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Method for detection of bleomycin based on double-circular hairpin probe and enzyme-mediated cascade amplification strategy

A hairpin probe, bleomycin technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as the inability to meet the requirements of method sensitivity

Active Publication Date: 2019-11-22
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, according to literature reports, the plasma concentration of bleomycin varies from 1.4nM to 2.7μM within 24 hours under intravenous injection, and this method still cannot meet the sensitivity requirements of clinical sample monitoring.

Method used

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  • Method for detection of bleomycin based on double-circular hairpin probe and enzyme-mediated cascade amplification strategy
  • Method for detection of bleomycin based on double-circular hairpin probe and enzyme-mediated cascade amplification strategy
  • Method for detection of bleomycin based on double-circular hairpin probe and enzyme-mediated cascade amplification strategy

Examples

Experimental program
Comparison scheme
Effect test

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 app...

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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Abstract

The invention discloses a kit for detecting bleomycin based on a double-ring hairpin probe and an enzyme-mediated cascade amplification strategy and a detection method of the kit. The double-ring hairpin probe can effectively silence a trigger sequence and inhibit non-specific hybridization, and therefore a background signal of the detection method is reduced; an enzyme-mediated cascade amplification reaction can achieve multiple amplification of a detection signal, therefore, the sensitivity of the detection method is improved, high-sensitivity detection of bleomycin is achieved, the linear range is 40 pM to 10 nM, and the detection limit is 35 pM. The sensitivity is improved by about two orders of magnitudes compared with a previously established label-free fluorescence detection method, and the requirements of clinical sample detection can be met.

Description

technical field [0001] The invention relates to a method for detecting bleomycin based on a double-ring hairpin probe and an enzyme-mediated cascade amplification strategy. Background technique [0002] Bleomycin is an anticancer drug commonly used in clinical practice. The monitoring of its therapeutic drug concentration is of great significance for guiding clinical rational drug use, improving anticancer efficacy and reducing toxic and side effects. The earlier established detection methods for bleomycin include microbial analysis, RIA, HPLC and enzyme immunoassay. The most widely used methods are RIA and HPLC. However, the above two methods have their own defects, such as low sensitivity and complicated operation of HPLC, and RIA radioactive isotopes are harmful to human health. Therefore, it is urgent to explore a new, sensitive and specific detection method to achieve simple and rapid detection of bleomycin. [0003] According to literature reports, the antitumor eff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/119C12Q2563/107
Inventor 王磊姜玮王慧娟
Owner SHANDONG UNIV
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