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Nucleic acid fluorescence probe capable of being repeatedly used and corresponding nucleic acid detection method

A fluorescent probe and nucleic acid technology, applied in the biological field, can solve problems such as bottlenecks in testing capabilities, failure to save costs, and time-consuming and labor-intensive problems

Active Publication Date: 2021-06-08
BEIJING UNIV OF CHEM TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Commercial quantitative RT-PCR kits contain reagents such as polymerases and fluorescent probes (Taqman probes), where the synthesis of fluorescent probes is not only costly but also time-consuming
Shortages of these reagents have caused severe bottlenecks in testing capacity during the pandemic
For the prevention and control of infectious diseases and sustainable development, it is imperative to develop a reusable probe system. The current reusable probe system is usually based on a heterogeneous solution system, and additional cleaning agents are required to complete the probe. recycling, which is time-consuming and labor-intensive, and does not achieve efficient cost savings

Method used

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  • Nucleic acid fluorescence probe capable of being repeatedly used and corresponding nucleic acid detection method
  • Nucleic acid fluorescence probe capable of being repeatedly used and corresponding nucleic acid detection method
  • Nucleic acid fluorescence probe capable of being repeatedly used and corresponding nucleic acid detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0077] Embodiment 1 uses the detection method of linear DNA probe and DNA hairpin probe

[0078] This embodiment is based on linear DNA probes or DNA hairpin probes to detect the oligonucleotide target shown in Sequence 1 of the Sequence Listing, using a single-stranded oligonucleotide synthesized according to Sequence 1 (synthesized by Shanghai Sangong Co., Ltd.) as Simulated samples to be tested.

[0079] Specific steps are as follows:

[0080] S1. Design nucleic acid fluorescent probes according to the oligonucleotide target shown in Sequence 1 of the sequence table. The nucleic acid fluorescent probes used in this example are linear DNA probes or DNA hairpin probes. The specific sequences are shown in Table 1:

[0081] Table 1 each nucleic acid sequence

[0082]

[0083] Note: * in Table 1 means thioxo. The sequence indicated by the underline in sequence 5 is the B1 target recognition domain, the sequence indicated by the wavy line is the target binding domain labele...

Embodiment 2

[0126] Embodiment 2 uses the detection method of three-way junction (TWJ) DNA nanoprobe

[0127] This embodiment was carried out in the virus detection laboratory of the Clinical Laboratory Department of the China-Japan Friendship Hospital.

[0128] In order to verify the effect of the detection method of the present invention, this embodiment 2 is set up for SARS-CoV-2, and the sample acquisition method used is shown in Figure 4 :

[0129] Nasopharyngeal swabs (from Kangwei Century Biotechnology Co., Ltd.) were taken from a total of 110 healthy people and COVID-19 patients (nucleic acid test positive) (of which 55 were positive and 55 were negative). The virus was inactivated and extracted with nucleic acid Kits (Tianlong Technology, qEX-DNA / RNA virus) were used to extract viral RNA, and RNA was reverse-transcribed and amplified by RT-PCR kit (Qiagen, 210212), and lambda exonuclease (NEB, M0262S) was used to ) to generate a single strand as the sample to be tested in the d...

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Abstract

The invention discloses a nucleic acid fluorescence probe capable of being repeatedly used. The nucleic acid fluorescence probe comprises a fluorescent probe, a quenching probe and DNA containing a target object binding region. The fluorophore of the nucleic acid fluorescence probe is initially in a non-activated state, a target object can be combined to a target object combined area of the nucleic acid fluorescence probe through strand displacement to enable the target object to be allosteric into a substrate compound with high affinity with exonuclease, and at the moment, the fluorophore is activated; and after the exonuclease cuts the substrate compound, the target object is degraded, and the nucleic acid fluorescence probe returns to an initial non-activated state through re-hybridization, so that automatic resetting is realized, and the effect of repeated use is achieved. The probe system disclosed by the invention can be repeatedly used for at least five times, and can operate in a homogeneous solution, so that the reagent cost is effectively saved, and a solution is provided for a nucleic acid detection scene in which a large number of clinical samples need to be provided within a short time. The method disclosed by the invention is widely applied to nucleic acid detection and other types of liquid biopsy.

Description

technical field [0001] The invention relates to a reusable nucleic acid fluorescent probe and a corresponding nucleic acid detection method in the field of biotechnology. Background technique [0002] Reliable, economical and rapid nucleic acid detection methods are essential for the prevention, control, diagnosis and treatment of SARS-CoV-2. Nucleic acid detection methods based on quantitative RT-PCR (RT-qPCR) are widely used worldwide. Commercial quantitative RT-PCR kits contain reagents such as polymerase and fluorescent probes (Taqman probes), where the synthesis of fluorescent probes is not only costly but also time-consuming. Shortages of these reagents have caused severe bottlenecks in testing capacity during the pandemic. For the prevention and control of infectious diseases and sustainable development, it is imperative to develop a reusable probe system. The current reusable probe system is usually based on a heterogeneous solution system, and additional cleaning ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6816C12N15/11
CPCC12Q1/701C12Q1/6816C12Q2600/166C12Q2537/1373C12Q2563/107C12Q2521/319C12Q2525/301C12Q2545/113Y02A50/30
Inventor 苏昕李娜喻长远刘宇张凌昊
Owner BEIJING UNIV OF CHEM TECH
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