Method for detecting SARS-CoV-2 by combining reverse transcription multi-cross displacement amplification with nano-biology sensing

A cross-replacement and reverse transcription technology, applied in the field of microbiology, can solve the problems affecting the efficiency of emergency detection, false negatives, complicated operation steps, etc., and achieve the effect of excellent detection sensitivity and fast detection speed.

Active Publication Date: 2020-08-11
贵州省疾病预防控制中心
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Problems solved by technology

However, this method has the following disadvantages: (1) expensive fluorescent quantitative RT-PCR instruments are required, and general laboratories cannot meet the requirements for equipment; (2) the method takes 4 to 5 hours from nucleic acid extraction to report issuance, which affects Emergency detection efficiency; (3) Novel coronavirus infection cannot be ruled out even if the detection result of this method is negative, there is a possibility of false negative due to insufficient sensitivity of the technology itself
However, this kind of constant temperature technology requires multiple enzymes to work simultaneously to achieve nucleic acid amplification, relies on expensive reagents, and has complicated operation steps. Its practicability, convenience and operability need to be improved

Method used

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  • Method for detecting SARS-CoV-2 by combining reverse transcription multi-cross displacement amplification with nano-biology sensing
  • Method for detecting SARS-CoV-2 by combining reverse transcription multi-cross displacement amplification with nano-biology sensing
  • Method for detecting SARS-CoV-2 by combining reverse transcription multi-cross displacement amplification with nano-biology sensing

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The feasibility of embodiment 1.MCDA-LFB amplification

[0061] Standard MCDA reaction system: the concentration of cross primer CP1 and CP1* is 30 pmol, the concentration of cross primer CP2 is 60 pmol, the concentration of displacement primer F1 and F2 is 10 pmol, the concentration of amplification primer R1, R2, D1 and D2 is 30 pmol, The concentration of amplification primers C1 and C2 is 20pmol, 10mM Betain, 6mM MgSO 4 , 0.1 mM Biotin-14-dATP and Biotin-14-dCTP, 1.4 mM dNTP, 12.5 μL of 10×Bst DNA polymerase buffer, 10 U of strand-displacing DNA polymerase, 1 μL of template, supplemented with deionized water to 25 μl. The entire reaction was thermostated at 64°C for 35 minutes.

[0062] After MCDA amplification, the MCDA product can be detected by LFB, positive two red bands (TL and CL) appear in the detection area, negative only one red band (CL). Since the hapten labeled with the MCDA primer designed for the F1ab of SARS-CoV-2 is Dig (digoxin). Therefore, when ...

Embodiment 2

[0064] Example 2. Feasibility of Multiplex MCDA-LFB Amplification

[0065] Multiplex MCDA reaction system: the concentration of cross primer F1ab-CP1 and F1ab-CP1* is 10 pmol, the concentration of cross primer F1ab-CP2 is 30 pmol, the concentration of displacement primer F1ab-F1 and F1ab-F2 is 5 pmol, and the concentration of amplification primer F1ab- The concentration of R1, F1ab-R2, F1ab-D1 and F1ab-D2 is 15 pmol, the concentration of amplification primers F1ab-C1 and F1ab-C2 is 10 pmol, the concentration of cross primer N-CP1 and N-CP1* is 10 pmol, cross primer The concentration of N-CP2 is 30pmol, the concentration of displacement primers N-F1 and N-F2 is 5pmol, the concentration of amplification primers N-R1, N-R2, N-D1 and N-D2 is 15pmol, and the concentration of amplification primers N- The concentration of C1 and N-C2 is 10 pmol, 10 mM Betain, 6 mM MgSO4, 0.1 mM Biotin-14-dATP and Biotin-14-dCTP, 1.4 mM dNTP, 12.5 μL of 10×Bst DNA polymerase buffer, 10U of strand-dis...

Embodiment 3

[0068] Embodiment 3. determine the optimal reaction temperature of MCDA technology

[0069] Under standard reaction system conditions, add the corresponding MCDA primers designed for the SARS-CoV-2 virus F1ab plasmid and N plasmid templates, and the template concentration is 5000 copies. The reaction was carried out under constant temperature conditions (60-67°C), and the results were detected by a real-time turbidimeter, and different dynamic curves were obtained at different temperatures, see Figure 6 (F1ab sequence amplification) and Figure 7 (N gene sequence amplification). 64-65°C is recommended as the optimal reaction temperature for MCDA primers. In the follow-up verification of the present invention, 64° C. was selected as the constant temperature condition for MCDA amplification. Figure 6 and 7 Represents the temperature dynamic curve of MCDA primers designed for the detection of SARS-CoV-2 virus for F1ab and N gene sequences.

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Abstract

The invention discloses a method for detecting SARS-CoV-2 by combining reverse transcription multi-cross displacement amplification with nano-biology sensing. The method aims at an open reading frame1a / b(F1ab) fragment and two targets of an N gene of the SARS-CoV-2, a uniquely designed multi-cross displacement amplification primer is used, a digoxin half antigen is marked on the 5'-end of a F1absequence cross primer CP1 in the multi-cross displacement amplification, and a FITC (fluorescein) half antigen is marked on the 5'-end of an N gene cross primer CP1. The method aims at a situation that the Flab fragment of the 2019-novel coronavirus and a product amplified by the N gene can be visually detected through a macromolecule nano-biology sensor. The method is convenient, quick, sensitive and specific and is suitable for the specific detection of the 2019-novel coronavirus.

Description

technical field [0001] The invention discloses a detection method for a novel coronavirus (SARS-CoV-2), belonging to the field of microbiology. Background technique [0002] Novel coronavirus is the pathogen that causes severe acute respiratory syndrome (severe acute respiratory syndrome) viral pneumonia. On January 12, 2020, the World Health Organization will name this virus 2019-novel coronavirus (2019-novel coronavirus), It was later renamed as severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2). The viral pneumonia that appeared this time was named the new type of coronavirus pneumonia (COVID-19), referred to as "new coronary pneumonia". [0003] Coronaviruses (coronavirus, CoVs) are a large class of viruses, named for the corona-like spikes found on the virus envelope by electron microscope observations, which can infect mammals and birds and cause a variety of diseases. There are 6 known human coronaviruses, 4...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/107C12Q2565/607Y02A50/30
Inventor 李世军蒋维佳黄俊飞
Owner 贵州省疾病预防控制中心
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