数字农科院2.0

Multiplex qPCR for simultaneous quantification of the main psychrotrophic bacteria and their enzymes in raw milk

文献类型： 外文期刊

作者： Jie Wu;Lu Meng;Wenjun Xu;Yankun Zhao;Huimin Liu;Nan Zheng;Ashikur Rahman;Jiaqi Wang

作者机构：

关键词： multiplex qPCR;Protease and lipase;psychrotrophic bacteria;Raw milk;Spoilage

期刊名称： LWT

ISSN： 0023-6438

年卷期： 2025 年 222 卷

页码：

收录情况： SCIE（2025版） ; ; EI（2025版）

摘要： Milk spoilage is caused by proteolytic enzymes produced by psychrotrophic bacteria during storage at low temperatures. However, the current enumeration method is cultured-based and time-consuming. In this study, we developed a dual-quadruplex qPCR assay targeting both housekeeping and enzyme-coding genes of key psychrotrophic bacteria, including Pseudomonas spp., Acinetobacter spp., Stenotrophomonas maltophilia, and Bacillus cereus. The results showed that all primers and probes exhibited high specificity. A linear relationship between Ct values and colony counts in both singleplex and multiplex qPCR was confirmed, with R2 values > 0.98 and amplification efficiencies ranging between 95% and 115%. The multiplex qPCR assay had detection limits of approximately 101–102 CFU/mL in pure culture and spiked milk samples. The detection of psychrotrophic bacterial loads in 30 raw milk samples further validated the assay's efficacy. This study provides a time-efficient approach for monitoring four main psychrotrophic bacteria and their enzymes in raw milk, enabling the prediction of spoilage potential.

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