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Development of Multiplex qPCR Method for Accurate Detection of Enzyme-Producing Psychrotrophic Bacteria
Kidane Yalew1,2, Shuwen Zhang1, Solomon Gebreyowhans3
1Key Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institution of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
This study developed a specific and sensitive multiplex quantitative PCR (qPCR) method for detecting spoilage genes lipA and aprX in milk. This advancement enables simultaneous microbial detection, improving food safety and dairy product quality.
Area of Science:
- Food Microbiology
- Molecular Biology
- Dairy Science
Background:
- Microbial contamination in milk poses risks to food safety and dairy product quality.
- Accurate detection and quantification of microorganisms are essential for preventing spoilage.
- Existing methods may lack the specificity or efficiency for simultaneous detection of multiple spoilage indicators.
Purpose of the Study:
- To develop and validate a multiplex quantitative PCR (qPCR) assay for the simultaneous detection of lipA and aprX genes in milk.
- To assess the specificity, sensitivity, and efficiency of the developed assay.
- To establish a reliable molecular tool for identifying spoilage-related microorganisms in dairy products.
Main Methods:
- Design and optimization of specific primer pairs and hydrolysis probes for lipA and aprX genes.
- Application of singleplex and multiplex quantitative PCR (qPCR) assays.
- Validation using cultured isolates and artificially contaminated ultra-high-temperature (UHT) milk samples.
Main Results:
- The multiplex qPCR assay demonstrated high specificity, amplifying only target strains carrying lipA and aprX genes.
- The assay achieved a limit of detection (LOD) as low as 1.2 × 102 CFU/mL in milk samples.
- High efficiency (95-102%) and correlation coefficient (R2 ≥ 0.9908) confirmed the assay's reliability for DNA quantification.
Conclusions:
- Multiplex TaqMan qPCR is an effective method for simultaneous detection of lipA and aprX genes in a single reaction.
- This method offers a significant advancement for rapid and accurate microbial analysis in raw milk.
- The developed assay paves the way for future multi-gene detection panels to identify spoilage psychrotrophic bacteria.
