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A technology of strand displacement amplification and hairpin probe, which is applied in the field of label-free strand displacement amplification mediated by double-loop hairpin probes for the detection of bleomycin, which can solve the problems of high background signal, false positive signal, etc.
Active Publication Date: 2016-03-09
SHANDONG UNIV
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However, since the trigger sequence is located in the loop of the probe, non-specific hybridization between the uncleaved DNA molecular probe and
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[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0023] Materials and Instrumentation Bicyclic 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 Institute 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. Klenowfragment (3'-5'exo-) polymerase and Nt.AlwI were purchased from NewEngland Biolabs Ltd. (Beijing, China). dNTPs were purchased from ThermoFisher 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 are of analyti...
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Abstract
The invention discloses a dual-ring hairpin probe mediate label-free strand displacement amplification method for detecting bleomycin. The method includes the steps that when bleomycin exists, a dual-ring hairpin probe fractures at a recognition site, and a triggering sequence is released; the triggering sequence and a ring part of a signal probe are combined and subjected to a strand displacement amplification reaction under the effect of a polymerase and a nicking enzyme, and finally a great number of G-four-chain sequences are generated. At the same time, a primer chain extends to open the signal probe, and therefore the G-four-chain sequences packaged in the neck of the signal probe can be exposed. Finally, NMM molecules are bound to the G-four-chain sequences to generate fluorescence signals, and bleomycin is quantified through the detected fluorescence signals. As the triggering sequence is designed at the neck of the dual-ring hairpin probe, background signals of a detection system are reduced; by combining bleomycin cutting and the strand displacement amplification reaction, bleomycin can be detected sensitively, and the detection limit is 0.34 nm. The method has the advantages of being easy to operate, free of labeling and good in specificity.
Description
technical field [0001] The invention relates to a method for detecting bleomycin, in particular to a method for label-free strand displacement amplification mediated by a bicyclic hairpin probe for detecting bleomycin. Background technique [0002] Bleomycin is a class of glycopeptide antibiotics secreted by Verticillium streptomycin. Clinically, bleomycin is an important anticancer drug, mainly used for the treatment of squamous cell carcinoma and macrophage tumor and other diseases (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, CriticalReviewsinOncology / 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. Rish...
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