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Detection reagent and detection method for Zika virus

A detection method and virus technology are applied in the field of analysis and detection to achieve the effects of sensitive on-site detection and improved detection sensitivity

Pending Publication Date: 2022-05-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is well known in the art that there are generally false positives in the method based on the detection of nucleic acid, and the LAMP in the flow measurement method also needs to be heated, which has certain requirements on the temperature; and the use of antibodies as recognition elements is easy to interact with other antigens cause cross reaction

Method used

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  • Detection reagent and detection method for Zika virus
  • Detection reagent and detection method for Zika virus
  • Detection reagent and detection method for Zika virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The construction of biotin-SA scaffold on the membrane of embodiment 1

[0048] The nylon membrane is first cut into circles using a custom steel die. The circular membranes were then rinsed three times in 0.1 M NaOH and deionized water, respectively. Subsequently, with NH 2 - The capped surface-modified membrane was gently shaken for 20 minutes in freshly prepared EDCI solution (150 mg / L) containing 15 mg / L biotin. Thus, biotin was immobilized on the membrane. Next, the biotin-labeled membrane was washed twice with deionized water and once with PBST, and incubated with SA (10 mg / L in PBST) for 30 minutes at 4°C with gentle shaking. After three washes with PBST to remove excess SA, the membrane was incubated with 40 μL cApt for 30 min at 4 °C. To reduce non-specific adsorption on the membrane, wash the membrane three times with PBST, then incubate with fatty acid-free BSA dissolved in PBST (1%, m / v) for 1 h at 4 °C, wash three times in PBST to remove residual blocki...

Embodiment 2

[0049] The preparation of embodiment 2 probe

[0050] The preparation of the probes can be briefly summarized as the surface of AuNPs was first immobilized with SA, where dApt and biotin-HRP molecules were immobilized sequentially.

[0051] First, 100 μL of SA (1 mg / mL) was added to 1 mL of AuNP, followed by gentle shaking at 4 °C for 1 h to immobilize SA molecules on the AuNP surface through electrostatic interaction. Then, the mixture was centrifuged at 8000 g for 10 min at 4°C to remove excess reagents and dissolved in deionized water (pH 7.5). Subsequently, 10 μL aliquots of dApt and 8 μL aliquots of biotin-HRP were added to the mixture and incubated at 4° C. for 30 minutes, respectively. Then, the mixture was centrifuged at 8000g for 10 minutes at 4°C to remove reagents. Finally, the centrifuged mixture was dissolved in deionized water (pH 7.5) containing 1% BSA (m / v) and stored at 4°C.

Embodiment 3

[0052] Example 3 Immunoassay and colorimetric reaction

[0053] The sensors prepared by the above method were incubated in PBST and real samples containing different concentrations of ZIKV-NS1 at room temperature for 30 min with slight shaking, and then rinsed three times with PBST. Subsequently, the antigen-conjugated sensor was incubated with the probe for 10 minutes, which was used as a detection probe to form a sandwich immunoassay. Finally, the reacted membrane-based sensor was rinsed three times with PBST and immersed in SA, TMB one solution (containing TMB and H 2 o 2 ) for 10 minutes for a sufficient colorimetric reaction. With the catalysis of the HRP loaded on the probe, the TMB molecule in the H 2 o 2 oxidized to TMB under the combined action of 2+ , indicating the presence of ZIKV-NS1.

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Abstract

The invention belongs to the field of analysis and detection, and relates to a Zika virus detection reagent and a Zika virus detection method. According to the membrane-based sensor and the detection method disclosed by the invention, viruses can be rapidly and sensitively detected by naked eyes. Target viruses are recognized and captured by specific capture elements and detection elements fixed on the membrane and the probe, so that a sandwich form is formed on the membrane-based sensor. Through colorimetric reaction, the color change of the sensor capturing the probe can be observed, and the existence of the target virus is indicated. The whole detection process can be completed within one hour. Based on the common influence of on-membrane pre-concentration and dual signal amplification, the sensor disclosed by the invention can realize the ng / mL-level detection limit. Due to high sensitivity and convenience, the technical scheme has a wide prospect in rapid and on-site virus detection in clinical and environmental applications.

Description

technical field [0001] The invention belongs to the field of analysis and detection, and relates to a detection reagent and a detection method for Zika virus. Background technique [0002] Detection of virus using a variety of techniques including conventional virus culture methods, molecular detection techniques, and immunoassays [3] . Currently, the commonly used virus detection method is based on reverse transcription polymerase chain reaction (RT-PCR) [4] ; And the basic requirements for using these methods are huge equipment and skilled technicians. Immunoassays are suitable for rapid in situ detection of viruses due to their less training and time costs. Lateral flow assay (lateral flow) is a membrane-based immunoassay and has many advantages such as user-friendly operation, short detection time, and does not require sample pretreatment or instrumentation [5] . However, its sensitivity is generally lower than that of laboratory-based immunoassays or RT-PCR. There...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/58G01N33/543G01N21/78
CPCG01N33/56983G01N33/581G01N33/587G01N33/54346G01N21/78G01N2469/10G01N2333/185G01N2333/183Y02A50/30
Inventor 陈若虹陈保卫栾天罡刘洪涛
Owner SUN YAT SEN UNIV
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