Method for detecting live bacteria of Listeria monocytogenes based on LA-PMA-qPCR

The LA-PMA-qPCR method, combined with lactic acid and PMA pretreatment and high-temperature treatment, solved the problem that traditional PCR technology could not distinguish between live and dead bacteria, achieved efficient detection of live Listeria monocytogenes, and improved the specificity and accuracy of detection.

CN120758651AInactive Publication Date: 2025-10-10JILIN UNIVERSITY
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Patent Information

Application Number
CN202511278248.9
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-09-09
Publication Date
2025-10-10
Estimated Expiration
Not applicable · inactive patent

AI Technical Summary

Technical Problem

Traditional PCR technology and real-time fluorescence PCR technology cannot effectively distinguish between live and dead bacteria in food samples, and the existing PMA-qPCR method has low staining efficiency.

Method used

The LA-PMA-qPCR method, including lactic acid pretreatment (LA pretreatment), PMA pretreatment, and high-temperature metal bath treatment, was used in combination with real-time fluorescence quantitative PCR detection. The ability to distinguish between live and dead bacteria was improved by optimizing the concentrations of lactic acid and PMA, dark incubation time, and exposure time.

Benefits of technology

It achieves effective distinction between live and dead Listeria monocytogenes bacteria, improves the specificity and accuracy of detection, and ensures that the amplification of live bacteria is not affected.

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Abstract

The invention is applicable to the technical field of microbiological detection, and provides a method for detecting live bacteria of Listeria monocytogenes based on LA-PMA-qPCR, which comprises the following steps: preparing a sample suspension; lA pretreatment: under a dark condition, adding lactic acid into the sample suspension, fully oscillating, uniformly mixing and incubating; pMA pretreatment: under a dark condition, taking a PMA solution, adding the PMA solution into the sample suspension subjected to LA pretreatment, fully mixing, incubating in a dark condition, and performing exposure treatment; sample DNA extraction: carrying out metal bath high-temperature treatment on the sample suspension subjected to PMA pretreatment, and then centrifugally extracting DNA; and carrying out real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection by taking the extracted sample DNA as a template. According to the embodiment of the invention, the method for detecting the live bacteria of the listeria monocytogenes through LA-PMA-qPCR is established, and the live bacteria and dead bacteria of the listeria monocytogenes can be effectively distinguished.
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