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Immune colloidal gold test strips for detection of fruit spot fungus and its monoclonal antibody and hybridoma cell line

A technology for detecting fruit spot disease and test paper, which is applied in the field of bioengineering, can solve the problem of low sensitivity of melon bacterial fruit spot disease, achieve remarkable technical progress, and achieve high detection specificity and sensitivity

Active Publication Date: 2016-06-29
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a rapid detection test strip of colloidal gold immune colloidal gold for bacterial fruit spot disease of melons. The paper strip solves the technical problem that the sensitivity of the test strip in the prior art to detect melon bacterial fruit spot is not high

Method used

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  • Immune colloidal gold test strips for detection of fruit spot fungus and its monoclonal antibody and hybridoma cell line
  • Immune colloidal gold test strips for detection of fruit spot fungus and its monoclonal antibody and hybridoma cell line
  • Immune colloidal gold test strips for detection of fruit spot fungus and its monoclonal antibody and hybridoma cell line

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specific Embodiment approach

[0027] At first introduce the reagent and instrument used in the embodiment of the present invention:

[0028] Main reagents:

[0029] PEG and TWEEN-20 were from Sigma; BSA was from Shanghai Broad Biotechnology Co., Ltd.; colloidal gold solution was from Shanghai Gold Standard Biotechnology Co., Ltd.; nitrocellulose membrane (NC membrane) 135S was from Millipore; goat anti-mouse, HRP-goat anti Mouse and goat anti-rabbit were from Sangon Bioengineering Co., Ltd.

[0030] Main instruments:

[0031] Microplate reader SpectraMaxM2 was purchased from Molecular Devices; Nanodrop2000C was purchased from ThermoScientific; spotting instrument AD6010 was purchased from BIO-DOT; scanner PhantomV9 was purchased from MICROTEK; constant temperature culture shaker SPH-100B was purchased from Shanghai Shiping; TM LP358BR5057 was purchased from BIO-RAD; single-person purification bench SW-SJ-2D was purchased from Suzhou Purification.

Embodiment 1

[0032] Example 1 Preparation of Monoclonal Antibody to Phytophthora spp.

[0033] 1. Immunogen Preparation

[0034] The 8 kinds of spot bacteria strains of watermelon were enriched in the brain-heart infusion liquid medium first, and then streaked on the solid medium, and single colonies were cultured in the brain-heart infusion liquid medium for PCR identification, and the 600nm At the OD value, the faster-growing strain was selected as the immune strain. Centrifuge and resuspend in sterilized normal saline 3 times, and adjust the bacterial concentration to 10 8 CFU / mL.

[0035] 2. Preparation process of monoclonal antibody

[0036] 1) Experimental animals: Three 8-week-old, weighing about 20 g female Balb / c mice were selected as experimental animals.

[0037] 2) Immunization method: each mouse was intraperitoneally injected with 0.4 mL of immunogen, and the same dose was boosted once every two weeks.

[0038] 3) Blood collection: Blood was collected from the tail vein a...

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Abstract

The invention relates to a melon bacterial fruit blotch immune colloidal gold rapid detection test strip. The test strip comprises a sample pad, a colloidal gold sample closely connected with the sample pad, a cellulose membrane arranged on the colloidal gold pad and closely connected with the colloidal gold pad, and a water absorption pad closely connected with the other end of the cellulose membrane, one end of the cellulose membrane away from the colloidal gold pad is provided with a quality control line, and the cellulose membrane between the quality control line and the colloidal gold pad is provided with a detection line, wherein the colloidal gold pad is provided with a monoclonal antibody coupling with colloidal gold, the monoclonal antibody is secreted by a hybridoma cell strain with the preservation number of CGMCC NO.10413, and the detection line is formed by the monoclonal antibody. The fruit blotch immune colloidal gold rapid detection test strip adopts the colloidal gold pad and the detection lime to be respectively sprayed with the new screened monoclonal antibody and has high detection specificity and sensitivity on fruit blotch. The invention also relates to monoclonal antibody for the test strip, and the hybridoma cell strain thereof.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a melon bacterial fruit spot fungus, specifically a colloidal gold rapid detection test strip for the melon bacterial fruit spot fungus and its monoclonal antibody and hybridoma cell strain. Background technique [0002] Melon fruit spot disease is a serious bacterial disease that received widespread attention in the late 1980s when a destructive outbreak occurred on watermelons in several states in the United States. spread within the range. The pathogen is Gram-negative bacteria Acidovorax avenae subsp.citrulli (Aacidovorax avenae subsp.citrulli, Aac), which is a highly destructive species-borne pathogen. So far, there are no commercial cultivars that are completely resistant to the pathogen. [0003] Melon fruit spot bacteria, also known as fruit spot bacteria, can cause diseases in Cucurbitaceae plants such as watermelons, melons, and pumpkins. According to reports...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/558C07K16/12C12N5/20C12R1/91
CPCC07K16/12G01N33/558G01N33/56911G01N33/577G01N2333/195
Inventor 曾海娟刘箐
Owner UNIV OF SHANGHAI FOR SCI & TECH
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