Rapid test paper for pathogenic Vibrio parahaemolyticus

A technology for Vibrio haemolyticus and detection test paper, which is applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of heavy workload, accurate determination of detection results, and increased screening difficulty for common antigens, and achieves the effect of accurate distinction.

Active Publication Date: 2017-05-10
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the similar taxonomic status, Vibrio and even Vibrio and other pathogenic bacteria often have common antigenic proteins, resulting in cross-immune reactivity between different bacteria, which interferes with the accurate determination of test results. Therefore, Excluding the interference of cross-reaction of other bacteria to accurately determine the infection of a certain pathogenic bacteria is the key problem to be solved in the detection of pathogens by immunological techniques
The usual solution is to screen the unique antigenic components or specific antibodies of pathogenic bacteria, but the existence of common antigens increases the difficulty of screening and the workload is large

Method used

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  • Rapid test paper for pathogenic Vibrio parahaemolyticus
  • Rapid test paper for pathogenic Vibrio parahaemolyticus
  • Rapid test paper for pathogenic Vibrio parahaemolyticus

Examples

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preparation example Construction

[0029] One, the preparation method of test paper of the present invention is as follows:

[0030] (1) Preparation of gold standard pad

[0031] ① Preparation of colloidal gold and gold-labeled rabbit antibody

[0032] Adopt microwave oven-trisodium citrate reduction method to make particle size and be the colloidal gold of 20nm, adjust colloidal gold pH value to be 8.4 with 0.1mol / L potassium carbonate solution; Then add BSA, centrifuge and purify, and suspend with gold-labeled preservation solution, which is the gold-labeled rabbit anti-liquid.

[0033] ② Preparation of gold standard pad

[0034] The prepared gold standard rabbit anti-liquid was sprayed on the glass fiber and freeze-dried.

[0035] (2) Design and preparation of detection layer of nitrocellulose membrane

[0036] The detection layer is designed according to the principle of antigen chip. Using the principle of antigen chip method, a variety of antigenic components of pathogenic bacteria are arranged toget...

Embodiment 1

[0053] Embodiment 1: the preparation of Vibrio parahaemolyticus rapid detection test paper gold standard pad

[0054] 1. Preparation, purification and potency determination of rabbit anti-Vibrio parahaemolyticus antibody

[0055] (1) Preparation of rabbit anti-Vibrio parahaemolyticus antibody

[0056] ①Expand culture Vibrio parahaemolyticus at 37°C for 24 hours, wash 3 times with 0.01mol / L sterile PBS, resuspend the bacteria, and adjust to 1×10 8 CFU / mL. A certain amount of formalin was added to the bacterial solution and inactivated for 24 hours. The next day, take the bacteria liquid plate and streak to check the degree of inactivation.

[0057] ②The New Zealand white rabbits were immunized four times with one week interval between each time. For the first immunization, the bacterial solution was mixed with Freund's complete adjuvant at a ratio of 1:2 (v:v), and injected at 6 points on the back, 0.2 mL per point. For the second immunization, the bacterial solution was m...

Embodiment 2

[0077] Example 2: Preparation of the detection layer of Vibrio parahaemolyticus rapid detection test paper and assembly of the test paper

[0078] 1. Preparation of Vibrio parahaemolyticus outer membrane protein

[0079] ① Expand culture of Vibrio parahaemolyticus at 37°C, centrifuge at 8,000×g for 15 min at 4°C, wash away the medium, resuspend and wash with 0.01mol / L PBS for 3 times, and finally add PBS to concentrate to 1 / 25 of the original volume.

[0080] ② Add a small amount of 10% sucrose solution to the collected bacterial liquid, mix well, and treat at -20°C for 15 minutes. Add an equal volume of 0.4% Triton X-100 (PBS), mix well at room temperature, and act for 10 minutes. Centrifuge at 12,000×g for 15 min at 4°C, and take the supernatant. Add 2.5 times volume of ice ethanol, mix well, and place overnight at -20°C. The next day, centrifuge at 12,000×g at 4°C for 15 minutes, and obtain the precipitate by high-speed centrifugation.

[0081] ③ After the precipitate w...

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Abstract

The invention provides a rapid test paper for pathogenic Vibrio parahaemolyticus. A cellulose nitrate membrane is provided with four test lines, i.e., T1, T2, T3 and T4, and a control line C which are successively arranged from the side close to a colloidal-gold pad, wherein the four test lines T1, T2, T3 and T4 are the outer membrane protein, flagellin, an extracellular product and whole bacterium disruption protein of Vibrio parahaemolyticus, respectively, and the control line C close to a water absorbing pad is goat anti-rabbit IgG. According to the invention, a plurality of antigen proteins of pathogenic Vibrio parahaemolyticus are extracted and specifically arrayed, and simultaneously react with a to-be-tested sample on the test paper, and in particular, the test line T4 is provided with the whole bacterium disruption protein of Vibrio parahaemolyticus; thus, the problem that immunological cross-reactions due to common antigens of other bacteria interfere in result determination is overcome, and positive results and cross reactions can be accurately distinguished.

Description

technical field [0001] The invention belongs to the technical field of pathogenic microorganism detection products, in particular to a rapid detection test paper for pathogenic Vibrio parahaemolyticus (Vibrio parahaemolyticus) in aquatic animals. Background technique [0002] Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is widely distributed in salt lakes and seafood such as fish, shrimp, crab, and shellfish. Eating food contaminated by Vibrio parahaemolyticus can cause gastrointestinal dysfunction and acute gastroenteritis , septicemia and other diseases are common pathogenic bacteria that cause food poisoning or foodborne diseases. Vibrio parahaemolyticus belongs to the genus Vibrio, and there are more than 20 kinds of vibrio pathogens reported in marine cultured animals. Due to the ecological changes in the breeding waters in recent years, Vibrio has become one of the main pathogenic bacteria of marine cultured animals, and often occurs in multiple...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/558
CPCG01N33/558G01N33/56911G01N2333/28
Inventor绳秀珍李嘉文战文斌唐小千邢婧
OwnerOCEAN UNIV OF CHINA