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

IF 6 1区农林科学 Q1 FOOD SCIENCE & TECHNOLOGY

LWT - Food Science and Technology Pub Date : 2025-03-09 DOI:10.1016/j.lwt.2025.117628

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

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引用次数: 0

Abstract

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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来源期刊

LWT - Food Science and Technology 工程技术-食品科技

CiteScore

11.80

自引率

6.70%

发文量

1724

审稿时长

65 days

期刊介绍： LWT - Food Science and Technology is an international journal that publishes innovative papers in the fields of food chemistry, biochemistry, microbiology, technology and nutrition. The work described should be innovative either in the approach or in the methods used. The significance of the results either for the science community or for the food industry must also be specified. Contributions written in English are welcomed in the form of review articles, short reviews, research papers, and research notes. Papers featuring animal trials and cell cultures are outside the scope of the journal and will not be considered for publication.