Method of analyzing and quantifying thallus quantity of probiotics and pathogenic bacteria by means of molecular biological technique

Abstract

The invention relates to a method of analyzing and quantifying thallus quantity of probiotics and pathogenic bacteria by means of a molecular biological technique. The method comprises the following steps: S1, dissolving a test article in sterile water, performing high speed centrifugation to separate thalli, and adding the sterile water to dilute as a to-be-detected bacterial sample; S2, matching the to-be-detected bacterial sample with a target bacterial special primer set with a real-time quantification polymerase chain reaction, wherein the target bacterial special primer set is a conserved gene of the target bacteria, including an inner transcription region or a housekeeping gene sequence fragment of 16S rDNA, 23S rDNA and 16S-23S rDNA as an amplification target, and the target bacteria are probiotics and pathogenic bacteria; and determining whether the to-be-detected bacterial sample contains a non-target culture or not according to the condition that whether a melting curve of the amplification fragment has a specific wave crest or not; and S3, introducing the obtained Ct value of the test article into a regression equation of a standard curve of a standard substance to obtain the thallus quantity of the to-be-detected bacterial sample.

Description

technical field [0001] The invention relates to a method for analyzing and quantifying the number of probiotics and pathogenic bacteria by using Real-time PCR technology, and using this technology to analyze the number of bacteria contained in derivative products. Background technique [0002] Polymerase chain reaction (English: Polymerase chain reaction, abbreviation: PCR) mainly uses the three-step cycle of DNA denaturation, bonding and extension to continuously amplify target DNA fragments to amplify a very small amount of DNA. Real-time quantitative PCR (Real-time quantitative PCR) -time PCR) is the use of fluorescent probes or dyes in the PCR process, and then through the optical system to achieve real-time detection (Real-time) effect. After each cycle of PCR amplification, record the number of reaction products (fluorescent substances) generated. After the reaction is complete, draw a graph with the number of amplification cycles (cycle number) and the amount of gener...

AI Technical Summary

Problems solved by technology

[0027] The technical problem to be solved in the present invention is to detect the bacterial count of probiotics (heat-deactivated bacteria) and pathogenic bacteria through heat-killing treatment, which cannot be counted through traditional selective medium culture, and lacks a reasonable amount of bacterial cells in quantification. , rapid, specific (or specificity) detection method, and the present invention uses real-time quantitative polymerase chain reaction (Real-time PCR) to provide probiotics (heat-deactivated bacteria) and pathogenic bacteria specificity Quantitative detection method and application, which has high sensitivity, strong specificity, good stability, simple operation, and can quickly detect the number of probiotics (heat-deactivated bacteria) and pathogenic bacteria in the desired test product.

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