Cotton swab product for grouper nervous necrosis virus and application

Abstract

The invention discloses a swab product of a grouper nervous necrosis virus and application of the swab product. The method comprises the following steps: modifying the surface of an activated swab with a grouper nervous necrosis virus NNV specific sequence capture probe Cp to prepare a recognition element; meanwhile, a signal probe Sp and a color signal molecule thymolphthalein TP are modified on the surface of the silver-loaded polydopamine nanoparticle to form TP-Sp-silver nanoparticle co-modified and wrapped polydopamine nanoparticle to obtain a color developing particle PDANS (at) Ag / Sp / TP, when NNV exists, the color developing particle PDANS (at) Ag / Sp / TP is connected to the surface of a cotton swab, then the cotton swab is placed in an alkaline reagent, and the color developing particle PDANS (at) Ag / Sp / TP is obtained. A color signal TP loaded on the surface of the color developing particle can be released into a solution for color developing, the color signal is measured by using a smart phone for analysis, and the product can be used for quantifying PRRSV (Porcine Reproductive and Respiratory Syndrome Virus).

Description

Technical Field

[0001] The invention discloses a cotton swab product for grouper neuronecrosis virus and application thereof, belonging to the field of life science technology. Background Art

[0002] With the continuous expansion of the scale of grouper farming in my country, the high density of farming and other reasons have led to more and more diseases in the farming process, which seriously threatens the normal development of the grouper farming industry. Among various diseases, nervous necrosis is one of the diseases that has the greatest impact on grouper farming. Nervous necrosis is also known as viral encephalopathy, "black body disease" and retinopathy. The pathogen is the nervous necrosis virus (NNV), which is one of the smallest animal viruses known to date and belongs to the second category of infectious diseases and parasitic diseases. After being infected with NNV, groupers float on the water surface and mainly on one side of the body, with slow reactions and poor vitality. The mortality rate after illness is high. In severe cases, it can cause the death of all farmed fry within a week. There is currently no specific medicine for this disease. Therefore, NNV poses a huge threat to the cultivation of fry of grouper and even marine fish.

[0003] At present, enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) are mostly used to detect NNV. The former can use specific antigens and antibodies for detection, and has the characteristics of high accuracy, strong specificity, and automated high throughput. However, due to the influence of certain external factors, the specific binding of ELISA is often unstable and prone to false positive results. In addition, the antibody preparation process is complicated, time-consuming, and costly, making it impossible to perform low-cost rapid detection. The RT-PCR method uses nucleic acid sequences as probes or recognition beacons to make up for the shortcomings of low sensitivity and high cost of the antigen-antibody detection system, but it has the disadvantages of cumbersome operation, easy contamination, medium sensitivity, and can only perform qualitative analysis. Therefore, it is very necessary to develop a simple, rapid, low-cost, and ideally specific and sensitive NNV detection method.

[0004] Among various detection technologies, colorimetric biosensor detection methods stand out in the detection fields of pathogens, chemicals, heavy metals, foods, etc. due to their simplicity, convenience, rapidity, and high efficiency. Cotton swabs are a cheap and readily available tool that uses the imine product generated by the Schiff base reaction to specifically separate and concentrate targets in complex environments, showing great potential in on-site analysis and instant diagnosis. At the same time, the simplicity of the colorimetric detection result analysis platform and the portability of the equipment are the key to achieving rapid on-site detection. With the increasing power and popularity of smartphone functions, smartphones are combined with colorimetric biosensor technology, and the image information of colorimetric detection results is collected through mobile phone software, and intelligent analysis is performed on it. Accurate quantification of the target can be achieved without complex instruments and professional operators. Summary of the invention

[0005] In view of the shortcomings of current detection technology, the purpose of the present invention is to provide a cotton swab for grouper neuronecrosis virus and an application method. When the target virus is present, the color-developing nanoparticles loaded with the indicator are coupled to the cotton swab through a sandwich method, and the image information of the color contrast detection results is collected and intelligently analyzed by mobile phone software to achieve ultra-sensitive detection of the target virus.

[0006] The technical solution of the present invention is:

[0007] A cotton swab for grouper neuronecrosis virus and application thereof, comprising the following steps:

[0008] (1) Preparation of polydopamine-loaded silver nanoparticles

[0009] Dissolve 50 mg of dopamine hydrochloride in 200 μL of 1.25 M FeSO 4 .7H 2 O, 0.2MH 2 O 2 , 25 mL Tris-buffer and 25 mL anhydrous ethanol, stirred at room temperature in the dark for 45 min, centrifuged at 10000 rpm for 10 min and washed three times to obtain a precipitate, which was freeze-dried to obtain PDANS. 2 mg PDANS was ultrasonically dispersed in 5 mL H 2 O, 1 mL of 0.278 g / mL sodium citrate solution and 20 mL of freshly prepared 0.025 M silver nitrate solution were added, stirred at room temperature for 3 h, then centrifuged at 10000 rpm for 10 min, and washed with ultrapure water for 3 times to obtain PDANS@Ag.

[0010] (2) Modification of signal probe Sp

[0011] 100 μL of 2 μM Sp was added to 1000 μL of 2 mg / mL PDANS@Ag solution, incubated at 37 °C with shaking for 12 h, washed three times by centrifugation with ultrapure water, and resuspended in 1 mL of water to obtain PDANS@Ag / Sp.

[0012] (3) Loading of thymolphthalein TP

[0013] 400 μL of 10 mM TP solution was added to 1 mL of PDANS@Ag / Sp solution, incubated with shaking at 37 °C for 2 h, washed three times by centrifugation with ultrapure water, and resuspended in 1 mL of water to obtain PDANS@Ag / Sp / TP.

[0014] (4) Activation of cotton swabs

[0015] Soak a cotton swab in 2 mM KIO 4 The cotton swab was immersed in a mixed solution of 100 μL concentrated sulfuric acid and a volume of 800 μL, kept in dark place at room temperature overnight, and washed three times with a large amount of ultrapure water to obtain an activated cotton swab.

[0016] (5) Modification of capture probe Cp

[0017] Take 400 μL of 0.25 μM capture probe Cp and place it in an activated cotton swab for overnight incubation at 37°C. Wash it three times with 1 / 15 M phosphate buffer at pH 7.4 and transfer the cotton swab to a total volume of 400 μL 400 mM NaCNBH 3 The cells were incubated in a solution of a 1:1 mixture of a reducing agent and 1 / 15 M phosphate buffer at 37° C. for 2 h, then blocked with 400 μL 1% BSA for 1 h and washed three times with 1 / 15 M phosphate buffer.

[0018] (6) Virus Detection

[0019] The cotton swab was placed in a mixed solution of 50 μL NNV virus RNA extract and 350 μL 1 / 15M phosphate buffer, incubated at 37°C for 30 min, washed three times with 1 / 15M phosphate buffer, and then placed in a mixed solution of 200 μL PDANS@Ag / Sp / TP and 200 μL PB buffer, reacted at 37°C for 30 min. After washing three times, 200 μL alkaline NaOH solution with a pH value of 13 was added to make the color-developing nanoparticles enriched on the swab undergo a color reaction and release a large amount of color signals. The color signal of the color developer TP was measured using a smartphone, and the amplified color signal could be acquired and analyzed. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The bar graph and linear relationship diagram are shown for NNV detection at different concentrations.

[0021] Figure 2 This is the result of NNV virus detection in grouper samples. DETAILED DESCRIPTION

[0022] The following examples are intended to better illustrate the present invention, but the present invention is not limited by the following examples.

[0023] Example 1. Detection experiment of viral nucleic acid at different concentrations

[0024] The target virus nucleic acid standard solutions with concentrations of 0.01, 0.1, 1, 10, 100, and 1000 nM were selected for detection. The detection method of this embodiment includes the same steps as those in the invention (1) to (6). Figure 1 As shown in the figure, as the concentration of target virus nucleic acid in the solution increases, the color signal of the cotton swab increases. According to the linear result graph obtained by the B / R ratio generated by different concentrations of NNV, the biosensor detection system can achieve the target virus within 10 -2 -10 3 The linear equation for the detection within the nM linear range is: Y = 0.23927lgC + 1.50617 (R 2 =0.996), and the minimum detection limit was 10.6 pM. This shows that the method of the present invention has good specificity and high sensitivity.

[0025] Example 2. Detection experiment of NNV virus in real grouper samples

[0026] Take the brain and eye tissues of the suspected NNV grouper, extract RNA for testing, the detection method of this embodiment includes the same steps as (1)-(6) in the invention content, and use RT-PCR for verification. Figure 2 As shown, sample No. 3 was positive, and samples No. 1, 2, and 4 were negative, which were consistent with the results of RT-PCR, indicating that the biosensor detection system has high accuracy and can be used for actual NNV detection.

Claims

1. A cotton swab product and application of grouper neuronecrosis virus, characterized in that: The method comprises the following steps: (1) dopamine hydrochloride and FeSO4·7H2O, H2O2, Tris-buffer and anhydrous ethanol were mixed and fully dissolved, stirred at room temperature in the dark for 45 minutes, and then centrifuged and freeze-dried to obtain PDANS; PDANS was ultrasonically dispersed in H2O, sodium citrate solution and freshly prepared silver nitrate solution were added, stirred for 3 hours, then centrifuged and washed with ultrapure water to obtain silver-loaded polydopamine nanoparticles PDANS@Ag, wherein the concentration of FeSO4·7H2O was 1.25M, the concentration of H2O2 was 0.2M, the concentration of sodium citrate solution was 0.278 g / mL, and the concentration of silver nitrate solution was 0.025M; (2) The PDANS@Ag prepared in step (1) was ultrasonically dispersed in ultrapure water, a signal probe Sp was added, and the mixture was incubated by oscillation, and the mixture was washed by centrifugation with ultrapure water and then resuspended to obtain Sp-silver nanoparticle co-modified polydopamine nanoparticles PDANS@Ag / Sp, to which thymolphthalein TP solution was added for oscillation and incubation, and then the mixture was centrifuged and washed with ultrapure water to obtain PDANS@Ag / Sp / TP, wherein the concentration of the signal probe Sp was 2 μM, and the concentration of the TP solution was 10 mM; (3) Soak the cotton swab in a mixed solution of KIO4 and concentrated sulfuric acid overnight, and wash the activated cotton swab with a large amount of ultrapure water; take the capture probe Cp and put it into the activated cotton swab for incubation overnight, and after washing, transfer the cotton swab to a phosphate buffer solution PB containing a reducing agent NaCNBH3 for incubation, and then block it with 1BSA and wash it with PB buffer solution, wherein the KIO4 concentration is 2mM, the capture probe Cp concentration is 0.25μM, the NaCNBH3 reducing agent concentration is 400mM, the PB concentration is 1 / 15M, and the BSA concentration is 1%; (4) placing the cotton swab in step (3) in a mixed solution of the target virus RNA extract and PB buffer and incubating for 30 minutes, and after washing, placing the cotton swab in a mixed solution of the PDANS@Ag / Sp / TP prepared in step (2) and PB buffer for reaction and then washing; (5) The cotton swab in step (4) is placed in an alkaline reagent to color the TP loaded on the surface. A standard curve is drawn using a smartphone for analysis, thereby achieving sensitive detection of the target virus content. By replacing the target virus-specific sequence and the corresponding probe, universal detection of other viruses can be achieved.

2. A cotton swab product and application of grouper neuronecrosis virus according to claim 1, characterized in that The sequence of the signal probe Sp modified on the surface of the color-developing nanoparticles is: 5′-NH2-(CH2)6-CCAGCAATACGACCGTCGATTGTGGCAAGC-3′, and the sequence of the capture probe Cp modified on the surface of the cotton swab is: 5′-GCGTACTCACGAGCGTAACACTTGCCCTGC-(CH2)6-NH2-3′.

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