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SARS-CoV-2 delta variant detection method based on primer design and copper nanoclusters

A detection method, copper nano-cluster technology, applied in the field of biosensors, can solve problems such as complicated operation steps, and achieve the effect of increasing ΔCt value and high specificity

Active Publication Date: 2022-01-11
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above method is either complicated in operation steps, or requires base modification, which increases the complexity and cost of detection

Method used

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  • SARS-CoV-2 delta variant detection method based on primer design and copper nanoclusters
  • SARS-CoV-2 delta variant detection method based on primer design and copper nanoclusters
  • SARS-CoV-2 delta variant detection method based on primer design and copper nanoclusters

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Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Design principles for the detection of SARS-CoV-2 delta variants based on rational primer design and copper nanoclusters

[0050] In order to more sensitively detect the variant strain of the new coronavirus, this study developed a precise detection method for the delta variant of SARS-CoV-2 based on primer design and CuNCs. The primer design includes two parts of PCR upstream primer and downstream primer. First, in order to improve the specificity of detection, DPO primers were used to identify the mutation sites, wherein the DPO primers were specific primers linked by polyisoxanthine, and the mutation sites with single base deletion were designed at the 3' end, and then passed Non-specific complementary hypoxanthine-linked primers with complementary bases at the 5' and 3' ends. At the same time, the AT base content of the primer was artificially increased at the 5' end, and the AT primer was constructed to increase the fluorescence value of CuNCs, making i...

Embodiment 2

[0054] Embodiment 2. Experimental feasibility verification

[0055] The method of PCR was used to prove the feasibility of DPO primers to distinguish the wild type and the delta mutant strains with single base deletion. A 25 μL PCR reaction system contains 1×SuperMix, 600 nM primers, and distilled water. The amplification program was 93°C for 3 min; 30 cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s. Divided into 6 pairs of primers: F-1 and R-1, F-2 and R-2, F-1 and R-1-down (PT), F-2-up (PT) and R-2, F- 1 and R-1-down (DPO), F-2-up (DPO) and R-2. Among them, F-1 and R-1, F-2 and R-2 are common PCR primers designed by NCBI; F-1 and R-1-down (PT), F-2-up (PT) and R-2 It is a common primer, and the length of the primer is exactly the same as that of F-1 and R-1-down (DPO), F-2-up (DPO) and R-2, except that the middle part of the latter is a hypoxanthine link, while the former is linked with Targets are linked by bases that are perfectly complementary. The main dif...

Embodiment 3

[0056] Embodiment 3 Concrete experimental condition optimization

[0057] 1. Optimization of annealing temperature

[0058] Annealing temperature is the key factor to achieve specific detection, so in order to distinguish the above phenomena more clearly, two pairs of F-1 and R-1-down (DPO), F-2-up (DPO) and R-2 Primers were optimized for annealing temperature. The result is as image 3 , 3A-3F is the amplification curve optimized by the annealing temperature of F-1 and R-1-down (DPO), and it can be seen from the figure that as the temperature increases, the specificity of the detection method is better; Figure 4 Among them, 4A-4F are the amplification curves optimized by the annealing temperature of F-2-up (DPO) and R-2. It can be seen from the figure that the specificity of the detection method is better as the temperature increases; and image 3 3G and 3H in the middle show the change of the ΔCt value of the two pairs of primers with the change of temperature. Although...

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Abstract

The invention discloses an SARS-CoV-2 delta variant detection method based on a primer design and copper nanoclusters. According to the method, a DPO primer and an AT primer are combined to successfully distinguish the SARS-CoV-2 delta variant with deletion of a single basic group from the SARS-CoV-2 wild strain. Moreover, PCR products of the DPO primer and the AT primer can be used as a generation template of the CuNCs, and visual detection of the SARS-CoV-2 delta variant is realized under ultraviolet irradiation. According to the method, the DPO primer and the AT primer are combined and amplified by virtue of a PCR instrument under conventional experimental conditions, so that the SARS-CoV-2 delta variant detection has the advantages of specificity, high sensitivity and visualization.

Description

technical field [0001] The invention belongs to the field of biosensors, and in particular relates to a method for detecting a SARS-CoV-2 delta variant strain based on rational primer design and copper nanoclusters. Background technique [0002] SARS-Cov-2 is a type of single-stranded RNA virus. The virus particles are mostly round, with a capsule, and there are coronal-arranged filaments around the periphery, so it is named coronavirus. After the virus enters the human body, it first binds to cells rich in "angiotensin-converting enzyme 2 (ACE2) receptors on the surface of the human body, and then affects the human lungs, cardiovascular system, kidneys, and even the brain. It can also be transmitted from person to person through aerosols. Although the fatality rate is lower than Ebola and MERS, its transmissibility and pathogenicity are difficult to overcome. At present, the variant strains worthy of attention announced by the World Health Organization include Alpha, Beta, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11
CPCC12Q1/701C12Q1/6858C12Q2600/156C12Q2531/113C12Q2523/313C12Q2563/107C12Q2563/137Y02A50/30
Inventor 许文涛杜再慧朱龙佼
Owner CHINA AGRI UNIV
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