Unlock instant, AI-driven research and patent intelligence for your innovation.

Semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV), and preparation method of test paper

A technology for detecting test strips and virus detection, which is applied in the fields of virology and diagnostic reagents, and immunology. It can solve the problems of polyclonal antibodies prone to cross-reaction, affecting the detection sensitivity of test strips, and unable to quantitatively analyze, achieving rapid detection and easy operation. , The effect of low detection cost

Inactive Publication Date: 2015-02-18
OCEAN UNIV OF CHINA
View PDF7 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional immunochromatographic test strips can only perform qualitative detection of pathogens, but cannot quantitatively analyze them, and cannot meet the needs of rapid quantification of pathogens at the breeding site and real-time evaluation of disease development; in addition, the use of the same antibody for capture antibodies and detection antibodies will cause As a result, the two compete for the same epitope, which affects the detection sensitivity of the test paper, and the polyclonal antibody is prone to cross-reaction, resulting in false positives

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV), and preparation method of test paper
  • Semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV), and preparation method of test paper
  • Semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV), and preparation method of test paper

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Preparation of test paper for semi-quantitative rapid detection of WSSV in prawns

[0038] 1. Preparation of anti-WSSV monoclonal antibody A and monoclonal antibody B and ascites preparation and purification

[0039] (1) Cell fusion: BALB / c mice were immunized with purified WSSV and Freund’s complete adjuvant in equal amounts (V / V) as an antigen. After 2 weeks, WSSV was mixed with Freund’s incomplete adjuvant in equal amounts (V / V V) Boost immunization once after mixing; then boost immunization once every week with purified WSSV by tail vein injection, and 3 days after the third boost immunization, aseptically remove the mouse spleen and P3- X63-Ag8U1 myeloma cells were fused with polyethylene glycol solution, and the fused cell suspension was mixed evenly and dropped into a 96-well culture plate, and placed in CO 2 The concentration was 4.5% in a 37°C incubator. Cell growth was observed with an inverted microscope, and hybridoma cell culture supernatant ...

Embodiment 2

[0066] Example 2: Determination of detection limit and quantitative range of semi-quantitative test paper for WSSV in prawn

[0067] 1. Purification of WSSV

[0068] The gills of Penaeus prawns suffering from white spot disease were taken, and 5 mL of TNE buffer (Tris-HCl 1.566 g, NaCl 5.844 g, EDTA-Na 2 0.372 g, H 2(O 1 000 mL, pH 7.5) prepared 25% sucrose solution, ground thoroughly in an ice bath for 3–5 min, filtered the grinding liquid into a 50 mL centrifuge tube, and centrifuged at 3 000, 5 000, and 8 000 rpm at 4 °C For 20 min, the supernatant was centrifuged at 20 000 rpm for 1.5 h in a Hitachi ultracentrifuge, and the precipitate was resuspended by adding an appropriate amount of 25% sucrose solution, then centrifuged at 25 000 rpm at 4 °C for 2 h through a discontinuous sucrose density gradient, and sucked out with a syringe for 46 h. For the virus band between the %~52% sucrose layer, add TNE buffer at 1:5 (V / V), centrifuge at 20 000 rpm for 1.5 h, and resuspe...

Embodiment 3

[0071] Example 3: Detection method of semi-quantitative test paper for WSSV in prawns

[0072] (1) Preparation of test samples

[0073] Take the gills of Penaeus prawns suffering from white spot disease, and add 5 mL of TNE buffer solution (Tris-HCl 1.566 g, NaCl 5.844 g, EDTA-Na 2 0.372 g, H 2 O 1 000 mL, pH 7.5) prepared 25% sucrose solution, fully ground in an ice bath for 3-5 min, filtered the grinding solution with gauze, and took the supernatant as the test sample solution.

[0074] (2) Application of test paper for semi-quantitative detection of prawn WSSV

[0075] Hold one end of the absorbent pad 4 of the test paper of the present invention, immerse the end of the sample pad 1 in the test sample for about 10 seconds, the immersion depth is about 0.5 cm, and lay the test paper flat for about 10 minutes, observe the test results with the naked eye, and quantify according to the color development of the test lines T1, T2 and T3 viral content.

[0076] (3) Resu...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
concentrationaaaaaaaaaa
Login to View More

Abstract

The invention discloses a semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV). The semi-quantitative rapid detection test paper comprises a carrier plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad and the water absorption pad are respectively arranged at the two ends of the carrier plate; the nitrocellulose membrane is arranged in the middle of the carrier plate; the gold-labeled pad loaded with a gold-labeled monoclonal antibody A is arranged at the position where the sample pad intersects with the nitrocellulose membrane; the edge of one end of the gold-labeled pad is overlapped under the sample pad, and the edge of the other end of the gold-labeled pad is overlapped above the nitrocellulose membrane; the nitrocellulose membrane is provided with three detection lines (T1, T2 and T3) which are sequentially arranged, and a quality control line; the three detection lines are respectively enveloped with a monoclonal antibody B, and the monoclonal antibodies B have different concentrations; the concentrations of the monoclonal antibodies B on the T1, T2 and T3 are sequentially reduced from the side of the gold-labeled pad; the quality control line close to the water absorption pad is enveloped with goat anti-mouse IgG. The semi-quantitative rapid detection test paper is rapid in detection; after the test paper is used, the result visible to the naked eyes can be obtained after about 10 minutes, and a pathogen can be detected in a way of relative quantification; the test paper is simple and convenient to operate and low in detection cost, thus being suitable for field test of WSSV in the aquaculture process of prawns, crabs, etc.

Description

Technical field [0001] The invention involves a detection method for the White Spot Syndrome Virus (WSSV), and it also involves a semi -quantitative test paper and its preparation methods for shrimp white spots.Technical field. Background technique [0002] Park white spots virus is an outbreak epidemic caused by shrimp WSSV. The mortality rate can reach 100%within 3 to 12 days after shrimp infection WSSV.The disease has frequently occurred since the first outbreak in 1993. It has restricted the healthy and sustainable development of the shrimp breeding industry and effectively controlling the outbreak of the disease has become the most prominent and urgent need to solve the problem.Due to the lack of effective treatment measures, in the early stage of WSSV infection, rapid and quantitative detection of WSSV is of great significance to prevent the ongoing outbreak and early warning forecast of the disease and reduce economic losses. [0003] At present, the commonly used WSSV det...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/558
CPCG01N33/577
Inventor 绳秀珍张玲唐小千战文斌邢婧
Owner OCEAN UNIV OF CHINA