During the past decades food safety concerns have steadily increased and are presently one of the most important challenges for food authorities. Growing emergence of food safety scares on one side and increased consumer preferences for minimally processed foods on the other side pose major challenges to food companies in controlling supply chains safety. In particular industrially processed foods represent a complex and challenging matrix for the detection of food-derived pathogens such as Salmonella and Listeria in routine food safety testing. With the requirement to detect down to one viable pathogen in 25 g of food, the majority of food safety testing laboratories still routinely apply classical, but extremely time-consuming, microbiological culturing methods to verify bacterial growth. For example salmonella, which is a major cause for food-borne bacterial infections throughout the world, requires five days for a classical microbiological confirmation. A classical microbiological salmonella confirmation comprises several steps. First, a potentially salmonella-contaminated food sample is incubated in an initial 18 hour overnight pre-enrichment culture, followed by an additional 24 hour selective enrichment phase. The culture is subsequently streaked on a selective nutrient agar (24 hour incubation) and characteristic colonies are further cultivated for final bacteria detection (24 hour incubation). Confirmation is carried out on the fifth day either biochemically or serologically.
Such time consuming methods can not be integrated in the regular flow of the food supply chain. A clear gap exists between microbiological product approval processes in the range of several days to weeks and food factory realities dealing with raw and perishable materials, creating an urgent need for short, flexible and easy-to-use testing methods. In this context, a workflow consisting of efficient pathogen nucleic acid extraction in combination with highly selective detection such as by real-time PCR has gained considerable importance in routine pathogen testing for food production and safety chains.
In food diagnostics methods that are based on molecular biological detection methods the pathogen detection workflow also starts from a food enrichment culture, in which the potentially contaminated food type is mixed with an enrichment medium and incubated at an elevated temperature for a certain amount of time. Under these conditions the number of contaminating pathogens is increased above an analytically detectable number. The preparation of an enrichment culture is necessary to process only viable pathogens in the subsequent extraction and detection steps and to guarantee a safely detectable pathogen concentration within the highly inhibitory food matrix. To prepare a food enrichment culture for the subsequent nucleic acid isolation usually an amount of potentially contaminated food (usually 25 g) is mixed with a defined volume of an enrichment medium. This food/medium mixture is incubated at raised temperature for a specific time (e.g. 18 h for Salmonella) to grow and enrich the contaminating pathogen above an analytically detectable level. The resulting enrichment culture comprises a high concentration of the food-borne pathogens but at the same time a dense matrix of food-derived inhibitors. Thus, the food sample is often highly inhibitory to the subsequent isolation and detection process. This is, because food samples differ in their nature and regularly comprise a multitude of different food-derived inhibitors in a high concentration. Therefore, even after enrichment, the isolation of a sufficient amount of nucleic acids with acceptable purity that allows to perform the subsequent pathogen detection steps is challenging.
Thus, the successful detection of food-borne pathogens in a food sample strongly depends on efficient protocols for the extraction and purification of pathogen nucleic acids such as bacteria DNA from complex food matrices. Here, the efficient depletion of most of the food-borne inhibitors is a crucial prerequisite for the reliable isolation of nucleic acids that can be subsequently used for molecular biological pathogen detection e.g. using a PCR based detection method.
To provide a nucleic acid method that is suitable for isolating pathogen nucleic acids from various food samples is particularly challenging, if a simple, rapid protocol is supposed to be provided that is also suitable for automation. Automation is a key requirement of the food industry. For highly standardized and safe routine food safety testing, the industry strongly requires a complete workflow line, applying a minimum of manual interactions from sample preparation to pathogen detection. A fully automated pathogen detection workflow is required to offer maximal process safety and rapid high throughput protocols while allowing for flexible sample volume and sample type. Furthermore, as discussed above with the emergence of a globalized market, food industry is in demand for reliable, accurate, and stable pathogen detection systems.
Thus, it is the object of the present invention to provide a method for isolating nucleic acids from a food sample that overcomes at least one of the prior art drawbacks. In particular, it is an object to provide an improved nucleic acid isolation protocol that allows to isolate pathogen derived nucleic acids, in particular DNA, from various food samples.
USPTO Backgrounds
USPTO Backgrounds