Quantitative detection method of live bacteria based on FISH technique

Abstract

The invention belongs to the field of microbiological detection, and relates to a quantitative detection method of live bacteria based on an FISH technique. The method comprises the following steps: adding a PMA nucleic acid dye for pre-treating before an implementation process of a conventional fluorescence in-situ hybridization method; carrying out covalent binding of nucleic acid in dead bacterium cells and PMA in the PMA treatment process, and sending out orange fluorescence under blue light excitation of a fluorescence microscope, so as to know that the bacteria in the orange fluorescence are dead bacteria in an original system; combining rRNA in live bacterium cells with a fluorescence probe in a hybridization step, and sending out green fluorescence under blue light excitation of the fluorescence microscope by the fluorescence probe, so as to know that the bacteria in the green fluorescence are the live bacteria in the original system. Through improvement of a fluorescent in-situ hybridization technique, the step of adding PMA dye for treating is increased, and effective distinguishing of the dead bacteria and the live bacteria in a sample system is achieved; and a set of method capable of rapidly and quantitatively detecting the live bacteria in the sample is built. The method has relatively high universality in bacterial quantization.

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...

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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

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