Molecular detection and characterization of foodborne bacteria: Recent progresses and remaining challenges

Abstract

The global food demand is expected to increase in the coming years, along with challenges around climate change and food security. Concomitantly, food safety risks, particularly those related to bacterial pathogens, may also increase. Thus, the food sector needs to innovate to rise to this challenge. Here, we discuss recent advancements in molecular techniques that can be deployed within various foodborne bacteria surveillance systems across food settings. To start with, we provide updates on nucleic acid-based detection, with a focus on polymerase chain reaction (PCR)-based technologies and loop-mediated isothermal amplification (LAMP). These include descriptions of novel genetic markers for several foodborne bacteria and progresses in multiplex PCR and droplet digital PCR.The next section provides an overview of the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins systems, such as CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13a, as tools for enhanced sensitive and specific detection of foodborne pathogens. The final section describes utilizations of whole genome sequencing for accurate characterization of foodborne bacteria, ranging from epidemiological surveillance to model-based predictions of bacterial phenotypic traits through genome-wide association studies or machine learning.

Development of a Taxon-Specific Real-Time Polymerase Chain Reaction Method to Detect Trichoderma reesei Contaminations in Fermentation Products

“…However, for such GMMs used to produce microbial fermentation products, both bacterial and fungal strains, genetic information, including sequencing data, is confidential, which critically hampers the controls performed by enforcement laboratories to guarantee the traceability of commercial microbial fermentation products. Therefore, without publicly available information on the GMM strains of interest, this isolation step is particularly challenging due to the enormous list of microbial growth conditions to be tested, including possible auxotrophic mutations [15,48,[50][51][52][53][54]. In the absence of prior knowledge, a high-throughput sequencing strategy, like metagenomics, represents an interesting and promising option, as recently demonstrated [49,[55][56][57][58][59][60].…”

An Evaluation of the Sensitivity and Applicability of a Droplet Digital Polymerase Chain Reaction Assay to Simultaneously Detect Pseudomonas aeruginosa and Pseudomonas fragi in Foods

“…In the sensitivity evaluation of the bacterial suspension template, the detection limits of P. fragi and P. aeruginosa were all presented in single digits (Figure 5). At present, CRISPR technology is used to detect P. aeruginosa with a minimum of 50 cfu/mL [6]. Wang et al detected Salmonella using digital PCR with a sensitivity of 10 −4 ng/µL or 10 2 cfu/mL, which are lower values than the results detected in this assay [47].…”

“…Spoilage bacteria have strong protein and lipid hydrolysis ability, making it easy to reduce meat quality and cause resource waste [5]. Therefore, it is necessary to identify different genera of Pseudomonas, which is similar to the identification of different genera of Listeria, in order to distinguish their pathogenicity [6]. It is well known that P. aeruginosa is a common bacterium that is widely found in water and other environments in nature [7].…”

Combination of nucleic acid amplification and CRISPR/Cas technology in pathogen detection

“…Specific primers are designed to detect different targets, and the target DNA amplification products accumulate exponentially within 25–30 PCR cycles. Due in part to its “amplification” nature, PCR has become a multifunctional tool with improved specificity and sensitivity compared to immunological assays, and can be used to detect a variety of pathogenic bacteria ( Malorny et al, 2003 ; Hadi et al, 2023 ). To improve the detection performance of PCR, multiplex PCR (mPCR) and quantitative real-time PCR (qPCR) have also been established with the advancement of molecular biology and PCR technology.…”