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Method for rapidly detecting RNA viruses based on CCP-FRET

A nucleic acid and sample technology, applied in the field of rapid detection of RNA viruses, can solve the problems of restricting large-scale application, high detection cost, complicated preparation process, etc.

Inactive Publication Date: 2021-02-05
INST OF CHEM CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these detection techniques have certain sensitivity and selectivity, their high detection cost, complicated preparation process and special requirements for instruments limit the large-scale application of these methods.

Method used

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  • Method for rapidly detecting RNA viruses based on CCP-FRET
  • Method for rapidly detecting RNA viruses based on CCP-FRET
  • Method for rapidly detecting RNA viruses based on CCP-FRET

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1. Design and preparation of primers for detecting COVID-2019

[0103] The primers were preliminarily designed through a large number of sequence analysis, and the primers with the best performance were obtained through pre-experimental screening.

[0104] The primer pair for detecting the ORF1ab region gene (O gene) of COVID-2019 is as follows (amplification length 119bp):

[0105] Upstream primer (sequence 1): 5'-CCCTGTGGGTTTTACACTTAA-3';

[0106] Downstream primer (SEQ ID NO: 2): 5'-ACGATTGTGCATCAGCTGA-3'.

[0107] The primer pair for detecting the nucleocapsid protein gene (N gene) of COVID-2019 is as follows (amplification length 99bp):

[0108] Upstream primer (SEQ ID NO: 3): 5'-GGGGAACTTCTCCTGCTAGAAT-3';

[0109] Downstream primer (SEQ ID NO: 4): 5'-CAGACATTTTGCTCTCAAGCTG-3'.

[0110] The primer pair for detecting the envelope protein gene (E gene) of COVID-2019 is as follows (amplification length 113bp):

[0111] Upstream primer (SEQ ID NO: 5): 5-AC...

Embodiment 2

[0114] Embodiment 2, establishment of method

[0115] 1. Prepare a reaction solution in a 200 μl PCR tube (see Table 1 for the components and their addition amounts, and the total volume is 25 μl) for reaction.

[0116] Table 1

[0117] components Amount added For sample solution 2μl dNTP Mixture 2μl upstream primer solution 1μl Downstream Primer Solution 1μl 5×Prime Script II Buffer 4μl PrimeScript II RTase 1μl HotMaster Taq DNA polymerase 0.5μl 10×HotMaster Taq Buffer 2.5μl RNase Free dH 2 o

Make up to 25μl

[0118] Active ingredients contained in dNTPs mixture and their concentrations: 100 μM dATP, 100 μM dCTP, 87.5 μM dGTP, 87.5 μM dTTP, 12.5 μM Fl-dGTP and 12.5 μM Fl-dUTP. Fl-dGTP is fluorescein-labeled dGTP (PerkinElmer, NEL429001EA, Fluorescein-12-dGTP). Fl-dUTP is fluorescein-labeled dUTP (PerkinElmer, NEL413001EA, Fluorescein-12-dUTP).

[0119] The upstream primer solution provides a...

Embodiment 3

[0132] Sample solution for the test: take armored RNA1 and dilute it with PBS buffer so that the concentration of armored RNA1 is 10 1 Copy number / mL, 10 2 Copy number / mL, 10 3 Copy number / mL, 10 4 Copy number / mL, 10 5 Copy number / mL, 10 6 Copy number / mL, 10 7 copy number / mL or 10 8 Copy number / mL. Control (CK): Use sterile distilled water instead of the sample solution for the test.

[0133] Adopt the method of embodiment 2 to detect. During detection, the primer pair prepared in Example 1 for detecting the O gene of COVID-2019 was used. For detection, use step 3 (1) to detect fluorescence resonance energy transfer.

[0134] see results figure 1 . The minimum virus concentration in the sample solution that can be detected is 10 6 Copy number / mL.

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Abstract

The invention discloses a method for rapidly detecting RNA viruses based on CCP-FRET. The invention provides a method for detecting whether a to-be-detected sample contains target nucleic acid. The method sequentially comprises the following steps: (1) taking the to-be-detected sample or nucleic acid extracted from the to-be-detected sample as a template, taking a target nucleic acid as a target fragment for PCR amplification, and carrying out PCR amplification in a system containing dye-labeled dNTP to obtain a dye-labeled PCR amplification product; and (2) mixing the dye-labeled PCR amplification product with a conjugated polymer, and judging whether the to-be-detected sample contains target nucleic acid or not by detecting fluorescence energy resonance transfer. According to the invention, a conjugated polymer-based fluorescence energy resonance transfer technology is adopted to establish the method for detecting whether the to-be-detected sample contains the target nucleic acid ornot, such that rapid, visual and low-cost detection can be achieved.

Description

technical field [0001] The invention relates to a method for rapid detection of RNA viruses based on CCP-FRET. Background technique [0002] In recent years, public health problems caused by pathogenic microorganisms have attracted more and more attention. Bacterial, viral and fungal infections are often very serious and even life-threatening. [0003] Typical methods for identifying microorganisms include culturing on selective media, analysis of visual morphological structures, and detection of immunological features. These techniques require well-trained microbiologists to operate, and have the disadvantages of variable sensitivity and long time consumption. [0004] Pathogen detection technologies include immunological methods for detecting pathogen antigen-antibodies, PCR methods based on specific nucleic acid probes, DNA microarray methods, mass spectrometry methods, biosensing methods, and surface plasmon resonance methods. Although these detection techniques have ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6818
CPCC12Q1/701C12Q1/6818C12Q2531/113C12Q2563/107Y02A50/30
Inventor 王树刘礼兵吉列尔莫·巴赞
Owner INST OF CHEM CHINESE ACAD OF SCI
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