Method for detecting escherichia coli based on immunomagnetic separation technique and bioluminescence technique

Abstract

The invention discloses a method for detecting escherichia coli based on an immunomagnetic separation technique and a bioluminescence technique. The method comprises the following steps of (1) coupling biotin and an escherichia coli antibody, so as to obtain the biotinylated escherichia coli antibody; (2) mixing the biotinylated escherichia coli antibody and streptavidin-modified magnetic nanoparticles, so as to prepare a magnetic nanometer probe; (3) flushing the magnetic nanometer probe by a PBS (phosphate buffer saline) buffer solution, adding the magnetic nanometer probe into an escherichia coli solution, and enabling the magnetic nanometer probe to capture the escherichia coli; (4) putting the solution after capturing the escherichia coli into a centrifuge tube, and enriching the magnetic nanometer probe captured with the escherichia coli; (5) cleaning the enriched sample by a cleaning solution, so as to obtain a solution for ATP (adenosine triphosphate) light emitting; enrichingand separating the magnetic nanometer probe by a magnetic separating frame; adding fluorescein and Mg<2+> into the solution after cracking, and detecting the fluorescent intensity of the solution, soas to obtain the number of the escherichia coli.

Description

technical field [0001] The invention relates to the field of food safety detection, in particular to a method for detecting the number of Escherichia coli in food based on immunomagnetic separation technology and ATP bioluminescence technology. Background technique [0002] The current detection methods for Escherichia coli include plate dilution method, filter membrane method, fluorescence quenching method, etc. These detection methods have high sensitivity, but there are certain shortcomings. For example, these traditional methods often need 1-3 days Moreover, strict experimental environment is required to cultivate bacteria, which determines that these methods cannot meet the requirements of rapid detection. [0003] Immunomagnetic separation technology is one of the important components of the rapid screening technology for food-borne pathogens. This technology can efficiently capture and concentrate the target bacteria in the enrichment solution and improve the detectio...

AI Technical Summary

Problems solved by technology

However, the current separation technology based on micron-sized immunomagnetic beads has low capture efficiency of magnetic beads, takes longer to specifically capture bacterial cells in the food matrix, and has poor monodispersity of micron magnetic beads. Self-aggregation or precipitation is easy to occur, the nature of the food matrix is ​​complex and the concentration of non-target pathogenic bacteria is large, micron magnetic beads are prone to non-specific adsorption, and it is difficult to achieve specific separation of target bacteria in food samples. If the concentration of beads is too high, the bacterial cells will be damaged, leading to the failure of separation, and the distance between the antibody and the surface of the magnetic beads is too close. leading to the decrease of antibody biological activity and other limitations

At present, there is still a lack of rapid and accurate technical means for the quantitative detection of Escherichia coli

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