Double-loop hairpin probe-mediated label-free strand displacement amplification method for detection of bleomycin
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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 bicyclic hairpin probes for detecting bleomycin, and can solve the problems of high background signal and false positive signal.
Active Publication Date: 2018-09-28
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 the subsequent reaction chain will cause relatively high background signals and false positive signals, which limits the application of the probe
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[0022] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.
[0023] 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 analyt...
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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 detection method for bleomycin, in particular to a double-loop hairpin probe-mediated label-free strand displacement amplification method for detection of bleomycin. Background technique [0002] 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...
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