Rapid test card for simultaneously detecting PEDV and TGEV and preparation and use methods thereof

A rapid test card and detection line technology, applied in the field of animal pathogen detection, can solve the problems of long time required, high operator requirements, complex processing, etc., and achieve short detection time, on-site and sensitive detection methods, and simple operation. Effect

Active Publication Date: 2020-08-28
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the routine detection method of PEDV and TGEV is PCR, but this method requires a special laboratory site, special instruments and equipment, and has high requirem

Method used

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  • Rapid test card for simultaneously detecting PEDV and TGEV and preparation and use methods thereof
  • Rapid test card for simultaneously detecting PEDV and TGEV and preparation and use methods thereof
  • Rapid test card for simultaneously detecting PEDV and TGEV and preparation and use methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the preparation of anti-PEDV specific monoclonal antibody and anti-TGEV specific monoclonal antibody

[0037] Hybridoma cell line FL011-26 (for convenience of description, the corresponding antibody obtained hereinafter will be referred to as PEDV antibody 1) secreting anti-PEDV specific monoclonal antibody with the deposit number of CCTCC NO: C2019170 hybridized with CCTCC NO: C2019194 Tumor cell line FL011-33 (for the convenience of description, the corresponding antibody obtained hereinafter is referred to as PEDV antibody 2) and the hybridoma cell line FL111-01 ( For ease of description, the corresponding antibody obtained hereinafter is referred to as TGEV antibody 1) and the hybridoma cell line F011-07 of CCTCC NO:C2019196 (for ease of description, the corresponding antibody obtained hereinafter is referred to as TGEV antibody 2), respectively Expand culture in cell culture flasks.

[0038] BALB / c mice aged 6-8 weeks were taken, and each mouse was...

Embodiment 2

[0041] Embodiment 2: the manufacture of quick test card

[0042] 1. Prepare the NC membrane containing the first detection line T1, the second detection line T2 and the quality control line C

[0043] First, prepare the antibody solution required for coating. The PEDV antibody 1 obtained in Example 1 was diluted to 1.0 mg / mL with PBS buffer solution with a concentration of 10 mM and a pH value of 7.4 to obtain a PEDV antibody 1 solution, which was used as a coating antibody for forming the first detection line T1. The TGEV antibody 1 prepared in Example 1 was diluted to 1.0 mg / mL with a PBS buffer solution with a concentration of 10 mM and a pH value of 7.4 to obtain a TGEV antibody 1 solution, which was used as a coating antibody for forming the second detection line T2. Goat anti-mouse IgG secondary antibody (purchased from Hangzhou Longji Biotechnology Co., Ltd.) was diluted to 1 mg / mL with PBS buffer solution with a concentration of 10 mM and a pH value of 7.4, and used...

Embodiment 3

[0061] Embodiment 3: PEDV and TGEV virions in the solution detected by the quick test card of embodiment 2

[0062] The following three solutions containing PEDV and / or TGEV virions prepared with PBS buffer were detected with the quick test card prepared in Example 2 to evaluate the effect of the quick test card: solution containing 1 μg / ml PEDV virion , a solution containing 1 μg / ml TGEV virions, and a solution containing both 1 μg / ml PEDV virions and 1 μg / ml TGEV virions. At the same time, dissolve with 0.2mol / L PBS buffer solution (pH 7.4) (the specific formula is: 8g sodium chloride, 3.35g disodium hydrogen phosphate dodecahydrate, 0.2g potassium dihydrogen phosphate, 0.2g potassium chloride, double distilled water Dilute to 1L) as a blank control.

[0063] Add 100uL of the solution to be tested into the sample holes of each quick test card, react at room temperature for 8 minutes, observe the color development results from the result observation window and record the t...

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Abstract

The invention discloses a rapid test card for simultaneously detecting PEDV and TGEV, and preparation and application methods thereof. The rapid test card comprises a card shell and a test strip arranged in the card shell. The test strip comprises a PVC bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad, the sample pad, wherein the combination pad, the nitrocellulose membrane and the absorption pad are sequentially connected and pasted to the PVC bottom plate; and a sample adding hole and a result observation window are formed in the card shell. An anti-PEDV specific monoclonal antibody and an anti-TGEV specific monoclonal antibody are fixed on the nitrocellulose membrane and are used for respectively forming a first detection line and a second detection line; and the anti-PEDV specific monoclonal antibody marked by colloidal gold and the anti-TGEV specific monoclonal antibody marked by colloidal gold are fixed on the combination pad. According to the present invention, with the rapid detection card, the PEDV and the TGEV in a pig tissue excrement sample can be simultaneously detected, the sample adding only needs one time, the reaction is performed under the unified condition, and the rapid detection card has advantages of rapidness, high accuracy, high sensitivity, simple operation and the like.

Description

technical field [0001] The invention relates to the technical field of animal pathogen detection, in particular to an immune colloidal gold quick test card for simultaneously detecting porcine epidemic diarrhea virus (PEDV) and porcine gastroenteritis virus (TGEV) and its preparation and use methods. Background technique [0002] Porcine epidemic diarrhea virus (PEDV) can cause diarrhea, vomiting, dehydration, and eventually death in infected pigs. Pigs of all ages can be infected, but suckling piglets are most susceptible. Since the outbreak of a new porcine viral diarrhea (PED) outbreak in 2010, it has caused serious economic losses to the global swine industry. Studies have confirmed that the mutant strain of PEDV is the culprit of this outbreak, and its pathogenicity is stronger, which can cause 100% death of newborn piglets. Due to the mutation and recombination characteristics of the virus, infection outbreaks of different genotype strains have occurred in many countr...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/577G01N33/569G01N33/558
CPCG01N33/587G01N33/577G01N33/56983G01N33/558G01N2333/165G01N2469/10Y02A50/30
Inventor 李彬范宝超郭容利周金柱赵永祥何孔旺倪艳秀常新见时丹怡
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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