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Fluorescent sensor for detecting HBV, and preparation and applications thereof

A fluorescent sensor and fluorescent signal technology, applied in the field of HBV detection, can solve the problems of reaction efficiency, limiting the versatility of nucleic acid analysis, and unfavorable promotion.

Active Publication Date: 2021-08-31
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite the great potential of CRISPR / Cas12a in nucleic acid detection, certain limitations still exist
First, there are sequence constraints on CRISPR effectors, which strictly require a characteristic PAM on the dsDNA target sequence, limiting the generalizability of nucleic acid analysis
Secondly, most of the current methods rely on PCR or RPA to amplify target substances, which require multiple enzymes, complex primer design and thermal cycling and other strict reaction conditions, which is very unfavorable for bedside detection or the promotion of scenes with more difficult conditions
The most important thing is that the Cas protein is combined with other enzyme systems, and the reaction efficiency is affected due to the incompatibility of different systems

Method used

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  • Fluorescent sensor for detecting HBV, and preparation and applications thereof
  • Fluorescent sensor for detecting HBV, and preparation and applications thereof
  • Fluorescent sensor for detecting HBV, and preparation and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Fabrication of fluorescent sensors and detection of miRNA

[0046] 1. Materials and methods

[0047] 1.1 Materials

[0048] HPLC-purified oligonucleotides were synthesized by Shanghai Sangong. EnGen Lba Cas12a and NEBuffer 3.0 solutions were purchased from New England Biolabs. DEPC water, enzyme-free water and RNase inhibitors were purchased from Shanghai Sangong.

[0049] 1.2 Testing instruments

[0050] Edinburgh Integrated Steady State Transient Fluorescence Spectrometer FS5.

[0051] 1.3 Detection principle

[0052] Such as figure 1 As shown, in the homogeneous system, the target substance binds to the toehold in the HP probe, and the HP probe is opened by the toehold-mediated strand displacement reaction, thereby exposing the toeholds located in the HP probe that are closed. Subsequently, the preassembled Cas12a / crRNA hybridizes to the exposed toehold domain via crRNA, displacing the target with base-pairing extension and forming the Cas12a activator. Then,...

Embodiment 2

[0070] Validation of the feasibility of detecting miRNA fluorescent sensors

[0071] 1. The verification of foothold-mediated cascade strand displacement reaction and Cas12a cleavage, the process is as follows:

[0072] 1) Preparation of HP probe: NEBuffer 3.0 buffer was used to prepare HP into a 20 μL solution with a concentration of 10 μM, denatured at 95° C. for 5 minutes, slowly cooled to room temperature and placed at 4° C. for later use. 2) Fluorescence spectrophotometer to verify the feasibility of the experiment:

[0073]

[0074]

[0075] Add the above liquid accordingly to obtain 100 μL of working solution, mix it on a shaker, centrifuge it quickly with a hand-held centrifuge for a short time, and place it in a 37°C incubator for 1 hour of reaction. After the reaction was completed, the fluorescence intensity of the solution was measured using a fluorescence spectrophotometer. The result is as figure 2 shown. figure 2 Among them, the red curve is the expe...

Embodiment 3

[0077] Molar ratio of HP probe, Cas12a, crRNA

[0078] In order to investigate the influence of the molar ratio of HP probe, Cas12a, and crRNA on the stability and reaction speed of the developed fluorescent sensor, this example further studies the optimal molar ratio of HP probe, Cas12a, and crRNA.

[0079] Depend on Figure 4 It can be seen that when the molar ratio is 1:1.5:2, the signal-to-noise ratio is the best. Therefore, a molar ratio of 1:1.5:2 was selected as the optimum ratio.

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Abstract

The invention discloses a fluorescent sensor for detecting HBV, and preparation and applications thereof. The sensor includes a signal identification portion and a signal conversion portion. The signal identification portion is used for achieving strand displacement reaction mediated by a foothold triggered by a target material; and the signal conversion portion converts the target material into a double stranded DNA containing a PAM sequence that can be identified by Cas12a. The principles of the sensor are as follows: the target material is combined with the foothold in an HP probe, and the HP probe is opened by TMSDR, so that the foothold that can be identified by crRNA is exposed; the Cas12a / crRNA is hybridized with the exposed foothold through the crRNA and replaces the target material, so as to form a Cas12a activator; the replaced target material triggers the next circular reaction by combining with other HP probes, so as to generate lots of the Cas12a activators; and a Cas12a activator trans-cleavage reporter probe generates a fluorescence signal, and the content of the target material can be learned by detecting the fluorescence signal. The fluorescent sensor is low in cost, high in sensitivity, fast in reaction speed, and good in reproducibility.

Description

technical field [0001] The invention relates to the technical field of HBV detection, in particular to a fluorescent sensor for detecting HBV and its preparation and application. Background technique [0002] Hepatitis B virus (HBV) is the main cause of diseases such as hepatitis B virus. It is mainly transmitted through blood and is very harmful. Therefore, strengthening the screening of hepatitis B virus in the blood of the population is of great significance for the control of hepatitis B diseases . In the past, enzyme-linked immunosorbent assay was often used to screen hepatitis B virus in clinical practice. This detection method has the advantages of low cost and easy operation. of missed diagnoses. In recent years, nucleic acid detection method has been used clinically to detect hepatitis B virus. This detection method belongs to molecular biology detection method, which can directly measure the DNA content of hepatitis B virus, and then interpret whether there is he...

Claims

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Application Information

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IPC IPC(8): C12Q1/6825C12Q1/70G01N21/64C12R1/93
CPCC12Q1/6825C12Q1/706G01N21/6428G01N2021/6432C12Q2525/301C12Q2537/1373C12Q2521/327C12Q2525/161C12Q2525/207
Inventor 丁世家程小雪龚瑶
Owner CHONGQING MEDICAL UNIVERSITY
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