Method for detecting dot-ELISA of principally outer membrane protein of vibrio vulnficus gene

A technology of the main outer membrane protein and Vibrio vulnificus, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of less quantitative detection research, insufficient attention, insufficient sensitivity of the method, and insufficient consideration of feasibility, etc., to achieve The effect of high sensitivity and specificity, convenient preparation and simple operation

Inactive Publication Date: 2009-06-10
张振冬
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Problems solved by technology

[0004] 1. At present, microorganisms in seawater quality testing indicators mainly detect Escherichia coli, while ignoring other important pathogenic bacteria such as Vibrio vulnificus, so it has not received enough attention
[0005] 2. In practical application, there are few studies on the quantitative detection of the main outer membrane proteins ...

Method used

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  • Method for detecting dot-ELISA of principally outer membrane protein of vibrio vulnficus gene
  • Method for detecting dot-ELISA of principally outer membrane protein of vibrio vulnficus gene

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Embodiment 1

[0045] 1. Preparation of Vibrio vulnificus antiserum: inactivate the purely cultured Vibrio vulnificus cells with 0.5% formalin at 4°C, adjust the concentration by 10% after centrifugation and washing 8CFU / mL, inject male New Zealand white rabbits into the ear vein, once a week, take blood from the heart after 6 weeks, let stand at room temperature for 1 hour, let stand at 4°C overnight, centrifuge at 3000rpm at 4°C for 15 minutes, and carefully separate the supernatant. The titer of antiserum obtained from New Zealand white rabbits immunized with Vibrio vulnificus was as high as 1:2048000.

[0046] 2. Preparation of Vibrio vulnificus outer membrane protein: collect the Vibrio vulnificus thallus freshly cultured on the TSA plate for 48 hours, wash 3 times with PBS buffer, and resuspend the bacteria in 2 mL of PBS buffer (containing 10 mmol / EDTA and 2 mmol / L PMSF), in a 45°C water bath for 30 minutes, then placed in an ice bath, and ultrasonically crushed (power 200W, super 5s,...

Embodiment 2

[0069] Wash 25-50 mL of Vibrio vulnificus liquid cultured for 24 hours with PBS buffer for 3 times, resuspend the bacteria in 2 mL of PBS buffer, add final concentrations of 10 mmol / EDTA (ethylenediaminetetraacetic acid) and 2 mmol / L PMSF (phenylmethylsulfonyl fluoride), in a water bath at 45°C for 30 minutes, then placed in an ice bath, and ultrasonically crushed (power 200W, super 5s, stop 5s). Centrifuge at 14000rpm at 4°C for 15min to collect the supernatant, and store at -20°C after aliquoting. The total protein content was determined by the Bradford method, and the supernatant was serially diluted 10 times as a positive control sample. Quantitative detection is then performed as follows:

[0070] (1) Make a small square of 0.5cm×0.5cm on the PVDF film (polyvinylidene fluoride film, the same below) with a pencil, and cut out the required number of squares according to the number of samples.

[0071] (2) After removing the protective layers on both sides of the PVDF memb...

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Abstract

Disclosed is dot-ELISA detection method of main outer-membrane protein of vibrio vulnficus. Cultured vibrio vulnficus is crashed (power 200w, beyond 5s stopping 5s) through ultrasonic, supernatant is centrifugally collected. Spotting 1.0 mu l is on a polyvinylidene fluoride film (PVDF film), after the spotting PVDF film is closed by 2% BSA (bovine serum albumin) under 37 DEG C, respectively reacted with rabbit anti vibrio vulnficus serum and goat anti-rabbit IgG whole serum marked by HRP (horseradish peroxide), finally reacted with substrate solution, macroscopic brown yellow round sedimentation is formed on the PVDF film, namely masculine result. The inventive method has detection sensitivity 15 ng/ml for main outer-membrane protein of vibrio vulnficus, is 3-5 times higher than enzyme linked immunosorbent assay (ELISA), the result is obtained after 2-3 hours, therefore vibrio vulnficus in the marine environment is detected fast and sensitively.

Description

technical field [0001] The invention relates to a rapid detection method for the main outer membrane protein of Vibrio vulnificus in marine environment. Background technique [0002] Vibrio vulnificus (Vibrio vulnificus) is a marine bacterium that is widely distributed in seawater and sediments in coastal waters, estuaries and bays. Vibrio vulnificus can cause serious diseases such as human food poisoning and sepsis through intestinal and trauma infection. Shellfish such as oysters are the main medium of Vibrio vulnificus infection, which is mostly caused by eating contaminated shellfish raw. It has been found in coastal areas of my country in recent years The cases of acute diarrhea and sepsis caused by Vibrio vulnificus are gradually increasing. At the same time, Vibrio vulnificus is also the main pathogen that endangers marine shellfish and fish farming in my country, causing serious economic losses to related industries. However, the qualitative and quantitative detectio...

Claims

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Application Information

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IPC IPC(8): G01N33/543
Inventor 张振冬王秀娟朱琳王淑芬邹亚男
Owner 张振冬
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