Ultrasensitive electrochemical detection of Kouanxi carp spring viremia virus based on gold nanoparticle signal amplification

Abstract

The invention discloses a method for ultrasensitive electrochemical detection of port carp spring viremia virus based on gold nanoparticle signal amplification, comprising the following steps: S1. preparing MBs-probe 1; S2. preparing AuNPs-probe 2; S3. preparing AuNPs-probe 4; S4. gold-plating the surface of the screen-printed electrode and covalently modifying the nucleic acid probe on the surface, then adsorbing hexaamine ruthenium, performing electrochemical determination, and obtaining the concentration of SVCV. The present invention establishes an ultrasensitive portable electrochemical detection method for SVCV based on the principle of gold nanoparticle signal amplification adsorbed by hexaamine ruthenium by combining high-affinity probes, which can amplify the signal geometrically, greatly improve the sensitivity, and the detection limit of SVCV is 0.0646nM, the recovery rate is 95%-102%, the specificity is high, the stability and reproducibility are good, the operation is simpler, the time required for detection is shortened, and the detection efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and more specifically, to an ultra-sensitive electrochemical detection method for Kougang carp spring viremia virus based on gold nanoparticle signal amplification. Background Art

[0002] Spring viremia of carp virus (SVCV) belongs to the genus Vesiculovirus and is a rhabdovirus that is mainly prevalent in Europe, the Middle East and Russia, and has spread to America and Asia in recent years. SVCV has a very wide host range and can cause large-scale outbreaks of spring viremia of carp (SVC) in fish. It is an animal disease that must be reported to the World Organization for Animal Health and is also a Class I animal disease listed in the "List of Class I and Class II Infectious Diseases and Parasitic Diseases of Animals Entering the People's Republic of China". It is a key disease to be prevented and controlled in freshwater fish farms. As the first line of defense to protect the country's borders, customs animal quarantine work plays a vital role in the prevention and control of this type of disease, and it is necessary to quickly and accurately detect and identify SVCV in daily monitoring work.

[0003] Traditional virus detection methods are reliable, but time-consuming and complex to operate. They have certain limitations in specific identification, sensitivity and rapid response. They are incapable of meeting the requirements in emergency situations and work scenarios with high customs clearance efficiency. For example, there are many interfering substances in actual biological detection samples, such as animal serum, frozen aquatic products, aquatic fish products and complex feed products, as well as mixed breeding of different types of fish and stacking of aquatic products in cold storage. These situations put high demands on the detection methods. Therefore, it is particularly important to develop a new, efficient, rapid and highly specific electrochemical detection method for carp spring viremia virus (SVCV) to achieve rapid and sensitive screening of aquatic animals at the port for this pathogen. Summary of the invention

[0004] The purpose of the present invention is to provide a method for ultrasensitive electrochemical detection of port carp spring viremia virus based on gold nanoparticle signal amplification.

[0005] According to one aspect of the present invention, a method for ultrasensitive electrochemical detection of Kougang Carp Spring Viremia Virus based on gold nanoparticle signal amplification is provided, comprising the following steps:

[0006] S1. Immobilizing the biotin-modified carp spring viremia virus SVCV probe 1 on the surface of streptavidin-modified magnetic beads to form a capture element 1, namely MBs-probe 1;

[0007] S2. immobilizing the thiol-SH-modified carp spring viremia virus SVCV probe 2 on the surface of gold nanoparticles to form a capture element 2, namely, AuNPs-probe 2;

[0008] S3. MBs-probe 1 and AuNPs-probe 2 specifically recognize and bind to the conserved sequence of SVCV gene to form a sandwich structure of MBs-probe 1 / virus / AuNPs-probe 2. The AuNPs end in the sandwich structure is cut by using the property of exonuclease III to cut the 3' recessed end of double-stranded DNA, and the supernatant is collected by magnetic separation to obtain the signal element AuNPs-probe 4.

[0009] S4. Drop AuNPs-probe 4 onto the surface of the screen-printed electrode that is gold-plated and covalently modified with SVCV probe 3, and use silver deposition or hexaamineruthenium adsorption to amplify the signal, and perform electrochemical determination by square wave voltammetry to obtain the concentration of SVCV.

[0010] In some embodiments, the sequence of SVCV probe 1 in step S1 is as shown in SEQ ID NO: 1: 5'-CAGTTTACTCCTCTTCATAAA-Biotin-3'.

[0011] In some embodiments, the sequence of SVCV probe 2 in step S2 is as shown in SEQ ID NO: 2: 5'-SH-AAAATGCCGATGGCCTTCGTGTGATTCTGTTGC-3'

[0012] In some embodiments, the sequence of SVCV probe 3 in step S4 is as shown in SEQ ID NO: 3: 5'-SH-AAAGGCCATCGGCAT-3'.

[0013] In some embodiments, the conserved sequence of the SVCV gene in step S3 is as shown in SEQ ID NO: 4: 5'-ATGAAGAGGAGTAAACTGCCTGCAACAGAATCACACGAA-3'.

[0014] In some embodiments, the preparation step of MBs-probe 1 is specifically to mix the streptavidin-modified magnetic beads with probe 1, incubate with shaking at 37°C, and further treat with bovine serum albumin to obtain MBs-probe 1. The preparation step of AuNPs-probe 2 is specifically to mix SVCV probe 2 and AuNPs evenly, freeze at -20°C for 1.5h, then thaw, and centrifuge at 12000rpm at 4°C for 30min to obtain AuNPs-probe 2.

[0015] In some embodiments, the preparation steps of AuNPs-probe 4 are specifically to mix MBs-probe 1, AuNPs-probe 2 and the conserved sequence of the SVCV gene, and incubate them with shaking at 37°C, magnetically separate and wash, add nuclease exonuclease III to perform enzymatic reaction at 37°C, then terminate the reaction at 70°C, and then magnetically separate and collect the supernatant to obtain AuNPs-probe 4.

[0016] In some embodiments, the surface modification step of the screen-printed electrode in step S4 is specifically to add chloroauric acid solution to the surface of the activated screen-printed electrode for electrodeposition, and then drop the SVCV probe 3 on the surface of the gold-plated screen-printed electrode and incubate at 37°C, and then place it in MCH solution for 30 minutes to remove non-specific adsorption, rinse with deionized water to remove the unadsorbed MCH on the electrode surface, and finally drop the AuNPs-probe 4 obtained in step S3 on the electrode surface and incubate at 37°C.

[0017] In some embodiments, the step of adsorbing hexaamineruthenium in step S4 is specifically to drop 10 μL of 5 μM hexaamineruthenium solution on the surface of the screen-printed electrode treated in the surface modification step of the screen-printed electrode, and incubate for 5 minutes, so that the hexaamineruthenium is adsorbed on the negatively charged DNA phosphate backbone through strong electrostatic interaction, thereby achieving the adsorption of hexaamineruthenium on the surface of the screen-printed electrode.

[0018] In some embodiments, the electrochemical determination step in step S4 is to immerse the screen-printed electrode obtained after the hexaamineruthenium adsorption in a solution containing 50 mM Tris-HCl, 5 mM [Ru(NH 3 ) 6 3+ ], square wave voltammetry scanning was performed at a potential between -0.15 V and -0.4 V in a scanning solution with a pH value of 7.0, and the parameters were set as: potential increment 2.0 mV, amplitude 25 mV, and frequency 15 Hz.

[0019] The beneficial effects of the present invention are as follows: the present invention realizes highly selective capture and identification of SVCV by combining high-affinity probes, and establishes an ultrasensitive portable electrochemical detection method for SVCV based on the signal amplification principle of gold nanoparticles adsorbed by hexaamineruthenium. This method is different from the low sensitivity of colloidal gold test paper and the time-consuming and high equipment requirements of electron microscope detection. The method can amplify the signal geometrically and greatly improve the sensitivity. The detection limit of SVCV is 0.0646nM, the recovery rate is 95%-102%, the specificity is high, the stability and reproducibility are excellent, the operation is simpler, the detection time is shortened, and the detection efficiency is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is a working principle diagram of the detection method established in Example 1 of the present invention.

[0021] Figure 2 It is a feasibility verification diagram of the detection method of Example 2 of the present invention, wherein curve a is measured without adding SVCV standard solution, and curve b is measured with adding SVCV standard solution.

[0022] Figure 3 It is a line graph showing the change of current intensity with binding time in Example 3 of the present invention.

[0023] Figure 4 It is a line graph showing the change of current intensity with the concentration of exonuclease III in Example 3 of the present invention.

[0024] Figure 5 It is a linear relationship diagram between the SVCV concentration and the corresponding current intensity of Example 4 of the present invention.

[0025] Figure 6 This is a specificity verification diagram of Example 5 of the present invention. DETAILED DESCRIPTION

[0026] The present invention is further described in detail through specific implementation cases, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art are all limited by the claims attached to this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0027] Example 1 An ultrasensitive electrochemical detection method for Kougang carp spring viremia virus based on gold nanoparticle signal amplification comprises the following steps:

[0028] S1. Preparation of MBs-probe 1

[0029] Take 10 µL of 10 mg / mL streptavidin-modified magnetic beads, add 990 µL of 50 mM Tris-HCl buffer to wash three times, dilute 5 times with 50 mM Tris-HCl buffer, add 20 µL of 10 µM SVCV probe 1 and 230 µL of 50 mM Tris-HCl buffer, mix well, incubate at 37 °C with shaking for 90 min, wash by magnetic separation and discard all the liquid, add 300 µL of 50 mM Tris-HCl buffer, repeat washing three times, add 500 µL of 2% BSA solution, stand at 37 °C for 1 h, magnetic separation and discard the supernatant, wash three times, and resuspend in 1.5 mL of 50 mM Tris-HCl buffer to obtain MBs-probe 1;

[0030] S2. Preparation of AuNPs-probe 2

[0031] Add 50 mL of 5 mM HAuCl 4 The solution was heated to boiling and 5 mL of 38.8 mM Na 3 C 6 H 5 O 7 ·2H 2 O, continue heating and stirring, stop heating when the solution turns wine red, continue stirring until cooling, and prepare AuNPs, which are stored at 4°C away from light for later use;

[0032] Take 6 µL of 100 µM SVCV probe 2 in an Ep tube, add 294 µL of AuNPs, mix by pipetting, freeze at -20°C for 1.5 h, thaw, centrifuge at 4°C, 12000 rpm, 30 min, wash three times with 50 mM Tris-HCl buffer, and finally redisperse in 300 µL of 50 mM Tris-HCl buffer to obtain AuNPs-probe 2, which is stored at 4°C in the dark.

[0033] S3. Magnetic separation and enzyme catalysis reaction

[0034] Take 150 μL MBs-probe 1, 100 μL AuNPs-probe 2, and 10 μL SVCV gene conserved sequence and mix them in an Ep tube, incubate at 37℃ with shaking for 1 h, perform magnetic separation on the above solution, discard 250 μL supernatant, add 250 μL 50mM Tris-HCl buffer and mix well, repeat washing 3 times, add 1 μL 10U Exo Ⅲ, shake gently at 37℃ for 30min, terminate the reaction after heating at 70℃ for 20 min, perform magnetic separation, collect the supernatant, and obtain AuNPs-probe 4;

[0035] S4. Surface modification of screen-printed electrodes

[0036] S4.1 Pretreatment of electrode surface

[0037] The screen-printed carbon electrodes were placed in 0.5 MH 2 SO 4 Cyclic voltammetry was performed in the solution for 30 cycles to remove surface impurities, with the scanning potential set at −0.3 V and +1.50 V, and the electrode was dried at room temperature;

[0038] S4.2 Surface modification of electrodes

[0039] 100 µL of 5 mM HAuCl 4Drop it on the surface of the activated screen-printed electrode for electrochemical deposition, the deposition potential is -200 mV, the time is 120 s, wash and blow dry, drop 10 μL 1 μMSVCV probe 3 on the gold-plated screen-printed electrode surface, incubate at 37°C for 1.5 h, rinse with ultrapure water, incubate in 100 μL 1.0 mM MCH solution for 30 min to remove non-specific adsorption, rinse with deionized water to remove the unadsorbed MCH on the electrode surface, dry at room temperature, take 10 μL AuNPs-probe 4 and drop it on the electrode surface, incubate at 37°C for 1.5 h, rinse the electrode surface with deionized water for 10 s, and dry at room temperature;

[0040] S4.3 Hexaammineruthenium adsorption

[0041] 10 μL of 5 μM hexaamineruthenium solution was added dropwise to the surface of the screen-printed electrode modified in S4.2, and incubated for 5 min, so that the hexaamineruthenium was adsorbed on the negatively charged DNA phosphate backbone through strong electrostatic interaction, thereby achieving the adsorption of hexaamineruthenium on the surface of the screen-printed electrode;

[0042] S4.4 Electrochemical determination

[0043] The screen-printed electrode obtained in S4.3 was immersed in a solution containing 50 mM Tris-HCl, 5 mM [Ru(NH 3 ) 6 3+ ], square wave voltammetry scanning was performed at a potential between -0.15 V and -0.4 V in a scanning solution with a pH value of 7.0, and the parameters were set as: potential increment 2.0 mV, amplitude 25 mV, and frequency 15 Hz.

[0044] The working principle diagram of the detection method in this embodiment is as follows Figure 1 shown.

[0045] Example 2 Feasibility Verification

[0046] 20 μL of 1 μM SVCV standard solution was selected for feasibility verification. The detection method of this embodiment refers to Example 1, and the verification results are shown in Figure 2 .

[0047] like Figure 2 As shown, when the detection is carried out without adding the SVCV standard solution (curve a), the current intensity is relatively weak. However, when the SVCV standard solution is added (curve b), it can be found that the current is significantly enhanced. Through the comparative verification experiment of this embodiment, it is fully proved that the ultrasensitive electrochemical detection method of Kougang Carp Spring Viremia Virus based on gold nanoparticle signal amplification of the present invention is feasible.

[0048] Example 3 Performance optimization of detection method

[0049] 3.1 Optimization of the binding time between the conserved sequence of SVCV gene and MBs-probe 1 and AuNPs-probe 2

[0050] In order to improve the detection efficiency of the method, the binding time of the conserved sequence of the SVCV gene, MBs-probe 1 and AuNPs-probe 2 was optimized, and the binding time was set to 15min, 30min, 45min, 60min, 75min and 90min, respectively, and then the method of Example 1 was referred to for comparative determination. The specific results are shown in Figure 3 .

[0051] like Figure 3 As shown in the figure, when the binding time of the SVCV gene conservative sequence with MBs-probe 1 and AuNPs-probe 2 increases from 15 min to 60 min, the current signal gradually increases; after more than 60 min, the current signal tends to be stable and no longer increases. This indicates that at 60 min, the binding between the SVCV gene conservative sequence and MBs-probe 1 and AuNPs-probe 2 has reached saturation, and 60 min is selected as the optimal binding time.

[0052] 3.2 Optimization of exonuclease III concentration

[0053] In order to ensure that the surface of the screen-printed electrode is modified with a sufficient amount of AuNPs-probe 4, it is necessary to optimize the amount of exonuclease III (ExoIII) to ensure sufficient enzyme cleavage reaction. The concentrations of exonuclease III were set to 2.5U, 5.0U, 10U, 15U, 20U, and 40U, respectively, and then the comparative determination was carried out according to the method of Example 1. The determination results are shown in Figure 4 .

[0054] like Figure 4 As shown in the figure, as the amount of Exo III gradually increases, the electrical signal increases; however, when the amount of Exo III reaches 20U, the electrical signal no longer increases, indicating that the AuNPs-probe 4 modified on the surface of the screen-printed electrode has reached saturation, so 20U is selected as the optimal amount of Exo III.

[0055] Example 4 Determination of the linear detection range and detection limit of the method

[0056] 1 nM, 10 nM, 100 nM, and 1000 nM SVCV standard solutions were selected and linear detection was performed according to the detection method of Example 1, with three replicates for each concentration. Figure 5 .

[0057] like Figure 5As shown in the figure, there is a good linear relationship between the peak current signal of SWV and the logarithm of SVCV concentration. The linear equation is: I(µA)=-0.19401+3.0024lg[C] (C represents SVCV concentration), and the correlation coefficient R 2 The detection range is 1-1000 nM, and the minimum detection limit is 0.0646 nM, which shows that the detection method of the present invention has the characteristics of wide linear range and low detection limit when detecting SVCV.

[0058] Example 5 Specificity Verification

[0059] In view of the common mixed culture of different types of fish and stacking of aquatic products in cold storage in the detection of inbound and outbound ornamental fish and aquatic products, four treatments were set up, including goldfish hematopoietic organ necrosis disease suspension (GFHNV group), fish neural necrosis virus suspension (NNV group), fish infectious spleen and kidney necrosis disease suspension (ISKNV group) and SVCV group. The specificity of the method for detecting SVCV was verified by referring to the detection method of Example 1. The results are shown in Table 1. Figure 6 .

[0060] like Figure 6 As shown, the current signal intensity of the GFHNV group, NNV group, and ISKNV group was significantly lower than that of the SVCV group, indicating that an obvious current signal can be generated only in the presence of SVCV, indicating that the detection method of the present invention has good specificity for SVCV.

[0061] Example 6 Sample Measurement

[0062] There are many interfering substances in the actual biological detection samples at the port, such as animal serum, frozen aquatic products, aquatic fish products and complex feed products. In order to be closer to the actual working scene, three different concentrations of SVCV standard solutions of 1.0nM, 10.0nM and 100nM were added to the 10-fold diluted carp serum, and the detection method of Example 1 was used for detection, and the recovery rate was calculated to evaluate the performance of the detection method of the present invention in complex matrices. The results are shown in Table 1.

[0063] Table 1 Recovery rate and relative standard deviation

[0064]

[0065] As can be seen from Table 1, the recovery rate of the detection method of the present invention is in the range of 95% to 102%, indicating that the detection method of the present invention can accurately capture SVCV in serum, and the relative standard deviation (RSD) is between 2% and 6.94%, proving that the detection method has good stability.

[0066] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. An ultrasensitive electrochemical detection method for non-disease diagnosis of Kougang carp spring viremia virus based on gold nanoparticle signal amplification, characterized in that: The following steps are involved: S1. Immobilizing the biotin-modified carp spring viremia virus SVCV probe 1 on the surface of streptavidin-modified magnetic beads to form a capture element 1, namely MBs-probe 1; S2. immobilizing the thiol-SH-modified carp spring viremia virus SVCV probe 2 on the surface of gold nanoparticles to form a capture element 2, namely, AuNPs-probe 2; S3. The MBs-probe 1 and AuNPs-probe 2 specifically recognize and bind to the conserved sequence of the SVCV gene to form a sandwich structure of MBs-probe 1 / virus / AuNPs-probe 2, and the AuNPs end in the sandwich structure is cut by utilizing the property of exonuclease III to cut the 3' recessed end of double-stranded DNA, and the supernatant is collected by magnetic separation to obtain the signal element AuNPs-probe 4; S4. The AuNPs-probe 4 is dropped onto the surface of the screen-printed electrode which is gold-plated and covalently modified with the SVCV probe 3, and the signal is amplified by hexaammineruthenium adsorption. The concentration of SVCV is obtained by electrochemical determination by square wave voltammetry. The sequence of the SVCV probe 1 is: 5'-CAGTTTACTCCTCTTCATAAA-Biotin-3', the sequence of the SVCV probe 2 is: 5'-SH-AAAATGCCGATGGCCTTCGTGTGATTCTGTTGC-3', the sequence of the SVCV probe 3 is: 5'-SH-AAAGGCCATCGGCAT-3', and the conserved sequence of the SVCV gene is: 5'-ATGAAGAGGAGTAAACTGCCTGCAACAGAATCACACGAA-3'. The hexaammineruthenium adsorption step in step S4 is specifically to add 10 μL A 5 μM hexaamineruthenium solution was added dropwise to the surface of the screen-printed electrode treated in the above surface modification step and incubated for 5 min, so that the hexaamineruthenium was adsorbed on the negatively charged DNA phosphate backbone through strong electrostatic interaction, thereby achieving the adsorption of hexaamineruthenium on the surface of the screen-printed electrode.

2. The ultrasensitive electrochemical detection method for non-disease diagnosis of Kougang Carp Spring Viremia Virus based on gold nanoparticle signal amplification according to claim 1, characterized in that: The preparation steps of the MBs-probe 1 are specifically to mix the streptavidin-modified magnetic beads with the probe 1, incubate with shaking at 37°C, and further treat with bovine serum albumin to obtain MBs-probe 1. The preparation steps of the AuNPs-probe 2 are specifically to mix the SVCV probe 2 with AuNPs evenly, freeze at -20°C for 1.5h, then thaw, and centrifuge at 12000rpm for 30min at 4°C to obtain AuNPs-probe 2.

3. The ultrasensitive electrochemical detection method for non-disease diagnosis of Kougang Carp Spring Viremia Virus based on gold nanoparticle signal amplification according to claim 1, characterized in that: The specific preparation steps of the AuNPs-probe 4 are to mix the MBs-probe 1, the AuNPs-probe 2 and the SVCV gene conserved sequence, and incubate them with shaking at 37°C. After magnetic separation and washing, add nuclease exonuclease III to perform enzyme cleavage reaction at 37°C, then terminate the reaction at 70°C, and then perform magnetic separation, collect the supernatant to obtain AuNPs-probe 4.

4. The ultrasensitive electrochemical detection method for non-disease diagnosis of Kougang Carp Spring Viremia Virus based on gold nanoparticle signal amplification according to claim 1, characterized in that: The surface modification step of the screen-printed electrode in step S4 is specifically to add chloroauric acid solution to the surface of the activated screen-printed electrode for electrodeposition, and then drop the SVCV probe 3 on the surface of the gold-plated screen-printed electrode and incubate at 37°C, and then place it in MCH solution for 30 minutes to remove non-specific adsorption, rinse with deionized water to remove the unadsorbed MCH on the electrode surface, and finally drop the AuNPs-probe 4 obtained in step S3 on the electrode surface and incubate at 37°C.

5. The ultrasensitive electrochemical detection method for non-disease diagnosis of Kougang Carp Spring Viremia Virus based on gold nanoparticle signal amplification according to claim 1, characterized in that: The electrochemical determination step in step S4 is to immerse the screen-printed electrode obtained after the hexaamineruthenium adsorption in a solution containing 50mM Tris-HCl, 5mM [Ru(NH3)6 3+ ], square wave voltammetry scanning was performed at a potential between -0.15 V and -0.4 V in a scanning solution with a pH value of 7.0, and the parameters were set as: potential increment 2.0 mV, amplitude 25 mV, and frequency 15 Hz.

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