CN116042792A - Telomerase activity gene detection kit, primer set, probe primer and detection method - Google Patents
Telomerase activity gene detection kit, primer set, probe primer and detection method Download PDFInfo
- Publication number
- CN116042792A CN116042792A CN202211075535.6A CN202211075535A CN116042792A CN 116042792 A CN116042792 A CN 116042792A CN 202211075535 A CN202211075535 A CN 202211075535A CN 116042792 A CN116042792 A CN 116042792A
- Authority
- CN
- China
- Prior art keywords
- reverse
- sample
- mmol
- telomerase activity
- primer
- 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 107
- 238000001514 detection method Methods 0.000 title claims abstract description 48
- 108010017842 Telomerase Proteins 0.000 title claims abstract description 47
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 35
- 230000000694 effects Effects 0.000 title claims abstract description 34
- 239000007788 liquid Substances 0.000 claims abstract description 36
- 239000003480 eluent Substances 0.000 claims abstract description 17
- 239000011324 bead Substances 0.000 claims abstract description 11
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 10
- 239000000725 suspension Substances 0.000 claims abstract description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 6
- 238000010791 quenching Methods 0.000 claims description 33
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 26
- 239000013642 negative control Substances 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 19
- 239000013641 positive control Substances 0.000 claims description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims description 15
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 108020004707 nucleic acids Proteins 0.000 claims description 12
- 102000039446 nucleic acids Human genes 0.000 claims description 12
- 150000007523 nucleic acids Chemical class 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 238000000605 extraction Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 239000011734 sodium Substances 0.000 claims description 11
- 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 claims description 10
- 238000011144 upstream manufacturing Methods 0.000 claims description 10
- 238000012545 processing Methods 0.000 claims description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 239000013074 reference sample Substances 0.000 claims description 8
- 108091035539 telomere Proteins 0.000 claims description 8
- 108010067770 Endopeptidase K Proteins 0.000 claims description 7
- 102100034343 Integrase Human genes 0.000 claims description 7
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 7
- 229920004890 Triton X-100 Polymers 0.000 claims description 7
- 239000013504 Triton X-100 Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 210000003411 telomere Anatomy 0.000 claims description 7
- 102000055501 telomere Human genes 0.000 claims description 7
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 5
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 241001481296 Malus spectabilis Species 0.000 claims description 4
- 229930195725 Mannitol Natural products 0.000 claims description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 4
- 229960003237 betaine Drugs 0.000 claims description 4
- 238000007664 blowing Methods 0.000 claims description 4
- 235000019441 ethanol Nutrition 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
- 239000000594 mannitol Substances 0.000 claims description 4
- 235000010355 mannitol Nutrition 0.000 claims description 4
- 238000003908 quality control method Methods 0.000 claims description 4
- 239000011541 reaction mixture Substances 0.000 claims description 4
- 238000003753 real-time PCR Methods 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- 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 claims description 4
- -1 vanadyl riboside Chemical class 0.000 claims description 4
- 241000713869 Moloney murine leukemia virus Species 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 239000001046 green dye Substances 0.000 claims description 3
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 239000012487 rinsing solution Substances 0.000 claims description 3
- 238000003149 assay kit Methods 0.000 claims 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 abstract description 12
- 108020004999 messenger RNA Proteins 0.000 abstract description 10
- 102100032938 Telomerase reverse transcriptase Human genes 0.000 abstract description 6
- 206010028980 Neoplasm Diseases 0.000 abstract description 4
- 201000011510 cancer Diseases 0.000 abstract description 4
- 239000012472 biological sample Substances 0.000 abstract description 2
- 230000015556 catabolic process Effects 0.000 abstract description 2
- 238000006731 degradation reaction Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 48
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 12
- 201000005202 lung cancer Diseases 0.000 description 12
- 208000020816 lung neoplasm Diseases 0.000 description 12
- 238000010586 diagram Methods 0.000 description 5
- 210000000349 chromosome Anatomy 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 206010008342 Cervix carcinoma Diseases 0.000 description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 2
- 201000010881 cervical cancer Diseases 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 108010081734 Ribonucleoproteins Proteins 0.000 description 1
- 102000004389 Ribonucleoproteins Human genes 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000032677 cell aging Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000002558 medical inspection Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 108010057210 telomerase RNA Proteins 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a telomerase activity gene detection kit, a primer group, a probe primer and a detection method, which comprise sample treatment liquid, rinsing liquid, eluent, magnetic bead suspension, PCR buffer, primer group and enzyme reaction mixed liquid, the problem of easy degradation of RNA is solved, the operation is simple and convenient, compatibility among all components is considered, the kit can ensure that a detected sample can obtain a stable and complete targeted RNA complex through sample treatment, the enriched RNA complex can be directly used for detecting hTERT mRNA genes of a subsequent telomerase reverse transcriptase subunit, the experimental period can be controlled within 30 minutes in a direct-amplification enzyme reaction system in the kit, full-automatic detection is realized, and the detection method of the kit can sensitively detect the existence of hTERT mRNA genes in a biological sample and can be used for qualitatively and quantitatively detecting cancer cells with the telomerase activity of more than 90 percent clinically.
Description
Technical Field
The invention relates to the technical field of molecular biology and medical inspection, in particular to a telomerase active gene detection kit, a primer group, a probe primer and a detection method.
Background
Telomeres are special structures of which the chromosome ends consist of repeated sequence DNA and protein, and the DNA sequences are highly conserved and play an important role in the processes of cell proliferation, aging and the like. The telomeres can reduce the shortening of chromosome ends, can avoid the fusion of adjacent chromosome ends, gradually shorten the length of the telomeres along with the increase of the replication times of cells, enable the cells to exit a cycle and start aging when reaching a certain critical point, and finally lead the cells to die and age when the length of the telomeres is shortened to a critical value of damaging DNA and the cells cannot compensate the length reduction, so that the stability of the chromosomes is poor and the cells finally die and age.
Telomerase is a ribonucleoprotein complex, is a special reverse transcriptase, and when activated, can synthesize telomere DNA by taking RNA of itself as a template, can prevent the loss of telomeres, maintain the length of the telomeres, and enable cells to develop into immortalized cells or cancer cells. Telomerase activity is detectable in over 90% of immortalized and cancerous cells, but not in most normal somatic cells, suggesting that activation of telomerase is an important event in immortalization and carcinogenesis.
Telomerase is composed of 3 subunits, namely telomerase RNA (hTR), telomerase binding protein (TP 1) and catalytic subunit (hTERT). Telomerase catalytic subunit (hTERT) is a critical part of telomerase activity, and its overexpression plays a key role in cell immortalization and the occurrence and development of human malignant tumors, so telomerase can be used as an effective marker of tissue canceration and precancerous lesions.
Currently, the most common methods for detecting telomerase activity are classified into direct detection of telomerase (protein and RNA binding complex) activity and indirect detection of telomerase (mRNA amount of catalytic subunit) activity. When the activity of telomerase (a combination compound of protein and RNA) is directly detected, whether the telomerase can extend a DNA template or not is mainly detected, then gel is run or a probe is hybridized, and the extension length of the DNA is detected, so that the activity of the telomerase is judged; labeling with a radioisotope; poor repeatability and long period. The activity sensitivity of indirect detection of telomerase (the mRNA amount of catalytic subunit) is high, and the operation is relatively simple; however, in most cells, expression of the telomerase catalytic subunit is difficult to detect.
Conventional RNA detection reagents have reagent compatibility problems, and various inhibitors affecting PCR amplification remain in the reagent, so that trace RNA samples are difficult to detect. And the complicated experimental steps can cause RNA in the sample to be degraded by RNase, so that detection fails.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a telomerase active gene detection kit, a primer group, a probe primer and a detection method, so as to solve the technical problems.
The technical scheme of the invention is realized as follows: a primer set for a telomerase activity gene detection kit, comprising an upstream primer, the upstream primer sequence number comprising:
Forward primer1：CTGGACGATATCCACAGGGC
Forward primer2：TGTCAAGGTGGATGTGACGG
Forward primer3：GTGGCACGGCTTTTGTTCAG
Forward primer4：GTGGCACGGCTTTTGTTCAG
Forward primer5：CAAGCTGTTTGCGGGGATTC
Forward primer6：CATCAGGGGCAAGTCCTACG
Forward primer7：AAACCTTCCTCAGCTATGCCC
Forward primer8：AACATGGACTACGTCGTGGG
Forward primer9：CGGAAGAGTGTCTGGAGCAA
Forward primer10：GTGCTACGGCGACATGGAGAAC
Forward primer11：CGACGTCTTCCTACGCTTCA
Forward primer12：AAACCTTCCTCAGCTATGCCC
Forward primer13：GTCACGGAGACCACGTTTCA
Forward primer14：TGGCACGGCTTTTGTTCAGA
Forward primer15：CGTGGTGAACTTGCGGAAGACAG
Forward primer16：TTCAGCGTGCTCAACTACGA
Forward primer17：CATGGACTACGTCGTGGGAG
Forward primer18：AGCAGAGCTCCTCCCTGAAT
Forward primer19：CAAGCTGTTTGCGGGGATTC
Forward primer20：CTGGACGATATCCACAGGGC
Forward primer21：AAACCTTCCTCAGCTATGCCC
Forward primer22：TCAGCGTGCTCAACTACGAG
Forward primer23：CATGGACTACGTCGTGGGAG。
the invention is further provided with: also included is a downstream primer, the downstream primer sequence number comprising:
Reverse primer1：AAGTTCACCACGCAGCCATA 20
Reverse primer2：TGTCTTCCGCAAGTTCACCA 20
Reverse primer3：AGTTCACCACTGTCTTCCGC 20
Reverse prime:4：AATGGCGAATCTGGGGATGG 20
Reverse primer5：ATCTGAACAAAAGCCGTGCC 20
Reverse primer6：ATACTCAGGGACACCTCGGA 20
Reverse primer7：CCTATGTGGGGAGTGGAAGC 20
Reverse primer8：GGCCTCGTCTTCTACAGGGA 20
Reverse primer9：CCATACTCAGGGACACCTCG 20
Reverse primer10：GTTGAAGGTGAGACTGGCTCTGATG 25
Reverse primer11：CCAGGGCCTCGTCTTCTACA 20
Reverse primer12：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer13：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer14：AGGGGTGAACAATGGCGAAT 20
Reverse primer15：TGACGCGCAGGAAAAATGTG 20
Reverse primer16：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer17：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer18：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer19：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer20：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer21：CTATGTGGGGAGTGGAAGCC 20。
the invention is further provided with: further comprises:
10～100mmol Tris-HCl(pH 8.0～8.8)；
0.1-2. Mu. Mol probe primer or 0.5-1 XSYBR Green dye;
0.1～10μmol Oligo dT15；
0.1～10μmol Random；
0.05～1mmol EDTA；
0.2～4mmol dNTP；
1～20mmol dUTP；
wherein the content of the upstream primer is 0.1-2 mu mol, and the content of the downstream primer is 0.1-2 mu mol.
A probe primer for a telomerase activity gene detection kit, the probe primer sequence number comprising:
fluorescent group-AACGCAGGAGCAGCCCGTCCCGCCG-quenching group
Fluorescent group-CGAGGGTGAAGGCACTGTTCAGCG-quenching group
Fluorescent group-GCTGCTCCTGCGTTTGGTGGATGAT-quenching group
Fluorescent group-TTCCCCTGGTGCGGCCTGCTGCTG-quenching group
Fluorescent group-CGAGGGTGAAGGCACTGTTCAGCG-quenching group
Fluorescent group-GACTACGTCGTGGGAGCCAGAACG-quenching group
Fluorescent group-GGCCTCCCTCTGCTACTCCATCCTGA-quenching group
Fluorescent group-CAGCCCGTCCCGCCGAATCCCC-quenching group
Fluorescent group-GACAGCCCAGACGCAGCTGAGTCGG-quenching group
Fluorescent group-TATTCCCCTGGTGCGGCCTGCTG-quenching group
Fluorescent group-TCGTGGTGAACTTGCGGAAGACAGTG-quenching group.
A telomerase activity gene detection kit comprises a sample treatment solution, a rinsing solution, an eluent, a magnetic bead suspension, a PCR buffer, a primer group and an enzyme reaction mixed solution.
The invention is further provided with: the sample processing liquid comprises: 20-200 mmol Na 2 HPO 4 、20～200mmol NaH 2 HPO 4 10-100 mmol NaAC, 2%2-ME, 0.1-1 mmol DTT (dithiothreitol), 2-6 mol GuHCl, 0.05% Triton X-100, 0.01-5 mg/ml proteinase K.
The invention is further provided with: the sample processing liquid comprises: 10-200 mmol Na 2 HPO 4 、10～200mmol NaH 2 HPO 4 2 to 50mmol of sodium citrate, 2 percent of beta-mercaptoethanol, 0.1 to 1mmol of DTT (dithiothreitol), 2 to 6mol of GuHCl or 2 to 6mol of guanidine isothiocyanate, 1 to 50mmol of vanadyl riboside complex, 1 percent of Triton X-100 and 0.01 to 5mg/ml of proteinase K.
The invention is further provided with: the rinse solution comprises: 0.05 to 0.1mol NaH 2 PO 4 、0.05～0.1mol Na 2 HPO 4 0.1-1 mmol DTT (dithiothreitol) or 0.1-10 mmol beta-mercaptoethanol, 60-80% ethanol or 30-60 isopropanol.
The invention is further provided with: the eluent comprises: 10-50 mmol Tris-HCl (pH 8.0), 10-100 mmol KCl, 20-100 mmol NaCl, 10-50 mmol beta-mercaptoethanol, 3.5mmol MgCl 2 、0.1～10mmol DTT。
The invention is further provided with: the enzyme reaction mixture comprises: DNA polymerase 5U/sample (5U/ul) 50ul, MMLV reverse transcriptase 200U/sample or hot start reverse transcriptase 200U/sample, 0.2U-1U UNG enzyme, 0.1-10 mg/BSA, 15% -50% glycerol, 0.1-1M Malus spectabilis, 2-20% mannitol, 0.5-1.5 mol/L betaine.
The invention is further provided with: the PCR Buffer includes: 10-50 mmol Tris-HCl (pH 7.5), 20-200 mmol NaCl, 10-50 mmol KCl, 0.1-1 mmol DTT (dithiothreitol), 1-10% formamide, 0.01-0.1% NP-40, 1-10 mmol MgCl 2 。
A detection method of a telomerase activity gene detection kit comprises the following steps:
s1, preparation before experiment: ensuring that the experimental facility equipment is provided;
s2, preparation of a reagent: before using, adding corresponding absolute ethyl alcohol according to the mark of the reagent bottle, and marking after the absolute ethyl alcohol is added for standby;
s3, preparation of a reagent: preparing 96-hole deep hole plate, adding sample treating liquid, magnetic bead suspension, rinsing liquid and eluent into corresponding holes of deep hole plate
S4, sample adding: adding 0.2-0.3 ml of sample to be measured into the 1 st hole site;
s5, sample extraction: the full-automatic nucleic acid extractor extracts samples or manually extracts samples;
s6, quality control products:
negative control: taking a 1.5ml centrifuge tube, and adding 0.5ml sample treatment solution as a negative control; a reference sample processing mode is processed in a subsequent mode;
positive control: taking 50 μl of positive control, adding 450ul of sample treatment solution, and subsequently treating the reference sample treatment mode;
s7, PCR amplification: taking 8ul eluted samples in a PCR tube, adding 12ul 2 XPCR buffer, 3ul PN mix and 2ul EN mix, lightly blowing by a liquid-transfering device for 3-5 times, uniformly mixing, and sealing the tube cover on a machine;
s8, performing the following procedures on a fluorescence quantitative PCR instrument: 50 ℃ for 10min; 3min at 95 ℃;95℃for 10s,65℃for 30s,40cycles; the threshold setting principle is that the threshold line exceeds the highest point of the negative control and is in the initial stage of the exponential amplification phase of the positive control.
The invention is further provided with: the step of extracting the sample by the full-automatic nucleic acid extractor in the step S5 is as follows:
a1, placing the 96-hole deep-hole plate with the well sample in a full-automatic nucleic acid extractor, and mounting a magnetic rod sleeve;
a2, starting the equipment, running the extraction program, and setting parameters as follows:
a3, after the extraction is finished, the equipment is automatically reset, the deep hole plate is taken out, the magnetic rod sleeve is taken out, and the automatic extraction is finished;
a4, transferring the eluted nucleic acid solution into a new 1.5ml centrifuge tube, and preserving the sample to be measured at 4 ℃ in a short time.
The invention is further provided with: the step of manually extracting the sample in the step S5 is as follows:
d1, adding 200 μl of a sample to be detected into a 1.5ml centrifuge tube, adding 300 μl of sample treatment fluid, adding 15 μl of magnetic beads, uniformly mixing and shaking for about 10 seconds, carrying out warm bath at 60 ℃ for 5-10 minutes, and carrying out warm bath on the eluent for 5-10 minutes;
d2, placing the sample on a magnetic rack for magnetic attraction for 5-30 seconds, and sucking liquid;
d3, adding 500 mul of rinsing liquid, shaking uniformly by hand for 5-10 seconds, and placing the rinsing liquid on a magnetic rack to perform magnetic attraction for 5-30 seconds to suck liquid;
d4, adding 500 mul of rinsing liquid, shaking uniformly by hand for 5-10 seconds, placing on a magnetic rack for magnetic attraction for 5-30 seconds, sucking off liquid, and airing at room temperature for 3-5 minutes or 60 ℃ for 1 minute;
d5, adding 30-100 mu l of eluent, and carrying out warm bath for 1-5 minutes at 60 ℃.
The invention has the beneficial effects that:
1. the method solves the problem of easy degradation of RNA, is simple and convenient to operate, can finish sample extraction in 5-10 minutes, and shortens the traditional RNA detection period from a few hours to 1 hour.
2. Compatibility among all components is considered, and an efficient steady-state and rapid RNA detection method is realized.
3. The kit can enable the detected sample to obtain a stable and complete targeted RNA complex through sample treatment, and the enriched RNA complex can be directly used for detecting the hTERT mRNA gene of the subsequent telomerase reverse transcriptase subunit.
4. The experimental period can be controlled within 30 minutes in a direct-amplification enzyme reaction system in the kit, so that the full-automatic detection is realized.
5. The kit can qualitatively and quantitatively detect whether the hTERT mRNA gene expression exists in the sample, and the expression of the hTERT mRNA has important value for indicating the grading of the precancerous lesions of the tumor.
6. The detection method of the kit can sensitively detect the existence of the gene of hTERT mRNA in a biological sample, and can be used for qualitatively and quantitatively detecting more than 90% of cancer cells with telomerase activity clinically.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a schematic diagram showing the results of cervical cancer cell detection according to an embodiment of the present invention;
FIG. 2 is a schematic diagram showing the detection results of lung cancer H1299 cells according to the embodiment of the present invention;
FIG. 3 is a schematic diagram showing the detection results of lung cancer A431 cells according to an embodiment of the present invention;
FIG. 4 is a schematic diagram showing the detection results of lung cancer A549 cells according to the embodiment of the invention;
FIG. 5 is a schematic diagram showing the detection results of lung cancer A549 cells according to the embodiment of the invention;
FIG. 6 is a schematic representation of a clinical sample of lung cancer according to an embodiment of the present invention;
FIG. 7 is a schematic representation of a clinical sample of lung cancer according to an embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As shown in fig. 1 to 7, the invention discloses a primer set for a telomerase activity gene detection kit, which comprises an upstream primer, wherein the sequence number of the upstream primer comprises:
Forward primer1：CTGGACGATATCCACAGGGC
Forward primer2：TGTCAAGGTGGATGTGACGG
Forward primer3：GTGGCACGGCTTTTGTTCAG
Forward primer4：GTGGCACGGCTTTTGTTCAG
Forward primer5：CAAGCTGTTTGCGGGGATTC
Forward primer6：CATCAGGGGCAAGTCCTACG
Forward primer7：AAACCTTCCTCAGCTATGCCC
Forward primer8：AACATGGACTACGTCGTGGG
Forward primer9：CGGAAGAGTGTCTGGAGCAA
Forward primer10：GTGCTACGGCGACATGGAGAAC
Forward primer11：CGACGTCTTCCTACGCTTCA
Forward primer12：AAACCTTCCTCAGCTATGCCC
Forward primer13：GTCACGGAGACCACGTTTCA
Forward primer14：TGGCACGGCTTTTGTTCAGA
Forward primer15：CGTGGTGAACTTGCGGAAGACAG
Forward primer16：TTCAGCGTGCTCAACTACGA
Forward primer17：CATGGACTACGTCGTGGGAG
Forward primer18：AGCAGAGCTCCTCCCTGAAT
Forward primer19：CAAGCTGTTTGCGGGGATTC
Forward primer20：CTGGACGATATCCACAGGGC
Forward primer21：AAACCTTCCTCAGCTATGCCC
Forward primer22：TCAGCGTGCTCAACTACGAG
Forward primer23：CATGGACTACGTCGTGGGAG。
also included is a downstream primer, the downstream primer sequence number comprising:
Reverse primer1：AAGTTCACCACGCAGCCATA 20
Reverse primer2：TGTCTTCCGCAAGTTCACCA 20
Reverse primer3：AGTTCACCACTGTCTTCCGC 20
Reverse prime:4：AATGGCGAATCTGGGGATGG 20
Reverse primer5：ATCTGAACAAAAGCCGTGCC 20
Reverse primer6：ATACTCAGGGACACCTCGGA 20
Reverse primer7：CCTATGTGGGGAGTGGAAGC 20
Reverse primer8：GGCCTCGTCTTCTACAGGGA 20
Reverse primer9：CCATACTCAGGGACACCTCG 20
Reverse primer10：GTTGAAGGTGAGACTGGCTCTGATG 25
Reverse primer11：CCAGGGCCTCGTCTTCTACA 20
Reverse primer12：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer13：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer14：AGGGGTGAACAATGGCGAAT 20
Reverse primer15：TGACGCGCAGGAAAAATGTG 20
Reverse primer16：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer17：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer18：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer19：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer20：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer21：CTATGTGGGGAGTGGAAGCC 20。
the primer group comprises the following components:
10～100mmol Tris-HCl(8.0～8.8)；
0.1 to 2. Mu. Mol probe primer;
0.1～10μmol Oligo dT15；
0.1～10μmol Random；
0.05～1mmol EDTA；
0.2～4mmol dNTP；
1～20mmol dUTP；
wherein the content of the upstream primer is 0.1-2 mu mol, and the content of the downstream primer is 0.1-2 mu mol.
Or the components of the primer group are
10～100mmol Tris-HCl(pH 8.0～8.8)；
0.5-1 XSYBR Green dye;
0.1～10μmol Oligo dT15；
0.1～10μmol Random；
0.05～1mmol EDTA；
0.2～4mmol dNTP；
1～20mmol dUTP；
wherein the content of the upstream primer is 0.1-2 mu mol, and the content of the downstream primer is 0.1-2 mu mol
A probe primer for a telomerase activity gene detection kit, the probe primer sequence number comprising:
fluorescent group-AACGCAGGAGCAGCCCGTCCCGCCG-quenching group
Fluorescent group-CGAGGGTGAAGGCACTGTTCAGCG-quenching group
Fluorescent group-GCTGCTCCTGCGTTTGGTGGATGAT-quenching group
Fluorescent group-TTCCCCTGGTGCGGCCTGCTGCTG-quenching group
Fluorescent group-CGAGGGTGAAGGCACTGTTCAGCG-quenching group
Fluorescent group-GACTACGTCGTGGGAGCCAGAACG-quenching group
Fluorescent group-GGCCTCCCTCTGCTACTCCATCCTGA-quenching group
Fluorescent group-CAGCCCGTCCCGCCGAATCCCC-quenching group
Fluorescent group-GACAGCCCAGACGCAGCTGAGTCGG-quenching group
Fluorescent group-TATTCCCCTGGTGCGGCCTGCTG-quenching group
Fluorescent group-TCGTGGTGAACTTGCGGAAGACAGTG-quenching group.
A telomerase activity gene detection kit comprises a sample treatment solution, a rinsing solution, an eluent, a magnetic bead suspension, a PCR buffer, a primer group and an enzyme reaction mixed solution.
The sample processing liquid comprises: 20-200 mmol Na 2 HPO 4 、20～200mmol NaH 2 HPO 4 10-100 mmol NaAC, 2%2-ME, 0.1-1 mmol DTT (dithiothreitol), 2-6 mol GuHCl, 0.05% Triton X-100, 0.01-5 mg/ml proteinase K.
The sample processing liquid comprises: 10-200 mmol Na 2 HPO 4 、10～200mmol NaH 2 HPO 4 2 to 50mmol of sodium citrate, 2 percent of beta-mercaptoethanol, 0.1 to 1mmol of DTT (dithiothreitol), 2 to 6mol of GuHCl, 1 to 50mmol of vanadyl riboside complex, 1 percent of Triton X-100 and 0.01 to 5mg/ml of proteinase K;
or the components of the sample treatment solution are 10-200 mmol Na 2 HPO 4 、10～200mmol NaH 2 HPO 4 2 to 50mmol of sodium citrate, 2 percent of beta-mercaptoethanol, 0.1 to 1mmol of DTT (dithiothreitol), 2 to 6mol of guanidine isothiocyanate, 1 to 50mmol of vanadyl riboside complex, 1 percent of Triton X-100 and 0.01 to 5mg/ml of proteinase K.
The rinse solution comprises: 0.05 to 0.1mol NaH 2 PO 4 、0.05～0.1mol Na 2 HPO 4 0.1 to 1mmol of DTT (dithiothreitol), 60 to 80 percent of ethanol or 30 to 60 isopropanol;
or the components of the rinsing liquid are 0.05 to 0.1mol NaH 2 PO 4 、0.05～0.1mol Na 2 HPO 4 0.1 mmol-10 mmol beta-mercaptoethanol, 60-80% ethanol or 30-60% isopropanol.
The eluent comprises: 10-50 mmol Tris-HCl (pH 8.0), 10-100 mmol KCl, 20-100 mmol NaCl, 10-50 mmol beta-mercaptoethanol, 3.5mmol MgCl 2 、0.1～10mmol DTT。
The enzyme reaction mixture comprises: DNA polymerase 5U/sample (5U/ul) 50ul, MMLV reverse transcriptase 200U/sample, 0.2U-1U UNG enzyme, 0.1-10 mg/BSA, 15% -50% glycerol, 0.1-1M Malus spectabilis, 2-20% mannitol, 0.5-1.5 mol/L betaine;
or the components of the enzyme reaction mixture are DNA polymerase 5U/sample (5U/ul) 50ul, heat-started reverse transcriptase 200U/sample, UNG enzyme 0.2U-1U, BSA 0.1-10 mg/BSA, glycerol 15% -50%, malus spectabilis 0.1-1M, mannitol 2-20% and betaine 0.5-1.5 mol/L.
The PCR Buffer includes: 10-50 mmol Tris-HCl (pH 7.5), 20-200 mmol NaCl, 10-50 mmol KCl, 0.1-1 mmol DTT (dithiothreitol), 1-10% formamide, 0.01-0.1% NP-40, 1-10 mmol MgCl 2 。
Example 1
A detection method of a telomerase activity gene detection kit comprises the following steps:
s1, preparation before experiment: ensuring that experimental facility equipment is provided, and the temperature in a water bath kettle is required to be calibrated and verified;
s2, preparation of a reagent: before using, adding corresponding absolute ethyl alcohol according to the mark of the reagent bottle (24.82 ml absolute ethyl alcohol is needed to be added to a 32T/box, 36.5ml absolute ethyl alcohol is needed to be added to a 48T/box, 71.54ml absolute ethyl alcohol is needed to be added to a 96T/box), and marking after the absolute ethyl alcohol is added for standby;
s3, preparation of a reagent: preparing 96-hole deep hole plate, adding sample treating liquid, magnetic bead suspension, rinsing liquid and eluent into corresponding holes of deep hole plate
S4, sample adding: adding 0.2-0.3 ml of sample to be measured into the 1 st hole site;
s5, sample extraction:
a1, placing the 96-hole deep-hole plate with the well sample in a full-automatic nucleic acid extractor, and mounting a magnetic rod sleeve;
a2, the following processes are recommended parameters of the kit 32T/box, 48T/box and 96T/box in an Au Cheng Quan automatic nucleic acid extractor or a Haifei instrument HP-Pure32 type full-automatic nucleic acid extractor, starting equipment, running an extraction program and setting parameters as follows:
a3, after the extraction is finished, the equipment is automatically reset, the deep hole plate is taken out, the magnetic rod sleeve is taken out, and the automatic extraction is finished;
a4, transferring the eluted nucleic acid solution into a new 1.5ml centrifuge tube, and preserving the sample to be detected for a short time at 4 ℃;
s6, quality control products:
negative control: taking a 1.5ml centrifuge tube, and adding 0.5ml sample treatment solution as a negative control; a reference sample processing mode is processed in a subsequent mode;
positive control: taking 50 μl of positive control, adding 450ul of sample treatment solution, and subsequently treating the reference sample treatment mode;
s7, PCR amplification: taking 8ul eluted samples in a PCR tube, adding 12ul 2 XPCR buffer, 3ul PN mix and 2ul EN mix, lightly blowing by a liquid-transfering device for 3-5 times, uniformly mixing, and sealing the tube cover on a machine;
s8, performing the following procedures on a fluorescence quantitative PCR instrument: 50 ℃ for 10min; 3min at 95 ℃;95℃for 10s,65℃for 30s,40cycles; the threshold setting principle is that the threshold line exceeds the highest point of the negative control and is in the initial stage of the exponential amplification phase of the positive control.
Interpretation of test results:
negative control (-): no Ct value or Ct value is more than or equal to 40
Positive control (+): ct value is less than or equal to 35.
Positive (+): ct value < 40; negative (-). No Ct value or Ct value is more than or equal to 40.
Invalidation: negative control Ct value <40 or positive control Ct value >35. The investigation and the re-experiment are needed. If the problem still exists, the lot number product should be taken out of service and contacted with the local supplier.
Example 2
A detection method of a telomerase activity gene detection kit comprises the following steps:
s1, preparation before experiment: ensuring that experimental facility equipment is provided, and the temperature in a water bath kettle is required to be calibrated and verified;
s2, preparation of a reagent: before using, adding corresponding absolute ethyl alcohol according to the mark of the reagent bottle (24.82 ml absolute ethyl alcohol is needed to be added to a 32T/box, 36.5ml absolute ethyl alcohol is needed to be added to a 48T/box, 71.54ml absolute ethyl alcohol is needed to be added to a 96T/box), and marking after the absolute ethyl alcohol is added for standby;
s3, preparation of a reagent: preparing 96-hole deep hole plate, adding sample treating liquid, magnetic bead suspension, rinsing liquid and eluent into corresponding holes of deep hole plate
S4, sample adding: adding 0.2-0.3 ml of sample to be measured into the 1 st hole site;
s5, sample extraction:
d1, adding 200 μl of a sample to be detected into a 1.5ml centrifuge tube, adding 300 μl of sample treatment fluid, adding 15 μl of magnetic beads, uniformly mixing and shaking for about 10 seconds, carrying out warm bath at 60 ℃ for 5-10 minutes, and carrying out warm bath on the eluent for 5-10 minutes;
d2, placing the sample on a magnetic rack for magnetic attraction for 5-30 seconds, and sucking liquid;
d3, adding 500 mul of rinsing liquid, shaking uniformly by hand for 5-10 seconds, and placing the rinsing liquid on a magnetic rack to perform magnetic attraction for 5-30 seconds to suck liquid;
d4, adding 500 mul of rinsing liquid, shaking uniformly by hand for 5-10 seconds, placing on a magnetic rack for magnetic attraction for 5-30 seconds, sucking off liquid, and airing at room temperature for 3-5 minutes or 60 ℃ for 1 minute;
d5, adding 30-100 mu l of eluent, and carrying out warm bath for 1-5 minutes at 60 ℃.
S6, quality control products:
negative control: taking a 1.5ml centrifuge tube, and adding 0.5ml sample treatment solution as a negative control; a reference sample processing mode is processed in a subsequent mode;
positive control: taking 50 μl of positive control, adding 450ul of sample treatment solution, and subsequently treating the reference sample treatment mode;
s7, PCR amplification: taking 8ul eluted samples in a PCR tube, adding 12ul 2 XPCR buffer, 3ul PN mix and 2ul EN mix, lightly blowing by a liquid-transfering device for 3-5 times, uniformly mixing, and sealing the tube cover on a machine;
s8, performing the following procedures on a fluorescence quantitative PCR instrument: 50 ℃ for 10min; 3min at 95 ℃;95℃for 10s,65℃for 30s,40cycles; the threshold setting principle is that the threshold line exceeds the highest point of the negative control and is in the initial stage of the exponential amplification phase of the positive control.
Interpretation of test results:
negative control (-): no Ct value or Ct value is more than or equal to 40
Positive control (+): ct value is less than or equal to 35.
Positive (+): ct value < 40; negative (-). No Ct value or Ct value is more than or equal to 40.
Invalidation: negative control Ct value <40 or positive control Ct value >35. The investigation and the re-experiment are needed. If the problem still exists, the use of the lot number product should be stopped.
Experimental example 1
Results of detection of telomerase reverse transcriptase subunit hTERT mRNA gene in cervical cancer (Hela) cells:
| hela cells | Ct value | Judgment result |
| 500 cells | 34.3 | Positive and negative |
| 500 cells | 28.6 | Positive and negative |
| 5000 cells | 24.5 | Positive and negative |
| 50000 cells | 20.7 | Positive and negative |
| Negative control | NO.Ct | Negative of |
Experimental example 2
Detection of telomerase reverse transcriptase subunit hTERT mRNA gene in lung cancer H1299 cells:
| h1299 cells | Ct | Judgment result | |
| 10 cells | 35.3 | Positive and negative | |
| 100 cells | 31.4 | Positive and negative | |
| 1000 cells | 28.1 | Positive and negative | |
| 10000 cells | 24.0 | Positive and negative | |
| 100000 cells | 19.5 | ||
| Negative control | NO.Ct | Negative of |
Experimental example 3
Detection of lung cancer A431 cells
| A431 cells | Ct value | Judgment result |
| 500 cells | 29.2 | Positive and negative |
| 5000 cells | 26.3 | Positive and negative |
| 50000 cells | 24.1 | Positive and negative |
| 500000 cells | 20.2 | Positive and negative |
| Negative control | NO.Ct | Negative of |
Experimental example 4
Lung cancer a549 cells
| A549 cells | Ct value | Judgment result |
| 100000 cells | 22.84 | Positive and negative |
| 10000 cells | 25.27 | Positive and negative |
| 1000 cells | 28.86 | Positive and negative |
| 100 cells | 31.88 | Positive and negative |
| 100 cells | 31.97 | Positive and negative |
Experimental example 4
Lung cancer a549 cells
| A549 cells | Ct value | Judgment result |
| 100000 cells | 21.91 | Positive and negative |
| 10000 cells | 25.27 | Positive and negative |
| 1000 cells | 28.86 | Positive and negative |
| Negative control | NO.CT | Negative of |
Sample example 1
Clinical specimens of lung cancer
| Sample of | Ct value |
| Sample 1 | |
| Sample | |
| 2 | 21.91 |
| Sample 3 | NO.CT |
| Sample 4 | 23.56 |
| Sample 5 | 31.24 |
| Sample 6 | NO.CT |
| Sample 7 | NO.CT |
| Negative control | 20.73 |
Sample example 2
Clinical specimens of lung cancer
| Sample of | Ct value |
| Sample 1 | 26.83 |
| |
27.58 |
| Sample 3 | 29.53 |
| Sample 4 | 28.69 |
| Sample 5 | 28.44 |
| Sample 6 | 29.66 |
| Sample 7 | 29.9 |
| Sample 8 | NO.CT |
| Negative control | 24.48 |
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (14)
1. A primer set for a telomerase activity gene detection kit, which is characterized in that: comprising an upstream primer, the upstream primer sequence number comprising:
Forward primer1：CTGGACGATATCCACAGGGC
Forward primer2：TGTCAAGGTGGATGTGACGG
Forward primer3：GTGGCACGGCTTTTGTTCAG
Forward primer4：GTGGCACGGCTTTTGTTCAG
Forward primer5：CAAGCTGTTTGCGGGGATTC
Forward primer6：CATCAGGGGCAAGTCCTACG
Forward primer7：AAACCTTCCTCAGCTATGCCC
Forward primer8：AACATGGACTACGTCGTGGG
Forward primer9：CGGAAGAGTGTCTGGAGCAA
Forward primer10：GTGCTACGGCGACATGGAGAAC
Forward primer11：CGACGTCTTCCTACGCTTCA
Forward primer12：AAACCTTCCTCAGCTATGCCC
Forward primer13：GTCACGGAGACCACGTTTCA
Forward primer14：TGGCACGGCTTTTGTTCAGA
Forward primer15：CGTGGTGAACTTGCGGAAGACAG
Forward primer16：TTCAGCGTGCTCAACTACGA
Forward primer17：CATGGACTACGTCGTGGGAG
Forward primer18：AGCAGAGCTCCTCCCTGAAT
Forward primer19：CAAGCTGTTTGCGGGGATTC
Forward primer20：CTGGACGATATCCACAGGGC
Forward primer21：AAACCTTCCTCAGCTATGCCC
Forward primer22：TCAGCGTGCTCAACTACGAG
Forward primer23：CATGGACTACGTCGTGGGAG。
2. the primer set for a telomerase activity gene detection kit as claimed in claim 1, wherein: also included is a downstream primer, the downstream primer sequence number comprising:
Reverse primer1：AAGTTCACCACGCAGCCATA 20
Reverse primer2：TGTCTTCCGCAAGTTCACCA 20
Reverse primer3：AGTTCACCACTGTCTTCCGC 20
Reverse prime:4：AATGGCGAATCTGGGGATGG 20
Reverse primer5：ATCTGAACAAAAGCCGTGCC 20
Reverse primer6：ATACTCAGGGACACCTCGGA 20
Reverse primer7：CCTATGTGGGGAGTGGAAGC 20
Reverse primer8：GGCCTCGTCTTCTACAGGGA 20
Reverse primer9：CCATACTCAGGGACACCTCG 20
Reverse primer10：GTTGAAGGTGAGACTGGCTCTGATG 25
Reverse primer11：CCAGGGCCTCGTCTTCTACA 20
Reverse primer12：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer13：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer14：AGGGGTGAACAATGGCGAAT 20
Reverse primer15：TGACGCGCAGGAAAAATGTG 20
Reverse primer16：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer17：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer18：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer19：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer20：GGGCATAGCTGAGGAAGGTTT 21
Reverse primer21：CTATGTGGGGAGTGGAAGCC 20。
3. the primer set for a telomerase activity gene detection kit according to claim 1 or 2, further comprising:
10～100mmol Tris-HCl(pH8.0～8.8)；
0.1-2. Mu. Mol probe primer or 0.5-1 XSYBR Green dye;
0.1～10μmol Oligo dT15；
0.1～10μmol Random；
0.05～1mmol EDTA；
0.2～4mmol dNTP；
1～20mmol dUTP；
wherein the content of the upstream primer is 0.1-2 mu mol, and the content of the downstream primer is 0.1-2 mu mol.
4. A probe primer for a telomerase activity gene detection kit, characterized in that the probe primer sequence number comprises:
fluorescent group-AACGCAGGAGCAGCCCGTCCCGCCG-quenching group
Fluorescent group-CGAGGGTGAAGGCACTGTTCAGCG-quenching group
Fluorescent group-GCTGCTCCTGCGTTTGGTGGATGAT-quenching group
Fluorescent group-TTCCCCTGGTGCGGCCTGCTGCTG-quenching group
Fluorescent group-CGAGGGTGAAGGCACTGTTCAGCG-quenching group
Fluorescent group-GACTACGTCGTGGGAGCCAGAACG-quenching group
Fluorescent group-GGCCTCCCTCTGCTACTCCATCCTGA-quenching group
Fluorescent group-CAGCCCGTCCCGCCGAATCCCC-quenching group
Fluorescent group-GACAGCCCAGACGCAGCTGAGTCGG-quenching group
Fluorescent group-TATTCCCCTGGTGCGGCCTGCTG-quenching group
Fluorescent group-TCGTGGTGAACTTGCGGAAGACAGTG-quenching group.
5. A telomerase activity gene assay kit according to any of claims 1 to 4, wherein: comprises sample treatment solution, rinsing solution, eluent, magnetic bead suspension, PCR buffer, primer group and enzyme reaction mixed solution.
6. The telomerase activity gene assay kit of claim 6, wherein: the sample treatment liquid bagThe method comprises the following steps: 20-200 mmol Na 2 HPO 4 、20～200mmol NaH 2 HPO 4 10-100 mmol NaAC, 2%2-ME, 0.1-1 mmol DTT (dithiothreitol), 2-6 mol GuHCl, 0.05% Triton X-100, 0.01-5 mg/ml proteinase K.
7. The telomerase activity gene assay kit of claim 6, wherein: the sample processing liquid comprises: 10-200 mmol Na 2 HPO 4 、10～200mmol NaH 2 HPO 4 2 to 50mmol of sodium citrate, 2 percent of beta-mercaptoethanol, 0.1 to 1mmol of DTT (dithiothreitol), 2 to 6mol of GuHCl or 2 to 6mol of guanidine isothiocyanate, 1 to 50mmol of vanadyl riboside complex, 1 percent of Triton X-100 and 0.01 to 5mg/ml of proteinase K.
8. The telomerase activity gene assay kit of claim 6, wherein: the rinse solution comprises: 0.05 to 0.1mol NaH 2 PO 4 、0.05～0.1mol Na 2 HPO 4 0.1-1 mmol DTT (dithiothreitol) or 0.1-10 mmol beta-mercaptoethanol, 60-80% ethanol or 30-60 isopropanol.
9. The telomerase activity gene assay kit of claim 6, wherein: the eluent comprises: 10-50 mmol Tris-HCl (pH 8.0), 10-100 mmol KCl, 20-100 mmol NaCl, 10-50 mmol beta-mercaptoethanol, 3.5mmol MgCl 2 、0.1～10mmol DTT。
10. The telomerase activity gene assay kit of claim 6, wherein: the enzyme reaction mixture comprises: DNA polymerase 5U/sample (5U/ul) 50ul, MMLV reverse transcriptase 200U/sample or hot start reverse transcriptase 200U/sample, 0.2U-1U UNG enzyme, 0.1-10 mg/BSA, 15% -50% glycerol, 0.1-1M Malus spectabilis, 2-20% mannitol, 0.5-1.5 mol/L betaine.
11. A telomere according to claim 6The enzyme activity gene detection kit is characterized in that: the PCR Buffer includes: 10-50 mmol Tris-HCl (pH 7.5), 20-200 mmol NaCl, 10-50 mmol KCl, 0.1-1 mmol DTT (dithiothreitol), 1-10% formamide, 0.01-0.1% NP-40, 1-10 mmol MgCl 2 。
12. The detection method of the telomerase activity gene detection kit is characterized by comprising the following steps of:
s1, preparation before experiment: ensuring that the experimental facility equipment is provided;
s2, preparation of a reagent: before using, adding corresponding absolute ethyl alcohol according to the mark of the reagent bottle, and marking after the absolute ethyl alcohol is added for standby;
s3, preparation of a reagent: preparing 96-hole deep hole plate, adding sample treating liquid, magnetic bead suspension, rinsing liquid and eluent into corresponding holes of deep hole plate
S4, sample adding: adding 0.2-0.3 ml of sample to be measured into the 1 st hole site;
s5, sample extraction: the full-automatic nucleic acid extractor extracts samples or manually extracts samples;
s6, quality control products:
negative control: taking a 1.5ml centrifuge tube, and adding 0.5ml sample treatment solution as a negative control; a reference sample processing mode is processed in a subsequent mode;
positive control: taking 50 μl of positive control, adding 450ul of sample treatment solution, and subsequently treating the reference sample treatment mode;
s7, PCR amplification: taking 8ul eluted samples in a PCR tube, adding 12ul 2 XPCR buffer, 3ul PN mix and 2ul EN mix, lightly blowing by a liquid-transfering device for 3-5 times, uniformly mixing, and sealing the tube cover on a machine;
s8, performing the following procedures on a fluorescence quantitative PCR instrument: 50 ℃ for 10min; 3min at 95 ℃;95℃for 10s,65℃for 30s,40cycles; the threshold setting principle is that the threshold line exceeds the highest point of the negative control and is in the initial stage of the exponential amplification phase of the positive control.
13. The method according to claim 12, wherein the step of extracting the sample by the fully automatic nucleic acid extractor in the step S5 is as follows:
a1, placing the 96-hole deep-hole plate with the well sample in a full-automatic nucleic acid extractor, and mounting a magnetic rod sleeve;
a2, starting the equipment, running the extraction program, and setting parameters as follows:
a3, after the extraction is finished, the equipment is automatically reset, the deep hole plate is taken out, the magnetic rod sleeve is taken out, and the automatic extraction is finished;
a4, transferring the eluted nucleic acid solution into a new 1.5ml centrifuge tube, and preserving the sample to be measured at 4 ℃ in a short time.
14. The method according to claim 12, wherein the step of manually extracting the sample in step S5 is as follows:
d1, adding 200 μl of a sample to be detected into a 1.5ml centrifuge tube, adding 300 μl of sample treatment fluid, adding 15 μl of magnetic beads, uniformly mixing and shaking for about 10 seconds, carrying out warm bath at 60 ℃ for 5-10 minutes, and carrying out warm bath on the eluent for 5-10 minutes;
d2, placing the sample on a magnetic rack for magnetic attraction for 5-30 seconds, and sucking liquid;
d3, adding 500 mul of rinsing liquid, shaking uniformly by hand for 5-10 seconds, and placing the rinsing liquid on a magnetic rack to perform magnetic attraction for 5-30 seconds to suck liquid;
d4, adding 500 mul of rinsing liquid, shaking uniformly by hand for 5-10 seconds, placing on a magnetic rack for magnetic attraction for 5-30 seconds, sucking off liquid, and airing at room temperature for 3-5 minutes or 60 ℃ for 1 minute;
d5, adding 30-100 mu l of eluent, and carrying out warm bath for 1-5 minutes at 60 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211075535.6A CN116042792A (en) | 2022-09-05 | 2022-09-05 | Telomerase activity gene detection kit, primer set, probe primer and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211075535.6A CN116042792A (en) | 2022-09-05 | 2022-09-05 | Telomerase activity gene detection kit, primer set, probe primer and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116042792A true CN116042792A (en) | 2023-05-02 |
Family
ID=86112208
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211075535.6A Pending CN116042792A (en) | 2022-09-05 | 2022-09-05 | Telomerase activity gene detection kit, primer set, probe primer and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116042792A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000046601A1 (en) * | 1999-02-02 | 2000-08-10 | Frank Larsen | Detecting telomerase activity |
| CN102816867A (en) * | 2012-08-30 | 2012-12-12 | 湖南圣湘生物科技有限公司 | Method for detecting coxsackievirus A16 RNA (ribose nucleic acid) and reagent kit |
| CN103642941A (en) * | 2013-12-12 | 2014-03-19 | 东北制药集团辽宁生物医药有限公司 | Quantitative detection kit of hepatitis B virus (HBV) nucleic acid |
| CN104593505A (en) * | 2015-01-23 | 2015-05-06 | 上海迪安医学检验所有限公司 | Kit for detecting activity degree of telomerase in whole blood or serum sample |
| CN110117644A (en) * | 2019-04-18 | 2019-08-13 | 王金固 | A kind of gene tester for RNA in biological sample |
| CN112921068A (en) * | 2021-02-25 | 2021-06-08 | 中国人民解放军军事科学院军事医学研究院 | Method for detecting telomerase and special kit thereof |
| CN114634925A (en) * | 2020-12-15 | 2022-06-17 | 深圳市帝迈生物技术有限公司 | Nucleic acid extraction kit and method for extracting nucleic acid |
| CN114657231A (en) * | 2022-04-27 | 2022-06-24 | 珠海宝锐生物科技有限公司 | Rapid DNA extraction kit with magnetic beads for fluorescent quantitative PCR detection and extraction method thereof |
-
2022
- 2022-09-05 CN CN202211075535.6A patent/CN116042792A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000046601A1 (en) * | 1999-02-02 | 2000-08-10 | Frank Larsen | Detecting telomerase activity |
| CN102816867A (en) * | 2012-08-30 | 2012-12-12 | 湖南圣湘生物科技有限公司 | Method for detecting coxsackievirus A16 RNA (ribose nucleic acid) and reagent kit |
| CN103642941A (en) * | 2013-12-12 | 2014-03-19 | 东北制药集团辽宁生物医药有限公司 | Quantitative detection kit of hepatitis B virus (HBV) nucleic acid |
| CN104593505A (en) * | 2015-01-23 | 2015-05-06 | 上海迪安医学检验所有限公司 | Kit for detecting activity degree of telomerase in whole blood or serum sample |
| CN110117644A (en) * | 2019-04-18 | 2019-08-13 | 王金固 | A kind of gene tester for RNA in biological sample |
| CN114634925A (en) * | 2020-12-15 | 2022-06-17 | 深圳市帝迈生物技术有限公司 | Nucleic acid extraction kit and method for extracting nucleic acid |
| CN112921068A (en) * | 2021-02-25 | 2021-06-08 | 中国人民解放军军事科学院军事医学研究院 | Method for detecting telomerase and special kit thereof |
| CN114657231A (en) * | 2022-04-27 | 2022-06-24 | 珠海宝锐生物科技有限公司 | Rapid DNA extraction kit with magnetic beads for fluorescent quantitative PCR detection and extraction method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 郑怀竞: "临床基因诊断实验指南", 25-26, pages: 25 - 26 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104099425B (en) | A kind of test kit for detecting B-raf transgenation | |
| CN110724741B (en) | Primer, probe and kit for detecting minimal residual leukemia related fusion gene | |
| CN106399462A (en) | BCR-ABL fusion gene amplification kit and BCR-ABL fusion gene detection kit | |
| CN103333958B (en) | Method for detecting telomerase through washing-free anchored-extension and telomeric-binding amplification and kit | |
| JP2022130509A (en) | Analytical method and kit | |
| CN102094074A (en) | Fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting leukemia fusion gene TEL-AML1 | |
| CN113718021A (en) | Primer, probe and kit for quantitatively detecting BCR-ABL1 fusion gene | |
| CN106148484B (en) | A kind of kit that diagnosis Y chromosome is micro-deleted | |
| CN108624697A (en) | A kind of 21 gene detecting kit of breast cancer and its detection method | |
| CN109251964B (en) | Circulating microRNAs detection kit, method for specifically detecting circulating microRNAs and application | |
| US20230304081A1 (en) | Primer and probe design method, detection composition, and kit for mirna detection | |
| JPWO2018199137A1 (en) | Method for detecting minor BCR-ABL1 gene | |
| CN117448456A (en) | Molecular target, probe combination and kit for identifying benign and malignant salivary gland tumor and application of molecular target and probe combination | |
| CN116042792A (en) | Telomerase activity gene detection kit, primer set, probe primer and detection method | |
| CN106868217A (en) | A kind of detection primer of zika virus and application | |
| CN106148500B (en) | Integrated micro RNA fluorescent quantitative detection kit and application thereof | |
| CN1840660B (en) | Method of determining dihydropyrimidine dehydrogenase gene expression | |
| CN108728538B (en) | ALK gene fusion detection primer, method and kit | |
| CN114525349A (en) | Method for identifying semen by using digital PCR and special kit thereof | |
| CN107012241B (en) | Triple RT-qPCR detection method and kit for U6, miR92a and miR21 | |
| CN108531598A (en) | ROS1 Gene Fusions detection primer, method and kit | |
| CN104087665B (en) | A kind of universal primer and real-time fluorescence tHDA test kit detecting flavus and Aspergillus fumigatus | |
| CN106811537A (en) | One kind detection epidermal growth factor receptor gene T790M low frequencies mutant primer and its application | |
| CN102899390A (en) | Small cell lung cancer markers and their detection | |
| US11021756B2 (en) | MiRNA markers for the diagnosis of osteosarcoma |
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 |