CN105385770B - The Dual-ring hairpin probe for detecting bleomycin mediates label-free strand displacement amplification method - Google Patents
The Dual-ring hairpin probe for detecting bleomycin mediates label-free strand displacement amplification method Download PDFInfo
- Publication number
- CN105385770B CN105385770B CN201510961410.7A CN201510961410A CN105385770B CN 105385770 B CN105385770 B CN 105385770B CN 201510961410 A CN201510961410 A CN 201510961410A CN 105385770 B CN105385770 B CN 105385770B
- Authority
- CN
- China
- Prior art keywords
- probe
- bleomycin
- sequence
- neck
- dual
- 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.)
- Expired - Fee Related
Links
- 239000000523 sample Substances 0.000 title claims abstract description 99
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 title claims abstract description 61
- 108010006654 Bleomycin Proteins 0.000 title claims abstract description 54
- 229960001561 bleomycin Drugs 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 30
- 230000003321 amplification Effects 0.000 title claims abstract description 29
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 29
- 238000006073 displacement reaction Methods 0.000 title claims abstract description 23
- 238000006243 chemical reaction Methods 0.000 claims abstract description 33
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 230000009471 action Effects 0.000 claims abstract description 7
- 125000002619 bicyclic group Chemical group 0.000 claims description 12
- 239000007850 fluorescent dye Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 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 5
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000000975 dye Substances 0.000 claims description 2
- 230000001960 triggered effect Effects 0.000 claims description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims 2
- 108091008146 restriction endonucleases Proteins 0.000 claims 2
- 238000003776 cleavage reaction Methods 0.000 abstract description 12
- 108091081406 G-quadruplex Proteins 0.000 abstract description 9
- 230000001404 mediated effect Effects 0.000 abstract description 5
- 238000011896 sensitive detection Methods 0.000 abstract description 5
- 102000007260 Deoxyribonuclease I Human genes 0.000 abstract description 4
- 108010008532 Deoxyribonuclease I Proteins 0.000 abstract description 4
- 230000007017 scission Effects 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 description 28
- 238000002474 experimental method Methods 0.000 description 8
- 239000000126 substance Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 5
- 239000013256 coordination polymer Substances 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 239000003068 molecular probe Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 3
- 238000004891 communication Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 108010092160 Dactinomycin Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 229930192392 Mitomycin Natural products 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 101710147059 Nicking endonuclease Proteins 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 229960000640 dactinomycin Drugs 0.000 description 2
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 2
- 229960000975 daunorubicin Drugs 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000011898 label-free detection Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 229960004857 mitomycin Drugs 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical class NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 1
- 108010052418 (N-(2-((4-((2-((4-(9-acridinylamino)phenyl)amino)-2-oxoethyl)amino)-4-oxobutyl)amino)-1-(1H-imidazol-4-ylmethyl)-1-oxoethyl)-6-(((-2-aminoethyl)amino)methyl)-2-pyridinecarboxamidato) iron(1+) Proteins 0.000 description 1
- 244000064895 Cucumis melo subsp melo Species 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 206010029155 Nephropathy toxic Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 206010061924 Pulmonary toxicity Diseases 0.000 description 1
- 241000082085 Verticillium <Phyllachorales> Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 108700004675 bleomycetin Proteins 0.000 description 1
- QYOAUOAXCQAEMW-UTXKDXHTSA-N bleomycin A5 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCCNCCCCN)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C QYOAUOAXCQAEMW-UTXKDXHTSA-N 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012938 design process Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 231100000371 dose-limiting toxicity Toxicity 0.000 description 1
- 229960002089 ferrous chloride Drugs 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000007274 generation of a signal involved in cell-cell signaling Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 231100000374 pneumotoxicity Toxicity 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 208000005069 pulmonary fibrosis Diseases 0.000 description 1
- 230000007047 pulmonary toxicity Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种检测博来霉素的双环发夹探针介导免标记链置换扩增方法,当博来霉素存在时,双环发夹探针在识别位点处断裂,并释放触发序列;然后,触发序列与信号探针的环部相结合,并在聚合酶和切刻酶的作用下,发生链置换扩增反应,最终产生大量的G‑四链体序列。与此同时,引物链延伸将信号探针打开,信号探针颈部封存的G‑四链体序列得以暴露。最后,G‑四链体序列绑定NMM分子,产生荧光信号,通过检测到的荧光信号对博来霉素进行定量。本发明通过将触发序列设计在双环发夹探针的颈部,降低检测体系的背景信号;将博来霉素切割与链置换扩增反应相结合,实现对博来霉素的灵敏检测,检测限为0.34nM。该方法具有操作简单,免标记以及特异性好的优势。
The invention discloses a double-loop hairpin probe-mediated label-free strand displacement amplification method for detecting bleomycin. When the bleomycin exists, the double-loop hairpin probe breaks at the recognition site and releases a trigger sequence; then, the trigger sequence is combined with the ring part of the signal probe, and under the action of polymerase and nickase, a strand displacement amplification reaction occurs, and finally a large number of G-quadruplex sequences are generated. At the same time, primer strand extension opens the signal probe, exposing the G-quadruplex sequence sequestered in the neck of the signal probe. Finally, the G-quadruplex sequence binds the NMM molecule, generating a fluorescent signal, which is used to quantify the bleomycin. The invention reduces the background signal of the detection system by designing the trigger sequence at the neck of the double-loop hairpin probe; combines the bleomycin cleavage with the strand displacement amplification reaction to realize the sensitive detection of the bleomycin, the detection The limit is 0.34nM. This method has the advantages of simple operation, label-free and good specificity.
Description
技术领域technical field
本发明涉及一种博来霉素的检测方法,尤其涉及一种检测博来霉素的双环发夹探针介导免标记链置换扩增方法。The invention relates to a detection method for bleomycin, in particular to a double-loop hairpin probe-mediated label-free strand displacement amplification method for detection of bleomycin.
背景技术Background technique
博来霉素是由链霉素轮枝杆菌分泌产生的一类糖肽类抗生素。临床上,博来霉素是一种重要的抗癌药物,主要用于治疗鳞状细胞癌以及巨噬细胞瘤等多种疾病(R.H.Blum,S.K.Carter and K.Agre,Cancer,1973,31,903;M.Froudarakis,E.Hatzimichael,L.Kyriazopoulou,K.Lagos,P.Pappas,A.G.Tzakos,V.Karavasilis,D.Daliani,C.Papandreou and E.Briasoulis,Critical Reviews in Oncology/Hematology,2013,87,90;Y.Akiyama,Q.Ma,E.Edgar,A.Laikhter and S.Hecht,Journal of the AmericanChemical Society,2008,130,9650;R.A.Giroux and S.M.Hecht,Journal of theAmerican Chemical Society,2010,132,16987;Z.Yu,R.Schmaltz,T.Bozeman,R.Paul,M.Rishel,K.Tsosie and S.Hecht,Journal of the American Chemical Society,2013,135,2883.)。但是,应用博来霉素治疗多种癌症,常常伴随产生剂量限制性毒性,比如肾毒性,肺毒性以及肺纤维化等(S.Sleijfer,Chest,2001,120,617;J.Hay,S.Shahzeidi andG.Laurent,Archives of Toxicology,1991,65,81.)。因此,为了监测博来霉素浓度,降低毒性,获得最好的治疗效果,如何构建一种博来霉素检测方法成为分析工作者研究的主要内容。目前,研究者们建立了多种检测方法用于博来霉素的定量分析,比如高效液相色谱法(R.P.Klett,J.P.Chovana and I.H.Danse,Journal of Chromatography:BiomedicalSciences and Application,1984,310,361;G.K.Shiu and T.J.Goehl,Journal ofChromatography B:Biomedical Sciences and Applications,1980,181,127.),酶免疫分析法(K.Fujiwara,M.Yasuno and T.Kitagawa,Cancer Research,1981,41,4121)、放射免疫分析法(J.Teale,J.Clough and V.Marks,British Journal of Cancer,1977,35,822;A.Broughton and J.E.Strong,Cancer Research,1976,36,1418.)、以及微生物分析法(T.Ohnuma,J.F.Holland,H.Masuda,J.A.Waligunda and G.A.Goldberg,Cancer,33,1230.)等。但是,这些方法通常具有费时,费力,危害人体健康以及成本较高等缺点。因此,需要建立一种新的、灵敏的、特异的检测方法,为临床博来霉素浓度监测提供一种更加简单便捷的分析方案。Bleomycin is a class of glycopeptide antibiotics secreted by Verticillium streptomycin. Clinically, bleomycin is an important anticancer drug, mainly used in the treatment of various diseases such as squamous cell carcinoma and macrophage tumor (R.H.Blum, S.K.Carter and K.Agre, Cancer, 1973, 31, 903; M. Froudarakis, E. Hatzimichael, L. Kyriazopoulou, K. Lagos, P. Pappas, A. G. Tzakos, V. Karavasilis, D. Daliani, C. Papandreou and E. Briasoulis, Critical Reviews in Oncology/Hematology, 2013, 87, 90; Y.Akiyama, Q.Ma, E.Edgar, A.Laikhter and S.Hecht, Journal of the American Chemical Society, 2008, 130, 9650; R.A.Giroux and S.M.Hecht, Journal of the American Chemical Society, 2010, 132, 16987; Z. Yu, R. Schmaltz, T. Bozeman, R. Paul, M. Rishel, K. Tsosie and S. Hecht, Journal of the American Chemical Society, 2013, 135, 2883.). However, the application of bleomycin to treat a variety of cancers is often accompanied by dose-limiting toxicities, such as nephrotoxicity, pulmonary toxicity, and pulmonary fibrosis (S.Sleijfer, Chest, 2001, 120, 617; J.Hay, S. Shahzeidi and G. Laurent, Archives of Toxicology, 1991, 65, 81.). Therefore, in order to monitor the concentration of bleomycin, reduce toxicity, and obtain the best therapeutic effect, how to construct a detection method for bleomycin has become the main research content of analysts. At present, researchers have established a variety of detection methods for the quantitative analysis of bleomycin, such as high performance liquid chromatography (R.P.Klett, J.P.Chovana and I.H.Danse, Journal of Chromatography:BiomedicalSciences and Applications, 1984,310,361 ; G.K.Shiu and T.J.Goehl, Journal of Chromatography B: Biomedical Sciences and Applications, 1980, 181, 127.), enzyme immunoassay (K.Fujiwara, M.Yasuno and T.Kitagawa, Cancer Research, 1981, 41, 4121) , radioimmunoassay (J.Teale, J.Clough and V.Marks, British Journal of Cancer, 1977, 35, 822; A.Broughton and J.E.Strong, Cancer Research, 1976, 36, 1418.), and microbial analysis (T.Ohnuma, J.F.Holland, H.Masuda, J.A.Waligunda and G.A.Goldberg, Cancer, 33, 1230.) and so on. However, these methods usually have the disadvantages of time-consuming, laborious, harmful to human health and high cost. Therefore, a new, sensitive and specific detection method needs to be established to provide a simpler and more convenient analysis solution for clinical bleomycin concentration monitoring.
据报道,在氧气以及具有氧化还原活性的金属离子存在时,博来霉素能够通过氧化脱氧核苷酸的方法切割DNA链,其主要识别位点为5’-GT-3’以及5’-GC-3’(Q.Ma,Y.Akiyama,Z.D.Xu,K.Konishi and S.M.Hecht,Journal of the American ChemicalSociety,2009,131,2013;J.Chen,M.K.Ghorai,G.Kenney and J.Stubbe,Nucleic AcidResearch,2008,36,3781.)。基于博来霉素切割DNA链这一性质,研究者们设计了各种识别博来霉素的DNA分子探针,以构建一种简单灵敏的博来霉素检测方法。目前,这些DNA分子探针主要分为两类。第一类是信号分子结合或标记的单链或发夹DNA(B.C.Yin,D.Wu andB.C.Ye,Analytical Chemistry,2010,82,8272;Y.Li,C.C.Huang,J.B.Zheng,H.L.Qi,W.Cao and Y.M.Wei,Biosensors and Bioeletronics,2013,44,177;Y.F.Qin,Y.F.Ma,X.Jin,L.L.Zhang,G.J.Ye and S.L.Zhao,Analytica Chimica Acta,2015,866,84;F.Li,Y.Feng,C.Zhao,P.Li and B.Tang,Chemical Communications,2012,48,127.)。这类探针与博来霉素作用以后,能够释放信号分子,并产生相应的信号响应。第二类是具有触发序列的功能颈-环DNA结构(F.L.Gao,J.P.Lei and H.X.Ju,Chemical Communications,2013,49,7561.)。这类探针与博来霉素作用以后,能够释放触发序列并触发信号扩增反应,将一次裂解事件表达为多次重复的信号产生事件。因此,这类DNA分子探针更有利于提高检测方法的信号强度和灵敏度。Gao等人设计了一个颈部带有两个5’-GC-3’识别位点,环部为一段触发序列的功能发夹探针,用于博来霉素的高灵敏检测(F.L.Gao,J.P.Lei and H.X.Ju,Chemical Communications,2013,49,7561.)。然而,由于触发序列位于探针的环部,未被切割的DNA分子探针与后续反应链之间的非特异性杂交,会引起相对较高的背景信号以及假阳性信号,这在一定程度上限制了该探针的应用。It is reported that in the presence of oxygen and metal ions with redox activity, bleomycin can cleave DNA strands by oxidizing deoxynucleotides, and its main recognition sites are 5'-GT-3' and 5'- GC-3'(Q.Ma, Y.Akiyama, Z.D.Xu, K.Konishi and S.M.Hecht, Journal of the American Chemical Society, 2009, 131, 2013; J.Chen, M.K.Ghorai, G.Kenney and J.Stubbe, Nucleic Acid Research, 2008, 36, 3781.). Based on the property that bleomycin cuts DNA strands, researchers designed various DNA molecular probes that recognize bleomycin to construct a simple and sensitive detection method for bleomycin. Currently, these DNA molecular probes are mainly divided into two categories. The first category is single-stranded or hairpin DNA bound or labeled by signaling molecules (B.C.Yin, D.Wu and B.C.Ye, Analytical Chemistry, 2010, 82, 8272; Y.Li, C.C.Huang, J.B.Zheng, H.L.Qi , W.Cao and Y.M.Wei, Biosensors and Bioeletronics, 2013, 44, 177; Y.F.Qin, Y.F.Ma, X.Jin, L.L.Zhang, G.J.Ye and S.L.Zhao, Analytica Chimica Acta, 2015, 866, 84; F.Li , Y. Feng, C. Zhao, P. Li and B. Tang, Chemical Communications, 2012, 48, 127.). After this type of probe interacts with bleomycin, it can release signal molecules and generate corresponding signal responses. The second category is a functional neck-loop DNA structure with a trigger sequence (F.L.Gao, J.P.Lei and H.X.Ju, Chemical Communications, 2013, 49, 7561.). After this type of probe interacts with bleomycin, it can release the trigger sequence and trigger the signal amplification reaction, expressing a cleavage event as multiple repeated signal generation events. Therefore, this kind of DNA molecular probe is more conducive to improving the signal intensity and sensitivity of the detection method. Gao et al. designed a functional hairpin probe with two 5'-GC-3' recognition sites in the neck and a trigger sequence in the loop for highly sensitive detection of bleomycin (F.L.Gao, J.P.Lei and H.X.Ju, Chemical Communications, 2013, 49, 7561.). However, since the trigger sequence is located in the loop of the probe, non-specific hybridization between the uncleaved DNA molecular probe and the subsequent reaction chain will cause relatively high background signals and false positive signals, which limits the application of this probe.
发明内容Contents of the invention
本发明的目的就是为了解决上述问题,提供一种检测博来霉素的双环发夹探针介导免标记链置换扩增方法。The object of the present invention is to solve the above problems and provide a double-loop hairpin probe-mediated label-free strand displacement amplification method for detecting bleomycin.
为了实现上述目的,本发明采用如下技术方案:In order to achieve the above object, the present invention adopts the following technical solutions:
一种识别和检测博来霉素的双环发夹探针,所述双环发夹探针包括:两个环部、识别位点和触发位点,所述两个环部为:具有一个DNA臂的末端环和具有两个DNA臂的颈凸环,所述识别位点位于双环发夹的颈部,所述触发序列用于触发后续扩增放大反应,所述触发序列中的碱基参与构成双环发夹探针的DNA臂和颈凸环。A double-ring hairpin probe that recognizes and detects bleomycin, the double-ring hairpin probe includes: two loops, a recognition site and a trigger site, the two loops are: having a DNA arm The terminal loop and the neck convex loop with two DNA arms, the recognition site is located at the neck of the double-loop hairpin, the trigger sequence is used to trigger the subsequent amplification reaction, and the bases in the trigger sequence participate in the formation of DNA arms and neck loops of a double loop hairpin probe.
一种检测博来霉素的双环发夹探针介导免标记链置换扩增方法,当目标物博来霉素存在时,双环发夹探针在识别位点处发生断裂,并释放触发序列;然后,触发序列与信号探针的环部相结合,并在聚合酶和切刻酶的作用下,发生链置换扩增反应,产生大量的荧光染料结合序列,与此同时,引物链延伸将信号探针打开,信号探针颈部封存的荧光染料结合序列得以暴露,最后,荧光染料结合序列绑定荧光分子,产生荧光信号,通过检测到的荧光信号对博来霉素进行定量;所述双环发夹探针如权利要求1中所述。A double-ring hairpin probe-mediated label-free strand displacement amplification method for detecting bleomycin. When the target bleomycin exists, the double-ring hairpin probe breaks at the recognition site and releases the trigger sequence Then, the trigger sequence is combined with the ring portion of the signal probe, and under the action of the polymerase and the nickase, a strand displacement amplification reaction occurs to generate a large amount of fluorescent dye binding sequences. At the same time, the primer chain extension will The signal probe is opened, and the fluorescent dye-binding sequence sealed in the neck of the signal probe is exposed. Finally, the fluorescent dye-binding sequence binds to the fluorescent molecule to generate a fluorescent signal, and the bleomycin is quantified by the detected fluorescent signal; The double loop hairpin probe is as described in claim 1.
优选:所述信号探针颈部的荧光染料结合序列为G-四链体序列,所述荧光分子为NMM。Preferably: the fluorescent dye binding sequence at the neck of the signal probe is a G-quadruplex sequence, and the fluorescent molecule is NMM.
优选:所述识别位点为5’-GTGC-3’。Preferably: the recognition site is 5'-GTGC-3'.
优选:所述触发序列为5’-GAGGAAGAAGAGAGGGAAGGA-3’(如SEQ ID No:1所示)。Preferably: the trigger sequence is 5'-GAGGAAGAAGAGAGGGAAGGA-3' (as shown in SEQ ID No: 1).
优选:所述双环发夹探针的序列为TCC TTC CCT CTC AAA ACC TCG CAC CAA AAGGTG CGA GGA AGA AGA GAG GGA AGG A(如SEQ ID No:2所示)。Preferably: the sequence of the double-loop hairpin probe is TCC TTC CCT CTC AAA ACC TCG CAC CAA AAGGTG C GA GGA AGA AGA GAG GGA AGGA (as shown in SEQ ID No: 2).
荧光探针的序列为CCC AAC CCG CCC TAC CCT TTT TGA TCC TTC CCT CTC TTCTTC CTC CCT CAA AAA GGG TAG GGC GGG TTG GG(如SEQ ID No:3所示)。The sequence of the fluorescent probe is CCC AAC CCG CCC TAC CCT TTT T GA TCC TTC CCT CTC TTCTTC CTC CCT CAA AAA GGG TAG GGC GGG TTG GG (as shown in SEQ ID No: 3).
优选:链置换扩增反应的具体步骤为:取1μL 2mM dNTPs,1U KF聚合酶,4UNt.AlwI,3μL 0.75μM SP and 2μL 10×NEB buffer2(10mM Tris-HCl,10mM MgCl2,50mMNaCl,1.0mM dithiothreitol,pH 7.9)加入上述反应体系,37℃下反应40分钟,即可。Preferably: the specific steps of the strand displacement amplification reaction are: take 1 μL 2mM dNTPs, 1U KF polymerase, 4UNt.AlwI, 3 μL 0.75 μM SP and 2 μL 10×NEB buffer2 (10mM Tris-HCl, 10mM MgCl 2 , 50mMNaCl, 1.0mM dithiothreitol, pH 7.9) was added to the above reaction system, and reacted at 37°C for 40 minutes.
优选:所述扩增方法中,双环发夹探针为100nM,信号探针为75nM,DNA聚合酶为1U,切刻内切酶为4U,NMM浓度为6μM。Preferably: in the amplification method, the bicyclic hairpin probe is 100 nM, the signal probe is 75 nM, the DNA polymerase is 1 U, the nicking endonuclease is 4 U, and the NMM concentration is 6 μM.
一种检测博来霉素的试剂盒,包括双环发夹探针和信号探针,所述双环发夹探针如权利要求1中所述;所述信号探针为发夹结构,所述信号探针的颈部具有荧光染料结合序列;所述双环发夹探针的触发序列与信号探针的环部序列配对,荧光分子,DNA聚合酶,切刻内切酶,10×NEB buffer2(10mM Tris-HCl,10mM MgCl2,50mM NaCl,1.0mMdithiothreitol,pH 7.9)。A test kit for detecting bleomycin, comprising a double-ring hairpin probe and a signal probe, the double-ring hairpin probe as described in claim 1; the signal probe is a hairpin structure, and the signal The neck of the probe has a fluorescent dye binding sequence; the trigger sequence of the double-loop hairpin probe is paired with the loop sequence of the signal probe, fluorescent molecules, DNA polymerase, nicking endonuclease, 10×NEB buffer2 (10mM Tris-HCl, 10 mM MgCl 2 , 50 mM NaCl, 1.0 mM dithiothreitol, pH 7.9).
本发明的有益效果:Beneficial effects of the present invention:
本发明构建了一种双环发夹探针,用于博来霉素的识别和检测。双环发夹探针主要包含两个区域:博来霉素5’-GTGC-3’识别位点和触发序列。此外,双环发夹探针具有两个环部,其中具有两个DNA臂的环称为颈凸环。与现有技术中的功能发夹探针不同,双环发夹探针的触发序列以半封闭的模式封存在探针的颈部,有效避免了非特异性杂交。半封闭模式,即触发序列中大部分碱基参与构成颈凸环两侧的DNA臂,仅有一小部分碱基构成颈凸环(例如本发明实施例中用到的双环发夹探针的触发序列为:5’-GAGGAAGAAGAGAGGGAAGGA-3’,仅仅下划线的部分参与颈凸环,其余构成两侧的DNA臂)。这种设计模式具有两个突出优势。一方面,双环发夹探针利用分子内杂交的方式沉默触发序列,有效避免非特异性杂交。另一方面,双环发夹探针发生切割反应以后,生成一个中间带有颈凸环的双螺旋结构;由于颈凸环降低了双螺旋结构的稳定性,触发序列与其不完全互补链发生解旋,然后以足够多的碱基数与后续反应链生成稳定的双螺旋,最终产生明显的阳性信号。基于这一双环发夹探针,并结合链置换扩增(SDA)反应,本发明构建了一个新型荧光传感平台用于博来霉素检测。该方法通过链置换扩增反应产生大量DNA单链以及G-四链体/NMM复合物发射荧光,首次实现了基于扩增技术的博来霉素免标记检测。通过单链DNA产物以及打开的信号探针绑定NMM染料分子产生增强的荧光信号,该方法表现出优越的灵敏度。方法的检测限为0.34nM,该方法具有操作简单,免标记以及特异性好的优势。此外,实验进行了人血清回收率实验,结果表明该检测方法具有较大的临床应用潜能。The invention constructs a double-circle hairpin probe for the recognition and detection of bleomycin. The double loop hairpin probe mainly contains two regions: bleomycin 5'-GTGC-3' recognition site and trigger sequence. In addition, a double-loop hairpin probe has two loop portions, and the loop with two DNA arms is called a neck loop. Different from the functional hairpin probes in the prior art, the trigger sequence of the double-loop hairpin probe is sealed in the neck of the probe in a semi-closed mode, effectively avoiding non-specific hybridization. Semi-closed mode, that is, most of the bases in the trigger sequence participate in forming the DNA arms on both sides of the neck convex loop, and only a small part of the bases constitute the neck convex loop (such as the triggering of the double-ring hairpin probe used in the embodiments of the present invention). The sequence is: 5'-GAGG AAGAA GAGAGGGAAGGA-3', only the underlined part participates in the neck ring, and the rest constitute the DNA arms on both sides). This design pattern has two prominent advantages. On the one hand, the bicyclic hairpin probe uses intramolecular hybridization to silence the trigger sequence, effectively avoiding non-specific hybridization. On the other hand, after the cleavage reaction of the double-loop hairpin probe, a double helix structure with a convex loop in the middle is generated; since the convex loop reduces the stability of the double helix structure, the trigger sequence unwinds with its incomplete complementary strand , and then generate a stable double helix with a sufficient number of bases and subsequent reaction chains, and finally produce an obvious positive signal. Based on the double-loop hairpin probe, combined with the strand displacement amplification (SDA) reaction, the present invention constructs a novel fluorescent sensing platform for the detection of bleomycin. In this method, a large number of DNA single strands are generated by strand displacement amplification reaction and the G-quadruplex/NMM complex emits fluorescence, and the label-free detection of bleomycin based on amplification technology is realized for the first time. Binding of NMM dye molecules by single-stranded DNA products and open signaling probes generates enhanced fluorescent signals, and the method exhibits superior sensitivity. The detection limit of the method is 0.34nM, and the method has the advantages of simple operation, label-free and good specificity. In addition, a human serum recovery experiment was carried out in the experiment, and the results showed that the detection method has great potential for clinical application.
附图说明Description of drawings
图1为基于双环识别探针以及链置换扩增反应构建的BLM检测体系实验原理图,A.博来霉素切割双环发夹探针,释放触发序列,并在聚合酶的作用下将信号探针打开,暴露出一段G-四链体序列,B.触发序列与信号探针结合以后,再聚合酶和切刻酶的作用下触发链置换扩增反应;Figure 1 is the experimental schematic diagram of the BLM detection system based on the bicyclic recognition probe and strand displacement amplification reaction. A. Bleomycin cuts the bicyclic hairpin probe, releases the trigger sequence, and detects the signal under the action of polymerase. The needle is opened to expose a G-quadruplex sequence, B. After the trigger sequence is combined with the signal probe, the strand displacement amplification reaction is triggered under the action of polymerase and nickase;
图2(A)为聚丙烯酰氨凝胶电泳验证博来霉素裂解以及链置换扩增放大反应;M:DNA ladder;(1)BHP;(2)SP;(3)BHP+BLM;(4)BHP+BLM+SP;(5)BHP+BLM+SP+KF polymerase;(6)BHP+BLM+SP+KF polymerase+Nt.AlwI;(7)BHP+SP+KF polymerase+Nt.AlwI;(B)检测体系荧光发射光谱:Curve a,BHP+SP+KF polymerase+Nt.AlwI+NMM;Curve b,BHP+BLM+SP+KFpolymerase+Nt.AlwI+NMM;Figure 2 (A) is polyacrylamide gel electrophoresis verification of bleomycin cleavage and strand displacement amplification amplification reaction; M: DNA ladder; (1) BHP; (2) SP; (3) BHP+BLM; ( 4) BHP+BLM+SP; (5) BHP+BLM+SP+KF polymerase; (6) BHP+BLM+SP+KF polymerase+Nt.AlwI; (7) BHP+SP+KF polymerase+Nt.AlwI; (B) Fluorescence emission spectrum of detection system: Curve a, BHP+SP+KF polymerase+Nt.AlwI+NMM; Curve b, BHP+BLM+SP+KF polymerase+Nt.AlwI+NMM;
图3(A)不同博来霉素浓度下的荧光光谱:曲线a→h依次为0nM,2nM,5nM,20nM,100nM,150nM,200nM,220nM;(B)荧光响应与博来霉素浓度之间的线性关系;Fig. 3 (A) Fluorescence spectra under different bleomycin concentrations: the curve a → h is sequentially 0nM, 2nM, 5nM, 20nM, 100nM, 150nM, 200nM, 220nM; (B) the relationship between fluorescence response and bleomycin concentration The linear relationship between;
图4为不同目标物对检测体系荧光响应的影响。Figure 4 shows the effect of different targets on the fluorescence response of the detection system.
具体实施方式Detailed ways
下面结合附图与实施例对本发明作进一步说明。The present invention will be further described below in conjunction with the accompanying drawings and embodiments.
材料和仪器双环发夹探针(BHP)和信号探针(SP)寡核苷酸序列由SangonBiotechnology Co.,Ltd.(上海,中国)合成和纯化。博来霉素A5,丝裂霉素以及柔红霉素购自中国食品药品检定研究院。放线菌素D购自Melone Pharmaceutical Co.,Ltd.(大连,中国)。氯化亚铁(FeCl2 ●H2O)购自天津光复精细化工研究所。Klenow fragment(3’-5’exo-)聚合酶和Nt.AlwI购自New England Biolabs Ltd.(北京,中国)。dNTPs购自Thermo FisherScientific Ltd.(中国)。NMM购自Frontier Scientific Inc.(Utah,USA)。临床血清样品取自山东大学附属医院。所有其他的化学试剂均为分析纯。实验中所用水溶液均用超纯水配置(电阻率>18.25MΩ●cm)。Materials and Instruments Biloop hairpin probe (BHP) and signal probe (SP) oligonucleotide sequences were synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China). Bleomycin A5, mitomycin and daunorubicin were purchased from China National Institutes for Food and Drug Control. Actinomycin D was purchased from Melone Pharmaceutical Co., Ltd. (Dalian, China). Ferrous chloride (FeCl 2 ● H 2 O) was purchased from Tianjin Guangfu Institute of Fine Chemicals. Klenow fragment (3'-5'exo-) polymerase and Nt.AlwI were purchased from New England Biolabs Ltd. (Beijing, China). dNTPs were purchased from Thermo Fisher Scientific Ltd. (China). NMM was purchased from Frontier Scientific Inc. (Utah, USA). Clinical serum samples were obtained from the Affiliated Hospital of Shandong University. All other chemical reagents were of analytical grade. All aqueous solutions used in the experiment were prepared with ultrapure water (resistivity>18.25MΩ ● cm).
15mM磷酸盐缓冲液:13.4mM Na2HPO4,1.6mM NaH2PO4和50mM NaCl(pH=8.0)。15 mM Phosphate Buffer: 13.4 mM Na 2 HPO 4 , 1.6 mM NaH 2 PO 4 and 50 mM NaCl (pH=8.0).
荧光光谱是利用荧光仪Hitachi F-2500(Japan)在室温条件下扫描获得。激发波长399nM,扫描范围550nM-680nM。激发和发射狭缝宽度均为10nM。实时电压700V。Fluorescence spectra were obtained by scanning at room temperature using a fluorometer Hitachi F-2500 (Japan). The excitation wavelength is 399nM, and the scanning range is 550nM-680nM. Both excitation and emission slit widths were 10 nM. The real-time voltage is 700V.
博来霉素活化以及双环发夹探针裂解反应:首先,双环发夹探针和信号探针分别于95℃退火5分钟,缓慢冷却到室温,备用;然后,博来霉素与氯化亚铁等浓度混合并在室温条件下孵育30分钟,得到活化的博来霉素;最后,将上述博来霉素和双环发夹探针充分混合,37℃下孵育30分钟,使得博来霉素充分裂解双环发夹探针。Bleomycin activation and bicyclic hairpin probe cleavage reaction: first, the bicyclic hairpin probe and the signal probe were annealed at 95°C for 5 minutes, cooled slowly to room temperature, and set aside; then, the bleomycin and chloroform Mix equal concentrations of iron and incubate at room temperature for 30 minutes to obtain activated bleomycin; finally, mix the above-mentioned bleomycin and double-ring hairpin probe thoroughly, and incubate at 37°C for 30 minutes to make bleomycin Fully cleaves double loop hairpin probes.
链置换扩增反应:待裂解反应完成以后,取1μL 2mM dNTPs,1U KF聚合酶,4UNt.AlwI,3μL 0.75μM SP and 2μL 10×NEB buffer2(10mM Tris-HCl,10mM MgCl2,50mMNaCl,1.0mM dithiothreitol,pH 7.9)加入上述反应体系,37℃下反应40分钟,即可。Strand displacement amplification reaction: After the cleavage reaction is completed, take 1μL 2mM dNTPs, 1U KF polymerase, 4UNt.AlwI, 3μL 0.75μM SP and 2μL 10×NEB buffer2 (10mM Tris-HCl, 10mM MgCl 2 , 50mMNaCl, 1.0mM dithiothreitol, pH 7.9) was added to the above reaction system, and reacted at 37°C for 40 minutes.
荧光测量:待链置换扩增反应完成以后,取1.2μL 150μM NMM,4.8μL 1M KCl加入反应体系,37℃下孵育30分钟。取最终反应所得溶液30μL置于荧光池,按照材料和仪器部分所述扫描参数进行光谱扫描。Fluorescence measurement: After the strand displacement amplification reaction was completed, 1.2 μL of 150 μM NMM and 4.8 μL of 1M KCl were added to the reaction system, and incubated at 37° C. for 30 minutes. Take 30 μL of the solution obtained from the final reaction and place it in a fluorescent cell, and perform spectral scanning according to the scanning parameters described in the Materials and Instruments section.
凝胶电泳:10%聚丙烯酰胺凝胶电泳验证双环发夹探针裂解反应以及链置换扩增反应。为了能够清楚的观察到DNA条带,我们采用高浓度的博来霉素,双环发夹探针以及信号探针进行实验。取最终反应得到的溶液10μL,与6×loading buffer 2μL充分混合,上样,跑胶。最后,将得到的DNA凝胶用溴化乙锭(EB)染色5分钟,清水冲洗后,置于紫外成像系统下成像。Gel electrophoresis: 10% polyacrylamide gel electrophoresis to verify the bicyclic hairpin probe cleavage reaction and strand displacement amplification reaction. In order to clearly observe the DNA bands, we used high concentrations of bleomycin, double-ring hairpin probes and signal probes for experiments. Take 10 μL of the solution obtained from the final reaction, mix well with 2 μL of 6×loading buffer, load the sample, and run the gel. Finally, the obtained DNA gel was stained with ethidium bromide (EB) for 5 minutes, rinsed with water, and imaged under an ultraviolet imaging system.
结果与讨论Results and discussion
实验原理Experimental principle
如图1所示,首先,博来霉素在识别位点处裂解双环发夹探针,产生一个新的短发夹结构以及中间带有颈凸环的双螺旋。由于颈凸环降低了双螺旋结构的稳定性,双螺旋解旋并释放出一段DNA序列,该序列命名为切割引物链(Cut-primer,简称CP)。然后,切割引物链与信号探针的环部结合,并在KF聚合酶的作用下,以信号探针为模板链发生延伸,形成一个带有切刻酶Nt.AlwI识别位点的双螺旋结构。接着,切刻酶在识别位点处切刻并产生一个新的聚合酶结合位点。于是,聚合酶延伸,复制链释放以及切刻酶切刻过程循环进行,最终产生了大量单链G-四链体序列;与此同时,引物链延伸打开信号探针,暴露出封存在信号探针颈部的G-四链体序列。最后,G-四链体序列绑定NMM,产生可检测的荧光信号。As shown in Figure 1, first, bleomycin cleaves the double-loop hairpin probe at the recognition site to generate a new short hairpin structure and a double helix with a collar in the middle. Since the neck loop reduces the stability of the double helix structure, the double helix unwinds and releases a DNA sequence, which is named Cut-primer (CP for short). Then, the cleavage primer strand binds to the ring portion of the signal probe, and under the action of KF polymerase, the signal probe is used as the template strand to extend to form a double helix structure with the recognition site of nickase Nt.AlwI . Next, the nicking enzyme nicks at the recognition site and creates a new polymerase binding site. Thus, polymerase extension, replication strand release, and nicking enzyme nicking process cycle, and finally generate a large number of single-stranded G-quadruplex sequences; at the same time, primer strand extension opens the signal probe, exposing G-quadruplex sequence at the neck of the needle. Finally, the G-quadruplex sequence binds the NMM, producing a detectable fluorescent signal.
可行性验证Feasibility verification
为了验证双环发夹探针是否能够按照上述设计过程发挥作用以及分析方案的可行性,我们进行了10%聚丙烯酰胺凝胶电泳实验。如图2A所示,同泳道1相比,泳道3在相同的位置出现了较暗的DNA带,并且在靠近20bp的位置出现一条新的DNA带。这说明双环发夹探针发生了裂解反应。将信号探针加入博来霉素和双环发夹探针反应完的溶液中,泳道4出现了一条迁移速度明显慢于信号探针的新的DNA带。这条新的DNA带说明,裂解反应发生以后,CP链被释放,并与SP的环部结合形成一个杂交双螺旋结构。加入KF聚合酶,泳道5在60bp的位置出现了一条明亮的DNA带,说明CP链以SP链为模板在聚合酶的作用下发生链延伸反应,生成一个更加稳定的DNA双螺旋。当KF聚合酶和切刻酶同时加入反应体系,泳道6出现一条迁移速度快于20bp的DNA带,说明SDA反应产生了大量的单链DNA分子。泳道7只有双环发夹探针和信号探针的DNA带,说明双环发夹探针双螺旋结构具有足够的稳定性,体系没有发生SDA反应。为了进一步证明双环发夹探针设计以及分析方案的可行性,我们进行了相应的荧光光谱扫描实验。如图2B,目标物不存在时,检测体系表现出低背景信号(Curve a)。这说明没有裂解反应发生时,双环发夹探针中的CP序列不能被用于链置换扩增反应产生信号。当目标物博来霉素加入反应体系时,检测体系表现出明显提高的响应信号(Curve b)。这说明裂解反应发生以后,CP序列被释放并用于后续的链置换扩增反应产生增强的荧光信号。以上结果均表明,该检测方案是可行的,双环发夹探针能够成功的沉默触发链,从而减低或消除非特异性杂交在均相分析中所带来的背景信号。In order to verify whether the bicyclic hairpin probe can function according to the above design process and the feasibility of the analysis scheme, we conducted a 10% polyacrylamide gel electrophoresis experiment. As shown in Figure 2A, compared with lane 1, a darker DNA band appeared in lane 3 at the same position, and a new DNA band appeared at a position close to 20 bp. This indicated that the cleavage reaction of the bicyclic hairpin probe occurred. When the signal probe was added to the reaction solution of bleomycin and bicyclic hairpin probe, a new DNA band with a migration speed significantly slower than that of the signal probe appeared in lane 4. This new DNA band indicates that after the cleavage reaction, the CP strand is released and combines with the SP loop to form a hybrid double helix structure. After adding KF polymerase, a bright DNA band appeared at the position of 60 bp in lane 5, indicating that the CP chain took the SP chain as a template to undergo a chain extension reaction under the action of the polymerase to generate a more stable DNA double helix. When KF polymerase and nicking enzyme were added to the reaction system at the same time, a DNA band with a migration speed faster than 20 bp appeared in lane 6, indicating that the SDA reaction produced a large number of single-stranded DNA molecules. Lane 7 has only the DNA bands of the double-loop hairpin probe and the signal probe, indicating that the double-helix structure of the double-loop hairpin probe has sufficient stability, and no SDA reaction has occurred in the system. In order to further prove the feasibility of the double-ring hairpin probe design and analysis scheme, we carried out corresponding fluorescence spectrum scanning experiments. As shown in Figure 2B, the detection system exhibited a low background signal (Curve a) in the absence of the target. This indicates that the CP sequence in the bicyclic hairpin probe cannot be used to generate a signal in the strand displacement amplification reaction when no cleavage reaction occurs. When the target bleomycin was added to the reaction system, the detection system showed a significantly improved response signal (Curve b). This indicated that after the cleavage reaction, the CP sequence was released and used in the subsequent strand displacement amplification reaction to generate an enhanced fluorescent signal. The above results show that the detection scheme is feasible, and the double-loop hairpin probe can successfully silence the trigger chain, thereby reducing or eliminating the background signal brought by non-specific hybridization in homogeneous analysis.
实验条件优化Experimental condition optimization
为了获得最好的传感效能,实验分别对双环发夹探针浓度,信号探针浓度,酶浓度以及NMM浓度等参数进行了优化。最终选择双环发夹探针为100nM,信号探针为75nM,KF聚合酶为1U,Nt.AlwI为4U,NMM浓度为6μM。In order to obtain the best sensing performance, parameters such as the concentration of the double-loop hairpin probe, the concentration of the signal probe, the concentration of the enzyme and the concentration of NMM were optimized in the experiment. Finally, 100 nM of the double-loop hairpin probe, 75 nM of the signal probe, 1 U of KF polymerase, 4 U of Nt.AlwI, and 6 μM of NMM were selected.
线性和范围Linearity and Range
在最优实验条件下,我们考察了检测体系的灵敏度以及线性范围。如图3所示,当博来霉素浓度从0nM到220nM逐渐增加时,荧光响应强度也随着增加。荧光强度与博来霉素浓度在0nM-220nM范围内成线性(R=0.998)。检测限0.34nM。Under the optimal experimental conditions, we investigated the sensitivity and linear range of the detection system. As shown in Figure 3, when the concentration of bleomycin gradually increased from 0 nM to 220 nM, the fluorescence response intensity also increased. The fluorescence intensity was linear with the concentration of bleomycin in the range of 0nM-220nM (R=0.998). Detection limit 0.34nM.
特异性specificity
为了考察了方法的特异性,实验选取博来霉素、丝裂霉素、柔红霉素以及放线菌素D分别作为目标物进行考察。结果如图4所示,在相同的实验条件下,检测体系只对博来霉素表现出较强的响应信号,表明该检测方法具有较好的特异性。In order to examine the specificity of the method, bleomycin, mitomycin, daunorubicin and actinomycin D were selected as targets for investigation. The results are shown in Figure 4. Under the same experimental conditions, the detection system only showed a strong response signal to bleomycin, indicating that the detection method has good specificity.
回收率考察Recovery inspection
为了考察本方法的实际应用能力,我们进行了人血清回收率实验。实验结果在95%-100%之间,说明该方法具有潜在的临床应用能力。In order to investigate the practical application ability of this method, we conducted a human serum recovery experiment. The experimental results are between 95%-100%, indicating that the method has potential clinical application.
总结Summarize
基于双环发夹探针介导的链置换扩增反应,本工作构建了一种免标记的荧光策略用于博来霉素的灵敏检测。据我们所知,该方法首次实现了基于DNA扩增技术的博来霉素免标记检测。检测限为0.34nM。人血清回收率考察符合要求,说明该方法在药物分析以及临床应用中具有潜在的实际应用能力。Based on the double-loop hairpin probe-mediated strand displacement amplification reaction, this work constructed a label-free fluorescent strategy for the sensitive detection of bleomycin. To the best of our knowledge, this method enables the label-free detection of bleomycin based on DNA amplification technology for the first time. The limit of detection was 0.34 nM. The recovery rate of human serum meets the requirements, indicating that the method has potential practical application ability in drug analysis and clinical application.
上述虽然结合附图对本发明的具体实施方式进行了描述,但并非对本发明保护范围的限制,所属领域技术人员应该明白,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。Although the specific implementation of the present invention has been described above in conjunction with the accompanying drawings, it does not limit the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical solution of the present invention, those skilled in the art do not need to pay creative work Various modifications or variations that can be made are still within the protection scope of the present invention.
Claims (9)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510961410.7A CN105385770B (en) | 2015-12-18 | 2015-12-18 | The Dual-ring hairpin probe for detecting bleomycin mediates label-free strand displacement amplification method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510961410.7A CN105385770B (en) | 2015-12-18 | 2015-12-18 | The Dual-ring hairpin probe for detecting bleomycin mediates label-free strand displacement amplification method |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105385770A CN105385770A (en) | 2016-03-09 |
CN105385770B true CN105385770B (en) | 2018-09-28 |
Family
ID=55418545
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510961410.7A Expired - Fee Related CN105385770B (en) | 2015-12-18 | 2015-12-18 | The Dual-ring hairpin probe for detecting bleomycin mediates label-free strand displacement amplification method |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105385770B (en) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105950755B (en) * | 2016-06-17 | 2020-05-08 | 山东大学 | Method for detecting microRNA based on split recognition mode combined with cascade signal amplification strategy |
CN105950757B (en) * | 2016-06-17 | 2019-11-22 | 山东大学 | A method for the detection of bleomycin based on a double-loop hairpin probe and an enzyme-mediated cascade amplification strategy |
CN106244703B (en) * | 2016-08-26 | 2019-12-13 | 山东大学 | Method for detection of UDG activity based on cohesive end-mediated strand displacement reaction combined with polymeric nicking isothermal amplification technique |
CN107151694B (en) * | 2016-09-30 | 2020-05-12 | 山东大学 | Loop-mediated cascade amplification strategy for highly sensitive detection of DNA methyltransferase activity |
CN106520764B (en) * | 2016-12-16 | 2019-02-22 | 福州大学 | A nano-bicyclic aptamer probe and its application |
CN108342456A (en) * | 2018-02-08 | 2018-07-31 | 中国农业大学 | A kind of visualization of dual heavy metal ion quantifies new detecting method |
CN117571982B (en) * | 2024-01-09 | 2024-04-09 | 德州学院 | Marker-free fluorescent aptamer sensor for detecting kanamycin with low background and application thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103439319A (en) * | 2013-09-03 | 2013-12-11 | 青岛科技大学 | Method for measuring bleomycins by utilizing electrochemical luminescence of carbon nanoparticle modified electrode |
-
2015
- 2015-12-18 CN CN201510961410.7A patent/CN105385770B/en not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103439319A (en) * | 2013-09-03 | 2013-12-11 | 青岛科技大学 | Method for measuring bleomycins by utilizing electrochemical luminescence of carbon nanoparticle modified electrode |
Non-Patent Citations (3)
Title |
---|
A bicyclo-hairpin probe mediated strand displacement amplificationstrategy for label-free and sensitive detection of bleomycin;Huijuan Wang et al.;《Sensors and Actuators B: Chemical》;20160715;第318-324页 * |
A Responsive Hidden Toehold To Enable Controllable DNA Strand Displacement Reactions;Yongzheng Xing et al.;《Angew. Chem. Int. Ed.》;20111231;第50卷;第11934-11936段 * |
Ultrasensitive fluorescence detection of bleomycin via exonuclease III-aided DNA recycling amplification;Fenglei Gao et al.;《Chem. Commun.》;20130628;第49卷;第7561-7563页 * |
Also Published As
Publication number | Publication date |
---|---|
CN105385770A (en) | 2016-03-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN105385770B (en) | The Dual-ring hairpin probe for detecting bleomycin mediates label-free strand displacement amplification method | |
Wang et al. | A fluorescent biosensor for protein detection based on poly (thymine)-templated copper nanoparticles and terminal protection of small molecule-linked DNA | |
Ye et al. | Ultrasensitive electrochemical DNA sensor for virulence invA gene of Salmonella using silver nanoclusters as signal probe | |
Hu et al. | Double-strand DNA-templated formation of copper nanoparticles as fluorescent probe for label free nuclease enzymedetection | |
Tao et al. | Metal nanoclusters combined with CRISPR-Cas12a for hepatitis B virus DNA detection | |
Chen et al. | Enzyme-free detection of DNA based on hybridization chain reaction amplification and fluorescence resonance energy transfer | |
Zhou et al. | Target-initiated autonomous synthesis of metal-ion dependent DNAzymes for label-free and amplified fluorescence detection of kanamycin in milk samples | |
Li et al. | Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification | |
CN104630363A (en) | Method for detecting activity of uracil-DNA glycosylase (UDG) based on fluorescence amplification strategy of label-free non-enzyme DNA machines | |
Zhu et al. | A ratiometric electrochemiluminescence strategy based on two-dimensional nanomaterial-nucleic acid interactions for biosensing and logic gates operation | |
Hu et al. | An electrochemical biosensor for sensitive detection of microRNAs based on target-recycled non-enzymatic amplification | |
Park et al. | A DNA-templated silver nanocluster probe for label-free, turn-on fluorescence-based screening of homo-adenine binding molecules | |
Ye et al. | Toehold-mediated enzyme-free cascade signal amplification for ratiometric fluorescent detection of kanamycin | |
CN104975079A (en) | 17beta-estradiol visualization detection method based on DNA nano-structure, and 17beta-estradiol visualization detection kit based on DNA nano-structure | |
CN108304932A (en) | The structure of logic gate based on silver nanoclusters and its application in intelligent measurement | |
CN111518874A (en) | Raman enhanced substrate, preparation method thereof and method for detecting miRNA (micro ribonucleic acid) | |
Jiang et al. | An aptasensing platform for simultaneous detection of multiple analytes based on the amplification of exonuclease-catalyzed target recycling and DNA concatemers | |
Peng et al. | A fluorescence light‐up silver nanocluster beacon modulated by metal ions and its application in telomerase‐activity detection | |
CN107151694A (en) | The Cascaded amplification strategy of ring mediation is used for highly sensitive detection dnmt rna activity | |
Li et al. | A Sensitive Fluorescence Biosensor for Silver Ions (Ag+) Detection Based on C‐Ag+‐C Structure and Exonuclease III‐Assisted Dual‐Recycling Amplification | |
Wang et al. | A metal ion-triggered and DNA-fueled molecular machine for amplified and sensitive fluorescent detection of Hg2+ | |
Song et al. | Electrochemical label-free biomolecular logic gates regulated by distinct inputs | |
Lu et al. | A dual-functional fluorescent biosensor based on enzyme-involved catalytic hairpin assembly for the detection of APE1 and miRNA-21 | |
Zhang et al. | Target-initiated synthesis of fluorescent copper nanoparticles for the sensitive and label-free detection of bleomycin | |
Wang et al. | Duplex featured polymerase-driven concurrent strategy for detecting of ATP based on endonuclease-fueled feedback amplification |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180928 |