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Clostridium perfringens alpha toxin visual detection method

A technology for Clostridium perfringens and alpha toxin, which is applied in chemical instruments and methods, biochemical equipment and methods, botanical equipment and methods, etc. Convenient and fast, high sensitivity, good stability

Pending Publication Date: 2020-11-24
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for visual detection of Clostridium perfringens α toxin, which is used to solve the problems of low detection limit, long time and making up for the lack of single detection technology in the prior art.

Method used

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  • Clostridium perfringens alpha toxin visual detection method
  • Clostridium perfringens alpha toxin visual detection method
  • Clostridium perfringens alpha toxin visual detection method

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Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1: the basic preparation of detection method

[0042] Step 1, type A standard strain (C57-1) was purchased from China Veterinary Drug Inspection Institute. Alpha toxin 4 amino acid mutant plasmid (CPA m4 ), and in alpha toxin (CPA C3 ) C-terminal three-copy tandem prokaryotic expression vector (pET30a) was constructed by Professor Chen Xiaoyun of China Institute of Veterinary Medicine [14,15] .

[0043] Because the site-directed mutagenesis toxin can greatly reduce the toxicity and lethality of CPA, it is convenient for subsequent preparation of non-lethal rabbit serum antibodies.

[0044] We then used the existing α-toxin 4 amino acid mutation plasmid (CPA m4 ) for N-terminal truncation construction, so the constructed expression vector contains two mutation sites (D56G, D130G). Construction of N-terminal expression vector PET30a-CPA of CPA using two primers N , according to Genbank search Clostridium perfringens alpha toxin gene (Genbank accession num...

Embodiment 2

[0049] Embodiment 2: Condition optimization of detection method

[0050] Step 1, determine the optimal coating conditions: the variable is the coating antibody (bound silica microsphere antibody CPA C3 ) concentration and time temperature, other conditions are fixed, the selected toxin concentration is 10LD 50 , (each condition was repeated 3 times) and then observe the fluorescence image by fluorescence microscope, and finally obtain the ratio by gray value analysis. Finally, the optimal coating condition was determined to be 0.1 mg / mL capture antibody, and the silica microspheres were mixed at 25°C for 2 hours. see attached results Figure 4 : Optimum condition for microsphere fluorescence detection experiment; (a) determine optimum antibody coating condition.

[0051] Step 2, determination of optimal blocking conditions: variable is the blocking agent (5% skimmed milk powder or 5% BSA) selected, other conditions are fixed, and the selected toxin concentration is 10LD 50...

Embodiment 3

[0053] Embodiment 3: the establishment of detection method

[0054] Step 1, the concentration gradient dilution of the toxin CPA was carried out, and the undiluted original toxin concentration was initially determined to be 10LD 50 , so we serially dilute it by 2 times to get 10LD 50 , 5LD 50 , 2.5LD 50 , 1.25LD 50 , 0.625LD 50 , three biological repetitions were carried out for each concentration gradient, and the corresponding fluorescence pictures were obtained by fluorescence microscope detection. see attached results Figure 5 , the CPA was serially diluted 10LD 50 , 5LD 50 , 2.5LD 50 , 1.25LD 50 , 0.625LD 50 After detecting the fluorescence intensity, three biological replicates were set for each concentration gradient. It is proved that the stability of the equation is good.

[0055] Step 2, use ImageJ software to analyze the gray value, and finally draw the obtained gray value into a regression curve, regression equation: Y=0.01915X+0.002875 (R 2 = 0.9972)...

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Abstract

The invention discloses a Clostridium perfringens alpha toxin visual detection method. A smartphone image processing technology and silicon dioxide microsphere antibody coupling are combined to detectfood-borne clostridium perfringens alpha toxin (CPA). According to a double-antibody sandwich principle, the prepared CPA polyclonal antibody is coupled with silicon dioxide microspheres, CPA fluorescence detection is performed through antibody-labeled FITC fluorescence, and finally, the CPA content is detected in real time in combination with an image processing technology of a smartphone APP. Compared with a traditional clostridial toxin detection method, the nano-microsphere combined intelligent mobile phone CPA detection system is a more sensitive, stable and convenient detection method,and a new method is provided for detection of natural CPA in food.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method of food-borne pathogenic bacteria-Clostridium perfringens alpha toxin. Background technique [0002] Clostridium perfringens, also known as foodborne gastroenteritis and other infectious diseases. As an opportunistic pathogen, it can contaminate many types of retail meat products and produces alpha toxins in the small intestines of livestock [1] , food poisoning may result when milk and dairy products contaminated with this bacterium or toxin are consumed. Clostridium perfringens causes a variety of diseases in various hosts due to the production of multiple toxins and extracellular enzymes [2] . So far, at least 20 exotoxins have been discovered, among which the main lethal toxins are α, β, ε and ι. Because all types of toxins can produce alpha toxins and cause hemorrhagic enteritis and acute death in livestock [3] , and α-toxin is the most important virulence ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N33/58C12N15/70C12N15/31C07K16/12C07K16/06G01N33/533
CPCG01N33/68G01N33/54313G01N33/582C12N15/70C07K14/33C07K16/1282C07K16/06G01N33/533G01N2333/33Y02A50/30
Inventor 黄金海郭艳余张丽琳曹爱萍
Owner TIANJIN UNIV
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