GB2469266A - Device for the isolation of nucleic acids in a sealed environment - Google Patents

Abstract

A device for the isolation of nucleic acid in a sample is provided, comprising an entry port for adding the sample to be analysed to the device, a compartment where the nucleic acid is liberated from the sample, a compartment where the nucleic acid is purified and a compartment where the nucleic acid is concentrated. The device also comprises a removable container for the isolated nucleic acids. The nucleic acids may then be used in amplification reactions. In a preferred embodiment, the nucleic acid is released from cells and is bound to species-specific or sequence-specific particles. Methods of use of the device are also provided. The device may be used for the purification of microbial nucleic acids in order to detect the contamination of foodstuffs.

Description

Method and Device for the Concentration of Micro-organisms and the Purification of their Nucleic Acids in order to enable the easy and Rapid Detection of Microbial Contaminants of Foodstuffs State of the art In microbiology used in food testing there are a lot of spoilage organisms that lead to great damage to food production or even pose serious health hazards to the consumer. Such organisms can be bacteria, moulds or yeast. Testing for these organisms involves labour and time consuming methods such as pre-enrichment and species specific culture methods on special agar plates for example. Time to result is, dependent on the organisms between 3 and 7 days. Some species are even harder to detect as they grow very slowly. Moulds in foodstuff can be detected by firstly filtration through a 5 pm prefilter to remove particles from the food and secondly collecting the mould cells in a 0.45 pm membrane. The filter is then incubated for 50 hours on a YM-1 1 agar plate at 25°C for mould detection (Food Spoilage Organisms, Blackburn, ed, Woodhead publishing ltd, Cambridge, p 46). Molecular methods include PCR, ELISA tests for mould biomolecules, DNA microarrays and RFLP analysis.

Problems of the state of the art In food processing facilities the use of microbiology is restricted to avoid contamination of the production process by micro-organisms. Therefore only simple pre-cultures are possible on-site. This method does not yield enough results to identify organisms spoiling the food production process. Further testing needs to be performed in a separate facility leading to a great loss of time. When molecular biology methods are used such as PCR, the extraction and enrichment of nucleic acids from the spoilage organisms can be a big problem, as the spoilage organism nucleic acid can be highly diluted in foodstuff nucleic acid. Furthermore the problem of DNA spillage both during the DNA purification stage and after PCR leads very often to contamination of the laboratory and to false results in testing. Also molecular detection methods currently in use are not designed to test quantitatively for spoilage yeasts and moulds (Food Spoilage Organisms, Blackburn, ed, Woodhead publishing ltd, Cambridge, p 46). PCR and real-time PCR are used by several groups to identify yeasts and moulds but have not yet reached a state where the technology is being used on site or even outside an analytical PCR lab with separation of nucleic acid extraction, amplification and detection. Another problem, occurring especially with complex food is, that components of the food might contain dead micro-organisms or parts of micro-organisms which still contain nucleic acids (in that case mostly DNA).

This nucleic acid will then subsequently lead to false positive identification using PCR based methods.

Solution to these problems The solution to this problem is that after pre-culture the micro-organisms are processed in a sealed environment to produce nucleic acid in analysis grade quality, especially RNA. In a pre-culture these organisms are grown for a short period of time to allow the growth of live cells which contain sufficient amounts of RNA The cells of the microbes are concentrated and lysed. The RNA (or DNA if preferred) is isolated by specific bonding to a solid phase or other isolation methods known to the skilled in the art. This solid phase can be paramagnetic beads with attached consensus probes for 28S rRNA for example. The solid support is subsequently washed and collected a separate compartment. This compartment can be an integral part of the system or may be removed from the analysis system for further processing.

Surprisingly it is possible to extract nucleic acid from bacteria, moulds and yeasts in one step. Thereafter the required nucleic acid is immediately separated from proteins to ensure the necessary stability. These nucleic acids are then analysed to show the presence of a specific dade of organisms, such as genera, families or higher orders of classification. Especially for the detection of live organisms the use of RNA as a template is of advantage, as RNA has a relatively short life and can therefore be used to assess the number of living organisms in a sample.

Description of the invention

The invention consists of a device where the sample potentially containing micro-organisms is pumped into an initial filter in which the micro-organisms are collected from pre-enrichment. In a following step, elution buffer is added from a reservoir.

After agitation/mixing the buffer is transferred via a valve to a second filter. That filter retains the micro-organisms. The used buffer in discarded in a waste reservoir and lysis buffer is applied to the second filter to destroy the micro-organisms and liberate the nucleic acid from these cells. Sonication, vibration or agitation could be used to facilitate the process. The lysis buffer is than pumped through a compartment filled with nucleic acid binding particles (silica microparticles for example). These particles bind the nucleic acid from the lysis buffer and allow for the removal of the remaining compounds (proteins and other biomolecules, lysis buffer components). The bound nucleic acids are then washed and removed from the matrix by en elution buffer. The elution buffer is than transferred to a new reaction chamber and nucleic acid binding particles are added. In a preferred embodiment these binding particles are magnetic or paramagnetic. These nucleic acid binding particles may bind nucleic acid species or nucleic acid species. In the case of nucleic acid species the term species can apply to a specific nucleic acid species or to a subgroup of a nucleic acid species defined by a consensus sequence. Consensus sequences of for example rRNA (or the corresponding DNA sequence) are known in the state of the art and are widely used for molecular biologic means to identify the presence of micro-organisms via PCR or RT-PCR. Nucleic acid sequences could also be degenerate sequences or sequences spaced with non-specific or non-binding moieties. Then the IPC (internal positive control) is added and the beads are next washed to remove unwanted nucleic acids and other substances. Then the beads are transferred to a sealed compartment for further use. If the nucleic acid sequence is RNA the amplification can be reverse transcription PCR or RNA based amplifications like NASBA.

The device for performing said nucleic acid isolation consists of a filter holder for the first filter. This compartment is sealed and is only accessible through an entry port. In a preferred embodiment that entry port has a unidirectional valve allowing liquid only to enter the system, therefore avoiding the risk of contamination. In the simplest version the microbial cell walls are lysed within that compartment with enzymes such as lysozyme (enzymes known to digest cell walls of different microorganisms are well known in the state of art). The digest can be supported by heating (e.g. resistance heating), ultrasound. In a more complex embodiment the bacteria are washed away from the first filter to be transferred to a second filter with a smaller filtering area.

Then the lysis takes place. After the cell walls have been weakened by the enzymatic digestion guanidiniumisothiocyate (GJTC) buffer is applied to completely remove the nucleic acids from the cells. The GITC buffer contains also the internal calibration standard nucleic acid in the applications where this is needed. GITC buffer and standard nucleic acids could be stored in a separate plug-in module that can be inserted into the system prior to use. This allows the refrigerated storage of sensitive material. In the simplest configuration the nucleic acids are then bound onto silica particles, washed and removed with suitable elution buffers. These buffers are for example available from Qiagen and known to a person skilled in the art of nucleic acid preparations. In a preferred embodiment the nucleic acid of choice could be purified by digestion of other unwanted nucleic acid species with nucleases (RNA can be digested by RNAses, DNA can be digested by DNAses). The purified nucleic acids are then collected in a sealable collector tube. All liquids could be applied via the entry port. The entry port could be a standard luer connection to which a syringe could be connected to pump liquids through. These liquids would then end up in a waste container that is part of the enclosed system. At least between the waste container and the collector tube is a two-way valve. All liquids except the elution liquid of the nucleic acid are transferred to the waste container. Only the desired nucleic acid is directed into the collector tubes. In another embodiment the nucleic acid is split into several (identical) sub fractions, which are directed to different collector tubes. In a preferred embodiment the nucleic acid is hybridized to paramagnetic particles after the isolation and silica particle purification to allow the concentration of desired nucleic acid sequences by hybrid isation to nucleic acid sequences attached to the magnetic beads

Detailed description of one possible solution

Precultu re Foodstuff like for example juices are filtered through a 0.45 pm membrane and then incubated in liquid standard growth medium for 6 hours to start a preculture of micro-organisms. Such growth media are for example listed in "Food Microbiology and Laboratory Practice" (Bell et al 2005, Blackwell Publishing, Oxford) Filtration After Preculture the broth (unspecific growth medium) is filtered through a 0.45 pm membrane, which is part of the integrated system. The filtered liquid is removed to a waste reservoir to avoid contamination.

Sample enrichment Buffer is pumped from a buffer reservoir via the first filter to a second filter. The second filter is much smaller in diameter than the first filter. The micro-organisms are collected in this filter and washed. After washing the lysis buffer is applied. The lysis buffer contains enzymes to break the cell walls of these organisms. This process can be facilitated for example (but not exclusively) by the application of heat, electricity, ultrasonic waves or pressure waves. The use of this step is optional for cases where further concentration is needed.

Sample preparation The nucleic acid liberated from the microbial cells is then purified by liquid chromatography or by absorption on solid particles. In case of bacterial RNA isolation relatively small molecules can be chosen (in a preferred embodiment 16s rRNA) and separated from other larger molecules such as genomic DNA by liquid electrophoresis. As all RNA molecules move with the same speed, to a buffer-only-filled electrophoretic track a t-junction could be used to insert RNA in a second electrophoretic system where the liquid buffer contains a molecular weight separation additive such as PVPP.

Electrophoretic buffer used in liquid electrophoresis is preferably a buffer that also allows hybridisation of nucleic acids. In a preferred embodiment this buffer is a phosphate buffer. In another preferred embodiment this buffer is a citrate buffer.

The isolated nucleic acid is then bound to magnetic or paramagnetic particles to allow magnetic dosage of the isolated NA into the reaction vessels. In case of the need to split the sample into several fractions, electrosmotic transport can be used to deliver exact volumes to the amplification tubes.

Definitions: Yeasts are eukaryotic microorganisms classified in the kingdom Fungi, with about 1,500 species currently described; they dominate fungal diversity in the oceans. Most reproduce asexually by budding, although a few do so by binary fission. Yeasts are unicellular, although some species with yeast forms may become multicellular through the formation of a string of connected budding cells known as pseudohyphae, or false hyphae as seen in most moulds. Yeast size can vary greatly depending on the species, typically measuring 3-4 pm in diameter, although some yeasts can reach over 40 pm.

The yeast species Saccharomyces cerevisiae has been used in baking and fermenting alcoholic beverages for thousands of years. It is also extremely important as a model organism in modern cell biology research, and is the most thoroughly researched eukaryotic microorganism. Researchers have used it to gather information into the biology of the eukaryotic cell and ultimately human biology. Other species of yeast, such as Candida albicans, are opportunistic pathogens and can cause infection in humans. Yeasts have recently been used to generate electricity in microbial fuel cells, and produce ethanol for the biofuel industry.

Yeasts do not form a specific taxonomic or phylogenetic grouping. At present it is estimated that only 1% of all yeast species have been described. The term "yeast" is often taken as a synonym for S. cerevisiae, but the phylogenetic diversity of yeasts is shown by their placement in both divisions Ascomycota and Basidiomycota. The budding yeasts (true yeasts) are classified in the order Saccharomycetales.

(Source: Wikipedia) Moulds include all species of microscopic fungi that grow in the form of multicellular filaments, called hyphae. In contrast, microscopic fungi that grow as single cells are called yeasts. A connected network of these tubular branching hyphae has multiple, genetically identical nuclei and is considered a single organism, referred to as a colony or in more technical terms a mycelium.

Moulds do not form a specific taxonomic or phylogenetic grouping, but can be found in the divisions Zygomycota, Deuteromycota and Ascomycota. Although some moulds cause disease or food spoilage, others are useful for their role in biodegradation or in the production of various foods, beverages, antibiotics and enzymes.

Nucleic acid: A nucleic acid is a macromolecule composed of chains of monomeric nucleotides. In biochemistry these molecules carry genetic information or form structures within cells. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are universal in living things, as they are found in all cells and viruses. Nucleic acids were first discovered by Friedrich Miescher.

Nucleic acid species: In this invention the different types of nucleic acids, RNA and DNA are described as nucleic acid species. Nucleic acids covered by this definition can be single stranded or double stranded.

Nucleic acid sequences: A oligomer or polymer consisting of nucleotides or desoxynucleotides where the position of the singe bases is homologous or showing 80% homology or greater.

Consensus sequence: A consensus sequence is a nucleic acid sequence that is identically found in different species or different genes having the same function in these species or genes.

Other literature: Casey & Dobson (2004) Potential of using real-time PCR-based detection of spoilage yeast in fruit juice -a preliminary study, mt J Food Microbiol, 91, 327-335