Patent Application
20090023175
The present invention relates to a device for sampling a fluid and detecting an analyte therein.
Devices suitable for detecting analytes in samples are known for many years in various applications. For instance, microbiological test methods for the determination of antibacterial compounds, particularly residues of antibiotics such as cephalosporin, penicillin, tetracycline and derivatives thereof and chemotherapeutics such as sulfadiazines and similar compounds (sulfa's), in fluids such as milk, meat juice, serum and urine have been described in CA 2056581, DE 3613794, EP 0005891, EP 0285792, EP 0611001, GB A 1467439 and U.S. Pat. No. 4,946,777. These descriptions all deal with ready to use tests that make use of a test microorganism and will give a result by the change indicated by an indicator molecule, for instance a change of color of a pH- and/or redox-indicator. The test systems described above usually include a test medium, such as agar in which test components are present. Such test components may be a test microorganism, indicator molecules and/or nutrients, but also other components are conceivable to the person skilled in the art. In most examples of test systems known today, the test medium is contained within a container such as a tube or a well in a microtiter plate. The sample to be analyzed then needs to be brought into said container onto the test medium. Usually, said containers are sealed in order to prevent contamination and in some cases auxiliary containers with required components such as nutrients are supplied from which said required components are to be transferred to the container with the test medium.
The problem with the test systems currently distributed on the market and/or described in literature is that they require multiple operations to be performed by the user. Since any operation is subject to the occurrence of errors, a high number of operations gives rise to a high number of possible errors and thus to a relatively unreliable and inconvenient test system. For example, a given test system may require operations such as opening an ampoule, addition of nutrients, attachment of a pipette to a syringe, withdrawal of a sample with the pipette/syringe, and application of the sample to the test medium. In most cases many or all these operations need to be performed under aseptic conditions. Yet another problem associated with the test systems currently distributed on the market and/or described in literature is that unwanted cross-contamination can easily occur.
Reducing the risk for errors in test systems by simplification of the equipment is a problem that has been addressed before. For instance, EP 0271102 describes a sample collection device wherein a liquid-absorbing material is used to absorb the required amount of sample without having to use complicated equipment. However, said improved test systems and methods still require multiple devices and process steps, each of which introduce risks for errors. Thus, there is still a need for an improved test system that does not have these problems.
It is an object of the present invention to provide a device and an improved method for the determination of analytes in fluids. The device of the present invention allows for such determination with an ease of handling that is unprecedented. The present invention provides a device for detecting an analyte in a fluid comprising a compartment (0) having a closed end (1.1) and an open end (2.2), wherein a test medium (1.3) comprising a substance with which said analyte can be detected is present in said compartment (0) at said closed end (1.1) and wherein said open end (2.2) has a means (2.3) for withdrawing a predetermined volume of said fluid.
Furthermore, the present invention provides a method for determining the presence or absence of an analyte in a fluid comprising:
Also, the present invention provides the use of a device as described above for sampling and analyzing a fluid.
The terms and abbreviations given below are used throughout this disclosure and are defined as follows.
The term ‘CFU’ is an abbreviation of Colony Forming Units and refers to the number of microorganisms spores of microorganisms, partially germinated spores of microorganisms, vegetative cells or spores of vegetative cells capable of producing colonies of microorganisms.
The term ‘drip ring’ refers to a means attached to the exterior of a device and fully enclosing the device which is designed such that any fluid which is present on the outside of the device is contained within the drip ring when the device is turned upside down. When the device is cylindrical, the drip ring is also cylindrical. Likewise, when the device has any other form, the drip ring has the same form as the device.
The term ‘fluid’ refers to a substance (as a liquid) tending to flow or conform to the outline of its container.
The term ‘gelling agent’ refers to a compound that assists in changing a mixture into or taking on the form of a gel.
The term ‘indicator’ refers to a substance used to show (for example by change of color or fluorescence) the condition of a mixture such as a solution or a gel with respect to the presence of a particular material (for example an acid, a base, oxidizing or reducing agents). For instance, the term ‘indicator’ may refer to one or more compounds that are known as pH-indicators, but also to one or more compounds that are known as redox-indicators. Also, the term ‘indicator’ may refer to mixtures of two or more different types of indicators, such as a combination of a pH- and a redox-indicator. In general, when two or more indicators are used, these indicators are co-operating to increase the indicator effect of each of the indicators when taken alone.
The term ‘nutrient’ refers to one or more nutritive substances or ingredients that promote and/or are required for the growth of microorganisms as used in an embodiment of the present invention.
The term ‘sampling device’ refers to a device with the aid of which a sample of a fluid can be added to a test medium. Such a device may be a container, optionally with volume markings. Such a container may be a capillary, a syringe, a pipette or an automated pipetting system. Such a syringe or pipette may be designed in such a fashion that with only one mode of operation a predetermined volume can be withdrawn from the fluid to be analyzed. Alternatively, a sampling device is a device suitable for obtaining fluid samples from solid materials such as meat. In the context of the present invention, the container of the sampling device may be equipped with cutting edges that are suitable for removing solid samples that can subsequently be pressed in the sampling device until a fluid sample is obtained.
The term ‘spore’ refers to a primitive usually unicellular often environmentally resistant dormant or reproductive body produced by plants or microorganisms and capable of development into such a plant or microorganism.
The term ‘test medium’ refers to a solid composition, preferably in the form of a sol or a gel, for instance comprising a gelling agent. Suitable examples of gelling agents are agar, alginic acid and salts thereof, carrageenan, gelatin, hydroxypropylguar and derivatives thereof, locust bean gum (Carob gum), processed eucheuma seaweed and the like. However, the person skilled in the art will understand that other types of solid test media may be based on carrier materials such as ceramics, cotton, glass, metal particles, paper, polymers in any shape or form, silicates, sponges, wool and the like. Usually, a test medium contains one or more indicators, however, these compounds may also be added during the test method. The test medium comprises one or more types of test microorganisms as detecting agents. Optionally, the test medium may also contain nutrients, stabilizers, and substances that change the sensitivity to certain antimicrobial compounds in a positive or negative way, and/or viscosity-increasing agents. Examples of substances that change the sensitivity to certain antimicrobial compounds are antifolates like ormethoprim, tetroxoprim and trimethoprim that improve the sensitivity of the test organism towards sulfa compounds or salts of oxalic acid or hydrofluoric acid, which improve the sensitivity towards tetracycline. Examples of viscosity-increasing agents are ascorbyl methylsilanol pectinate, carbomer, carboxymethyl cellulose, cetearyl alcohol, cetyl alcohol, cetyl esters, cocamide DEA, emulsifying wax, glucose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose, lauramide DEA, linoleamide DEA, magnesium aluminum silicate, maltodextrins, PEG-8 distearate, polyacrylamide, polyvinyl alcohol, PVP/hexadecene copolymer, sodium chloride, sodium sulfate, soyamidopropyl betaine, xanthan gum and the like. Alternatively, the optional ingredients of the test medium mentioned above may also be added exogenously.
The term ‘threshold’ refers to the concentration value above which a given analyte is to be regarded as present and below which said analyte is to be regarded as absent. Generally, a threshold value is given for particular analytes in particular samples by local, regional or interregional authorities but it can also be pre-set for certain research purposes.
In a first aspect of the invention, there is provided a device, an example of which is given in
Said other open end (2.2) of said second compartment (2) is optionally sealed with a cap (5) such as a screw cap, a breakable cap, a filmhinge or any other suitable sealing means. Preferably, the diameter of said other open end (2.2) is such that evaporation of sample and/or medium components is kept to a minimum. In many devices known in the art said evaporation leads to loss in accuracy of the test system. Suitable diameters of open end (2.2) in this respect are from 0.01-2 cm, preferably from 0.05-1 cm, more preferably from 0.1-0.5 cm, most preferably from 0.2-0.3 cm. Although different shapes are conceivable and by no means unsuited for the present invention, the usual shape of the first and second compartments is cylindrical with diameters ranging from 0.2-10 cm, preferably from 0.4-5 cm, more preferably from 0.6-2 cm, most preferably from 0.9-1.4 cm and lengths ranging from 0.5-20 cm, preferably from 1-10 cm, more preferably from 2-8 cm, most preferably from 3-5 cm. Normally the ratio between length and diameter is from 0.1-100, preferably from 1-50, more preferably from 2-20, most preferably from 5-10.
Suitable materials of which the device of the present invention may be constructed are plastics such as polyacrylics, polycarbonates, polyethylene, polyvinyl chloride and the like. The devices of the first aspect of the present invention can all be easily made using standard protocols known to the person skilled in the art. Traditional and well-known processes such as injection molding and blow-molding can be used for the production of all devices of the present invention. Production of these devices can be performed with many types of materials using standard molding equipment, which can be adapted to the required dimensions. Introduction of the test medium into the devices of the present invention can be performed in several ways. One option, as outlined in
Preferably the fluid that is to be withdrawn is a fluid that may or may not contain one or more analytes that are to be detected. Examples of such fluids are beverages, blood, cream, eggs, fruit juices, honey, meat juice, milk, milk products, urine and the like. The volume of fluid that is to be withdrawn usually ranges from 0.01-10 ml, preferably from 0.05-5 ml, more preferably from 0.1-1 ml, most preferably from 0.2-0.5 ml. Examples of analytes that are to be detected are antibiotics, carbohydrates, hormones, metals, microorganisms, nucleic acids, peptides, salts, toxic components and the like.
In an embodiment of the first aspect of the invention, the device allows for the incorporation of a medium comprising a substance with which an analyte can be detected. A device of this type has the advantage that sample withdrawal and measurement can be carried out in one and the same device as the sample analysis. This reduces the amount of operations to be performed by the user, and consequently reduces the occurrence of errors and increases the reliability. Thus there is provided a device that is partially filled with a test medium such as carrier material and/or a test medium comprising a substance with which an analyte can be detected in a fluid. The person skilled in the art will appreciate that the device of the second aspect may be filled with a wide variety of media that are suitable for detecting various analytes. Such a test medium may contain an indicator or may be an indicator itself. Preferably, the test medium is solid which has the advantage that the test medium stays in place when the device is rotated.
In a second embodiment, the device may be partially filled with a liquid such as a solution comprising a substance with which an analyte can be detected in a fluid. In order to prevent loss of this liquid, the closed end of the first compartment comprises an insert that is constructed such that said liquid cannot leave the device when the device is turned in order to withdraw sample fluid.
The test medium may be any medium suitable for the detection of an analyte in a fluid. It may be a medium comprising or consisting of an indicator. For the detection of antibiotics in fluids, for instance, the test medium or the liquid may also comprise a test organism, nutrients for the test organism, a substance that provides a solid state and at least one indicator. Suitable nutrients in this respect are carbon-sources and nitrogen-sources of which many commercially available variants exist. Typical constituents are amino acids, monosaccharides, vitamins and the like. Also oligosaccharides may be present as nutrient. Preferably the oligosaccharide is partly soluble in aqueous solutions and preferably the oligosaccharide contains one or more glucose units. More preferably the oligosaccharide is a relatively short-chained oligosaccharide such as a disaccharide, a trisaccharide, a tetrasaccharide or a pentasaccharide. Most preferably the oligosaccharide is a disaccharide such as cellobiose, gentiobiose, lactose, maltose, sucrose or trehalose. Suitable trisaccharides are maltotriose, melezitose and raffinose.
The person skilled in the art is well aware of methods available for the preparation of suitable test media. An example is given in U.S. Pat. No. 4,946,777. For reasons of clarity, the method described herein, which is only one of many examples, is repeated below.
A culture of Bacillus stearothermophilus var. calidolactis L.M.D. 74.1 was inoculated on a medium consisting of:
which was sterilized for 20 minutes at 120° C.
After inoculation, the medium was incubated at 60° C. for at least 48 hours until a good sporulation was observed. The spores were then collected, washed with distilled water and stored at 4° C. The amount of viable spores was detected by testing on a medium consisting of:
which was sterilized for 20 minutes at 120° C.
After inoculation, the medium was incubated for 48 hours at 60° C. after which the colonies were counted. Distilled water was added to, or water was removed from, the spore suspension until the suspension contained about 108 viable germs per ml. One percent of the above-mentioned spore suspension containing 108 germs per ml was added to the following solution:
The medium was liquefied by heating and then was cooled at 60° C. Devices of the dimensions of the present invention were each filled with 0.5 ml of the thus obtained medium and the contents of the devices were allowed to solidify with the devices being held in an upright position. The devices were stored at a temperature of 4° C.
In U.S. Pat. No. 4,946,777, there is also a description for the preparation of so-called Nutrient Discs, the purpose of which is to be added to the test medium upon start of the test (as i.e. envisioned with the additional compartment (3) of
Solutions (a) and (b) were sterilized by passage through a Seitz filter and medium (c) was sterilized at 110° C. for 30 minutes and medium (c) remained a suspension. Five parts of solution (a), two parts of solution (b) and three parts of suspension (c) were mixed together, and 0.04 ml of the solution obtained was contacted with filter paper discs having a cross-sectional dimension of about 8 mm, and the discs were then dried.
The skilled artisan will appreciate that not only Bromocresol Purple, as mentioned above, but also indeed many indicators are suitable for the purpose of the first aspect of the present invention. Particularly useful are indicators that, upon changing from one state to the other, provide a visually detectable signal such as a change in color or fluorescence. The amount of indicator in the test medium is between 0.01 and 50 g.l−1 test medium, preferably between 0.1 and 10 g.l−1, more preferably between 0.5 and 5 g.l−1, most preferably between 1 and 3 g.l−1. Such indicators may be selected from handbooks such as ‘H.J. Conn's Biological Stains’, R. D. Lillie ed., Baltimore, 1969. Preferred indicators are pH-indicators and/or redox indicators. Examples of suitable indicators are Acid Blue 120, Acid Orange 51, Acid Yellow 38, Alizarin acid, Alizarin Blue, Azure A, Azure B, Basic Blue 3, Brilliant Black, Brilliant Cresyl Blue, Brilliant Crocein MOO, Brilliant Yellow, Bromocresol Green, Bromocresol Purple, Bromophenol Blue, Bromophenol Red, Bromothymol Blue, Chlorocresol Green, Congo Red, m-Cresol Purple, Gallocyanine, Indigo Carmine, Janus Green B, Litmus, Methylene Blue, Nile Blue A, Nitrazol Yellow (also referred to as Nitrazine Yellow), o-Nitrophenol, p-Nitrophenol, 1-10 Phenanthroline, Phenolphthalein, Safranine O, Thionin, Thymol Blue, Toluidine Blue and Xylenol Blue.
Preferably, the substance providing for a solid state is a gelling agent and/or a carrier material. The amount of gelling agent in the test medium is between 1 and 200 g.l−1 test medium, preferably between 2 and 50 g.l−1, more preferably between 5 and 20 g.l−1, most preferably between 7 and 15 g.l−1. Preferred gelling agents are agar and gelatin.
The test organism preferably is a thermophilic test organism such as a Bacillus species, preferably Bacillus stearothermophilus, an Escherichia coli species, or a Streptococcus species, preferably Streptococcus thermophilus. These species may be introduced in the test as units capable of producing colonies, or Colony Forming Units (CFU's). Said CFU's may be spores, vegetative cells or a mixture of both. The concentration of said CFU's is expressed as Colony Forming Units per ml of test medium (CFU.ml−1) and is usually in the range of 1×105 to 1×1012 CFU.ml−1, preferably 1×106 to 1×1010 CFU.ml−1, more preferably 2×106 to 1×109 CFU.ml−1, most preferably 5×106 to 1×108 CFU.ml−1, or still more preferably 5×106 to 2×107 CFU.ml−1.
In a third embodiment of the first aspect of the invention, the first and second compartments are inseparable and form one compartment (0) having a closed end (1.1) and an open end (2.2).
In a fourth embodiment of the first aspect of the invention, said means for withdrawing a predetermined amount of fluid is an insert (2.3.2 in
In a fifth embodiment of the first aspect of the invention, said means for withdrawing a predetermined amount of fluid is an array of at least one volume marking on said second compartment (2.3.3 in
In a sixth embodiment, an example of which is given in
In a seventh embodiment, the first and second compartments are connected through a foldable member as exemplified in
In an eighth embodiment, the device of the first aspect of the invention may be constructed such that fluid samples can be obtained from solids. For instance, this may be achieved by supplying cutting edges at said other open end of said second compartment by means of which a sample can be cut from solid material such as meat or fish or fruit. The part of the device in which said sample will be present is made from flexible material so that fluid can be withdrawn from said sample by the application of pressure.
The devices of the first aspect of the present invention are illustrated in
In a second aspect of the invention, there is provided a method for determining the presence or absence of an analyte in a fluid comprising the steps of inserting the open end of the second compartment of the device of the first aspect of the invention into said fluid, withdrawing a fluid sample and contacting said fluid sample with a test medium comprising a substance with which an analyte can be detected.
In a first embodiment, for the detection of antibiotics, the test medium comprises CFU's of a microorganism and at least one indicator and optionally nutrients for the microorganism. Preferably, the test medium is a sol or gel comprising a gelling agent and/or a carrier material. Advantageously, the method also provides for conditions whereby there is minimal growth of a microorganism prior to the addition of fluid sample. Such conditions comprise an unfavorable temperature and/or an unfavorable pH-value and/or the absence of nutrients essential for growth, provided these conditions do not cause irreversible damage to all CFU's present. After addition of the fluid sample, growth of the microorganism is allowed to take place during a period sufficiently long for the microorganisms to grow in case no antibiotic is present. Growth is encouraged by adding nutrients, optionally before the contacting of said fluid sample, and/or raising the temperature, and/or providing for a pH-value at which the microorganism is able to grow. Alternatively, these conditions may be established prior to contact of the fluid sample with the test medium. Growth of the microorganism is detected by observing the presence or absence of a change of the indicator.
In a second embodiment of the second aspect of the present invention, the antibiotic is β-lactam antibiotic such as a cephalosporin or a penicillin derivative. Examples of such derivatives are amoxicillin, ampicillin, cefadroxil, cefradine, ceftiofur, cephalexin, penicillin G, penicillin V and ticarcillin, but of course many other similar β-lactam derivatives are known and applicable in the method of the present invention.
In a third embodiment of the second aspect of the invention, the test micro-organism is incubated for a predetermined period, preferably within a time span of 0.5 to 4 hours, more preferably between 0.75 to 3 hours, most preferably between 1.0 to 2.75 hours. Preferably the microorganism is incubated at a predetermined temperature, preferably the optimal growth temperature of the microorganism. When, for example, thermo stable microorganisms are used, said temperature is preferably between 40 and 70° C., more preferably between 50 and 65° C., most preferably between 60 and 64° C. Optionally said reaction could be carried out with the aid of a thermostatic device. Alternatively, the time required for growth of the microorganism is equal to the time that is required for a calibration sample with a known amount of analyte(s) to induce a change in the indicator. The latter change occurs when the concentration of analyte in the sample is below the threshold value.
In a fourth embodiment of the second aspect of the present invention, nutrients are added as a separate source, e.g. as a tablet, disc or a paper filter. Also other compounds such as the indicator(s), stabilizers and/or antifolates may be added as a separate source, or optionally incorporated in the nutrient medium.
The presence or absence of an antibiotic is determined by the presence or absence of a change of the indicator or indicators used. When, for example such a change is a color change, said color change may be observed visually. However in one embodiment of the invention said color change is determined using an arrangement that generates digital image data or an arrangement that generates analog image data and converts said analog image data into digital image data followed by interpretation of said digital image data by a computer processor. Such an arrangement, which may for instance be a sample-reading device such as a scanner coupled to a personal computer, is described in International Patent Application WO 03/033728, incorporated by reference, and briefly summarized below.
The arrangement can be used for detecting residues of antibiotics in milk. The commercially available Delvotest® and BR®-test are commonly used. Delvotest® comprises an agar matrix, CFU's of an acid forming microorganism, as well as a color indicator. With the arrangement mentioned above it is possible to automatically scan the bottom side of each of the samples in a test plate. The color and the brightness of the reflected light are registered in three variables, each describing one color component, for instance the so-called L*a*b* model. In the L*a*b* model, the color spectrum is divided in a two-dimensional matrix. The position of a color in this matrix is registered by means of the two variables “a” and “b”. The variable L indicates the intensity (for instance, from light blue to dark-blue). It is possible to make a criterion comprising the a-value, b-value and L-value to make a composite function as follows:
Z=w
L
.L+w
a
.a+w
b
.b
where wL, wa and wb are weighting factors for the L-value, a-value and b-value, respectively. The values of these weighting factors can be calculated by means of “discriminent analysis”, such that the group mean shows a maximum distance in relation to the spreading. By combining two or more of the color components in the L*a*b* model in a predetermined manner that depends on the type of residue and the sample, an accurate detection is possible. In practice, a certain value of Z at which a test should switch between positive and negative result (the threshold value) can be experimentally predetermined.
In a third aspect of the invention there is provided a kit for carrying out the method of the present invention. Such a kit comprises one or more devices according to the first aspect of the invention filled with test medium. The devices may be of any shape and size and of any material available, provided that observation of indicator changes is possible.
Optionally, said kit comprises a means for sealing of said devices filled with test medium during incubation and/or an insert with instructions for use and/or a means for setting the time needed for incubation.
In a first embodiment of the third aspect of the invention, said kit also comprises nutrients incorporated in the second compartment of the device of the first aspect of the invention. Preferably said nutrients are contained within a medium such as a tablet, disc or a paper filter. The amounts can be predetermined so as to avoid errors in dosing the required amounts. Also other compounds such as the indicator(s), stabilizers and/or antifolates may be added as a separate source, or optionally incorporated in the nutrient medium.
In a second embodiment of the third aspect, said kit comprises a thermostatic device, with the aid of which test samples can be kept at a pre-set temperature. The temperature may be one at which the microorganism shows sufficient growth. Preferably, said thermostatic device is designed in such a fashion that it can hold said containers filled with test medium. Optionally the thermostatic device is coupled to a means for setting the time needed for incubation such that heating and/or cooling is stopped after lapse of a pre-set period.
In a third embodiment of the third aspect of the invention, said kit comprises a data carrier loaded with a computer program suitable for instructing a computer to analyze digital data obtained from a sample-reading device. Said data carrier may be any carrier suitable for storing digital information such as a CD-ROM, a diskette, a DVD, a memory stick, a magnetic tape or the like. Advantageously, said data carrier loaded with a computer program also provides for easy access to the latest available computer programs suitable for use in the method of the present invention.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 04105186.3 | Oct 2004 | EP | regional |
| 0512674.8 | Apr 2005 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2005/055426 | 10/20/2005 | WO | 00 | 4/10/2007 |
