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Double-labelled immunofluorescence detection test paper for acidovorax avenae subsp.citrullii of melons

A technology for the detection of melon fruit spot bacteria and immunofluorescence, applied in the field of bioengineering, can solve the problems of many cross-reactions, short validity period of test strips, low sensitivity of test strips, etc., and achieve high detection sensitivity, high accuracy and sensitivity, and high sensitivity high effect

Inactive Publication Date: 2017-07-28
UNIV OF SHANGHAI FOR SCI & TECH
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0005] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a double-labeled immunofluorescence detection test paper for Melon fruit spot fungus, the double-label immunofluorescence test paper for Melon fruit spot disease It is necessary to solve the technical problems of low sensitivity, many cross-reactions, and short validity period of the test strips used in the prior art to detect melon fruit spot bacteria

Method used

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  • Double-labelled immunofluorescence detection test paper for acidovorax avenae subsp.citrullii of melons
  • Double-labelled immunofluorescence detection test paper for acidovorax avenae subsp.citrullii of melons
  • Double-labelled immunofluorescence detection test paper for acidovorax avenae subsp.citrullii of melons

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Experimental program
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specific Embodiment approach

[0024] At first introduce the reagent and instrument used in the embodiment of the present invention:

[0025] Main reagents:

[0026] BSA Shanghai Broad Biotechnology Co., Ltd.; FITC American SIGMA Company; Nitrocellulose Membrane (NC Membrane) American Millipore Company; Brain Heart Infusion Medium Beijing Land Bridge Co., Ltd.

[0027] Main instruments:

[0028] Microplate reader SpectraMax M2 was purchased from Molecular Devices; Nanodrop 2000C was purchased from ThermoScientific; spotting instrument AD6010 was purchased from BIO-DOT; constant temperature culture shaker SPH-100B was purchased from Shanghai Shiping; single-person clean bench SW-SJ-2D purchased from Suzhou Purification.

[0029] The hybridoma cell line 6D4C5E9B9 of the monoclonal antibody used in this experiment was deposited in the China General Microorganism Culture Collection Center (CGMCC, Beijing, China) in April 2015, and the preservation number is China General Microbiology Culture Collection Manage...

Embodiment 1

[0031] Example 1 Development of Immunofluorescence Detection Test Paper

[0032] 1. Fluorescein isothiocyanate (FITC) modified medium

[0033] Freshly prepare FITC mother liquor, add different volumes of FITC solution to brain-heart infusion medium (BHI) to make a modified BHI medium, and inoculate with Phytophthora spp. for 12 hours at 37°C in the dark. figure 1 The bacteria cultivated in the improved BHI medium were observed under a fluorescent microscope, and it can be seen that the bacteria cultivated by this method can emit strong yellow-green fluorescence.

[0034] 2. Determination of bacterial fluorescence intensity at different FITC concentrations by flow cytometry

[0035] Fluorescence intensity of bacteria in media with different concentrations of FITC was measured by flow cytometry to determine the optimal concentration of FITC. Depend on Figure 2A It can be seen that the labeling rates (M2+M3+M4) of different concentrations are all above 97%, but the distribut...

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Abstract

The invention provides a piece of double-labelled immunofluorescence detection test paper for acidovorax avenae subsp.citrullii of melons. The test paper comprises a support layer, a sample pad, a water absorbing pad and a nitrocellulose membrane, wherein a detection line and a quality control line are arranged on the nitrocellulose membrane; the detection line is close to one side of the sample pad; the quality control line is close to one side of the water absorbing pad; a conjugate pad is arranged on one side close to the sample pad, of the nitrocellulose membrane; the conjugate pad is tightly connected with the sample pad; the detection line consists of a monoclonal antibody which is secreted from a hybridoma cell strain of which the preservation number is No.10413; the concentration of the monoclonal antibody is 2mg / mL; the conjugate pad is coated by the monoclonal antibody which is labelled by fluorescein isothiocyanate and secreted from the hybridoma cell strain with the preservation number being No.10413. The double-labelled immunofluorescence detection test paper provided by the invention is capable of detecting most acidovorax avenae subsp.citrullii, is free of cross reaction, and has the characteristics of high sensitivity, good accuracy and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a method for detecting microorganisms, in particular to a double-labeled immunofluorescence detection test paper for melon fruit spot pathogen. 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 Acidovorax citrulli (Acidovorax citrulli, Ac), a Gram-negative bacterium, which is a highly destructive seed-borne pathogen. The fungus is very drought resistant and can survive for 35 years on dry seed surfaces. 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 report...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 曾海娟刘箐谢曼曼
Owner UNIV OF SHANGHAI FOR SCI & TECH
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