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： Wu, Jie 1 ; Meng, Lu 1 ; Xu, Wenjun 1 ; Zhao, Yankun 1 ; Liu, Huimin 1 ; Zheng, Nan 1 ; Rahman, Ashikur 1 ; Wang, Jiaqi 1 ;
作者机构： 1.Chinese Acad Agr Sci, Lab Qual & Safety Risk Assessment Dairy Prod, Minist Agr & Rural Affairs, Inst Anim Sci, Beijing 100193, Peoples R China
2.Chinese Acad Agr Sci, Key Lab Qual & Safety Control Milk & Dairy Prod, Minist Agr & Rural Affairs, Inst Anim Sci, Beijing 100193, Peoples R China
3.Xinjiang Acad Agr Sci, Inst Qual Stand & Testing Technol Agroprod, Urumqi 830091, Peoples R China
关键词： psychrotrophic bacteria; Protease and lipase; multiplex qPCR; Raw milk; Spoilage
期刊名称：LWT-FOOD SCIENCE AND TECHNOLOGY （ 影响因子：6.6； 五年影响因子：6.9 ）
ISSN： 0023-6438
年卷期： 2025 年 222 卷
页码：
收录情况： SCI
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 timeefficient approach for monitoring four main psychrotrophic bacteria and their enzymes in raw milk, enabling the prediction of spoilage potential.
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