WO2016026829A1

WO2016026829A1 - Method of extracting nucleic acids

Info

Publication number: WO2016026829A1
Application number: PCT/EP2015/068887
Authority: WO
Inventors: Wolfgang Weber; Christine Werner; Nadine NOAK
Original assignee: Ifp Privates Institut Fuer Produktqualitaet Gmbh
Priority date: 2014-08-18
Filing date: 2015-08-18
Publication date: 2016-02-25
Also published as: WO2016026829A9; EP3183344A1

Abstract

The method of isolating nucleic acids from a sample for further analysis by a DNA polymerase chain reaction, wherein the cells of the sample are lysed in aqueous solution using dielectric heating, which encompasses subjecting the sample to microwave electromagnetic radiation having wavelengths ranging from 11.8 to 12.6 cm, comprises the steps of providing a vessel made of microwave-transparent material with closable means and loading said vessel with a sample to be analyzed, a tablet comprising water insoluble hydrated magnesium silicate and crystalline phosphate buffer saline, and water; selecting a hollow receptacle made of microwave-reflecting material which cooperates with microwave electromagnetic radiation; placing one or more closed vessels containing said loaded mixture inside said hollow receptacle; introducing one or more said receptacles with one or more said closed vessels inside a microwave oven; and microwaving, so that the microwave electromagnetic radiation elicits a temperature rise of the mixture inside the closed vessel up to 95°C, and nucleic acids are released and a solution of nucleic acids suitable for PCR analysis is provided.

Description

METHOD OF EXTRACTING NUCLEIC ACIDS

FIELD OF THE INVENTION

[001] The invention relates to a method and a kit of parts for extraction of nucleic acids from complex matrices for subsequent PCR analysis.

BACKGROUND OF THE INVENTION

[002] Dissociation of samples for isolation of cellular components, such as nucleic acids for subsequent amplification and analysis by PCR, can be aided by application of heat. For example, the use of a heating block and the addition of chaotropic agents, detergents and oils for isolating nucleic acids are described in US 2014/0051088. These reagents represent, already at low concentration, a source of polymerase chain reaction inhibitors. Further, transfer of thermal energy using heating blocks or water baths occurs rather slowly. Fast heating of the sample is preferred for an accelerated dissociation process. Temperature increase is, however, difficult to control in small samples. US 6,623,945 B1 discloses cell lysis in low volumes using high frequency microwave radiation up to 26 GHz. Yet the microwave frequency used in conventional microwave ovens, namely 2.45 GHz, is not efficient. Moreover, the method requires the use of computer-controlled means and a complex wave-guide cavity for holding a single sample and subjecting it to radiation. This method can therefore neither be applied with a conventional microwave oven nor for processing of several samples simultaneously.

[003] The increase in temperature of an aqueous solution by microwave radiation is linked to the risk of bursting due to a sudden increase in vapour pressure. US 2006/0141556 A1 describes a method of cell disruption by treating a sample with microwaves and addition of zwitterionic surfactants. However, the disclosure fails to show a fast method by which cell lysis of difficult-to-dissociate samples, for example foodstuffs, can effectively be carried out inside closed vessels without bursting. Thus, the described method cannot prevent cross- contamination due to evaporation and spillage. Further, the mentioned zwitterionic surfactants are highly expensive and, thus, their use is economically disadvantageous for the analysis of large number of samples. The prior art therefore represents a problem.

[003a] WO 2005/103682 A1 discloses a process for extraction of intercellular organelles, components and nucleic acids from cells in a preliminary dried state and attached to a support. SUMMARY OF THE INVENTION

[004] The problem is solved by a method and kit according to claims 1 and 10. Preferred embodiments of the invention are disclosed in the dependent claims 2 to 10 and 12.

[005] The method of isolating nucleic acids from a sample for further analysis by a DNA polymerase chain reaction, wherein the ceils of the sample are lysed in aqueous solution using dielectric heating, which encompasses subjecting the sample to microwave electromagnetic radiation having wavelengths ranging from 11.8 to 12.6 cm, comprises the steps of providing a vessel made of microwave-transparent material with closable means and loading said vessel with (i) a sample to be analyzed, (ii) a tablet comprising water insoluble hydrated magnesium silicate and crystalline phosphate buffer saline, and (iii) water, to obtain a weight ratio of tablet to sample in the vessel from 1 :5 to 5:1 ; and closing the vessel; selecting a hollow receptacle made of microwave- reflecting material which cooperates with microwave electromagnetic radiation, wherein said receptacle displays cylindrical form having closed bottom side and open top side; the inner diameter of the receptacle having an inner diameter/microwave wavelength ratio ranging from 1 :2 to 1:4; and the side wall of the receptacle extending from the bottom side of the receptacle in a side wall/microwave wavelength ratio ranging from 1 :2 to 1 :3; placing upright one or more closed vessels containing said loaded mixture inside said hollow receptacle; introducing one or more said receptacles with one or more said closed vessels inside a microwave oven; and microwaving, so that the microwave electromagnetic radiation elicits a temperature rise of the mixture inside the closed vessel up to 95°C, and nucleic acids are released; and separating the water-insoluble components adsorbed on the magnesium silicate and removal of the supernatant containing soluble nucleic acids, followed by desalting to obtain a solution of nucleic acids suitable for PCR analysis.

[006] In a preferred embodiment, the method further comprises the steps of dissolving the buffer components of the tablet to obtain an aqueous phosphate buffered saline solution having a pH from 5.5 to 7.0 and a salt concentration of 0.4 to 1.2 mol/L; and microwaving for about 10 seconds to 10 minutes, minutes, preferably for about 15 seconds to 5 minutes, most preferred for about 20 seconds to 3 minutes;

[007] In another preferred embodiment, the method of isolating nucleic acids from a sample for further analysis by a DNA polymerase chain reaction comprises placing the vessel inside the receptacle so that the top edge of the vessel is located preferably at least 1 mm below the upper edge of the receptacle, more preferably at least 2.5 mm, most preferred at least 5 mm. In another embodiment, the vessel with closable means is capable of accommodating volumes up to about 2 mL, more preferably up to about 1.8 mL, most preferred up to about 1.5 mL.

[008] One aspect of the disclosure relates to a tablet for extracting nucleic acids from a sample, wherein the tablet is a mixture of solids comprising from 30 to 70 percent by weight crystalline phosphate buffer saline and from 10 to 40 percent by weight water insoluble hydrated magnesium silicate particles. In another embodiment, the tablet for extracting nucleic acids may further comprise from 15 to 45 percent by weight hydrophilic colloid, wherein the hydrophilic colloid is cellulose, carboxy-methyl cellulose, cellulose derivatives, alginate, starch, xantan gum, arabic gum, guar gum or mixtures thereof.

[009] In one embodiment, the receptacle for extracting nucleic acids from a sample has an inner diameter/microwave wavelength ratio ranging preferably from 1 :2.1 to 1 :3.8, more preferably from 1:2.2 to 1:3.6. In another embodiment, the side wall of the receptacle extends from the bottom side of the receptacle in a side wall/microwave wavelength ratio ranging preferably from 1:2.2 to 1:2.8, more preferably from 1:2.4 to 1:2.6. In a further embodiment, the side wall of the receptacle displays a side wall thickness ranging preferably from 1 to 4 mm, more preferably from 1.5 to 3 mm, most preferred from 2 to 2.5 mm. The disclosed receptacle is preferably made of metal, such as aluminium, or metal alloys, and has smoothened surface. In one aspect, the receptacle comprises one or more cavities at the inner side of the bottom part for accommodating one or more vessels.

[010] In another aspect, the disclosure provides a kit of parts for isolating nucleic acids from a sample for further analysis by a DNA polymerase chain reaction comprising one or more tablets for extracting nucleic acids from a sample, and one or more hollow receptacles for extracting nucleic acids from a sample. The disclosed kit of parts may further comprise a rack made of microwave-transparent material for accommodating a plurality of receptacles.

[011] In a preferred embodiment, the method and kit can be used for isolating and characterizing the type of nucleic acids from raw and/or processed animal and plants materials and processed products thereof, potential allergens present in cereals and products thereof, chickpea and products thereof, casein, almond and products thereof, cashew and products thereof, peanut and products thereof, hazelnut and products thereof, macadamia and products thereof, mustard and products thereof, soya and products thereof, sesame and products thereof, walnut and products thereof, pistachio and products thereof, lupin and products thereof, celery and products thereof, fish and products thereof, crustaceans and products thereof, genetically modified organisms, pathogens, Salmonella spp., Listeria spp. Shigella spp., Campylobacter spp., Cronobacter, Clostridium spp., Legionella spp., Enterobacteriaceae, Escherichia spp, fecal samples, preferably human and animal fecal samples.

[012] According to the present disclosure, there is no longer a requirement for the addition of surfactants, enzymes, detergents, organic solvents, etc. for dissociation of a complex matrix. Temperatures up to 95°C and high osmotic strength, due to the high salt concentration, cooperate to dissociate all cell structures and, thus, to promote a release of nucleic acids from the cells into the aqueous solution. The buffering agent contained in the tablet is responsible for creating an osmotic shock, which forces the cytoplasm and, in particular, the cell nucleus to release its content into the extraction solution. The combination of osmotic pressure and short exposure to high temperatures enhances the preservation of nucleic acid integrity for subsequent analysis. The nucleic acids strands are further stabilized by the acidic phosphate buffer, even in aqueous solution with temperatures up to 95°C. This method can be used with virtually all complex matrices, such as food samples, enabling an efficient extraction of nucleic acids from nearly all biological matrices. Efficient extraction means that i) the isolated nucleic acid solution is free of DNA polymerase specific inhibitors and ii) thereby the subsequent polymerase chain reaction (PCR) can be performed faster and with higher degree of reproducibility than conventional methods.

[013] By way of the present disclosure, the temperature of aqueous solutions confined in closed vessels which are exposed to microwaves can be tightly controlled. Until now, the rise of temperature in small volumes with the aid of microwave ovens was unpredictable. The combined used of a single tablet and a hollow receptacle according to the disclosure, and the exposure to microwave radiation generated by a conventional microwave oven yields high DNA quality and reduces extraction time. An important advantage is that the temperature of the aqueous solution is rapidly raised by the action of the microwaves; however, there is no longer vessel bursting due to uncontrolled increase of vapour pressure inside the vessel. This also allows the processing of numerous samples simultaneously without cross- contamination and spillage of the samples.

[014] Advantageous is the straightforward establishment of a routine procedure for the simultaneous analysis of a variety of samples regardless of their different physico-chemical properties. Such samples can be animal and plant foodstuffs rich in lipids, polysaccharides, proteins, and, in particular, mixtures thereof. There is no need for experienced personnel or sophisticated equipment. Any person with minor laboratory experience is thus able to carry out an efficient extraction of nucleic acids from any biological sample, even when handling complex matrices. Full standardisation of nucleic acid extraction is provided in an unprecedented short- term by way of the disclosed method, tablet and receptacle. [015] It is assumed that the disclosed microwaving-step leads to disruption of cellular structures and denaturation of peptidic DNA polymerase specific inhibitors. Further inhibitors with hydrophobic, lipophilic and acidic properties and at least a portion of lipids, proteins, polysaccharides and salts contained in the matrix become adsorbed on the magnesium silicate particles. Subsequent removal of the precipitated material from the aqueous fraction containing the genomic DNA, which can be subsequently desalted, enables the provision of a PCR inhibitor-free DNA solution in a fraction of the time needed following conventional procedures. Desalting can be performed by size exclusion chromatography, ultra filtration or conventional DNA binding chromatography. Optionally, the PCR sample may be simply diluted, so that the salt concentration is lowered to appropriate levels.

[016] Without wishing to be bound by theory, it is believed that the receptacle according to the present disclosure cooperates with the microwave radiation, so that the characteristics of microwave radiation within the receptacle are modified. The vessel according to the disclosure is made of microwave-transparent material, so that it barely interferes with the microwaves. It is well known that microwaves are heterogeneously distributed within the cooking chamber of a microwave oven. As a result, some areas have higher exposure to microwaves than others, and thus differential heating occurs depending on the spot within the oven. In order to partially compensate these differences, most ovens exhibit a rotating plate; however, microwaving and rotating a vessel loaded with an aqueous solution does not allow reaching high temperatures without vessel bursting. It is assumed that, according to the present disclosure, the incident microwaves adopt a homogenous pattern of microwave distribution field within the receptacle. It appears that, because of the particular shape and dimensions of the receptacle with respect to the wavelength of the generated microwaves, a particular wave pattern is formed. This specific wave pattern enables controlled homogenous heating within the receptacle; to a certain extent, irrespective of the irradiation time. Further, such a fast and homogenous heating leads to an evenly dissociation of the cellular components of the sample. Surprisingly, equivalent results are obtained regardless of the number of vessels; this effect is assessed by performance of a quick and efficient DNA extraction, and iack of vessel bursting. Notably, the effect conferred by the use of the disclosed receptacle can only be achieved in combination with the disclosed tablet. The amount of salt added to the sample by way of addition of the tablet appears to alter the dielectric heating pattern in the aqueous solution, leading to a faster molecular rotation and, thus, faster heat transfer to the sample solution. The synergistic effect of tablet and receptacle enables a homogenous dielectric heating pattern within the sample, which thus reduces the sudden explosive behaviour of liquids exposed to microwave radiation, even at longer periods of irradiation. [017] Evaluation of the quality of the extracted nucleic acids and/or the presence of inhibitors in a sample to be analyzed is performed based on the Ct value obtained by quantitative real-time polymerase chain reaction. Fewer cycles necessary to amplify target DNA extracted from the sample are illustrated by lower Ct values. In other words, low Ct values indicate low amount of PCR inhibitors present in the sample and, thus, an efficient DNA extraction. The method and kit of the disclosure allows for fast, reliable and economically advantageous nucleic acid extraction from complex matrices without the disadvantages of conventional approaches.

[018] The conventional methods require an exhaustive removal of added surfactants, reagents and/or solvents to avoid inhibition of the polymerase chain reaction. Also biological samples are known to contain inhibitors. Typical PCR inhibitors endogenous to biological samples are collagen, myoglobin, hemoglobin, immunoglobins and heme (meat and blood), complex and other polysaccharides (feces, plant materials), humic acid (soil, plant materials), melanin and eumelanin (hair, skin), calcium ions and proteinases (milk, bone), and bile salts (feces). The present disclosure overcomes the problems associated with endogenous and added PCR inhibitors. Firstly, expensive surfactants or reagents are not required, and, second, removal of inhibitors is provided by binding to hydrated magnesium silicate particles, which are supplied by the tablet. Further, the method has the advantage that it can be applied simultaneously to very different complex matrices, and allows for the isolation of DNA from different species with the same workflow.

[019] Further advantages, goals and embodiments of the invention can be understood from the following examples and drawings. The invention shall not be limited to the examples, but it has been defined in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[020] In the drawings:

Fig. 1 is a schematic representation of the receptacle and vessel according to the disclosure in a longitudinal section;

Fig. 2 is a schematic representation of the bottom part of the receptacle and corresponding perspective views of the receptacle holding vessels according to the disclosure;

Fig. 3 is a schematic representation of a rack holding several receptacles for use in a microwave oven according to the disclosure. DETAILED DESCRIPTION OF THE INVENTION

[021] By way of the disclosed method, nucleic acids are isolated from a complex matrix, such as a food sample, for further analysis by DNA polymerase chain reaction. The cells of the sample are lysed in aqueous solution using dielectric heating, which encompasses subjecting the sample to microwave electromagnetic radiation having wavelengths ranging from 11.8 to 12.6 cm. Microwaves are non-ionizing electromagnetic waves with frequencies ranging from 300 MHz up to 300 GHz, and wavelengths A from 1 meter to 1 millimetre, respectively. Following international regulations, microwave ovens operate at frequencies of about 2.45 GHz, which corresponds to a wavelength of 12.23 cm. Upon exposure to an electromagnetic field, molecular rotation occurs in materials containing polar molecules having an electrical dipole moment, such as water. As a consequence, these molecules align themselves in the electromagnetic field. If the field is oscillating, as it is the case in an electromagnetic wave or in a rapidly oscillating electric field, these molecules rotate continuously aligning with it. As the field alternates, the molecules reverse direction. Rotating molecules push, pull, and collide with other molecules through electrical forces, distributing the energy to adjacent molecules and atoms in the material. Once distributed, this energy appears as heat. Such process of thermal absorption is known as dielectric heating.

[022] Microwave-reflecting materials are those which reflect incident microwave energy. In other words, electrically conducting metals such as aluminium heat up marginally because they have high thermal conductivity and do not absorb the generated microwave field. They can thus modify the field intensity pattern of the microwaves inside a microwave cavity (i.e. cooking chamber of a microwave oven). On the other hand, microwave-transparent materials such as plastic are not electrically conductive and do not reflect microwaves. Thus, microwave-transparent materials do not interact with the microwaves and do not alter the field intensity pattern within the microwave cavity.

[023] According to the method of the present disclosure, the sample to be analysed can preferably be mechanically dissociated into particles, dispersion or solution. A portion of the sample ranging from 50 mg to 5 g can be weighted and loaded into a vessel (12) made of microwave-transparent material having closable means, such as a screw cap or safe lock. A predefined amount of a composition for extracting nucleic acids may be pressed or compressed to obtain a tablet; in this form, it may be added to the dissociated sample. Said tablet may be a mixture of solids comprising from 30 to 70 percent by weight crystalline phosphate buffer saline, and from 10 to 40 percent by weight water insoluble hydrated magnesium silicate particles. The phosphate buffer salt may be present in the mixture of solids as fine crystals or in granulated form, and its composition is preferably as follows: NaC1 137 mmol/L, Na₂HP0₄ · 2 H₂0 10 mmol/L, KCI 2.7 mmol/L, KH₂P0₄2 mmol/L. The phosphate buffer salt gives a hypertonic solution after dissolving in water, forcing a release of nucleic acids from the biological sample through the resulting osmotic shock. The water-insoluble hydrated magnesium silicate is preferably a hydrated magnesium silicate or fine talc in the form of powder or fine granules. Said hydrated magnesium silicate powder may have a median particle size in the range of 1.0 to 2.0 μηπ, preferably from 1.2 to 1.5 Mm; a median diameter D₅₀ in the range of 0.8 to 2.5 μηι; and a density of 2.6 to 2.8 g/cm3. The insoluble silicate powder has a large surface for adsorption of lipids, complex polysaccharides and other potential polymerase inhibitors. Said tablet may further comprise from 15 to 45 percent by weight swellable hydrophilic colloid, which can be selected from cellulose, carboxy-methyl cellulose, cellulose derivatives, alginate, starch, xantan gum, arabic gum, guar gum or mixtures thereof. The swellable hydrophilic colloid can both facilitate the compacting of the composition as well as the dispersion of the mixture of solids upon contact with an aqueous solution. The swellable material must be free of contaminants, in particular plant and animal nucleic acids, genetically modified organisms and allergens. A predefined amount of composition in form of a tablet may be added so as to obtain a weight ratio of tablet to sample in the vessel from 1:5 to 5:1.

[024] An amount of water may then be added to the dissociated sample and tablet in the vessel (12) to dissolve the buffer components and to obtain an aqueous phosphate buffered saline solution having a pH from 5.5 to 7.0 and a salt concentration of 0.4 to 1.2 mol/L. After closing the vessel (12), a vortexing or shaking step may be carried out from 1 to 120 seconds for proper dissolution of the tablet's components, followed by a short spin-down to avoid accumulation of the dispersion of sample and tablet at the lid of the vessel.

[025] See Figure 1 ; the hollow receptacle (10) according to the disclosure is made of microwave-reflecting material and cooperates with microwave electromagnetic radiation. Further, the receptacle (10) is selected based on its cylindrical shape and dimensions (18, 22, 24, 26) relative to the microwave 's wavelength generated by a conventional microwave oven (38). The receptacle (10) for extracting nucleic acids displays cylindrical form having closed bottom side (14) and open top side (16). Said receptacle (10) is preferably made of metal, such as aluminium, stainless steel, or metal alloys. Notably, the receptacle (10) has smoothened surface. The smoothening of the surface is required to avoid any risk of arcing upon microwaving. The inner diameter (18) of the receptacle (10) may preferably have an inner diameter/microwave wavelength ratio ranging from 1:2 to 1:4. In one embodiment, the inner diameter/microwave wavelength ratio may range from 1 :2.1 to 1 :3.8, more preferably from 1:2.2 to 1:3.6. The side wall (20) of the receptacle (10) extends from the bottom side (14) of the receptacle (10) in a side wall/microwave wavelength ratio ranging from 1:2 to 1 :3. In one embodiment, the side wall (20) may extend from bottom side (14) of the receptacle (10) in a side wall/microwave wavelength ratio ranging preferably from 1:2.2 to 1 :2.8, more preferably from 1:2.4 to 1:2.6. Further, the side wall (20) of the receptacle (10) may display a side wall thickness (22) ranging preferably from 1 to 4 mm, more preferably from 1.5 to 3 mm, most preferred from 2 to 2.5 mm.

[026] See Figures 1 and 2; a closed vessel (12) or a plurality of closed vessels (12) containing said loaded mixture of sample, tablet and water may be placed upright inside the hollow receptacle (10). The hollow receptacle (10) may comprise one or more cavities (30) at the inner side of the bottom part (14) for accommodating one or more vessels (12). The cavities (30) may be configured to accommodate vessels (12) capable of receiving volumes up to about 2 mL, more preferably up to about 1.8 mL, most preferred up to about 1.5 ml_. Said cavities (30) may display a depth (32) from 2 to 10 mm, and a width from 5 to 20 mm; alternatively, a holder (34) made of microwave-transparent material such as plastic having one or more cavities can be used for holding the vessels inside the receptacle (10). Notably, the top edge of the vessel (12) may be located preferably at least 1 mm below the upper edge (16, 28) of the receptacle (10), more preferably at least 2.5 mm, most preferred at least 5 mm. According to the disclosure, one or more hollow receptacles (10) holding one vessel (12) or a plurality of vessels (12) may be placed inside the cooking chamber (40) of a microwave oven (38). In one embodiment, a plurality of receptacles (10) can be set in a rack (36) made of microwave- transparent material such as plastic, and placed on the turning plate (42) of the microwave (38); see Figure 3.

[027] According to the present method, an increase in the temperature of the sample is achieved by microwaving the mixture of sample, tablet and water contained in the vessel (12), which is located inside said hollow receptacle (10). The microwaving step can be applied for about 10 seconds to 10 minutes, preferably for about 15 seconds to 5 minutes, most preferred for about 20 seconds to 3 minutes. The microwave oven (38) may be operated at a power from 150 to 1200 Watt, preferably 300 to 900 Watt. The microwave electromagnetic radiation elicits a temperature rise of the mixture inside the closed vessel up to 95°C. After the microwaving step, the vessel (12) may be vortexed to further the homogenization of the heated mixture. The synergistic activity of the hollow receptacle (10) and the tablet allows for fast temperature increase without solution bursting and, thus, an efficient release of nucleic acids; the integrity of the vessel (12) and sample is thereby not compromised. The stability of the extracted DNA, assessed by subsequent PCR analysis, is preserved even if treated at 95 degrees Celsius for long periods because of the salt and phosphate provided by addition of the tablet. A release of nucleic acids occurs when cellular structures (membranes, organelles, etc..) are so disrupted that no interaction of nucleic acids with any proteins, lipids and polysaccharides takes place. A release of the nucleic acids from the cell nuclei is effected through a large osmotic difference created by the hypertonic solution. The rapid increase in temperature leads to efficient denatu ration of proteins and disruption of cell walls. Also, an increased solubilisation of lipids and polysaccharides is promoted. These cellular components are further adsorbed and precipitated through binding on the water-insoluble magnesium silicate particles, reducing the binding to the inner walls of the vessel (12), which in turn eliminates subsequent carry over into the analytical steps. The water-insoluble components adsorbed on the magnesium silicate may be separated from the aqueous phase by centrifugation or filtration. The aqueous supernatant or filtrate containing soluble nucleic acids may be desalted or diluted to lower the salt concentration so as to obtain a solution of nucleic acids suitable for PCR analysis. Desalting can be carried out by affinity chromatography such as commercial silica-based nucleic acid extraction columns, size exclusion chromatography or ultra-filtration.

[028] Microwave ovens are so designed that microwaves generated at the magnetron are conveyed through a waveguide to the cooking chamber, where they are then reflected by the metal walls of the cooking chamber. Whereas the refractive index of metal is close to 100 %, a penetration depth δ for microwaves is given. For example, the penetration depth 6 for microwaves with wavelength (λ) = 12.2 cm incident on aluminium is approximately 1.2 μm. The receptacle (10) according to the disclosure may display a side wall (20) of about 1 mm and wider, so that microwaves hitting the outer part of the side wall (20) will effectively be reflected away from the receptacle. However, it is assumed that a portion of the microwaves reflected by the cooking chamber enters the receptacle (10) through its open top end (16). It is assumed that geometry of the wall structure in a microwave-reflecting chamber influences the field distribution of microwaves. Without wishing to be bound by a theory, it is believed that a receptacle (10) according to the present disclosure acts as a microwave resonator, which upon exposure to microwaves at wavelengths typical for conventional microwave ovens cooperates in the generation of standing microwaves having different electromagnetic properties. Due to the cylindrical shape, dimensions (18, 22, 24, 26) and material of the receptacle (10), standing waves may be reflected, so that they interfere with each other, superposing and giving rise to frequencies, which are multiple of the fundamental wave, thus harmonics. It is believed that the development of harmonics results in the formation of a homogenous microwave field distribution within the receptacle (10). This phenomenon is different to such occurring in a waveguide transporting or guiding the microwaves from the magnetron to the cooking chamber. In fact, for a given perpendicular direction to the axis, the inner tube size of the waveguide must be at least maximum wavelength / 2 (= 6.1 cm), so that the microwave can effectively be transported into the cooking chamber. Microwaves are then reflected by the side walls of the cooking chamber, which is generally rectangular in shape. As a matter of fact, microwave intensity inside the cooking chamber is not homogenously distributed, leading to unevenly heated spots within the cooking chamber.

[029] The receptacle (10) and tablet for extracting nucleic acids according to the invention solve this problem. First, the high salt concentration promotes a faster molecular motion within the aqueous solution under the electromagnetic field and, thereby, facilitates dielectric heating. Second, the microwaves are forced to enter from the top (16) of the receptacle (10) and they are reflected by the inner side of the cylindrical wall (20). The microwaves reflect back and forth against the inner part of the side wall (20), so that the electromagnetic field in the microwave region is partially confined therein. This results in the enrichment of electromagnetic radiation within the receptacle (10) and a rapid heat-up of the water-containing sample. It is important to keep the edge of the tube (12) below the edge (16) of the receptacle (16), so that the sample is mainly subjected to the reflecting microwaves under the resonant conditions created within the receptacle (10). It has been ruled out that a homogenous sample heating, which ultimately allows for a fast and efficient DNA extraction, is cooperatively dependent on i) the amount of salt contained in the tablet and ii) the receptacle's dimensions (18, 22, 24, 26) relative to the wavelength of the irradiated microwaves. This effect is highly significant when a sample in aqueous solution is confined in a closed vial (12). It is known that water contained in a sample may be heated up by exposure to microwave radiation. However, the heating process of small volumes is rather long and unpredictable. Fine control of the increasing temperature without the risk of bursting of the vessel (12) was, until now, not possible. This is, in part, due to the uneven vapour pressure distribution within the aqueous solution. This phenomenon is characterized by the typical sudden explosion of liquids and foodstuffs inside microwave ovens. By placing the vial (12) containing the tablet and sample inside the disclosed receptacle (10), an optimum heat distribution within the vessel (12) is achieved. Surprisingly, this fine temperature control can be maintained during microwaving for periods longer than 10 minutes, whereas DNA stability is not compromised. With the disclosed method, tablet and receptacle (10), DNA of higher analytical quality, i.e. free of PCR inhibitors, can be quickly and efficiently isolated from complex matrices. A simple, reliable and economically advantageous DNA extraction than conventional methods can thus be achieved.

[030] Another aspect relates to a kit of parts for isolating nucleic acids from a sample for further analysis by DNA polymerase chain reaction and may comprise one or more tablets comprising a defined amount of the composition for extracting nucleic acids and one or more hollow receptacles (10) in accordance with the present disclosure. Optionally, the kit may comprise a rack made of microwave-transparent material for accommodating a plurality of receptacles (10) according to the disclosure.

[031] In a preferred embodiment of the disclosure, the method and kit of parts for extracting nucleic acids may be used for isolating and characterizing the type of nucleic acids from raw and/or processed animal and plants materials and processed products thereof, potential allergens present in cereals and products thereof, chickpea and products thereof, casein, almond and products thereof, cashew and products thereof, peanut and products thereof, hazelnut and products thereof, macadamia and products thereof, mustard and products thereof, soya and products thereof, sesame and products thereof, walnut and products thereof, pistachio and products thereof, lupin and products thereof, celery and products thereof, fish and products thereof, crustaceans and products thereof, genetically modified organisms, pathogens, Salmonella spp., Listeria spp. Shigella spp., Campylobacter spp., Cronobacter, Clostridium spp., Legionella spp., Enterobacteriaceae, Escherichia spp, fecal samples, preferably human and animal fecal samples.

[032] Further embodiments, objects and advantages of the invention will become apparent from the following examples.

EXAMPLES

Example 1 - Composition for DNA extraction and tablet

[033] Two variants of the composition for DNA extraction differing in the amount of phosphate buffered saline (PBS) were used in the preparation of DNA extraction tablets (see Table 1; tablet a and b). The first component of the composition was PBS as microcrystalline salt according to Dulbecco (1 x PBS = NaC1 137 mmol/L, Na₂HPO₄ · 2 H₂0 10 mmol/L, KCI 2.7 mmol/l, KH2PO4 2 mmol/L). Further, pharmaceutical grade talcum (Si0₂ (61.5 %), MgO (31.0 %), CaO (0.4 %), Fe₂0₃ (0.6 %), (AI₂O₃) 0.5 %; pH 8.8) as powder having median particle size of 1.2 μm, a median 5 diameter D50 of 0.65 μm and a density of 2.8 g/cm³ was used. Pharmaceutical grade microcrystalline cellulose free of contaminants was used as dispersion agent. All three components were compressed into a tablet using a stamping press. The "salt" tablet variants had a total unit weight of 83 mg (tablet a) and 117 mg (tablet b). Table 1 shows the composition and relative concentrations of the three components in both tablet variants, which were suitable for DNA extraction from food samples having about 200 mg.

Example 2 - Receptacle for DNA extraction from food samples

[034] A hollow receptacle was designed and produced in a range of sizes (see

Table 2 and Figures 1-2) for use in association with any tablet for DNA extraction of Example 1. In all variants, the receptacle was hollow and displayed cylindrical form having closed bottom side and open top side. The inner diameter of the receptacle ranged from 3 to 5 cm, whereas the side wail of the receptacle had a height of either 5 or 5.5 cm. The side wall of the receptacle had a thickness of about 2 mm in all variants, resulting in outer diameters ranging from 3.4 to 5.4 cm. The receptacle was entirely made of microwave- reflecting material such as aluminium/magnesium/silicon alloy. Similar results were obtained with receptacles made of stainless steel. Shortly, a metal sheet was rolled and welded to form a cylinder; a circular bottom part was then welded into one open side of said cylinder and, finally, one or more cavities of about 5 mm in depth were drilled at the bottom part of the inner side of said cylinder. A fine polishing step was performed in the production of the cylinders so that smoothening of all surfaces of the cylinder was achieved, with special focus of protruding edges. This step was required to eliminate any risk of arcing or sparking upon microwave oven operation.

[035] Alternatively, and with the purpose to adapt to different vessel sizes and brands, three holder variants which differed it their diameter were designed and produced for fitting in the above described microwavab!e receptacles (see Figure 1). The holder was made of microwave-transparent material such as microwavable plastic. For use, the holder was placed at the inner bottom of the corresponding receptacle to accommodate the vessels containing the food sample to be processed. The thickness of the holder was about 4-5 mm. The cavity present in the holder was designed to hold standard 1 to 2 ml_ plastic vials.

Example 3 - Microwave-mediated DNA extraction from food samples

[036] DNA extraction: 10 g of sample was obtained and mechanically homogenised using a grinder or mixer with rotating knifes. 200 mg homogenous sample was transferred into a 2 ml (microwave-transparent) plastic vial with screw cap or snap-lock using a spatula or pipette. A DNA extraction tablet containing either 34 mg PBS (tablet a) or 68 mg PBS (tablet b) according to Example 1 was added together with 1 ml aqua dest. After closing the vial, the tube was vortexed for 3 seconds and spinned down shortly to remove any liquid from the cap of the vessel. The PBS concentration in the resulting sample solution was about either 2.5 times (tablet a) or 5 times (tablet b). The vial containing sample and composition for DNA extraction was placed inside the receptacle according to Example 2. Up to 9 closed vials could be fitted into a single hollow receptacle. Importantly, the physical contact between the closed vials and the side wall of the metal receptacle was not prejudicial to the efficient DNA extraction, also in terms of temperature distribution and vessel bursting. Next, the receptacle was introduced into a conventional microwave oven provided with a rotating plate, whereby the receptacle was placed at the outer edge of the turntable, and was exposed to microwave radiation while rotating within the cooking chamber. When necessary, a circular rack capable of accommodating several receptacles was used, so that a large sample number was processed simultaneously; see Figure 3. In fact, more than 70 samples were processed at the same time using a conventional microwave oven. Importantly, the receptacles were located at the outer edge of the rotating table of the microwave oven, however, avoiding the contact with the walls of the cooking chamber. A gap of at least 3-4 centimetres to the walls of the chamber proved to be effective in eliminating any risk of sparking. At this stage, the microwave was operated at 300-900 Watts for a time period ranging from 30 seconds to 10 minutes. A time period of 3 min at 600 Watt were shown to be efficient conditions for DNA extraction. Also periods of 2 minutes at 900 Watt resulted in equivalent extraction efficiency. Notably, although the temperature of the extracted sample reached 95 °C, no bursting of the vials due to increased activity of vapour pressure occurred. Following microwave irradiation, the vials were centrifuged at 14.000 rpm, RT for 5 minutes. After centrifugation, two well defined phases were observed in the DNA preparations with added extraction tablet; say a clear supernatant and a defined precipitation pellet. The pellet with precipitated cell debris was discarded and the clear supernatant used for further analyses.

[037] Desalting: The supernatant was desalted using a DNA affinity column (Centrispin, Genaxxon) according to the manufacturer's instructions. First, 100 μL_ supernatant was added to 500 μΙ_ binding buffer and vortexed. The volume (600 μL,) loaded onto an equilibrated DNA spin column, followed by 14.000 rpm for 1 minute at RT. The flow-through was discarded, the column washed with 700 μL washing buffer and centrifuged 14.000 rpm for 1 minute at RT. The sample DNA was finally eluted with 50 μί elution buffer.

Example 4 - Real-time PCR and assessment of DNA extraction

[0381 RT-PCR was performed using the RotorGene thermocycler (Qiagen) in accordance with manufacturer's instructions. The PCR was performed in a 20 μΙ volume comprising 10 μΙ 2x SensiFAST™ Multiplex Master Mix (Bioline GmbH, Luckenwalde, DE), 10 μΙ DNA extract, 400 nM primers, and 200 nM reference DNA. The SensiFast Multiplex MasterMix consists of a buffer system, dNTPs, Mg²+, and DNA polymerase. The PCR thermocycler program consisted of an incubation step at 95°C for 5 min followed by 45 cycles of incubation at 95°C for 15 sec, 60°C for 15 sec and 72°C for 10 sec. PCR were performed in duplicates. The Ct value was determined using a threshold of 0.02 by means of the RotorGene software.

[039] For assessment of DNA extraction, 2 ng DNA extracted and prepared in accordance with Example 3 from animal feed and using tablet a (34 mg PBS) was added into a volume of 10 μL with specific primers for detection of target DNA from plant material. The relative PCR sensitivity can then be taken from the Ct values. A sample was prepared as described in Example 3 for each one of the microwavable receptacle variants according to Example 2 (see table 2). Briefly, the samples were exposed to microwave radiation for 3 min at 300 W. In order to figure out the influence of the arrangement of the vial inside the receptacle, the vial was placed either upright or inverted for every one of the receptacle variants. The results are shown in Table 3.

[040] As control, the samples were processed without the addition of the extraction tablet and the vessels placed outside the receptacles. In virtually all cases, this resulted in bursting of the vial; rarely, insufficient heating of the sample was observed (not shown). In sum, when a closed sample vial containing a food sample in aqueous solution is heated up in a microwave oven, the risk of vessel explosion and loss of the sample is inevitable. This demonstrated that a tightly controlled temperature rise can only be achieved with the disclosed method, tablet and receptacle, so that an effective dissociation of the sample and efficient nucleic acid release is carried out without bursting and subsequent spillage/loss of the sample.

Example 5 - DNA extraction from complex foodstuffs

[041] The result of the extraction experiments showed in Table 3 showed similar Ct values regardless of the receptacle variant and the orientation of the vial within the receptacle. Notably, these results indicate that the herein disclosed DNA extraction method allows for reproducible purification of high quality DNA in an unprecedented short time period. This is the case when performing the extraction with the disclosed receptacle in combination with the disclosed tablet for DNA extraction. Consistently, the average Ct value (about 15.8) among the different receptacles coincides in both trials. With the disclosed method, homogenous heating of samples for DNA extraction by means of microwave radiation can thus be achieved, without the disadvantage of vial bursting. In addition, many samples can be processed very rapidly and, notably, without the risk of cross-contamination due to sudden opening of lids and/or vial explosion.

[042] This confirms the feasibility of a high performance DNA extraction protocol in extremely reduced time periods (extraction and RT-PCR can be performed on the very same day). Importantly, the disclosed method renders DNA extraction a less laborious procedure with the further advantage that it does not require the use of expensive and/or hazardous chemicals. No chaotropic chemicals or organic solvents are required, so that the requirement of a laboratory fume hood is dispensed. The use of the disclosed extraction composition also contributes to the reduction of the risks of carrying over potential exogenous PCR polymerase inhibitors. Although not essential, the salt contained in the supernatant following extraction was effectively removed by DNA affinity columns.

[043] After exposure to microwave radiation, the receptacles 1.1, 1.2, 2.1 and 2.2 could be handled without the use of gloves as they were just slightly warm but not hot. On the other hand, although DNA was efficiently extracted, the temperature of receptacles 1.3 and 2.3, which both had an inner diameter of 5 cm, increased so that the use of gloves was advisable to avoid unpleasant handling. This demonstrates that, upon exposure to microwaves, there is a direct relationship between the dimensions of the receptacle and the increase in temperature.

Example 6 - Influence of salt concentration and receptacle's dimension

[044] Animal feed was homogenised and sample DNA extracted according to

Examples 3 and 4, with the difference that here tablet b (PBS 68 mg) was used and all vials were place upright during microwave oven operation. For comparison, 3 different DNA extraction trials were earned out. Real time PCR using primers directed to plant DNA was performed as described in Example 4.

[045] The experiments resulted in very similar Ct values among receptacle variants; see table 4. While no major differences in temperature were observed in the experiments performed with receptacles with inner diameters up to 4 cm, the bursting of vials during microwave radiation exposure occurred when using receptacles having 5 cm inner diameter These observations suggest that there is a direct relationship between the salt amount of the tablet and the increase in temperature effected by interaction of microwaves and receptacle The receptacle's shape and dimensions proved to play essential roles in the heat distribution. The degree of radiation absorption by the sample could be thus controlled by specifying salt concentration and inner diameter of the receptacle. These experiments therefore suggest a diameter of about 4 cm for reliable DNA extraction and comfortable handling, regardless of the salt concentration (34 mg/rnL or 68 mg/mL) in the sample.

[046] Animal feed was homogenised and sample DNA extracted and isolated according to Examples 3 and 6 with the difference that here the microwave oven was operated at 600 W for 2 minutes For comparison. 2 different DNA extraction trials were carried out. Real time PGR using primers directed to plant DNA was performed as described in Example 4.

[047] Table 5 shows that obtained Ct values were again comparable when the receptacles had an inner diameter of 3 or 4 cm (1,1 and 1 2, respectively). The use of the receptacles 1.1 and 1.2 gave consistent Ct values, indicating an efficient and reproducible DNA extraction following the disclosed method, even at higher power (600 W) and shorter microwave exposure time (2 min). This highlights the flexibility of the method, which can be easily optimized to reduce even further the time required for the extraction protocol Once again, the performance of DNA extraction using a receptacle having 5 cm inner diameter (1 ,3) and PBS 68 mg/ml (tablet b) compromised the stability of the sample upon exposure to microwave radiation, leading to vial bursting.

[048] Animal feed was homogenised and sample DNA extracted according to Examples 3 and 6, with the difference that here the microwave oven was operated at 600 W for 5 minutes. For comparison, 3 different DNA extraction trials were carried out Real time PGR using primers directed to plant DNA was performed as in Example 4.

[049] Table 6 shows consistent Ct values obtained when the receptacles had an inner diameter of 3 or 4 cm (1.1 and 1 2. respectively) These results indicated an efficient and reproducible DNA extraction, even at higher power (600 W) and longer microwave exposure time (5 min), This illustrates the adaptability of the method to long microwaving steps at high power, without compromising the stability of both DNA and vessel. This experiment further corroborated the correlation of salt concentration in the sample and temperature increase within the receptacle, in particular when the inner diameter was broader (1.3).

[050] Various complex matrices (hazelnut pulp, wheat flour, soy lecithin, mustard flour, rodent feed, corn flour and sausage) were homogenised and sample DNA extracted and isolated according to Example 3 In this case, however, the experimental conditions comprised receptacles having 3 or 4 cm diameter, tablet b (68 mg/mL), 600 W and microwaving 10 minutes long. The use of receptacles with 5 cm inner diameter was omitted. Real time PCR using primers directed to either plant or animal DNA was performed as described in Example 4,

[051] Notably, this experiment showed that different complex matrices can be processed simultaneously. Also, the extracted DNAs were neither degraded nor the occurrence of PCR-inhibitors increased in the sample by prolonged exposure to microwave radiation; see table 7. It appeared that the receptacles of the disclosure effectively established a homogenous temperature distribution even for very long time periods, obtaining consistent Ct values among different receptacle's dimensions. Importantly, no bursting was observed under these conditions.

TABLE 7

Example 10 - DNA extraction from complex vegetable/animal matrices

[052] Complex matrices as in Example 9 (hazelnut pulp, wheat flour, soy lecithin, mustard flour, rodent feed, corn flour and sausage) were homogenised, and DNA extracted and isolated according to Example 3. In this case, however, the experimental conditions comprised a single receptacle having 4 cm diameter (1.2), tablet b (PBS 68 mg/mL), 600 W and microwaving for 1, 2, 3, 4 or 5 minutes. The use of receptacles with 5 cm inner diameter was omitted. Real time PCR using primers directed to either plant or animal DNA was performed as described in Example 4.

[053] See table 8; microwaving for only 1 minute yielded very good DNA quality in all complex matrices, assessed by comparison with Ct values of samples exposed to microwaves for longer time. In all cases, comparable Ct values were observed regardless of the microwaving periods, notably, both upon analysis of plant and animal DNA. This highlights the unprecedented short time of efficient extraction from a variety of samples with different composition and physical properties, whereas the isolated DNA sample was always PCR inhibitor-free. Importantly, the method has the advantage that it can be applied simultaneously to very different complex matrices and allows for the isolation and analysis of DNA from different species with the same workflow.

Example 11 - influence of salt on DNA extraction from complex samples

[054] Complex matrices (hazelnut pulp, wheat flour, soy lecithin, rodent feed, com flour and sausage) were processed as in Example 10, with the difference that here tablet a (PBS 34 mg/mL) was used and the microwave step was only 3 minutes.

[055] See table 9; in most cases, the obtained Ct values were even lower compared to those in Example 10 (PBS 68 mg/mL). This experiment demonstrated that a lowered salt amount (PBS 34 mg) is equally effective for an efficient DNA extraction. Also, a reduced salt concentration enabled the dilution of the sample prior to PCR analysis, rendering a desalting step dispensable.

Reference sign list

10 Receptacle

12 Vessel

14 Receptacle; bottom side

16 Receptacle; upper edge

18 Receptacle; inner diameter

20 Receptacle; side wall

22 Receptacle; side wall thickness

24 Receptacle; outer diameter

26 Receptacle; side wail height

28 Gap between vessel and upper edge of receptacle

30 Cavity

32 Cavity; depth

34 Holder

36 Rack

38 Microwave

40 Microwave; cooking chamber

42 Rotating plate

Claims

CLAIMS 1 . Method of isolating nucleic acids from a sample for further analysis by a DNA polymerase chain reaction wherein the cells of the sample are lysed in aqueous solution using dielectric heating, which encompasses subjecting the sample to microwave electromagnetic radiation having wavelengths ranging from 11.8 to 12.6 cm, comprising the steps of

providing a vessel made of microwave-transparent material with closable means and loading said vessel with (i) a sample to be analyzed, (ii) a tablet comprising water insoluble hydrated magnesium silicate and crystalline phosphate buffer saline, and (iii) water to obtain a weight ratio of tablet to sample in the vessel from 1 :5 to 5:1 ; and closing the vessel;

selecting a hollow receptacle made of microwave-reflecting material which cooperates with microwave electromagnetic radiation, wherein said receptacle displays cylindrical form having closed bottom side and open top side; the inner diameter of the receptacle having an inner diameter/microwave wavelength ratio ranging from 1 :2 to 1 :4; and the side wall of the receptacle extending from the bottom side of the receptacle in a side wall/microwave wavelength ratio ranging from 1 :2 to 1 :3;

placing upright one or more closed vessels containing said loaded mixture inside said hollow receptacle;

introducing one or more said receptacles with one or more said closed vessels inside a microwave oven; and microwaving, so that the microwave electromagnetic radiation elicits a temperature rise of the mixture inside the closed vessel up to 95°C and nucleic acids are released; and

separating the water-insoluble components adsorbed on the magnesium silicate and removal of the supernatant containing soluble nucleic acids, followed by desalting to obtain a solution of nucleic acids suitable for PCR analysis. 2. Method according to claim 1, further comprising the steps of

dissolving the buffer components of the tablet to obtain an aqueous phosphate buffered saline solution having a pH from 5.5 to 7.0 and a salt concentration of 0.4 to 1.

2 mol/L; and

microwaving for about 10 seconds to 10 minutes, minutes, preferably for about 15 seconds to 5 minutes, most preferred for about 20 seconds to 3 minutes;

3. Method according to any claim 1 or 2, wherein the top edge of the vessel is located preferably at least 1 mm below the upper edge of the receptacle, more preferably at least 2.5 mm, most preferred at least 5 mm, wherein the vessel with closable means is capable of accommodating volumes up to about 2 ml_, more preferably up to about 1.8 ml_, most preferred up to about 1.5 ml_.

4. Use of a tablet for extracting nucleic acids from a sample in a method according to claim 1 , wherein the tablet is a mixture of solids comprising

from 30 to 70 percent by weight crystalline phosphate buffer saline; and from 10 to 40 percent by weight water insoluble hydrated magnesium silicate particles.

5. Use of a tablet according to claim 4, wherein the tablet for extracting nucleic acids further comprises from 15 to 45 percent by weight hydrcphilic colloid, wherein the hydrophilic colloid is cellulose, carboxy-methyl cellulose, cellulose derivatives, alginate, starch, xantan gum, arabic gum, guar gum or mixtures thereof.

6. Use of a receptacle in a method for extracting nucleic acids from a sample according to claim 1 , wherein the inner diameter has an inner diameter/microwave wavelength ratio ranging preferably from 1 :2.2 to 1 :2.8, more preferably from .1 :2.4 to 1 :2.6.

7. Use of a receptacle in a method according to 6, wherein the side wall of the receptacle extends from the bottom side of the receptacle in a side wall/microwave wavelength ratio ranging preferably from 1 :2.2 to 1:2.8.

8. Use of a receptacle in a method according to claim 6 or 7, wherein the side wall of the receptacle displays a side wall thickness ranging from 1 to 4 mm.

9. Use of a receptacle according to any claim 6 to 8, wherein the receptacle is preferably made of metal or metal alloys and has smoothened surface.

10. Use of a receptacle according to any claim 6 to 9, wherein said receptacle comprises one or more cavities at the inner side of the bottom part for accommodating one or more vessels.

1 1. Kit of parts for isolating nucleic acids from a sample for further analysis by a DNA polymerase chain reaction, comprising

one or more tablets as claimed in any claim 1 , 4 and 5; and

one or more hollow receptacles as claimed in any claim 1 and 6 to 10;

optionally, a rack made of microwave-transparent material for accommodating a plurality of receptacles as claimed in any claim 1 and 6 to 10.

12. Use of the method and kit according to any claim 1 to 11 for isolating and characterizing the type of nucleic acids from raw and/or processed animal and plants materials and processed products thereof, potential allergens present in cereals and products thereof, chickpea and products thereof, casein, almond and products thereof, cashew and products thereof, peanut and products thereof, hazelnut and products thereof, macadamta and products thereof, mustard and products thereof, soya and products thereof, sesame and products thereof, walnut and products thereof, pistachio and products thereof, lupin and products thereof, celery and products thereof, fish and products thereof, crustaceans and products thereof, genetically modified organisms, pathogens, Salmonella spp., Listeria spp. Shigella spp., Campylobacter spp., Cronobacter, Clostridium spp., Legionella spp., Enterobacteriaceae, Escherichia spp, fecal samples, preferably human and animal fecal samples.