC2 gene methylation is respectively 0.4uM, detects the probe of NDRG4 gene methylation
Concentration be 0.4uM, the concentration of β-actin primer is respectively 0.4uM, and the concentration of β-actin probe is 0.4uM.
Preferably, in the SDC2 reaction solution, the concentration of 10 × PCRbuffer is 1 × PCRbuffer, the concentration of dNTP
For 0.25mM, the concentration for detecting the primer of SDC2 gene methylation is respectively 0.3uM, detects the probe of SDC2 gene methylation
Concentration is 0.3uM, and the concentration of β-actin primer is respectively 0.4uM, and the concentration of β-actin probe is 0.4uM.
Preferably, in the BMP3 reaction solution, the concentration of 10 × PCRbuffer is 1 × PCRbuffer, the concentration of dNTP
For 0.25mM, the concentration for detecting the primer of BMP3 gene methylation is respectively 0.4uM, detects the probe of BMP3 gene methylation
Concentration is 0.4uM, and the concentration of β-actin primer is respectively 0.4uM, and the concentration of β-actin probe is 0.4uM.
Preferably, the kit further include: Taq enzyme, positive control and negative control.
Preferably, the positive control is colorectal cancer cell lines, and the negative control is human peripheral genomic DNA.
The present invention provides a kind of primer combination of probe and its application for detecting NDRG4, SDC2 and BMP3 gene methylation
And kit, the methylation of the promoter of NDRG4, SDC2 and BMP3 gene and the occurrence and development of colorectal cancer are closely related,
It is the special molecular labeling of colorectal cancer, passes through the methylation of the promoter of NDRG4, SDC2 and BMP3 gene in detection human faecal mass
To detect colorectal cancer.
The embodiment of the present invention is as the result is shown: using detection NDRG4, SDC2 and BMP3 gene methylation provided by the invention
Primer combination of probe improve the sensitivity to colorectal cancer, sensitivity 88.1% can be examined in colorectal cancer early stage
It measures, it is convenient without invasive.
Detailed description of the invention
Fig. 1 is NDRG4 methylation positive sample results figure；
Fig. 2 is methylation negative sample result figure；
Fig. 3 is the human genome sample results figure for not carrying out sulphite conversion；
Fig. 4 is SDC2 methylation positive sample results figure；
Fig. 5 methylation negative sample result figure；
Fig. 6 is the human genome sample results figure for not carrying out sulphite conversion；
Fig. 7 is BMP3 methylation positive sample results figure；
Fig. 8 is methylation negative sample result figure；
Fig. 9 is the human genome sample results figure for not carrying out sulphite conversion.
Specific embodiment
The present invention provides a kind of primer combination of probe for detecting NDRG4, SDC2 and BMP3 gene methylation, the primers
Probe includes detection target gene methylated primers probe and β-actin primed probe；
The sequence of the primer of NDRG4 gene methylation is detected as shown in No.1~2 SEQ ID, detects NDRG4 gene methyl
The sequence of the probe of change is as shown in SEQ ID No.3；
The sequence of the primer of SDC2 gene methylation is detected as shown in No.4~5 SEQ ID, detects SDC2 gene methylation
Probe sequence as shown in SEQ ID No.6；
The sequence of the primer of BMP3 gene methylation is detected as shown in No.7~8 SEQ ID, detects BMP3 gene methylation
Probe sequence as shown in SEQ ID No.9；
The sequence of the β-actin primer is as shown in No.10~11 SEQ ID, and the sequence of the β-actin probe is such as
Shown in SEQ ID No.12.
In the present invention, the primed probe of NDRG4, SDC2 and BMP3 gene methylation be according to NDRG4, SDC2 and
The promoter region design of BMP3 gene.
In the present invention, the primer of the detection NDRG4 gene methylation includes the upstream for detecting NDRG4 gene methylation
Primer and downstream primer, the sequence of the upstream primer are specific as follows shown as shown in SEQ ID No.1:
5`-GGTTTTTGGTTGTTCGTATTTGT-3`；
The sequence of the downstream primer is specific as follows shown as shown in SEQ ID No.2:
5`-GAAAATACGAACGAAACAAACGC-3`；
The sequence of the probe of the detection NDRG4 gene methylation is specific as follows shown as shown in SEQ ID No.3:
5`-AAACGAAACTTCGACGCC-3`。
The present invention preferably in the end the 5` addition FAM label of the probe of detection NDRG4 gene methylation, adds MGB at the end 3`
Label.
In the present invention, the primer of the detection SDC2 gene methylation includes upstream primer and downstream primer, it is described on
The sequence of primer is swum as shown in SEQ ID No.4, specific as follows:
5`-AAGCGAGCGTTTTCGAGTTTC-3`；
The sequence of the downstream primer is specific as follows as shown in SEQ ID No.5:
5`-CACACGAATCCGAAACAAAATAC-3`；
The sequence of the probe of the detection SDC2 gene methylation is specific as follows as shown in SEQ ID No.6:
5`-AACGATTACGACTCAAAC-3`。
The present invention is preferably in the end the 5` addition FAM label of the probe of detection SDC2 gene methylation, in the end 3` addition MGB mark
Note.
In the present invention, the primer of the detection BMP3 gene methylation includes upstream primer and downstream primer, it is described on
The sequence of primer is swum as shown in SEQ ID No.7, specific as follows:
5`-ACTCCAAACCAACTAAAACGAAAAC-3`；
The downstream primer is specific as follows as shown in SEQ ID No.8:
5`-GTCGTTATTTCGTTGTATTCGGTC-3`；
The sequence of the probe of the detection BMP3 gene methylation is specific as follows shown as shown in SEQ ID No.9:
5`-CACGAAACCCGAAAC-3`。
The present invention preferably in the end the 5` addition FAM label of the probe of the detection BMP3 gene methylation, adds at the end 3`
MGB label.
In the present invention, the β-actin primer includes upstream primer and downstream primer, and the sequence of the upstream primer is such as
It is specific as follows shown in SEQ ID No.10:
5`-GGTTAGGAAGGAGGTTGTTTGTTTT-3`；
The sequence of the downstream primer is specific as follows shown as shown in SEQ ID No.11:
5`-ATCATCTTTCCCACCAAACTATAACC-3`；
The sequence of the β-actin probe is specific as follows shown as shown in SEQ ID No.12:
5`-CCCATTAACTAAACACAACCT-3`。
The present invention is preferably in the end 5` of β-actin probe addition VIC label, in the end 3` addition MGB label.
The present invention also provides the primer combination of probe described in above-mentioned technical proposal to detect Human colorectal carcinoma reagent in preparation
Application in box.
The present invention also provides a kind of kits, including NDRG4 reaction solution, SDC2 reaction solution and BMP3 reaction solution；
The NDRG4 reaction solution includes nuclease-free water, 10 × PCR buffer, dNTP, detection NDRG4 gene methylation
Primer, detect NDRG4 gene methylation probe, β-actin primer and β-actin probe；
The SDC2 reaction solution includes nuclease-free water, 10 × PCRbuffer, dNTP, detection SDC2 gene methylation
Primer, probe, β-actin primer and the β-actin probe for detecting SDC2 gene methylation；
The BMP3 reaction solution includes nuclease-free water, 10 × PCRbuffer, dNTP, detection BMP3 gene methylation
Primer, probe, β-actin primer and the β-actin probe for detecting BMP3 gene methylation.
In the present invention, in the NDRG4 reaction solution, the concentration of 10 × PCR buffer is preferably 1 × PCRbuffer,
The concentration of dNTP is preferably 0.25mM, and the concentration for detecting the primer of SDC2 gene methylation is respectively preferably 0.4uM, is detected
The concentration of the probe of NDRG4 gene methylation is preferably 0.4uM, and the concentration of β-actin primer is respectively preferably 0.4uM, β-
The concentration of actin probe is preferably 0.4uM.The present invention is not particularly limited the source of mentioned reagent, is using conventional method
It can.The present invention is not particularly limited placement amount of the NDRG4 reaction solution in kit, is placed using conventional kit anti-
Answer the amount of liquid.
In the present invention, in the SDC2 reaction solution, the concentration of 10 × PCRbuffer is preferably 1 × PCR buffer,
The concentration of dNTP is preferably 0.25mM, and the concentration for detecting the primer of SDC2 gene methylation is respectively preferably 0.3uM, detects SDC2
The concentration of the probe of gene methylation is preferably 0.3uM, and the concentration of β-actin primer is respectively preferably 0.4uM, and β-actin is visited
The concentration of needle is preferably 0.4uM.The present invention is not particularly limited the source of mentioned reagent, using conventional method.This hair
It is bright that placement amount of the SDC2 reaction solution in kit is not particularly limited, using the amount of conventional kit placing response liquid
?.
In the present invention, in the BMP3 reaction solution, the concentration of 10 × PCRbuffer is preferably 1 × PCR buffer,
The concentration of dNTP is preferably 0.25mM, and the concentration for detecting the primer of BMP3 gene methylation is respectively preferably 0.4uM, detects BMP3
The concentration of the probe of gene methylation is preferably 0.4uM, and the concentration of β-actin primer is respectively preferably 0.4uM, and β-actin is visited
The concentration of needle is preferably 0.4uM.The present invention is not particularly limited the source of mentioned reagent, using conventional method.This hair
It is bright that placement amount of the BMP3 reaction solution in kit is not particularly limited, using the amount of conventional kit placing response liquid
?.
In the present invention, it is also preferable to include Taq enzyme, positive control and negative controls for the kit.In the present invention,
The positive control is preferably colorectal cancer cell lines, and the negative control is preferably human peripheral genomic DNA.
In the present invention, the application method of the kit preferably includes following steps:
1) human faecal mass genomic DNA is extracted, the human faecal mass genomic DNA is subjected to sulphite conversion processing, is obtained
Handle DNA；
2) the processing DNA obtained the step 1) is anti-with NDRG4 reaction solution, SDC2 described in above-mentioned technical proposal respectively
Real-time fluorescent PCR amplification is carried out after answering liquid, the mixing of BMP3 reaction solution, then after mixing respectively with Taq enzyme, obtains amplification；
3) as the Ct≤38 of any gene of step 2) amplification the β-actin≤35, NDRG4, SDC2 and BMP3,
NDRG4, SDC2 and BMP3 gene methylation in human faecal mass is the positive,
As the Ct > 38 of any gene of amplification the β-actin≤35, NDRG4, SDC2 and BMP3, in human faecal mass
NDRG4, SDC2 and BMP3 gene methylation be feminine gender, as the amplification β-actin > 35, sample size is insufficient or has suppression
Object processed exists, and real-time fluorescent PCR amplification reaction is invalid.
In the present invention, the extracting method of the human faecal mass genomic DNA preferably includes following steps:
A, after being centrifuged human faecal mass, after the first obtained supernatant is mixed with extract A, 5min is incubated at 70 DEG C,
4000g is centrifuged 5min again, obtains the second supernatant；
B, it is incubated for 15min at 65 DEG C after mixing the second obtained supernatant with Proteinase K Solution, extract B, then
4000g is centrifuged 10min, obtains third supernatant；
C, 95 DEG C of incubation 5min of third supernatant that will be obtained, temperature drops to 65 DEG C and visits with containing capture at room temperature
The magnetic bead solution of needle mixes, and places 1h, successively respectively carries out 2 cleaning magnetic with washing lotion A, washing lotion B and washing lotion C after discarding supernatant liquid
Pearl elutes obtained cleaning magnetic bead with eluent, and human faecal mass genomic DNA is contained in obtained solution.
In the present invention, the volume ratio of first supernatant and extract A are preferably 1:1.In the present invention, the pumping
The EDTA of the Tris and 20mM of SDS, 0.05~0.1M that mass percentage is 1~2 are preferably comprised in extract A.
In the present invention, the volume ratio of second supernatant and Proteinase K Solution, extract B is preferably 5ml:30ul:
5ml.In the present invention, the concentration of the Proteinase K is preferably 20mg/mL.In the present invention, preferably contain in the extract B
There is polysorbas20 that the guanidinium isothiocyanate of 4~5M, the sodium citrate of 20~40mM, volume fraction are 0.5~1% and mass fraction is
1 ‰ dithiothreitol dithio.
In the present invention, the volume ratio of the third supernatant and the magnetic bead solution containing capture probe is preferably 10ml:
40ul.In the present invention, the concentration of magnetic bead is preferably 10mg/ml in the magnetic bead solution containing capture probe, the magnetic bead
On preferably comprised capture probe, the amount of the capture probe on every milligram of magnetic bead is preferably 500pmol.In the present invention, described
Capture probe includes NDRG4 probe, SDC2 probe, BMP3 probe and β-actin probe, and 4 kinds of probes etc. are than mixing.
In the present invention, the sequence of the NDRG4 probe is specific as follows as shown in SEQ ID No.13:
5`-GAGATGCGGACGAGACAGACGCGCAGGCG 3'。
The present invention preferably carries out Biotin-TEG label in the 5` of NDRG4 probe.
In the present invention, the sequence of the SDC2 probe is specific as follows as shown in SEQ ID No.14:
5`AGCCCGCGCACACGAATCCGGAGCAGAGTACCG 3'。
The present invention preferably carries out Biotin-TEG label in the 5` of SDC2 probe.
In the present invention, the sequence of the BMP3 probe is specific as follows as shown in SEQ ID No.15:
5`CGGGTGCAGCGAGATAGCGGCCAGCC 3'。
The present invention preferably carries out Biotin-TEG label in the 5` of BMP3 probe.
In the present invention, the sequence of the β-actin probe is specific as follows as shown in SEQ ID No.16:
5`TGTGAACCTGTGTCTGCCACTGTGTGCTGG 3'。
The present invention preferably carries out Biotin-TEG label in the 5` of β-actin probe.
In the present invention, preferably comprised in the washing lotion A 5~7M guanidine hydrochloride, mass percentage be 1~5% PEG,
The glacial acetic acid that 0.1~0.3M anhydrous sodium acetate and volumetric concentration are 4 ‰.
In the present invention, the guanidine hydrochloride of 0.2~0.6M is preferably comprised in the washing lotion B, mass percentage is 1~4%
CTAB, volume fraction be 75% ethyl alcohol and 20mM EDTA,
In the present invention, the washing lotion C is preferably ethanol solution, and the volume fraction of ethyl alcohol is preferably 70~80%.
In the present invention, the eluent is preferably TE buffer, the Tris-Hcl containing 10mM in the TE buffer
PH value with the EDTA of 1mM, the TE buffer is preferably 8.0.
In the present invention, the method that the human faecal mass genomic DNA carries out sulphite conversion processing preferably includes: by institute
It states after human faecal mass genomic DNA is mixed with nuclease-free water, conversion reaction solution and is converted.
In the present invention, the human faecal mass Genomic DNA solution and nuclease-free water, the volume ratio of conversion reaction solution are preferred
For (1~20): (0~19): (130).
In the present invention, the preparation method of the conversion reaction solution preferably includes: taking 1 pipe Solution 1,300uL is added
Solution 2 and 790uL Solution 3 fullys shake 8~10min of mixing and is completely dissolved to crystal；160uL is added
Solution4, concussion mix, and obtain conversion reaction solution.In the present invention, the Solution 1, Solution 2,
Solution 3 and Solution4 derives from conversion reagent box, is derive specifically from EZ DNAMethylationTM Kits。
In the present invention, the condition of the conversion includes: 98 DEG C of reaction 8min, and 64 DEG C of reaction 3.5h, 20 DEG C are at most reacted
20h。
In the present invention, the system of the real-time fluorescence PCR reaction, every 20uL includes NDRG4 reaction solution, SDC2 reaction solution
Or BMP3 reaction solution 18.3uL, Taq enzyme 0.2uL, handle DNA 1.5uL.
In the present invention, the program of the real-time fluorescence PCR reaction are as follows: 95 DEG C of 5min；95 DEG C of 15s, 60 DEG C of 30s (acquisitions
Fluorescence), 50 circulations.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
A kind of specific primer design for Human colorectal carcinoma detection
The PCR primer and probe of Human colorectal carcinoma specific methylation are according to NCBI (US National Biotechnology Information
Center) disclosed in mankind's whole genome sequence, set using Primer Premier3.0 and Methyl Primerpress v1.0
Meter, is shown in Table 1, is synthesized by Invitrogen (Shanghai) Trading Co., Ltd..
1 primed probe of table
| NDRG4-MF | 5`-GGTTTTTGGTTGTTCGTATTTGT-3` | SEQ ID No.1 |
| NDRG4-MR | 5`-GAAAATACGAACGAAACAAACGC-3` | SEQ ID No.2 |
| NDRG4-MP | 5`-FAM-AAACGAAACTTCGACGCC-3`MGB | SEQ ID No.3 |
| SDC2-MF | 5`-AAGCGAGCGTTTTCGAGTTTC-3` | SEQ ID No.4 |
| SDC2-MR | 5`-CACACGAATCCGAAACAAAATAC-3` | SEQ ID No.5 |
| SDC2-MP | 5`-FAM-AACGATTACGACTCAAAC-3`MGB | SEQ ID No.6 |
| BMP3-MF | 5`-ACTCCAAACCAACTAAAACGAAAAC-3` | SEQ ID No.7 |
| BMP3-MR | 5`-GTCGTTATTTCGTTGTATTCGGTC-3` | SEQ ID No.8 |
| BMP3-MP | 5`-FAM-CACGAAACCCGAAAC-3`MGB | SEQ ID No.9 |
| β-actin-MF | 5`-GGTTAGGAAGGAGGTTGTTTGTTTT-3` | SEQ ID No.10 |
| β-actin-MR | 5`-ATCATCTTTCCCACCAAACTATAACC-3` | SEQ ID No.11 |
| β-actin-MP | 5`-VIC-CCCATTAACTAAACACAACCT-3`MGB | SEQ ID No.12 |
Wherein detection target gene probe 5 ' end is adopted using FAM label as luminophore, the end of β-actin-MP probe 5 '
Use VIC label as luminophore, 3 ' ends use MGB as quenching group.
Embodiment 2
A kind of kit extracted for human faecal mass cast-off cells DNA
Human faecal mass cast-off cells DNA extract reagent include:
Sample preservation liquid: LiCl (2-6M), EDTA (0.05-0.2M), Tris (0.1-0.3M), TCEP.HCl (50mM),
PVPP (5%)
Extract A: the solution containing SDS (1-2%), Tris (0.05-0.1M), EDTA (20mM)；
Extract B: contain guanidinium isothiocyanate (4-5M), sodium citrate (20-40mM), polysorbas20 (0.5-1%), two thio Soviet Unions
The solution of sugar alcohol (1 ‰)；
Proteinase K: 20mg/mL；
Biotin labeling magnetic bead: 10mg/mL, wherein include capture probe (~500pmol biotinylated probes/mg magnetic bead),
Sequence capture probe such as table 2,5 end of capture probe use biotin labeling:
2 capture probe of table
| NDRG4-Bio | 5`Biotin-TEGGAGATGCGGACGAGACAGACGCGCAGGCG3' | SEQ ID No.13 |
| SDC2-Bio | 5`Biotin-TEGAGCCCGCGCACACGAATCCGGAGCAGAGTACCG3' | SEQ ID No.14 |
| BMP3-Bio | 5`Biotin-TEGCGGGTGCAGCGAGATAGCGGCCAGCC3' | SEQ ID No.15 |
| β-actin-Bio | 5`Biotin-TEGTGTGAACCTGTGTCTGCCACTGTGTGCTGG3' | SEQ ID No.16 |
Washing lotion A: hydrochloric guanidine (5-7M), PEG (1-5%), anhydrous sodium acetate (0.1-0.3M), glacial acetic acid (4 ‰)；
Washing lotion B: hydrochloric guanidine (0.2-0.6M), CTAB (1-4%), ethyl alcohol (75%), EDTA (20mM)；
Washing lotion C: (70-80%) containing ethyl alcohol；
Eluent: TE buffer (10mMTris-HCl, 1mM EDTA, PH=8.0).
Embodiment 3
A kind of kit for Human colorectal carcinoma DNA methylation assay, comprising:
NDRG4 reaction solution include: nuclease-free water, 10 × PCRbuffer, dNTP, NDRG4-MF, NDRG4-MR,
NDRG4–MP,β-actin-F,β-actin-R,β-actin-MP.Final concentration PCRbuffer (1 ×), the dNTP of various substances
(0.25mM)、NDRG4-MF(0.4uM)、NDRG4-MR(0.4uM)、NDRG4-MP(0.4uM)、、β-actin-F(0.4uM)、β-
actin-R(0.4uM)、β-actin-MP(0.4uM)。
SDC2 reaction solution include: nuclease-free water, 10 × PCRbuffer, dNTP, SDC2-MF, SDC2-MR, SDC2-MP,
β-actin-F,β-actin-R,β-actin-MP.The final concentration PCR buffer (1 ×) of various substances, dNTP (0.25mM),
SDC2-MF(0.3uM)、SDC2-MR(0.3uM)、SDC2-MP(0.3uM)、β-actin-F(0.4uM)、β-actin-R
(0.4uM)、β-actin-MP(0.4uM)。
BMP3 reaction solution includes: nuclease-free water, 10 × PCR buffer, dNTP, BMP3-MF, BMP3-MR, BMP3-
MP,β-actin-F,β-actin-R,β-actin-MP.Final concentration PCRbuffer (1 ×), the dNTP of various substances
(0.25mM)、BMP3-MF(0.4uM)、BMP3-MR(0.4uM)、BMP3-MP(0.4uM)、β-actin-F(0.4uM)、β-
actin-R(0.4uM)、β-actin-MP(0.4uM)。
HS Taq enzyme, methylate standard items DNA and non-methylation standard items DNA.
Embodiment 4
Application of the kit of embodiment 2~3 in diagnosis Human colorectal carcinoma
Nuclease-free water that the present invention uses, 10 × PCR Buffer, HS Taq enzyme, dNTPs are (big purchased from precious biology
Even) Co., Ltd；Conversion reagent box EZ DNA MethylationTMKits is purchased from ZYMO RESEARCH.
Positive control (colorectal cancer cell lines), negative control (healthy human peripheral blood genomic DNA).
Biomaterial of the present invention is all from the refined medical test institute in Hunan.
Method:
One, biological sample:
For during 10-2019 March in 2018 in the refined hospital admission in Hunan adenomas 90, colorectal polypus 104,
139,280 enteron aisle normal population samples of Colon and rectum inflammation, 361 fecal samples of colorectal cancer case.
Two, DNA is extracted:
This 10ml is sampled, takes supernatant 5ml after mixing centrifugation.
5ml extract A, 70 DEG C of incubation 5min are added, then 4000g is centrifuged 5min；
5ml supernatant is taken to be added in new centrifuge tube；
30ul PK is added in new centrifuge tube, 5ml extracts B liquid；Mix 65 DEG C of incubation 15min；
Centrifugation 10min is put into a centrifuge after incubation, and supernatant is taken to be transferred to new centrifuge tube；
95 DEG C of incubation 5min；
The magnetic bead 30ul with capture probe is added, is placed at room temperature for 1H after mixing；
Magnetically attractive removes supernatant after hybridization；
Washing lotion A, washing lotion B, washing lotion C are respectively cleaned 2 times in order；
40ul eluent is added to be eluted at 65 DEG C.
2-8 DEG C of DNA solution preservation, is placed in -20 DEG C or lower temperature if needing long-term preservation.
Three, the process of DNA conversion
The DNA configuration sulphite transformation system of extraction is configured:
By the DNA configuration transformation system configuration of extraction: (conversion reaction solution is prepared: taking 1 pipe Solution 1,300uL is added
Solution 2 and 790uL Solution 3 fullys shake and mixes 8-10min and be completely dissolved to crystal；160uL is added
Solution 4, concussion mix, for use；Unspent conversion reaction solution is please saved in 2-8 DEG C, is saved and is no more than 1 month)
3 sulphite transformation system of table
| Ingredient | Reaction volume (μ L) |
| The DNA to be transformed extracted | 1-20 μ L (200ng~2ug) |
| RNase-freeWater | Supply 20 μ L |
| Conversion reaction solution | 130uL |
| It amounts to | 150μL |
1. sulphite conversion operation:
4 sulphite conversion condition of table
| Reaction temperature | Reaction time |
| 98℃ | 8min |
| 64℃ | 3.5h |
| 20℃ | ≤20h |
2. DNA is purified after sulphite conversion:
1) it takes purification column that 600uLBufferA is added, then converted product is transferred in purification column, be mixed by inversion for several times；
2) 10000rpm is centrifuged 30s, abandons efflux；
3) plus 100 μ LBufferB, 10000rpm are centrifuged 30s, abandon efflux；
4) 200uLBufferC is added, is stored at room temperature 15min, 10000rpm is centrifuged 30s, abandons efflux；
5) plus 200 μ LBufferB, 10000rpm are centrifuged 30s, abandon efflux；
6) step 5 is repeated；
7) 10000rpm idle running centrifugation 2min；
8) purification column is inserted into a new EP pipe, 20uL ElutionBuffer is added, is stored at room temperature 1min；
9) 10000rpm is centrifuged 1min and collects the DNA solution that efflux has as converted, and is stored in -20 DEG C.It is described
BufferA, B and ElutionBuffer derive from conversion reagent box, are derive specifically from EZ DNAMethylationTM Kits。
Four, PCR process
1, the PCR instrument used is ABI 7500, reaction system 20ul；
2, PCR reaction system is prepared and condition is as shown in such as the following table 5 and table 6:
The preparation of 5 PCR reaction system of table
| Ingredient | Reaction volume (ul) |
| PCR reaction solution | 17.3ul |
| Taq enzyme | 0.2ul |
| DNA profiling after sulphite conversion | 2.5ul |
6 PCR response procedures of table
Five, interpretation of result
Kit test result meets Quality Control requirement, judges according to testing result sample.- actin≤35 β, sample
This testing result is wantonly 1 gene C t≤38, then determines sample for methylation positive；- actin≤35 β, pattern detection result are
The equal Ct > 38 of NDRG4, SDC2 and BMP3；It is negative for methylation then to sentence sample, β-actin > 35, then sample is insufficient or has inhibition
There are PCR reactions for object in vain, need to repeat to test or sample.
7 fluorescent PCR result interpretation of table
Six, colorectal cancer NDRG4, SDC2 and BMP3 gene methylation
NDRG4, SDC2 and BMP3 gene methylation in negative sample (90 adenomas, 104 colorectal polypus,
139 Colon and rectum inflammation, 280 enteron aisle normal population samples) recall rate is 7.35% positive, 361 Colon and rectum fecal samples
Middle recall rate is 88.1%.P < 0.001 compared with normal group, the methylation ratio of NDRG4, SDC2 and BMP3 gene have significant
Difference.
NDRG4, SDC2 and BMP3 gene methylation detection statistics result of 8 fecal cast-off cell of table
Sensitivity=A/ (A+B) × 100%=318/ (318+43)=88.1%
Specificity=D/ (C+D) × 100%=731/ (731+58)=92.6%
Total coincidence rate: (A+D)/(A+B+C+D) × 100%=(318+731)/1150=91.2%
Each stage recall rate of 9 colorectal cancer of table
Embodiment 5
Using the identical experimental method of embodiment 4 detect NDRG4 difference methylation positive proportional sample, the result is shown in Figure 1~
3；The DNA for not carrying out sulphite conversion is in order to which the genomic DNA that reference does not carry out sulphite conversion will not be to result
It is interfered.
SDC2 difference methylation positive proportional sample is detected using the identical experimental method of embodiment 4, as a result sees Fig. 4-6；
The DNA for not carrying out sulphite conversion is that the genomic DNA for not carrying out sulphite conversion for reference will not have result
It is interfered.
BMP3 difference methylation positive proportional sample is detected using the identical experimental method of embodiment 4, as a result sees Fig. 7-9；
The DNA for not carrying out sulphite conversion is that the genomic DNA for not carrying out sulphite conversion for reference will not have result
It is interfered.
By above embodiments it can be concluded that, using detection NDRG4, SDC2 and BMP3 gene methylation provided by the invention
Primer combination of probe improves the sensitivity to colorectal cancer.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>the primary gene technology Co., Ltd in Hunan hundred
<120>a kind of primer combination of probe for detecting NDRG4, SDC2 and BMP3 gene methylation and its application and kit
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ggtttttggt tgttcgtatt tgt 23
<210> 2
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gaaaatacga acgaaacaaa cgc 23
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aaacgaaact tcgacgcc 18
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aagcgagcgt tttcgagttt c 21
<210> 5
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cacacgaatc cgaaacaaaa tac 23
<210> 6
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
aacgattacg actcaaac 18
<210> 7
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
actccaaacc aactaaaacg aaaac 25
<210> 8
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gtcgttattt cgttgtattc ggtc 24
<210> 9
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cacgaaaccc gaaac 15
<210> 10
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ggttaggaag gaggttgttt gtttt 25
<210> 11
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
atcatctttc ccaccaaact ataacc 26
<210> 12
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cccattaact aaacacaacc t 21
<210> 13
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gagatgcgga cgagacagac gcgcaggcg 29
<210> 14
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
agcccgcgca cacgaatccg gagcagagta ccg 33
<210> 15
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
cgggtgcagc gagatagcgg ccagcc 26
<210> 16
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
tgtgaacctg tgtctgccac tgtgtgctgg 30
Claims (10)
1. a kind of primer combination of probe for detecting NDRG4, SDC2 and BMP3 gene methylation, which is characterized in that the primer is visited
Needle includes detection target gene methylated primers probe and β-actin primed probe；
The sequence of the primer of NDRG4 gene methylation is detected as shown in No.1~2 SEQ ID, detection NDRG4 gene methylation
The sequence of probe is as shown in SEQ ID No.3；
The sequence of the primer of SDC2 gene methylation is detected as shown in No.4~5 SEQ ID, detects the spy of SDC2 gene methylation
The sequence of needle is as shown in SEQ ID No.6；
The sequence of the primer of BMP3 gene methylation is detected as shown in No.7~8 SEQ ID, detects the spy of BMP3 gene methylation
The sequence of needle is as shown in SEQ ID No.9；
The sequence of β-actin primer is as shown in No.10~11 SEQ ID, the sequence of β-actin probe such as SEQ ID No.12 institute
Show.
2. primer combination of probe according to claim 1, which is characterized in that in the spy of the detection target gene methylation
The end the 5` addition FAM label of needle, in the end 3` addition MGB label.
3. primer combination of probe according to claim 1, which is characterized in that added at the end 5` of the β-actin probe
VIC label, in the end 3` addition MGB label.
4. the answering in preparation detection Human colorectal carcinoma kit of primer combination of probe described in claims 1 to 3 any one
With.
5. a kind of kit, which is characterized in that including NDRG4 reaction solution, SDC2 reaction solution and BMP3 reaction solution；
The NDRG4 reaction solution includes nuclease-free water, 10 × PCR buffer, dNTP, detection described in claim 1
The primer of NDRG4 gene methylation, the detection probe of NDRG4 gene methylation described in claim 1, described in claim 1
β-actin primer and β-actin probe described in claim 1；
The SDC2 reaction solution includes nuclease-free water, 10 × PCRbuffer, dNTP, detection SDC2 base described in claim 1
Because of the primer of methylation, probe, the β-actin described in claim 1 of detection SDC2 gene methylation described in claim 1
Primer and β-actin probe described in claim 1；
The BMP3 reaction solution includes nuclease-free water, 10 × PCRbuffer, dNTP, detection BMP3 base described in claim 1
Because of the primer of methylation, probe, the β-actin described in claim 1 of detection BMP3 gene methylation described in claim 1
Primer and β-actin probe described in claim 1.
6. kit according to claim 5, which is characterized in that in the NDRG4 reaction solution, 10 × PCRbuffer's
Concentration is 1 × PCRbuffer, and the concentration of dNTP is 0.25mM, and the concentration for detecting the primer of SDC2 gene methylation is respectively
0.4uM, the concentration for detecting the probe of NDRG4 gene methylation is 0.4uM, and the concentration of β-actin primer is respectively 0.4uM, β-
The concentration of actin probe is 0.4uM.
7. kit according to claim 5, which is characterized in that in the SDC2 reaction solution, 10 × PCRbuffer's is dense
Degree is 1 × PCRbuffer, and the concentration of dNTP is 0.25mM, and the concentration for detecting the primer of SDC2 gene methylation is respectively
0.3uM, the concentration for detecting the probe of SDC2 gene methylation is 0.3uM, and the concentration of β-actin primer is respectively 0.4uM, β-
The concentration of actin probe is 0.4uM.
8. kit according to claim 5, which is characterized in that in the BMP3 reaction solution, 10 × PCRbuffer's is dense
Degree is 1 × PCRbuffer, and the concentration of dNTP is 0.25mM, and the concentration for detecting the primer of BMP3 gene methylation is respectively
0.4uM, the concentration for detecting the probe of BMP3 gene methylation is 0.4uM, and the concentration of β-actin primer is respectively 0.4uM, β-
The concentration of actin probe is 0.4uM.
9. kit according to claim 5, which is characterized in that the kit further include: Taq enzyme, positive control and
Negative control.
10. kit according to claim 9, which is characterized in that the positive control is colorectal cancer cell lines, described
Negative control is human peripheral genomic DNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910379603.XA CN109943638A (en) | 2019-05-08 | 2019-05-08 | A kind of primer combination of probe detecting NDRG4, SDC2 and BMP3 gene methylation and its application and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910379603.XA CN109943638A (en) | 2019-05-08 | 2019-05-08 | A kind of primer combination of probe detecting NDRG4, SDC2 and BMP3 gene methylation and its application and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109943638A true CN109943638A (en) | 2019-06-28 |
Family
ID=67017197
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910379603.XA Pending CN109943638A (en) | 2019-05-08 | 2019-05-08 | A kind of primer combination of probe detecting NDRG4, SDC2 and BMP3 gene methylation and its application and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109943638A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923300A (en) * | 2019-11-18 | 2020-03-27 | 人和未来生物科技（长沙）有限公司 | Gene methylation detection method and application |
| CN113454243A (en) * | 2021-04-23 | 2021-09-28 | 华大数极生物科技(深圳)有限公司 | Composition for colorectal cancer detection, kit and application |
| CN115287352A (en) * | 2022-06-01 | 2022-11-04 | 上海市第一人民医院 | Primer probe combination for detecting colorectal cancer gene methylation through digital PCR (polymerase chain reaction), kit and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101680025A (en) * | 2007-01-09 | 2010-03-24 | 癌甲基化组科学公司 | Epigenetic change in selected genes and cancer |
| CN104975110A (en) * | 2015-06-02 | 2015-10-14 | 浙江诺辉生物技术有限公司 | Primer and probe for detecting methylation levels of BMP3 and NDRG4 in biological sample |
| CN105247071A (en) * | 2013-04-01 | 2016-01-13 | 基因特力株式会社 | Nucleic acid amplification method using allele-specific reactive primer |
| CN105452485A (en) * | 2013-08-14 | 2016-03-30 | 基因特力株式会社 | Method for detecting precancerous lesions |
| CN106755491A (en) * | 2017-01-24 | 2017-05-31 | 韩林志 | Primer pair, kit and method based on the detection of cervical carcinoma specific methylation |
| CN106893777A (en) * | 2017-03-02 | 2017-06-27 | 武汉艾米森生命科技有限公司 | For detecting many site methylating reagent boxes of colorectal cancer related gene and application |
| CN108103195A (en) * | 2018-01-22 | 2018-06-01 | 上海酷乐生物科技有限公司 | A kind of noninvasive polygenes for early stage colorectal cancer methylates primer pair and probe, the kit and its application of joint-detection |
| CN108350485A (en) * | 2015-10-30 | 2018-07-31 | 精密科学发展有限责任公司 | The multiplex amplification detection assay of plasma dna and separation and detection |
| CN108660209A (en) * | 2018-05-23 | 2018-10-16 | 杭州诺辉健康科技有限公司 | Colorectal cancer early detection method based on BMP3 gene methylation sequences |
-
2019
- 2019-05-08 CN CN201910379603.XA patent/CN109943638A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101680025A (en) * | 2007-01-09 | 2010-03-24 | 癌甲基化组科学公司 | Epigenetic change in selected genes and cancer |
| CN105247071A (en) * | 2013-04-01 | 2016-01-13 | 基因特力株式会社 | Nucleic acid amplification method using allele-specific reactive primer |
| CN105452485A (en) * | 2013-08-14 | 2016-03-30 | 基因特力株式会社 | Method for detecting precancerous lesions |
| CN104975110A (en) * | 2015-06-02 | 2015-10-14 | 浙江诺辉生物技术有限公司 | Primer and probe for detecting methylation levels of BMP3 and NDRG4 in biological sample |
| CN108350485A (en) * | 2015-10-30 | 2018-07-31 | 精密科学发展有限责任公司 | The multiplex amplification detection assay of plasma dna and separation and detection |
| CN106755491A (en) * | 2017-01-24 | 2017-05-31 | 韩林志 | Primer pair, kit and method based on the detection of cervical carcinoma specific methylation |
| CN106893777A (en) * | 2017-03-02 | 2017-06-27 | 武汉艾米森生命科技有限公司 | For detecting many site methylating reagent boxes of colorectal cancer related gene and application |
| CN108103195A (en) * | 2018-01-22 | 2018-06-01 | 上海酷乐生物科技有限公司 | A kind of noninvasive polygenes for early stage colorectal cancer methylates primer pair and probe, the kit and its application of joint-detection |
| CN108660209A (en) * | 2018-05-23 | 2018-10-16 | 杭州诺辉健康科技有限公司 | Colorectal cancer early detection method based on BMP3 gene methylation sequences |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923300A (en) * | 2019-11-18 | 2020-03-27 | 人和未来生物科技（长沙）有限公司 | Gene methylation detection method and application |
| CN113454243A (en) * | 2021-04-23 | 2021-09-28 | 华大数极生物科技(深圳)有限公司 | Composition for colorectal cancer detection, kit and application |
| CN115287352A (en) * | 2022-06-01 | 2022-11-04 | 上海市第一人民医院 | Primer probe combination for detecting colorectal cancer gene methylation through digital PCR (polymerase chain reaction), kit and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TWI741418B (en) | Multi-gene combined detection reagent | |
| CN110283911A (en) | Primer pair and probe and kit for fecal sample progress early stage colorectal cancer gene methylation detection | |
| CN109943638A (en) | A kind of primer combination of probe detecting NDRG4, SDC2 and BMP3 gene methylation and its application and kit | |
| WO2021004214A1 (en) | Genetic marker combination and application thereof | |
| EP2210942B1 (en) | Gene associated with liver cancer, and method for determination of the risk of acquiring liver cancer | |
| CN108728535A (en) | Applications of the hsa_circ_0049154 as prostate cancer molecular target in preparing drug and kit | |
| CN109055563A (en) | The related cyclic annular rna gene of colorectal cancer, colorectal cancer molecular marker and its application | |
| CN110484621A (en) | A kind of method of liver cancer early warning | |
| CN112553302A (en) | Method for extracting DNA sample from excrement and methylation detection method of colorectal cancer related gene | |
| CN107586842A (en) | A kind of biomarker for clear cell carcinoma of kidney diagnosis and treatment | |
| CN114875139A (en) | Application of exosome miR-106b-3p, et-7a and the like in lung cancer diagnosis | |
| ES2694284T3 (en) | Methods and uses of nucleic acids for the analysis of methylation and gene expression, in particular of NFATC3, associated with proliferative disorders of prostate cells | |
| CN108949969A (en) | Application of the long-chain non-coding RNA in colorectal cancer | |
| CN105463115B (en) | For detecting probe, primer and the kit of 7 kinds of mankind RET gene mutation | |
| TWI716882B (en) | Tumor marker, methylation detection reagent, kit and application thereof | |
| CN112575082A (en) | Fluorescent quantitative PCR detection kit for target gene methylation detection | |
| CN116004831A (en) | Reagent for diagnosing or assisting in diagnosing bladder cancer and detection kit | |
| CN113430272B (en) | Reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesion and application of reagent and kit | |
| CN112553341B (en) | Renal clear cell carcinoma SCNN1 primer, diagnostic kit and application thereof | |
| CN106011263B (en) | Kit for detecting gastric cancer-related NDRG4 gene methylation degree and application thereof | |
| CN110042159A (en) | A kind of polymerase spiral amplimer group and detection kit based on Human colorectal carcinoma related gene SEPT9 DNA methylation assay | |
| WO2024001602A1 (en) | Composition for detecting gastric cancer, kit, and use thereof | |
| CN116970705B (en) | Nucleic acid product for methylation detection of urothelial oncogene, kit and application | |
| CN114457156B (en) | Primer and probe combination for methylation detection of cervical cancer, kit and use method thereof | |
| CN114150065B (en) | Marker for colorectal cancer or precancerous lesion and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190628 |