Colorimetric Point-of-Care Detection of Clostridium tyrobutyricum Spores in Milk Samples.

Clostridium tyrobutyricum represents the main spoiling agent responsible for late blowing defects (LBD) in hard and semi-hard cheeses. Its spores are resistant to manufacturing procedures and can germinate during the long ripening process, causing the burst of the cheese paste with a consequent undesirable taste. The lower quality of blown cheeses leads to considerable financial losses for the producers. The early identification of spore contaminations in raw milk samples thus assumes a pivotal role in industrial quality control. Herein, we developed a point of care (POC) testing method for the sensitive detection of C. tyrobutyricum in milk samples, combining fast DNA extraction (with no purification steps) with a robust colorimetric loop-mediated isothermal amplification (LAMP) technique. Our approach allows for the sensitive and specific detection of C. tyrobutyricum spores (limit of detection, LoD: ~2 spores/mL), with the advantage of a clear naked-eye visualization of the results and a potential semi-quantitative discrimination of the contamination level. In addition, we demonstrated the feasibility of this strategy using a portable battery-operated device that allowed both DNA extraction and amplification steps, proving its potential for on-site quality control applications without the requirement of sophisticated instrumentation and trained personnel.

A Rapid Colorimetric Assay for On-Site Authentication of Cephalopod Species.

A colorimetric assay, exploiting the combination of loop-mediated isothermal amplification (LAMP) with DNA barcoding, was developed to address the authentication of some cephalopod species, a relevant group in the context of seafood traceability, due to the intensive processing from the fishing sites to the shelf. The discriminating strategy relies on accurate design of species-specific LAMP primers within the conventional 5' end of the mitochondrial COI DNA barcode region and allows for the identification of Loligo vulgaris among two closely related and less valuable species. The assay, coupled to rapid genomic DNA extraction, is suitable for large-scale screenings and on-site applications due to its easy procedures, with fast (30 min) and visual readout.

A Fast, Naked-Eye Assay for Varietal Traceability in the Durum Wheat Production Chain.

The development of a colorimetric mono-varietal discriminating assay, aimed at improving traceability and quality control checks of durum wheat products, is described. A single nucleotide polymorphism (SNP) was identified as a reliable marker for wheat varietal discrimination, and a rapid test for easy and clear identification of specific wheat varieties was developed. Notably, an approach based on the loop-mediated isothermal amplification reaction (LAMP) as an SNP discrimination tool, in combination with naked-eye visualization of the results, was designed and optimized. Our assay was proven to be effective in the detection of adulterated food products, including both substitution and mixing with different crop varieties.

Xylenol orange-based loop-mediated DNA isothermal amplification for sensitive naked-eye detection of Escherichia coli.

Loop-mediated isothermal amplification (LAMP) can amplify DNA specifically and sensitively. Under minimal buffering conditions, it produces hydrogen ions that lower the pH of the solution upon DNA amplification. This characteristic was applied to visually detect amplified DNA of Escherichia coli through the use of Xylenol Orange, a pH-dependent dye. Under the optimal conditions, 120 min at 63 °C, the Xylenol orange-dependent colorimetric LAMP revealed a detection limit as low as 1 CFU, namely 100,000 times more sensitive than typical multiplex PCR, and showed no cross-reactions with other foodborne pathogens. The colorimetric assay was successfully exploited to detect E. coli contaminations in milk samples, showing high reliability and the same high sensitivity with naked-eye readout. Together with robustness, simplicity, and visual detectability of amplification, this assay can serve as an alternative tool to PCR for detecting E. coli, which is suitable for both laboratory and on-field applications.