A pH-sensitive fluorescent sensor for high-throughput detection of active microorganisms and its construction method

Abstract

The invention relates to a pH-sensitive type fluorescence sensor for high-flux detection of an active microorganism and a construction method of the pH-sensitive type fluorescence sensor. The method comprises the following steps: (1) preparation of a fluorescent nanometer material: dissolving phospholipid polyethylene glycol and 5-(N-palmitoyl) amino fluorescein by using ethyl alcohol, mixing fully, slowly dropping the mixture into pure water to prepare fluorescent nano-micelle; (2) preparation of sensing gel: adding the fluorescent nano-micelle into 1-3% of a hot agar solution to mix fully, and condensing to prepare fluorescent sensing gel; and (3) construction of a sensor: dividing a container into two parts by using a water-phase filter membrane, loading the sensing gel into a lower part, and loading a liquid microorganism culture medium into an upper part. The pH-sensitive type fluorescence sensor has the advantages that a culture unit and a detection unit are separated, by detecting the changes of fluorescent signals in the sensing gel, fast and real-time detection of active acid-producing gas-producing microorganisms is realized, and compared with a traditional flat band method, the sensor is based on a traditional culture theory, convenient to operate and short in detection time, and can be used for realizing the high-flux screening of the microorganisms in different materials.

Description

technical field [0001] The invention belongs to the field of microorganism detection and relates to the detection of trace active microorganisms, in particular to a pH-sensitive fluorescent sensor and a construction method for high-throughput detection of active microorganisms. Background technique [0002] In recent years, food safety incidents at home and abroad have emerged one after another, which not only affected the market order, but also brought some panic to the public. For example, the recent microbial contamination incident of Fonterra milk powder, although it was finally proved that it was not botulism, it also indirectly Indicates society's concern about microbial contamination of food. [0003] The traditional plate counting method used for microbial detection has high accuracy and sensitivity, but involves many experiments, cumbersome operations, long time preparation and finishing, and the participation of a large number of personnel. Therefore, there is an ...

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Problems solved by technology

[0004] 3M's Perrifilm TM Plate series of microbial test pieces can detect the total number of colonies, coliform counts, mold and yeast counts, etc. The disk method and the national standard method are based on the traditional culture theory, and the consistency in the detection rate of microorganisms is good, and the colonies are typical , easy to determine, but the detection throughput is not high, and the sensitivity of the test paper still needs to be further improved

Due to the high sensitivity of PCR, it is theoretically possible to detect a copy of a bacterial gene. Therefore, in the detection of bacteria, only a short period of enrichment or no enrichment is required, and PCR can be used for screening, which saves a lot of time, but PCR technology is required. High personnel requirements and laboratory environment, and it is impossible to distinguish between live and dead bacteria, and there are certain difficulties in the extraction of target genes in complex matrices

[0006] ATP bioluminescence method uses ATP bioluminescence analysis technology and somatic cell clearance technology to measure bacterial ATP and somatic ATP. The amount of bacterial ATP is proportional to the number of bacteria, so as to realize the detection of microorganisms, but this method cannot distinguish the types of microorganisms , can only be used to monitor the total microbial

[0007] Flow cytometry uses specimen pretreatment to prepare single-cell suspension and analyze it after specific staining. The analysis speed is fast and multiple parameters can be analyzed at the same time, but the instrument is expensive, and the concentration of the analysis needs to be of a higher order of magnitude. Analytical error is large

[0008] To sum up, although there are many methods and technologies for microbial detection, most of them have certain problems, and are quite different from the traditional culture theory. There is a risk in the consistency of the results, and the detection of trace microorganisms still exists. certain challenges

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