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A kind of detection method of new coronavirus sars-cov-2 antigen

A detection method and virus antigen technology, applied in the biological field, can solve the problems of false negative and insufficient detection sensitivity, and achieve the effects of low preparation cost, good stability and fast detection speed.

Active Publication Date: 2022-05-13
厦门睿信诺生物科技有限公司
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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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  • A kind of detection method of new coronavirus sars-cov-2 antigen
  • A kind of detection method of new coronavirus sars-cov-2 antigen
  • A kind of 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 for a new coronavirus SARS-CoV-2 antigen, which belongs to the field of biotechnology. In the present invention, the new coronavirus SARS‑CoV‑2S protein immune magnetic beads captures nanoparticles and S protein for specific immunization, and incubates with immune gold signal nanoparticles to construct a sandwich structure; then, it can be directly measured by a Raman spectrometer Its SERS signal can specifically detect the new coronavirus SARS-CoV-2 protein within 5 minutes, and can also specifically detect SARS-COV-2 pseudoviruses, mutant pseudoviruses and pseudoviruses diluted in human saliva. The above methods do not require nucleic acid extraction or rely on antibodies for detection, and at the same time have the advantages of good stability, higher specificity and affinity, low immunogenicity, easy chemical modification, and simple operation, and can realize the detection of new coronavirus within five minutes. The rapid detection of SARS‑CoV‑2 antigens and mutant viruses has created conditions for the early detection of 2019 SARS‑CoV‑2 pathogens.

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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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/65G01N33/569
CPCG01N21/658G01N33/56983G01N2333/165
Inventor 李剑锋关鹏程宋彦龄张月皎
Owner 厦门睿信诺生物科技有限公司