Pseudomonas putida test strip and preparation method thereof

A technology for detecting Pseudomonas putida and test strips, which is applied in the field of immunochemical detection, can solve the problems of not disclosing Pseudomonas putida IgY, etc., and achieve the effect of rapid detection, high detection accuracy, and low temperature requirements

Active Publication Date: 2015-02-25
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are no reports about Pseudomonas putida IgY published at home and abroad

Method used

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  • Pseudomonas putida test strip and preparation method thereof
  • Pseudomonas putida test strip and preparation method thereof
  • Pseudomonas putida test strip and preparation method thereof

Examples

Experimental program
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Effect test

specific Embodiment 1

[0043] A test strip for detecting Pseudomonas putida, such as figure 1 As shown, the test strip consists of a PVC base plate 7 and a sample pad 1, a bonding pad 2, a nitrocellulose membrane 3, and a water-absorbing pad 4 that are sequentially overlapped on the PVC base plate 7; the bonding pad 2 is coated with an anti-odor dummy sheet Bacillus yolk antibody-colloidal gold marker, the detection line 5 coated with Pseudomonas putida antigen ultrasonic bacterial solution and the quality control coated with rabbit anti-chicken yolk antibody (IgY) are respectively set on the nitrocellulose membrane 3 Line 6.

[0044] The preparation method of the above-mentioned anti-Pseudomonas putida yolk antibody-colloidal gold marker is as follows: the anti-Pseudomonas putida yolk antibody and the colloidal gold solution with a diameter of 25-50nm of colloidal gold particles are mixed at 6-8μg: 1mL, and mixed in Combine by stirring and shaking for 30-60min at pH 5.4, add PBST buffer containing...

specific Embodiment 2

[0045] The preparation method of rapid detection pseudomonas putida colloidal gold test strip, such as figure 2 As shown, the specific operation steps are as follows:

[0046] (1) Preparation of Pseudomonas putida

[0047] The preparation method of Pseudomonas putida antigen is as follows: Take the Pseudomonas putida species stored at -80°C in the laboratory, activate it by streaking in LB solid medium, culture it upside down at 28°C for 24 hours, and then pick a single colony and inoculate it in a 5mL In the test tube of LB liquid medium, cultivate at 28°C and 200rpm for 12-18h, dilute the cultured bacterial liquid with LB liquid medium at 1:100 into the Erlenmeyer flask, expand the culture at 28°C and 200rpm, and measure its OD600 value, when the OD600 value reaches 0.4-0.6, stop culturing. Centrifuge the cultured bacterial solution at 5000rpm for 10min at 4°C, discard the supernatant, resuspend and wash the lower layer with 0.01M PBS with pH 7.2, centrifuge at 5000rpm fo...

specific Embodiment 3

[0065] Colloidal gold test strips for the detection of clinical samples, the specific steps are as follows:

[0066] Purchase eel, swimming crab, crucian carp, shrimp and other fish samples from local supermarkets, as well as water samples from various local ponds, dissect and take a total of 41 samples of intestines, gills and liver, add PBS buffer solution to the tissue samples, and crush Tissue, after centrifugation at 2000-3000rpm for 5 minutes, take the supernatant, insert the test strip into the sample to be tested, and observe the result after 10 minutes: if only one red line appears in the quality control area of ​​the test strip, it is a positive result, that is The sample contains Pseudomonas putida; if the detection line and quality control line of the test strip have red lines at the same time, it is a negative result, that is, the sample does not contain Pseudomonas putida. Each sample was repeated 5 times, and the test results of all clinical samples were compare...

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Abstract

The invention discloses a pseudomonas putida test strip and a preparation method thereof. The pseudomonas putida test strip comprises a sample pad, a jointing pad, a nitrocellulose membrane, a water absorbing pad and a PVC substrate; the pseudomonas putida test strip is characterized in that the sample pad, the jointing pad, the nitrocellulose membrane and the water absorbing pad are orderly bonded to the PVC substrate; the jointing pad is coated with an anti-pseudomonas putida yolk antibody-colloidal gold label; the nitrocellulose membrane is coated with a detection line formed by the ultrasonic broken liquid of the pseudomonas putida and a quality control line formed by rabbit anti-chicken yolk antibodies; the pseudomonas putida test strip has the advantages of high sensitivity, strong specificity, simplicity and convenience in operation, fast detection and high accuracy.

Description

technical field [0001] The present invention relates to immunochemical detection technology, in particular to a colloidal gold immunochromatography technology based on egg yolk antibody for rapid detection of Pseudomonas putida in various tissues of environment, food, human and animal, blood and feces samples Test strip and preparation method thereof. Background technique [0002] Pseudomonas is an important group of microorganisms and one of the most widely distributed microorganisms in nature. Pseudomonas putida belongs to the genus Pseudomonas (Pseudomonas), Pseudomonadaceae (Pseudomonadaceae), is a gram-negative bacterium, widely exists in soil, seawater, freshwater animal and plant surfaces and various In protein-containing foods, it is human or animal origin or opportunistic pathogenic bacteria. At present, some diseases of farmed fish and crustaceans caused by Pseudomonas putida have also been reported successively in China, such as gill rot disease of European eel ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/531
CPCG01N33/56916
Inventor 章礼平李登峰方静刘联国刘飞陈梅娟
Owner NINGBO UNIV
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