A quantitative detection method for living bacteria based on fish technology

Abstract

The invention belongs to the field of microorganism detection and relates to a method for quantitative detection of living bacteria based on FISH technology. Before the implementation of the conventional fluorescence in situ hybridization method, the method adds the pretreatment step of PMA nucleic acid dye; during the PMA treatment process, the nucleic acid in the dead bacteria cells is covalently combined with PMA, and it emits orange under the blue light excitation of the fluorescence microscope Fluorescence, so that it can be known that the bacteria with orange fluorescence are dead bacteria in the original system, and the rRNA in the living bacteria cells is combined with the fluorescent probe in the hybridization step, and the fluorescent probe emits green fluorescence under the blue light excitation of the fluorescence microscope, so that the green fluorescence can be known Bacteria are living bacteria in the original system. The invention realizes the effective distinction of dead and alive bacteria in the sample system by improving the fluorescence in situ hybridization technology, that is, adding the step of PMA dye treatment, and constructs a set of methods that can quickly quantify the active bacteria in the sample. This method has strong universality for bacterial quantification.

Description

technical field [0001] The invention belongs to the field of microorganism detection and relates to a method for quantitative detection of living bacteria based on FISH technology. Background technique [0002] The culture method is currently the most widely used specific detection method for active microorganisms, but the culture method has obvious disadvantages: on the one hand, the method takes a long time, the workload is heavy, and the detection results have a large lag; on the other hand, for feces Bacteria that are not culturable or in a viable non-culturable state (Viable but Non-culturable, VBNC) in the sample cannot be detected by culture methods. [0003] Fluorescence in situ hybridization (FISH) technology uses fluorescently labeled specific nucleic acid probes to hybridize with corresponding target DNA molecules or RNA molecules in cells, and observes fluorescent signals under a fluorescent microscope or confocal laser scanner to determine the specific probes th...

AI Technical Summary

Problems solved by technology

[0002] The culture method is currently the most widely used specific detection method for active microorganisms, but the culture method has obvious disadvantages: on the one hand, the method takes a long time, the workload is heavy, and the detection results have a large lag; on the other hand, for feces Bacteria that are not cultivable or in a viable non-culturable state (Viable but Non-culturable, VBNC) in the sample cannot be detected by culture methods

Images