Method for rapidly detecting SARS-CoV-2 based on DNA nano-scaffold

A nano-scaffold, sars-cov-2 technology, applied in the field of genes, achieves the effects of programmable sequence, simple operation and low cost

Active Publication Date: 2020-09-18
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] At present, there is a lack of an easy-to-operate and ultra...

Method used

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  • Method for rapidly detecting SARS-CoV-2 based on DNA nano-scaffold
  • Method for rapidly detecting SARS-CoV-2 based on DNA nano-scaffold
  • Method for rapidly detecting SARS-CoV-2 based on DNA nano-scaffold

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Embodiment 1

[0039] A method for rapidly detecting SARS-CoV-2 based on DNA nano-stents of the present invention comprises the following steps:

[0040] (1) A DNA nanoscaffold was constructed by DNA strand self-assembly and self-quenching probe H1, and the target RNA triggered the free hairpin probe H 2 and hairpin probe H 1 Rapid hybridization along the nanoscaffold enables signal amplification.

Embodiment 2

[0042] A method for rapidly detecting SARS-CoV-2 based on DNA nano-stents of the present invention comprises the following steps:

[0043] (1) by DNA hairpin probe H 1 Self-assembly with DNA nanowires to construct nanoscaffolds, in which RCA reaction by Pre-cirDNA and Primer produced DNA nanowires containing repeat sequence fragments, and hairpins were labeled with 5-carboxyfluorescein FAM dye and its fluorescent quencher BHQ1 both ends of probe H1 to form a self-quenching probe;

[0044] (2) When the target RNA is present, based on the H in the nanoscaffold 1Encoding design, which will hybridize with one H1 in the nanoscaffold, so that the hairpin probe H1 hairpin is opened and the fluorescence is restored, which in turn triggers the cascade of hairpin probe H2 and hairpin probe H1 along the DNA nanoscaffold hybridization; the Pre-cirDNA has the nucleotide sequence of SEQ ID No.4. The Primer has the nucleotide sequence of SEQ ID No.5. The target RNA is the conserved regio...

Embodiment 3

[0057] Optimize experimental conditions

[0058] In order to obtain better detection efficiency, we optimized some key factors in the experiment process. The first is the effect on the background signal of the length (foothold) at which H1 hybridizes to the DNA nanowire. The present invention optimizes the distance of the H1 probe on the nanowire, from Figure 5 It can be seen that there is the strongest fluorescent signal at 25 base pairs. In addition, the time of the RCA reaction affects the length of the generated DNA scaffold, which in turn changes the number of assembled H1 probes on the scaffold, ultimately affecting the sensitivity of the assay. like Image 6 As shown, the signal intensity increases with the prolongation of the RCA time from 0 min to 30 min, and decreases with the prolongation of the RCA time between 40 min and 100 min. Therefore, we choose 30 minutes as the optimal RCA response time. Finally, we explored the reproducibility of target RNA recogniti...

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Abstract

The invention discloses a method for rapidly detecting SARS-CoV-2 based on a DNA nano-scaffold. The method comprises the following steps of (1) constructing the DNA nano-scaffold through a DNA chain self-assembly and self-quenching probe H1, triggering free hairpin probe H2 and hairpin probe H1 to rapidly hybridize along the nano-scaffold by target RNA, and realizing amplification of signals. Theglobal coronavirus disease (COVID-19) in 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The method is high in detection sensitivity, programmable in sequence, high insignal gain, high in specificity, short in reaction time (10 minutes), low in cost and simple to operate.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to a method for rapidly detecting SARS-CoV-2 based on DNA nano-stents. Background technique [0002] In recent years, the continuous prevalence and outbreak of viral influenza has attracted worldwide attention, such as novel viruses (AIDS virus, SARS virus and MERS-CoV, H1N1 virus of the 2009 influenza pandemic, Ebola virus, etc. , Zika virus, and the recent SARS-COV-2) have caused serious harm to the physical and mental health of people all over the world. Almost all of these epidemics are caused by initial zoonotic transmission to humans, with clinically overt or occult spread into susceptible populations. Due to the lack of rapid, convenient and accurate molecular diagnostic technology, the public has many difficulties in the management and control of the spread of the epidemic. [0003] At present, the novel coronavirus (SARS-CoV-2) pneumonia (COVID-19) epidemic is spreading wid...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/682
CPCC12Q1/701C12Q1/682C12Q2531/125C12Q2525/301C12Q2563/107Y02A50/30
Inventor 项阳焦瑾
Owner NANJING UNIV
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