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A method for detecting pathogenic microorganisms

A pathogenic microorganism and bio-nano technology, applied in the field of nano-biotechnology detection, can solve the problems of complex preparation of antibody molecules, limited wide application and high cost, and achieve the effects of low price, good selectivity and small molecular weight

Inactive Publication Date: 2018-07-20
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, antibody molecules have disadvantages such as complex preparation, high cost, and poor stability, which greatly limit their wide application in the biological field.

Method used

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  • A method for detecting pathogenic microorganisms
  • A method for detecting pathogenic microorganisms
  • A method for detecting pathogenic microorganisms

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Experimental program
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Effect test

Embodiment 1

[0028] 1) Preparation of peptide ligands displayed on the surface of phage

[0029] Using the food-borne pathogen Vibrio parahaemolyticus as the target, the landscape phage f8 / 9 library was panned using biopanning technology, and through the specificity test, a phage monoclonal capable of specifically recognizing Vibrio parahaemolyticus was successfully obtained . The phages were further amplified in large quantities and the phage fusion pVIII displaying specific polypeptides were separated and purified by saturated phenol.

[0030] First, V. parahaemolyticus was inoculated into a 96-well plate and allowed to dry overnight. Take 10 μl of the f8 / 9 landscape phage library (approximately 10 13phage / ml) was mixed with 45 μl library blocking solution, left at room temperature for 1 h, and then the phage library that could not bind to the target cells after incubation was aspirated. Add 100 μl of washing buffer (containing 0.5% BSA, 0.1% Tween-20) to the Vibrio parahaemolyticus c...

Embodiment 2

[0043] Detection of Vibrio parahaemolyticus:

[0044] Using the MnO obtained above 2 Based on the peroxidase-like activity of NSs and the specific binding performance of fusion pVIII to Vibrio parahaemolyticus, a method for detecting Vibrio parahaemolyticus with an unlabeled enzyme-like nanoprobe based on the specific phage pVIII fusion protein was constructed.

[0045] Detection: 100 μl of 40 μg / ml fusion pVIII was added to a 96-well plate, and fixed overnight at 4°C. Wash with PBS buffer (10 mM, pH 7.4) 3 times to remove unfixed fusion pVIII. Block with 10mg / ml BSA solution for 1h, wash twice with PBS buffer (10mM, pH 7.4), add target microorganism Vibrio parahaemolyticus, incubate at 37°C for 2h, then add 100μl 0.5mg / ml multifunctional nano The probe was incubated at 37°C for 1 h, washed three times with PBS buffer (10 mM, pH 7.4), and then added 140 μl acetic acid-sodium acetate buffer (10 mM, pH 4), 20 μl 10 mM H 2 o 2 And 20μl 6mM TMB, react at room temperature for ...

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Abstract

The invention belongs to the field of nanobiological technology detection, and specifically refers to a method for detecting pathogenic microorganisms based on displaying polypeptide ligands and enzyme-like nanometer materials on the surface of phages. Use the multifunctional biological nanoprobe to incubate with the target pathogenic microorganism to be detected, and then realize its detection under the chromogenic system; among them, the multifunctional biological nanoprobe is screened from the landscape phage library by using the biopanning technology The phage monoclonal that specifically binds to the pathogenic microorganism is further isolated and purified to obtain the protein at the corresponding site of the phage displaying the specific polypeptide; then the phage capsid protein displaying the specific polypeptide is assembled on the imitation enzyme nanomaterial. The probe of the invention can not only specifically recognize and bind target microorganisms, but also can realize signal amplification due to its imitation enzyme activity, thereby realizing the immunocolorimetric quantitative detection of Vibrio parahaemolyticus. The method is simple in operation, low in cost, high in specificity and sensitivity, and provides an effective way for sensitive detection of pathogenic microorganisms.

Description

technical field [0001] The invention belongs to the field of nano-biotechnology detection, and specifically refers to a method for detecting pathogenic microorganisms based on assembling a multifunctional biological nano-probe based on displaying polypeptide ligands on the surface of phages and imitating enzyme nano-materials. Background technique [0002] Pathogenic microorganisms refer to organisms that can directly or indirectly cause damage to humans and animals. They are of various types and are very common in humans, threatening almost everyone. Foodborne diseases caused by foodborne pathogenic microorganisms are currently the most common threat to human beings and society. Vibrio parahaemolyticus (Vibrio parahaemolyticus) is one of the most prevalent pathogens in the public health care system worldwide, and it is also the most important microorganism that contaminates common food and aquatic products. Foodborne diseases caused by such pathogenic microorganisms, such ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
CPCG01N33/56911
Inventor 刘爱骅刘培
Owner QINGDAO UNIV
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