Corn bacterial wilting germ biotin-avidin ELISA detection method

An enzyme-linked immunoassay, bacterial technology, used in measuring devices, instruments, scientific instruments, etc.

Inactive Publication Date: 2009-08-05
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports on ELISA detection of corn bacterial wilt pathogen at home and abroad, and the establishment of a sensitive and stable biotin-avidin ELISA detection method for corn bacterial wilt pathogen (biotin-avidin ELISA) is an urgent requirement for corn quarantine

Method used

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Examples

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Embodiment Construction

[0037] In order to further illustrate the present invention, it will be specifically described in conjunction with the following examples:

[0038] 1. Preparation of immunogen

[0039]Test corn bacterial wilt disease bacteria were inoculated on liquid NBY medium and cultured at 28°C under constant temperature shaking. The bacteria were collected in the logarithmic phase of bacterial growth, centrifuged at 12000g for 10 minutes, and the supernatant was discarded; the pelleted bacteria should be used again. Bacteria phosphate buffered saline (PBS) reconstituted, repeated centrifugation and washing three times, then added sterile PBS to resuspend, turbidimetric method count and set the lysate concentration to 1x10 8-9 cfu / mL.

[0040] NBY medium:

[0041] Beef juice 8g

[0042] Yeast extract 2g

[0043] K 2 HPO 4 2g

[0044] KH 2 PO 4 , 0.5g

[0045] Glucose 5g

[0046] MgSO 4· 7H 2 O 0.25g

[0047] Double distilled water (ddH 2 O) 1000mL

[0048] PBS buffer: 0.01mol / L phosp...

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PUM

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Abstract

A biotin-avidin enzymoimmunoassay aiming at the corn bacterial wilt includes the following steps of preparing a polyclonal antibody, purifying the polyclonal antibody and performing biotin labeling; extracting bacterium in a sample and coating the extracting solution of the sample on a micro-perforated plate; then sequentially adding a biotin labeling antibody, an alkaline phosphatase labeled strepen avidin compound and a PNP zymolyte, developing and then reading the value of the absorbency with the wavelength below 405 nm by an enzyme-labeling instrument. The biotin-avidin enzymoimmunoassay aiming at the corn bacterial wilt has the advantages of strong specificity, high detecting sensitivity reaching 1*10 cfu/mL, simple and rapid operation, accurate result and high efficiency.

Description

Technical field [0001] The invention relates to an enzyme-linked immunoassay detection method, in particular to a corn bacterial wilt disease biotin-avidin enzyme-linked immunoassay method and a detection kit. Background technique [0002] Corn (Neizhou) wilt disease, also known as corn bacterial wilt and blight of maize (Clavibacter michiganensis subsp.nebraskensis), is a plant disease that can cause severe corn loss. In 1969, the disease first occurred in Dawson County, Nebraska, USA, and then spread to Colorado, Illinois, Iowa, Kansas, Michigan, Minnesota, and Neb. Las Vegas, South Dakota, and Wisconsin have spread to Ontario, Canada by 2000. The harmfulness of wilt disease and the spread of its distribution area have attracted people's attention. The European Union, Canada and other countries have established risk assessment systems for the disease. In May 2007, my country included the pathogen in the newly revised "List of Entry Phytosanitary Pests of the People's Republic o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/531G01N33/543
Inventor 田世民邹明强王岭哈斯马吉湘陈艳齐小花金涌
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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