Nanopore-based amplicon sequencing for rapid detection and identification of Bacillus spp. in plant-based products

Bacillus contamination in plant-based food products is a significant concern due\r\nto heat-resistant spores that can survive heating and proliferate during storage\r\nor handling, possibly leading to foodborne illnesses. Given the potential of highthroughput\r\nDNA sequencing to enhance microbial monitoring, we used a targeted\r\napproach by combining amplification of the Bacillus tuf gene with Oxford Nanopore\r\nsequencing. The ability of the MinION-based protocol to detect and identify closely\r\nrelated Bacillus species was first assessed using plant-based food samples spiked\r\nwith spore suspensions of five representative Bacillus strains. A DNA extraction\r\nmethod, relying on food enzymatic pre-processing combined with mechanical\r\ncell lysis, was implemented to maximize Bacillus spore DNA recovery, and a\r\ndroplet digital PCR (ddPCR) assay was used to evaluate extraction efficiency.\r\nAfterwards, the approach was applied to 72 different commercial plant-based\r\nfoods and supplements to compare nanopore-based tuf profiling with established\r\nnanopore-based 16S rRNA analysis and culture-based methods. ddPCR analysis\r\nshowed the high efficiency of the DNA extraction procedure for Bacillus spores from\r\nspiked samples. Sequencing of the tuf gene with the MinION device successfully\r\ndifferentiated the five different Bacillus species selected as reference strains for the\r\nartificial inoculation of food, when the resulting sequences were aligned against\r\nthe custom-made BacTufDB database. Tests on commercial products confirmed\r\nthe tuf gene ability (over the 16S rRNA gene) to highlight the presence of Bacillus\r\nspecies: Bacillus and closely related genera were detected in 35 of the tested\r\nplant-based products, out of which seven were contaminated by Bacillus cereus\r\ngroup. The study demonstrated that the tuf-based methodology more effectively\r\ndetects Bacillus species in plant-based products, offering potential applications\r\nin food safety and quality control.