Method for detecting bleomycin based on double-ring hairpin probe and enzyme-mediated cascade amplification strategy
A technology of hairpin probes and bleomycin, which is applied in biochemical equipment and methods, microbiological determination/inspection, etc., and can solve problems such as the inability to meet the sensitivity requirements of the method
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Embodiment 1
[0063]Example 1: Detection of bleomycin by double-loop hairpin probe and enzyme-mediated cascade amplification strategy
[0064] The specific method is as follows:
[0065] (1) Bleomycin cut double loop hairpin probe
[0066] Dilute the bicyclic hairpin probe with 15mM PBS, place it in a metal bath at 95°C for 5 minutes, then slowly cool down to about 25°C, take it out, centrifuge, and set aside. Mix bleomycin with freshly prepared ferrous chloride solution at a ratio of 1:1 for activation. The activated bleomycin was fully mixed with the annealed double-loop hairpin probe, and incubated in a 37°C incubator for 30 minutes, so that the bleomycin fully cleaves the double-loop hairpin probe.
[0067] (2) Enzyme-mediated cascade amplification reaction
[0068] After the above reaction is completed, add 1 μL 10mM dNTPs, 1.2 μL template strand HP 1 , 0.6 μL Template Strand HP 2 , 2μL 10×NEBuffer 2 (10mM Tris-HCl, 10mM MgCl 2 , 50mM NaCl, 1.0mM dithiothreitol, pH 7.9) and an ap...
Embodiment 2
[0071] Embodiment 2: Feasibility study of detection method of the present invention
[0072] In order to verify the feasibility and amplification capability of the detection method of the present invention, the fluorescence emission spectra of the reaction systems under different conditions were investigated. The result is as figure 2 As shown, the curves a, b and c represent the blank experiments of the three reactions of SDA2, EXPAR+SDA2 and SDA1+EXPAR+SDA2 respectively, and it can be seen that the intensity of the obtained fluorescence signal increases sequentially, indicating that with the increase of the number of amplification reactions, the amplification The background signal of the system is higher, but the fluorescence signal does not exceed 250a.u. Curves d, e, and f represent the positive experiments of the above three amplification reactions respectively, and the fluorescence intensity of curve d has increased to a certain extent compared with curve a, indicating...
Embodiment 3
[0075] Embodiment 3: Optimization of detection conditions
[0076] The present invention selects and optimizes the parameter conditions that have a relatively large impact on the detection performance, including the concentration of the template chain and the reaction time of each stage of the cascade amplification reaction mediated by the enzyme, etc., and conducts an optimization investigation. the result shows:
[0077] template chain HP 2 The optimal concentration is 20nM; template strand HP 3 The optimal concentration is 200nM; the optimal reaction time of stage one (SDA1+EXPAR) and stage two (SDA2) is 30min and 20min, respectively.
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