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Melon bacterial fruit blotch germs immunofluorescent quick detection test paper and application thereof

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

Inactive Publication Date: 2017-06-20
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 immunofluorescence detection test paper for bacterial fruit spot of melons and its application. Its application is to solve the technical problems of low sensitivity, many cross-reactions, and short validity period of test strips for detecting melon bacterial fruit spot disease in the prior art.

Method used

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  • Melon bacterial fruit blotch germs immunofluorescent quick detection test paper and application thereof
  • Melon bacterial fruit blotch germs immunofluorescent quick detection test paper and application thereof
  • Melon bacterial fruit blotch germs immunofluorescent quick detection test paper and application thereof

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Experimental program
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specific Embodiment approach

[0023] At first introduce the reagent and instrument used in the embodiment of the present invention:

[0024] main reagent

[0025] 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.

[0026] main instrument

[0027] 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 was purchased from Purified from Suzhou.

Embodiment 1

[0028] Example 1 Monoclonal Antibody to Phytophthora spp.

[0029] The hybridoma cell line 6D of the monoclonal antibody of fruit blotch used in this experiment was preserved in the China General Microorganism Culture Collection Center (CGMCC, Beijing, China) in April 2015, and the preservation number is China General Microbiology Culture Collection Management Center (CGMCC, Beijing, China) No.10413.

Embodiment 2

[0030] Example 2 Immunofluorescence detection test paper

[0031] 1. Fluorescein isothiocyanate (FITC) modified medium

[0032] 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.

[0033] 2. Fluorescence intensity of bacteria with different FITC concentrations was measured by flow cytometry

[0034] 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 distribution is different...

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Abstract

The invention provides a melon bacterial fruit blotch germs immunofluorescent quick detection test paper. The test paper comprises a supporting layer, wherein a sample pad and a water absorption pad are arranged on the supporting layer; a nitrocellulose (NC) membrane is arranged between the sample pad and the water absorption pad; a detection line is arranged on the nitrocellulose membrane and consists of monoclonal antibodies secreted by hybridoma cell strains with the preservation number of No. 10413. The invention also provides a method for detecting melon bacterial fruit blotch germs by using the test paper. A brain-heart infusion culture medium added with FITC (fluorescein isothiocyanate) is adopted to culture bacteria, and the cultured bacteria can emit yellow and green fluorescence, so that the bacteria are fluorescent probes; the monoclonal antibodies generated by the hybridoma cell strains with the preservation number of CGMCC No. 10413 are taken as detection antibodies and are sprayed onto the NC membrane to form the detection line. The immunofluorescent quick detection test paper is a mark-free single-line immunofluorescent test paper. In the whole preparation process, the antibodies do not require mark, so that the influence of a mark effect to a detection result is avoided.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a method for detecting microorganisms, in particular to a test paper for rapid detection of melon bacterial fruit spot pathogen immunofluorescence and the application thereof. 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, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/58
CPCG01N33/56911G01N33/582
Inventor 曾海娟刘箐
Owner UNIV OF SHANGHAI FOR SCI & TECH
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