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Detection method of new coronavirus SARS-CoV-2 antigen

A detection method and virus antigen technology, applied in the biological field, can solve problems such as false negatives and insufficient detection sensitivity, and achieve the effects of low preparation cost, good stability, and improved accuracy

Active Publication Date: 2021-09-28
杭州佑戈科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And the new coronavirus SARS-CoV-2 is constantly mutating, which puts more pressure on detection
However, the existing antigen detection kits have the problem of insufficient detection sensitivity leading to false negatives. There is an urgent need for a faster antigen detection method with better detection sensitivity and the ability to detect mutated viruses.

Method used

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  • Detection method of new coronavirus SARS-CoV-2 antigen
  • Detection method of new coronavirus SARS-CoV-2 antigen
  • Detection method of new coronavirus SARS-CoV-2 antigen

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preparation example Construction

[0038] In the preparation of the immune gold signal nanoparticles, the preparation of the washing solution PBS-T: Tween-20 with a concentration of 0.01%-1% is added to the PBS solution.

[0039] Preferably, the aptamer S Aptamer 2 modified by the sulfhydryl group is

[0040] 5-SH-CAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTTAATGGACA-3

[0041] Preferably, the preferred biotinylated aptamer S Aptamer 2 (or other novel coronavirus antigen biotinylated nucleic acid aptamers), is characterized in that the nucleic acid aptamer is thiol-labeled, preferably end-modified thiol-labeled.

[0042] Preferably, the method for detecting novel coronavirus SARS-CoV-2 antigen based on nucleic acid aptamer recognition surface-enhanced Raman spectroscopy comprises the step of capturing nanoparticles with the prepared immunomagnetic beads and novel coronavirus SARS-CoV- 2 After the antigen or virus sample is mixed, it is then mixed with immunogold signal nanoparticles (or other mixing order), incu...

Embodiment 1

[0053] 1) Preparation of nano-gold sol: get 200mL mass fraction of 0.01% chloroauric acid solution and heat to boiling under magnetic stirring; then get 4.5mL mass fraction of 1% sodium citrate solution, add boiling chloroauric acid solution After boiling for 20-30 minutes, the reaction is completed, and cooled to room temperature to prepare nano-gold sol.

[0054] 2) Preparation of immune gold signal nanoparticles: mix 100ul gold nanoparticles with 10ul 30uM Raman reporter NBA (Nile Blue A), incubate at room temperature for 10 minutes, centrifuge, discard the supernatant, and redisperse in PBS-T In the solution, mix with sulfhydryl-modified aptamer S Aptamer 2 (sequence as described above), freeze at -20°C for 2 hours, after thawing, wash with PBS-T for 3 times, add 500ul of 1% BSA solution to block The remaining active sites were incubated at room temperature for 2 hours, centrifuged, washed 3 times with PBS-T solution, and finally the supernatant was discarded and redispers...

Embodiment 2

[0061] 1) Preparation of nano-gold sol: get 200mL mass fraction of 0.01% chloroauric acid solution and heat to boiling under magnetic stirring; then get 4.5mL mass fraction of 1% sodium citrate solution, add boiling chloroauric acid solution After boiling for 20-30 minutes, the reaction is completed, and cooled to room temperature to prepare nano-gold sol.

[0062] 2) Preparation of immune gold signal nanoparticles: mix 100ul gold nanoparticles with 10ul 30uM Raman reporter NBA (Nile Blue A), incubate at room temperature for 10 minutes, centrifuge, discard the supernatant, and redisperse in PBS-T In the solution, mix with sulfhydryl-modified aptamer combinations S Aptamer 1 and S Aptamer 2, freeze at -20°C for 2 hours, after thawing, wash with PBS-T for 3 times, add 500ul of 1% BSA solution to block the remaining activity site, incubated at room temperature for 1 h, centrifuged, washed 3 times with PBS-T solution, and finally discarded the supernatant, redispersed in 500ul PBS...

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Abstract

The invention discloses a detection method of a new coronavirus SARS-CoV-2 antigen, and belongs to the technical field of biology. The method comprises the following steps of capturing nanoparticles by virtue of new coronavirus SARS-CoV-2S protein immunomagnetic beads, carrying out specific immunization on the nanoparticles and S protein, and incubating with immune gold signal nanoparticles so as to construct a sandwich structure; detecting an SERS signal through a Raman spectrometer, specifically detecting the new coronavirus SARS-CoV-2 protein within 5 minutes, and specifically detecting the pseudovirus and variant pseudovirus of the SARS-CoV-2 and the pseudovirus diluted in human saliva. Through the above mode, nucleic acid extraction or antibody-dependent detection is not needed, meanwhile, the method has the advantages of being good in stability, higher in specificity and affinity, low in immunogenicity, easy to chemically modify, simple and convenient to operate and the like, the rapid detection of the new coronavirus SARS-CoV-2 antigen and the variant virus can be achieved within five minutes, and a condition is created for the early detection of the 2019SARS-CoV-2 pathogen.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting SARS-CoV-2 antigens of a new coronavirus based on nucleic acid aptamer recognition surface-enhanced Raman spectroscopy. Background technique [0002] The etiological and serological examinations in the detection method of the new coronavirus pneumonia virus SARS-CoV-2 mainly include nucleic acid detection, antibody detection, and antigen detection. Among them, nucleic acid detection is the "gold standard" for SARS-CoV-2 detection, but it has the disadvantages of being time-consuming and unable to detect on-site. However, antibody detection requires a certain window period, making it difficult to achieve early diagnosis of infected persons. [0003] Existing detection methods such as: [0004] CN202011431411.8 discloses a kit for detecting novel coronavirus and its mutants, which is characterized in that it includes the following parts: (1) presubstrate A chai...

Claims

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Application Information

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IPC IPC(8): G01N21/65G01N33/569
CPCG01N21/658G01N33/56983G01N2333/165
Inventor 李剑锋关鹏程宋彦龄张月皎
Owner 杭州佑戈科技有限公司
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