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Port porcine epidemic diarrhea virus electrochemical detection method based on silver deposition signal amplification

A porcine epidemic diarrhea and signal amplification technology, applied in the field of life sciences, can solve problems such as low success rate, long time-consuming virus, and limited popularization, and achieve high stability and reproducibility, convenient disease prevention and control, and high equipment requirements Effect

Active Publication Date: 2022-07-29
NANJING AGRICULTURAL UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The colloidal gold test strip method is a common diagnostic method for PEDV, but typically, it requires high concentrations of labels to make the color visible, and its detection sensitivity is 1000 times lower than that of laboratory instrument detection methods; using Raman spectroscopy to read When taking the test strip signal, the nitrocellulose membrane in the test strip often produces interference signals, which affects the accuracy of the test results; the separation method takes a long time to detect the virus and the success rate is not high, so it can only be used as an auxiliary detection method; The high requirements for the method equipment and the long time for reporting the results limit its popularization; both PCR and ELISA will have false positive results, and they cannot be promoted in clinical use.

Method used

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  • Port porcine epidemic diarrhea virus electrochemical detection method based on silver deposition signal amplification
  • Port porcine epidemic diarrhea virus electrochemical detection method based on silver deposition signal amplification
  • Port porcine epidemic diarrhea virus electrochemical detection method based on silver deposition signal amplification

Examples

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Embodiment 1

[0025] Example 1. Construction of detection method

[0026] An electrochemical detection method for port porcine epidemic diarrhea virus based on silver deposition signal amplification, comprising the following steps:

[0027] (1) Preparation of MBs-probe 1

[0028] Take 10 μL of 10 mg / mL streptavidin-modified magnetic beads, wash 3 times with 50 mM Tris-HCl buffer, and dilute the magnetic beads by 5 times, then mix 20 μL of 10 μM probe 1, 50 μL MBs, and 230 μL Tris-HCl at 37 Shake at ℃ for 90 min, wash with Tris-HCl for 3 times, add 500 μL of 2% BSA for further treatment for 1 h, and finally resuspend in 3 mL of Tris-HCl;

[0029] (2) Preparation of AuNPs-probe 2

[0030] 20 μL of 10 μM probe 2 was heated to 95°C for 5 min, rapidly cooled to room temperature, and then mixed with 500 μL of AuNPs. After shaking for 12 h, sodium chloride solutions of different concentrations were added to the above solution. Centrifuge three times at 12000rpm for 30min at 4°C, and finally red...

Embodiment 2

[0039] Example 2. Feasibility verification

[0040] 20 μL of 1 μM PEDV standard solution was selected for feasibility verification, and the steps included in the detection method of this embodiment are the same as those of the content of the invention (1)-(6). like figure 2As shown, first of all, the current intensity is weaker when PEDV is not added (curve a), while when PEDV is added (curve b), it can be found that the current is significantly enhanced. Feasibility of electrochemical detection methods.

Embodiment 3

[0041] Example 3. Sensitivity experiment

[0042] The standard solutions of PEDV with concentrations of 1, 10, 100, and 1000 nM are selected for detection. The detection method of this embodiment includes the same steps as those of the content of the invention (1)-(6). like image 3 As shown in (left), with the continuous increase of PEDV concentration in the solution, the current on the electrode surface increases continuously. like image 3 As shown in (right), according to the linear results obtained by the current generated by different concentrations of PEDV solutions, the linear detection equation of the biosensor is: I=2.53775+3.50079lgC(R 2 =0.99999), the detection range is 1-1000nM, based on the calculation method that the signal-to-noise ratio is equal to 3, the minimum detection limit of the detection system is 0.4744nM. This shows that the method of the present invention can effectively and sensitively detect the content of PEDV in the solution. By replacing nu...

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Abstract

The invention discloses a port porcine epidemic diarrhea virus electrochemical detection method based on silver deposition signal amplification, and belongs to the technical field of life science. The detection principle is as follows: a biotin-modified porcine epidemic diarrhea virus PEDV probe 1 is fixed on the surface of a streptavidin-modified magnetic bead to form a capture element 1, namely MBs-probe1, a sulfydryl SH-modified PEDV probe 2 is fixed on the surface of a gold nanoparticle to form a capture element 2, namely AuNPs-probe2, and when a screened PEDVN gene conserved sequence exists, the capture element 1, namely MBs-probe1, and the capture element 2, namely AuNPs-probe2, is fixed on the surface of the magnetic bead; the two sections of capture elements are hybridized with the conserved sequence of the PDEVN gene to form an MBs-probe1 / AuNPs-probe2 / PEDV complex; the complex is cut by using the characteristic that exonuclease III cuts the sunken tail end of double-chain DNA 3 ', a signal element AuNPs-probe4 is collected through magnetic separation, then the collected signal element is dropwise added to the surface of a silk-screen printing electrode which is plated with gold on the surface and is covalently modified with a nucleic acid probe probe3, and signal amplification is performed by using silver deposition. And finally, the current intensity measured by square wave voltammetry (SWV) is positively correlated with the PEDV concentration.

Description

technical field [0001] An electrochemical detection method of port porcine epidemic diarrhea virus based on silver deposition signal amplification belongs to the field of life science technology. Background technique [0002] Port quarantine and testing capabilities are an important part of the core capabilities of port public health, and are also an important guarantee for the effective prevention and control of imported epidemics. Porcine epidemic diarrhea virus (PEDV) is also a single-stranded positive-stranded RNA virus of the family Coronaviridae, which is consistent with the family and genus of SARS-CoV-2. It poses a serious threat to animal health and causes huge economic losses to animal husbandry. It can be used as a model virus for SARS-CoV-2 and other coronaviruses for the development of detection methods. At present, there are six main detection methods for PEDV, namely colloidal gold test strip method, Raman spectroscopy, virus isolation and culture method, imm...

Claims

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Application Information

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IPC IPC(8): G01N27/327G01N27/48
CPCG01N27/3277G01N27/48Y02A50/30
Inventor 康伟任向阳董莹
Owner NANJING AGRICULTURAL UNIVERSITY
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