METHOD FOR DETECTING THE PRESENCE OR ABSENCE OF A TARGET MICRO-ORGANISM

专利摘要:
Method for detecting the presence or absence of a target microorganism and detection articles The present invention relates to a method of detecting a target microorganism. the method comprises providing a target microorganism culture device. the method comprises providing a culture device with a selective culture medium and a detection article comprising a first indicator system. the selective culture medium facilitates the growth of an indicator microorganism. when an indicator microorganism is detected in a sample in contact with the culture medium, the detection article is brought into contact with the culture medium to detect the target microorganism.
公开号:BR112013016757B1
申请号:R112013016757-2
申请日:2011-12-23
公开日:2020-02-18
发明作者:Takatoshi Moriyama；Akio Kitahara；Henry J. Lubrant；Patrick A. Mach
申请人:3M Innovative Properties Company；
IPC主号:

专利说明:

METHOD FOR DETECTING THE PRESENCE OR ABSENCE OF A TARGET MICROORGANISM ”
Reference to Related Deposit Applications [001] This application claims benefit under U.S. Provisional Patent Application No. 61 / 428,722, filed on December 30, 2010, which is hereby incorporated by reference in its entirety.
Background of the Invention [002] Testing samples of all foods, beverages and water for pathogenic microorganisms may not be practical due to cost and because pathogenic microorganisms are rarely found in, for example, properly processed foods. Therefore, tests to check for the presence of indicator microorganisms are routinely used to test food and water in order to determine the likelihood of contamination with human pathogens. The presence of one or more indicator microorganisms can be an indication of faecal contamination, for example and can indicate the potential presence of a pathogenic microorganism.
[003] Coliform bacteria (or just coliforms) represent an example of indicator microorganisms. The coliform group includes several genera (for example, Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella and Serratia) of rod-shaped gram-negative bacteria that are found in large numbers in the feces of warm-blooded animals and are characterized by their ability to ferment lactose for acid and gas by-products. Although most coliforms are only associated with opportunistic infections in humans, some coliform bacteria (for example, E. coli O157: H7 or another Shiga toxin that produces E. coli - STEC) are associated with a higher incidence of morbidity and mortality .
[004] Members of the micro Enterobacteriaceae family
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2/60 organisms represent another example of indicator microorganisms. In addition to coliform bacteria, this family also includes a large number of other gram-negative, rod-shaped bacteria. Like coliform bacteria, the presence of Enterobacteriaceae microorganisms in a food or water sample may indicate the presence of faecal contamination and thus the possible presence of human pathogens (for example, Salmonella enterica Enteritiditis, Salmonella enterica Typhimurium, species Shigella and Cronobacter species).
[005] There is a need for efficient methods to test the presence of pathogenic microorganisms in a sample.
Brief Description of the Invention [006] In general, the invention is concerned with a method for evaluating the microbiological content of a sample (for example, food or water sample, environmental sample). In particular, the method of the invention can detect the presence or absence of a target microorganism in a sample that contains an indicator organism that can indicate the presence of the target microorganism. The method of the invention includes culturing a sample in a culture device with a first indicator system to determine the presence of an indicator micro-organism and, if an indicator micro-organism is detected, by contacting the culture device with a detection article comprising a second indicator system for determining the presence or absence of a target microorganism.
[007] In one aspect, the present description presents a method of detecting the presence or absence of a target microorganism. The method may comprise providing a culture device including a culture medium comprising selected ingredients to facilitate the growth of a predetermined indicator microorganism, a detection article comprising a first indicator system and a sample. The first
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3/60 indicator system can be selected to detect a target microorganism. The method may additionally comprise inoculating the culture device with the sample, incubating the inoculated culture device long enough to allow the growth of indicator microorganisms, observing the culture device to indicate the presence of at least one microorganism indicator, contact the culture medium of the culture device incubated with the detection article and observe the culture device in contact with the article to detect a conversion of the first indicator system from a first state to a second state. In some embodiments, the conversion of the first indicator system from a first state to a second state, if present, may be indicative of the presence of at least one target microorganism. In some embodiments, the conversion of the first indicator system from a first state to a second state, if absent, is indicative of the presence of at least one target microorganism.
[008] In some embodiments, the method may additionally comprise providing a second indicator system and placing the second indicator system in fluid communication with the culture medium, in which observing the culture device for the presence of at least one indicator microorganism comprises detecting the presence or absence of a conversion of the second indicator system from a first state to a second state. In any of the above embodiments, providing a culture device may further comprise providing a culture device that includes the second indicator system. In any of the embodiments of the above method, providing the culture device may further comprise providing a culture device comprising a hydrogel or a dry gelling agent soluble in ice water.
[009] In any of the achievements of the method above, contacting the culture medium with the detection article is done only
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4/60 when the indication of the presence of at least one indicator microorganism is observed.
[010] In any of the above embodiments, providing a culture medium may comprise providing a selected culture medium to facilitate the growth of an Enterobacteriaceae microorganism, wherein providing a detection article may comprise providing a detection article to detect a microorganism of the genus Salmonella. In some embodiments, the first indicator system may comprise a reagent to detect the enzymatic activity of α-galactosidase or caprylate esterase.
[011] In any of the above embodiments, providing a culture medium may comprise providing a selected culture medium to facilitate the growth of an Enterobacteriaceae microorganism, wherein providing a detection article may comprise providing a detection article to detect a microorganism of the Shigella genus. In some embodiments, the first indicator system may comprise a reagent to detect β-glycosidase, β-fucosidase, N-acetyl-and-galactosaminidase or a combination of any two or more of the above-mentioned enzyme activities.
[012] In any of the above embodiments, providing a culture medium may comprise providing a selected culture medium to facilitate the growth of an Enterobacteriaceae microorganism, wherein providing a detection article may comprise providing a detection article to detect a microorganism of the Cronobacter genus. In some embodiments, the first indicator system may comprise a reagent to detect enzymatic activity α-glycosidase and / or βcelobiosidase.
[013] In any of the above embodiments, providing a culture medium may comprise providing a selected culture medium to facilitate the growth of an Enterobacteriaceae microorganism, in which
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5/60 providing a detection article may comprise providing a detection article to detect Escherichia coli. In any of the above embodiments, providing a culture medium comprises providing a selected culture medium to facilitate the growth of a coliform microorganism, wherein providing a detection article comprises providing a detection article to detect Escherichia coli. In some embodiments, the first indicator system may comprise a reagent to detect the enzymatic activity of β-glucuronidase.
[014] In some embodiments, providing a culture medium may comprise providing a selected culture medium to facilitate the growth of a microorganism of the genus Listeria, where providing a detection article may comprise providing a detection article to detect Listeria monocytogenes. In some embodiments, the first indicator system may comprise a reagent for detecting the enzymatic activity of α-mannopyranidasidase or phospholipase C specific for phosphatidyl inositol.
[015] In any of the above embodiments, observing the culture device or culture device in contact with the article may comprise observing the culture device visually. In any of the above embodiments, observing the culture device or culture device in contact with the article may comprise observing the culture device with the use of an automated reader. In any of the above embodiments, the method may further comprise enumerating a portion of the colony-forming units of the indicator microorganism. In any of the above embodiments, the method may further comprise enumerating a portion of the colony-forming units of the target microorganism.
[016] In any of the above achievements, contacting the culture medium with the detection article can comprise
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6/60 additionally come into contact with the culture medium at a predetermined temperature.
[017] In another aspect, the present description provides an article. The article may comprise a substrate with upper and lower main surfaces and a coating arranged on at least one of the main surfaces. The coating may comprise a first indicator system. The indicator system can be converted from a first state to a second state by the enzymatic activity of α-galactopyranoside or caprylate esterase. In some embodiments of the article, the first indicator system can be selected from the group consisting of 5-bromo-4-chloro-3indolyl-αD-galactopyranoside, 5-bromo-6-chloro-3-indolyl-caprylic acid and 5- bromo-4-chloro-3-indolyl-caprylic.
[018] In yet another aspect, the present description presents an article. The article may comprise a substrate with upper and lower main surfaces and a coating arranged on at least one of the main surfaces. The coating may comprise a first indicator system. The first indicator system can be converted from a first state to a second state by the enzymatic activity of β-glucuronidase. In some embodiments, the first indicator system can be selected from the group consisting of 5-bromo-4-chloro-3-indolyl-pD-glucuronic acid, pnitrophenyl-p-glucuronide, p-nitrophenyl-2,3,4- tri-O-acetyl-p-glucuronic methyl ester, phenolphthalein glycuronic acid, enolphthalein mono-P-glycuronic acid, naphthyl-AS-BI-pD-glucuronide and 4-methylumbelliferyl β-D-glucuronide, 8-hydroxy acid quinoline-beta-D -glycuronic, sodium salt, 2-naphthyl-beta-D-glucuronic acid, sodium salt, 4-nitrophenyl-beta-D-glucuronic acid, sodium salt, phenolphthalein-beta-D-glucuronic acid, sodium salt monohydrate, 5-acid -bromo-4-chloro-3-indoxylbeta-D-glucuronic, cyclohexylammonium salt, 3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt, 3-indoxyl-beta-D-glucuronic acid, sodium salt, 5
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7/60 bromo-6-chloro-3-indoxyl-beta-D-glucuronic, cyclohexylammonium salt, 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic, anhydrous sodium salt and 5-bromo acid -4chloro-3-indoxyl-beta-D-glucuronic, sodium salt trihydrate.
[019] In yet another aspect, the present description presents an article. The article may comprise a substrate with upper and lower main surfaces and a coating arranged on at least one of the main surfaces. The coating may comprise a first indicator system. The first indicator system can be converted from a first state to a second state by the enzymatic activity of α-mannopyranidasidase or phospholipase C specific to phosphatidyl inositol. In some embodiments, the first indicator system can be selected from the group consisting of 5-bromo-4-chloro-
3-indoxyl-myo-inositol-1-phosphate, 5-bromo-6-chloro-3-indoxyl-myo-inositol-1-phosphate, ammonium salt, 4-methylumbelliferyl-myo-inositol-1-phosphate, salt N-methyl-morpholine, 3indoxyl-aD-manopyranoside, 5-bromo-3-indoxyl-aD-manopyranoside, 4-chloro-3indoxyl-aD-manopyranoside, 5-iodo-3-indoxyl-aD-manopyranoside, 5-bromo-5 4chloro-3-indoxyl-aD-manopyranoside, chloro-3-indoxyl-aD-manopyranoside, 5-bromo-6-chloro-3-indoxyl-aD-manopyranoside, 6-bromo-3-indoxyl-a-Dmanopyranoside, 6- chloro-3-indoxyl-aD-manopyranoside, 6-fluoro-3-indoxyl-a-Dmanopyranoside, 4,6-dichloro-3-indoxyl-aD-manopyranoside, 6,7-dichloro-3-indoxyl-D-manopyranoside, 4,6,7-trichloro-3-indoxyl-aD-manopyranoside, 5-bromo-4chloro-N-methyl - aD-manopyranoside, 3-indoxyl-aD-manopyranoside and N-methyl-3indoxyl-aD-manopyranoside, 6 -bromo-2-naphthyl-aD-mannopyranoside, 4-methylumbelliferyl-aD-mannopyranoside and 4-nitrophenyl-aD-mannopyranoid.
[020] In any of the above embodiments, the coating can be arranged on the upper and lower main surfaces. In any of the above embodiments, the article may additionally comprise an adhesive layer. In some embodiments, at least a portion of the first indicator system can be arranged on or in the adhesive layer. In any
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8/60 one of the above embodiments, the first indicator system can be arranged on both main surfaces. In any of the above embodiments, the substrate can be selected from the group consisting of a polymeric film, paper, a nonwoven, a filtering membrane and derivatives of any of the above. In any of the above embodiments, the coating may additionally comprise a binder.
[021] The words preferential and preferably refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other achievements may also be preferred under the same or other circumstances. In addition, the recitation of one or more preferred realizations does not imply the disuse of other realizations and is not intended to exclude other realizations from the scope of the invention.
[022] The terms comprise and variations thereof do not have a limiting meaning, these terms appearing in the description and in the claims.
[023] For use in the present invention, one, one, o, a, at least one, at least one, one or more and one or more are used interchangeably. Thus, for example, a microorganism can be interpreted as one or more average microorganisms.
[024] The term and / or means one or all of the elements mentioned or a combination of any two or more of the elements listed.
[025] Culture device, as used here, refers to an article adapted to accommodate a nutrient medium that facilitates the growth of a microorganism. Optionally, the culture device may comprise a cover or cover to minimize exposure of the medium to external contamination and / or reduce the loss of moisture from the culture medium during incubation and / or storage. Some non-limiting examples of culture devices
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9/60 include flasks, beakers, tubes, Petri dishes, multi-well plates, PETRIFILM plates, COMPACT DRY media sheets, SANITA-KUN sheets and the like.
[026] Indicator system, as used herein, refers to one or more of any of the following and any combination of one or more of the following: a chromogenic enzyme substrate, a fluorogenic enzyme substrate, a redox indicator (for example , triphenyl tetrazolium chloride, methylene blue), a metabolizable nutrient, pH indicator. Metabolizable nutrient refers to any molecule that can be used by a predetermined indicator organism and / or a predetermined target microorganism to produce biomass and / or energy. The use of the metabolizable nutrient by the microorganisms results directly or indirectly in a pH or other ionic exchange detectable in an aqueous medium that is in fluid contact with the microorganism. A differentiating indicator system is an indicator system that can be used to distinguish two microorganisms not identical to the base of their respective reactivities with a component (s) of the indicator system (s).
[027] Indicating microorganism, as used herein, refers to a microorganism that belongs to a group of microorganisms that is known to be found in an environment (for example, a liquid or solid matrix) in which a target microorganism is also found. According to the present description, the indicator microorganisms and their corresponding target microorganisms are capable of being grown in the same culture medium. In addition, target microorganisms can react with the same indicator system that detects the corresponding indicator microorganisms. Indicator microorganisms may include groups of relatively large and diverse microorganisms (eg aerobic bacteria, yeast, filamentous fungi), groups of relatively large and relatively less diverse microorganisms (eg, a phylogenetically related group of micro -organisms like the family
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10/60
Enterobacteriaceae, for example or a physiologically related group of microorganisms such as coliform bacteria, for example) and even smaller and / or relatively less diverse groups of microorganisms (for example, a genus like Listeria, a species like Escherichia coli) .
[028] Target microorganism, as used herein, refers to a predetermined microorganism that can be found in one or more of the same environments as the indicator microorganisms are found. The target microorganism can be distinguished from one or more of a group of indicator microorganisms on the basis of its reactivity, or lack thereof, with a differentiating indicator system. In some embodiments, the target microorganism may belong to the group of indicator microorganisms (for example, an exemplary target microorganism, Escherichia coli, is a member of the Enterobacteriaceae family, which is a group of indicator microorganisms known in the art) . In certain embodiments, the target microorganism may refer to one or more strains of a specific species, one or more species of a specific genus, or more than one species of each two or more genera or one or more strains or species of a group non-taxonomic (ie, physiologically related).
[029] For use in the present invention, recitations of numeric ranges with extremes include all numbers contained in this range (for example, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4 , 5, etc.).
[030] The foregoing summary of the present invention is not intended to describe each of the embodiments presented or all implementations of the present invention. The following description more particularly exemplifies the illustrative achievements. In several places, during application, guidance is provided through lists of examples, in which examples can be used in various ways. In each instance, the list recited serves only with
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11/60 a representative group and should not be interpreted as an exclusive list.
[031] Additional details of these and other achievements are demonstrated in the attached drawings and in the description below. Other characteristics, objectives and advantages will become apparent from the description and drawings and from the claims.
Brief Description of the Figures [032] Figure 1 is a block diagram of an embodiment of a method for detecting a target microorganism according to the present description.
[033] Figure 2 is a top view of an embodiment of a culture device with colonies of indicator microorganisms arranged on it.
[034] Figure 3 is a perspective view of an embodiment of a detection article in accordance with the present description.
[035] Figure 4 is a top view of the culture device in Figure 3 after placing a detection article on it.
Detailed Description of the Invention [036] The invention is concerned with a method for evaluating the microbiological content of a sample. In particular, the method includes sequential detection processes that are advantageously conducted in a single culture device. The detection processes are distinct, but related in the sense that the first detection process identifies a group of indicator microorganisms and the second detection process identifies a target microorganism in the group. Advantageously, the result of the first identification process can be used to decide whether the use of the second detection process is indicated. Consequently, in some embodiments, when an indicator microorganism is not detected by the first identification process, an operator can avoid time,
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12/60 materials, labor and expenses for the second identification process.
[037] Figure 1 is a block diagram showing an embodiment of a method for detecting a target microorganism according to the present description. Methods of the present description are aimed at testing a sample for the presence or absence of a target microorganism. The method includes step 152 of providing a sample to be tested, a detection article and a culture device that includes a culture medium. Optionally, a second indicator system can be provided. The method further comprises step 154 of placing the second indicator system, if present, in fluid communication with the culture medium. The method further comprises step 156 of inoculating the culture device with the sample, step 158 of incubating the inoculated culture device and step 160 of observing the culture device for indicating an indicator microorganism. If an indicator microorganism is observed in step 160, the method further comprises the optional step 162 of contacting the culture medium with the detection article and step 164 of observing the first indicator system. Each of the steps in the method is described in more detail below. An advantage of the method is that the detection article may need to be applied only to the culture medium if an indication of an indicator microorganism is detected. Thus, in samples where no indication of an indicator microorganism is detected, it can be inferred that a target microorganism is not present and the detection article does not have to be used.
[038] Providing a sample to be tested may comprise providing a sample that is suspected to contain a target microorganism. The sample can be any sample that can include a target microorganism as defined here. Some sample non-limiting examples
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Suitable 13/60 include environmental samples (for example, surface pads / sponges, dirt, sediment, fomites), food (for example, raw materials, in-process samples and finished product samples), drinks, clinical / veterinary samples (for example , blood, serum, plasma, urine, sputum, tissue, mucus, feces, wound exudate, pus, cerebrospinal fluid) and water (eg surface water, drinking water, process water).
[039] In some embodiments, the presence or absence of a target microorganism can be analyzed in a test sample that is derived from a variety of food, drink or food - or environmental beverage processing sources. Non-limiting examples of food sources include raw or processed meat, raw or processed fruits or vegetables, non-fluid dairy products (for example, cheese, butter and ice cream), nuts, spices, ingredients and syrups. Non-limiting examples of drinking sources include drinking water, fruit or vegetable juices, milk and fermented drinks. Pasteurized food or drinks can also be suitable sources. Non-limiting examples of environmental food or beverage processing samples include food handling surface samples (for example, conveyor belts, sheets, cutting surfaces, mixing equipment surfaces, filters, storage containers), environment samples ( eg walls, floors, drains, ventilation equipment) and cleaning equipment (eg hoses, cleaning tools).
[040] In some embodiments, the presence or absence of a target microorganism can be analyzed in a sample that is derived from a variety of human or animal sources, such as a physiological fluid, for example, blood, saliva, lens fluid eye, synovial fluid, cerebrospinal fluid, pus, sweat, exudate, urine, mucus, breast milk or the like. In addition, the test sample can be derived from a location
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14/60 of the body, for example, injury, skin, nostrils, scalp, nails, etc.
[041] Samples of particular interest from human or animal sources include samples that contain mucus, such as nasal samples (eg from anterior nostrils, nasopharyngeal cavities, nasal cavities, anterior nasal vestibule, etc.), as well as samples from the external ear , middle ear, mouth, rectum, vagina or other similar tissue. Examples of specific mucosal tissues include buccal, gingival, nasal, ocular, tracheal, bronchial, gastrointestinal, rectal, urethral, ureteral, vaginal, cervical and uterine mucous membranes.
[042] In addition to physiological fluids, other test samples may include other liquids as well as solids (s) dissolved in a liquid medium. The samples of interest may include process flows, water, soil, plants or other vegetation, air, surfaces (for example, contaminated surfaces) and the like. Samples can also include cultured cells. Samples may also include samples on or in a device comprising cells, spores or enzymes (for example, a biological indicator device).
[043] Suitable samples of methods of the present description may include certain solid samples. Solid samples can be disintegrated (for example, by mixing, sonication, homogenization) and can be suspended in a liquid (for example, water, buffer, broth). In some embodiments, a sample collection device (for example, a pad, a sponge) that contains the sample material can be used in the method. Alternatively, the sample material can be eluted (e.g., rinsed, scraped, expressed) from the sample collection device prior to using the sample material in the method. In some embodiments, liquid or solid samples can be diluted in a liquid (for example, water, buffer, broth).
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15/60 [044] The sample can comprise an indicator microorganism, as described here. The indicator micro-organism can be indicative of contamination (for example, faecal contamination), infection (for example, infection with a pathogenic micro-organism) or an indicator of general sanitization (for example, any aerobic micro-organism). The indicator microorganism may also be a target microorganism.
[045] Microorganisms of particular interest, which may be of interest as an indicator organism or a target microorganism, include prokaryotic and eukaryotic organisms, particularly gram-positive bacteria, gram-negative bacteria, fungi, mycoplasma and yeast. Particularly relevant organisms include members of the Enterobacteriaceae family or the Micrococcaceae family or the genera Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Enterococcus spp., Salmonella spp., Legionella spp., Shigella spp. Yersinia spp., Enterobacter spp., Escherichia spp., Bacillus spp., Listeria spp., Vibrio spp., Corynebacteria spp. as well as herpes viruses, Aspergillus spp., Fusarium spp., and Candida spp. Particularly virulent organisms include methicillin-resistant Staphylococcus aureus (including resistant strains such as Staphylococcus aureus (MRSA)), S. epidermidis, Streptococcus pneumoniae, S. agalactiae, S. pyogenes, Enterococcus faecalis, Enterococcus (VRE) resistant to vancomycin, Staocylus Vancomycin-resistant VRSA), vancomycin-resistant Staphylococcus aureus (VISA), Bacillus anthracis, Pseudomonas aeruginosa, Escherichia coli, Aspergillus niger, A. fumigatus, A. clavatus, Fusarium solani, F. oxysporum, F. chlamydosporum, Lister monocytogenes, Listeria ivanovii, Vibrio cholera, V. parahemolyticus, Salmonella cholerasuis, S. typhi, S. typhimurium, Candida albicans, C. glabrata, C. krusei, Cronobacter sakazaki, E. coli O157 and multiple gram-negative rods (MDR) drug resistant.
[046] Gram-positive and gram-negative bacteria are of particular
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16/60 interest. Of particular interest are Gram-positive bacteria, such as Listeria monocytogeness. In addition, antibiotic-resistant microbes including MRSA, VRSA, VISA, VRE and MDR are of particular interest.
[047] With reference again to step 152 of figure 1, in addition to providing a sample to be tested, the method also includes providing a culture device, a first indicator system and an article with a second indicator system coated on it. The culture device is used in a broad sense and includes a variety of articles adapted to accommodate a nutrient medium that facilitates the growth of a microorganism. The selection of a specific nutrient medium to facilitate the growth of any specific indicator microorganism according to the method is within the scope of knowledge of a person who has basic knowledge in the technique.
[048] In some embodiments, the nutrient medium may comprise one or more selective inhibitors. Selective inhibitors, as used herein, refer to chemical compounds that are added to the nutrient medium to partially or completely inhibit the growth of certain susceptible microorganisms or groups of microorganisms, thus selectively favoring the growth of other microorganisms or groups of microorganisms. Selective inhibitors are known in the art and include, for example, bile salts, inorganic salts (for example, NaCl, LiCl, MgCl2) and antibiotics (fluoroquinolones, β-lactam, aminoglycosides). In certain preferred embodiments of the present description, the nutrient medium may include selective inhibitors at concentrations that are minimally selective (that is, at concentrations that are below the usual concentrations used in selective growth media). Advantageously, the use of a minimally selective nutrient medium can allow the recovery and detection of more target microorganisms using the method of the present description. Without sticking to the theory, this may be possible because the lowest concentration of inhibitors
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Selective 17/60 allows the growth of injured target microorganisms, subjected to stress and / or relatively susceptible in the selective medium, thus enabling their detection with the second indicator system.
[049] The first indicator system is used to indicate the presence or absence of a target microorganism. The second optional indicator system is used to indicate the presence or absence of an indicator microorganism. The first and / or second indicator system may comprise a chromogenic enzyme substrate, a fluorogenic enzyme substrate, a redox indicator, a metabolizable nutrient, a pH indicator or any combination of two or more of the above. In some embodiments, a combination of a pH indicator and a certain metabolizable nutrient can be provided from the method by providing one of the components in the culture medium of the culture device and providing the other component in the detection article.
[050] The choice of the first indicator system may depend on the target micro-organism and / or the culture medium used in the culture device and such choices are guided by the present disclosure, as will be recognized by a person with basic knowledge in the technique. In some embodiments of the method, a first highly differential indicator system (i.e., an indicator system that reacts with relatively few microorganisms, including the target microorganism) can be used in conjunction with a minimally selective culture medium. In these embodiments, the minimally selective culture medium can allow the recovery and growth of target microorganisms injured and / or subjected to stress, thus enabling the detection of such target organisms that can be inhibited by a highly selective culture medium.
[051] In some embodiments of the method, a relatively less differential first indicator system (ie, an indicator system that reacts with relatively many microorganisms, including the target microorganism) can be used in conjunction with a rather large culture medium. selective. That
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The 18/60 approach can be used with highly complex samples (for example, samples that require highly selective conditions, such as floor drain samples that tend to have highly diversified microbial content or samples of relatively non-selective pre-enrichment broth cultures). An example of this approach is the use of Demi-Fraser / UVM, a Fraser broth enrichment system with Modified Oxford agar detection of Listeria microorganisms.
[052] In either embodiment, the first or second indicator system may comprise an oxidation-reduction indicator (also called a redox indicator) suitable for biological oxidation-reduction reactions. The redox indicator dyes can be pH dependent or pH independent. Some non-limiting examples of redox indicator dye include 2,2'-bipyridine (Ru complex), Nitrophenanthroline (Fe complex), N-phenylanthranilic acid, 1,10-phenanthroline (Fe complex), N-ethoxycrisoidine, 2,2'-bipyridine (Fe complex), 5,6dimethylphenanthroline (Fe complex), o-dianisidine, sodium diphenylamine sulfonate, diphenylbenzidine, diphenylamine, Viologen, 2,6-dibromophenolindophenol sodium, Sodium 2,6-dichlorophenol -indophenol, o-Cresol of sodiumindophenol, thionine (syn. Lauth's violet), methylene blue, sulfonic indigotetraacid, sulfonic indigotriacid, sulfonic indigodiac acid, sulfonic indigomonoacid, phenosafranin, safranin T and neutral red.
[053] In either embodiment, the first and / or second indicator system may comprise a chromogenic enzyme substrate. Eligible chromogenic enzyme substrates include bromochloro-indolyl derivatives, nitrophenyl derivatives and phenophthalein derivatives, for example.
[054] Useful 5-bromo-4-chloro-3-indolyl derivatives include 5-bromo-6-chloro-3-indolyl acetate, 5-bromo-4-chloro-3-indolyl acetate, 5-bromo- 4-chloro-3-indoxyl-pD-galactopyranoside, 5-bromo-4-chloro-3-indolyl-
1,3-diacetate, 5-bromo-4-chloro-3-indolyl-p-D-fucopyranoside, 5-bromo-4
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19/60 chloro-3-indolyl-pD-glycopyranoside, 5-bromo-4-chloro-3-indolyl-p-Dglycuronic acid, 5-bromo-4-chloro-3-indolyl phosphate and 5-bromo- 4-chloro3-indolyl.
[055] Useful nitrophenyl derivatives include pnitrophenol and o-nitrophenol derivatives. Particularly useful P-nitrophenols include diethylp-nitrophenyl phosphate; di-p-nitrophenyl phosphate; p-nitrophenyl-2-acetamido-2-deoxy-3-O-pgalactopyranosyl-p-glycopyranoside; p-nitrophenyl-2-acetamido-2-deoxy-glycopyrananoside; p-nitrophenylacetate, p-nitrophenyl-N-acetyl-p-D-glucosaminide, pnitrophenyl-p-D-N, N'-diacetyl chitobiose; p-nitrophenyl-a-glycopyranoside, p-nitrophenylα-maltoside; p-nitrophenyl-p-maltoside; p-nitrophenyl-a-mannopyranoside; pnitrophenyl-p-mannopyranoside; p-nitrophenyl myristate; p-nitrophenyl palmitate; p-nitrophenyl phosphate; bis (p-nitrophenyl) phosphate; tris (p-nitrophenyl) phosphate; p-nitrophenylp-glycopyranoside; p-nitrophenyl-p-glucuronide; α-p-nitrophenyl glycerin; p-nitrophenylα-ramnopyranoside; p-nitrophenyl stearate; p-nitrophenyl sulfate; pnitrophenyl-2,3,4-tri-O-acetyl-p-glucuronic acid methyl ester; and p-nitrophenyl valerate.
[056] Particularly useful O-nitrophenols include onitrophenyl acetate, o-nitrophenyl-p-glycoside and o-nitrophenyl-p-D-glycopyranoside. Other particularly useful nitrophenyl derivatives include nitrophenyl-p-fucopyranoside, nitrophenyl-a-galactopyranoside, nitrophenyl butyrate, nitrophenyl caprate, nitrophenyl caprate, nitrophenyl caprylate, nitrophenyl laurate and nitrophenyl propionate.
[057] Useful indoxyl derivatives include indoxyl acetate; indoxyl-p-D-glycoside; 3-indoxyl sulfate; 3-indoxyl phosphate.
[058] Useful phenolphthalein derivatives include: phenolphthalein dibutyrate; phenolphthalein diphosphate; phenolphthalein disulfate; glycuronic phenolphthalein acid; phenolphthalein mono-P-glycosiduronic acid; phenolphthalein mono-P-glucuronic acid; and phenolphthalein monophosphate.
[059] All the chromogenic substrates described above
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20/60 will react directly with a suitable enzyme to produce a chromophore.
[060] Additional enzyme substrates containing derivatives of Inaftila, 2-naphthyl and Naftil-AS-BI are usefully used if the modified derived enzyme product is further reacted with a chromogenic reagent, such as diazotized dyes, for example, 1-diazo -4-benzoylamino2,5, diethoxybenzene, (commercially available as Fast Blue BB Salt from Sigma Chemical), 1-diazo-4-benzoylamino-2,5-diethoxybenzene, p-diazo-2,5dietoxi- N-benzoylalanine, chloride chloro-2-methylbenzene diazonic and oaminoazotoluene diazonic salt, to produce a chromophore.
[061] Particularly useful 1-naphthyl derivatives include 1-naphthyl-N-acetyl-P-D-glycosaminide.
[062] Particularly useful 2-naphthyl derivatives include 2-naphthyl phosphate; 2-naphthyl-butyrate; 2-naphthyl-caprylate; 2-naphthyl-myristate; L-leucyl-2-naphthylamide; L-valyl-2-naphthylamide; L-cystyl-2-naphthylamide; N-benzoyl-DL-arginine2-naphthylamide; N-glutaryl-phenylalanine 2-naphthylamine; 2-naphthyl phosphate; 6-bromo-2-naphthyl-a-D-galactopyranoside; 2-naphthyl-P D-galactopyranoside; 2-naphthyl-2-Dglycopyranoside; 6-bromo-2-naphthol-P-D-glycopyranoside; 6-bromo-2-naphthyl-2-Dmanopyranoside; and 2-naphthyl-a-L-fucopyranoside.
[063] Particularly useful naphthyl-AS-BI derivatives include naphthyl-AS-BI-phosphate and naphthyl-AS-BI-P-D-glucuronide.
[064] When the enzyme whose activity to be detected is alpha-Dglycosidase, a suitable chromogenic enzyme substrate, for example, is pnitrophenyl-a-glycopyranoside. When the enzyme activity to be detected is alpha-L-arabinofuranosidase, a suitable chromogenic enzyme substrate, for example, is p-nitrophenyl-a-L-arabinofuranoside. When the enzyme activity to be detected is beta-D-glycosidase, a suitable chromogenic enzyme substrate, for example, is p-nitrophenyl-P-D-glycopyranoside.
[065] In any of the achievements, the first and / or second
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The indicator system may comprise a fluorogenic enzyme substrate. Eligible fluorogenic enzyme substrates include derivatives of 4-methyl umbeliferone 7-starch-4-methylcoumarine, fluorescein, rhodamine and fluorescamine, for example.
[066] Suitable 4-methylumbelliferyl derivatives include, for example: 4-methylumbelliferyl-2-acetamido-4, 6-O-benzylidene-2-deoxy-and-D-glycopyranoside; 4-methylumbelliferyl acetate; 4-methylumbelliferyl-N-acetyl-βD-galactosaminide; 4-methylumbelliferyl-N-acetyl-a-D-glycosaminide; 4 methylumbelliferyl-N-acetyl-β-D-glycosaminide; 2 '- (4-methylumbelliferyl) -aD-N-acetyl neuraminic acid; 4-methylumbelliferyl a-L-arabinofuranoside; 4-methylumbeliferyl α-L-arabinoside; 4-methylumbelliferyl butyrate; 4methylumbeliferyl β-D-cellobioside; methylumbeliferyl β-D-N, N'diacetyl chitobioside; 4-methylumbelliferyl elaidate; 4-methylumbeliferyl-β-Dfucoside; 4-methylumbeliferyl-a-L-fucoside; 4-methylumbeliferyl-β-L-fucoside;
4-methylumbelliferyl-a-D-galactoside; 4-methylumbeliferyl-β-D-galactoside; 4 methylumbelliferyl-a-D-glycoside; 4-methylumbeliferyl-β-D-glycoside; 4-methylumbelliferyl-β-D-glucuronide; 4-methylumbelliferyl p-guanidinobenzoate; 4-methylumbelliferyl heptanoate; 4-methylumbelliferyl-a-D-mannopyranoside;
4-methylumbelliferyl-β-D-mannopyranoside; 4-methylumbelliferyl oleate; 4-methylumbelliferyl palmitate; 4-methylumbelliferyl phosphate; propionate
4-methylumbelliferyl; 4-methylumbelliferyl stearate; 4-methylumbelliferyl sulfate; 4-methylumbelliferyl-β-D-N, N ', N ”-triacetylquitotriose; 4methylumbeliferyl-2,3,5-tri-o-benzoyl-a-L-arabinofuranoside; 4-methylumbelliferyl-p-trimethylammonium chloride cinnamate; and 4-methylumbeliferyl-β-D-xyloside.
[067] Suitable derivatives of 7-starch-4-methylcoumarin include, for example: L-alanine-7-starch-4-methylcoumarin; L-proline 7-starch-4 methylcoumarin; L-tyrosine-7-starch-4-methylcoumarin; L-leucine-7-starch-4methylcoumarin; L-phenylalanine-7-starch-4-methylcoumarin; and 7-glutarylphenylalanine Petition 870190068023, of 7/18/2019, p. 28/143
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7-starch-4-methylcoumarin.
[068] Suitable peptide derivatives of 7-starch-4-methylcoumarin include, for example: N-t-BOC-Ile-Glu-Gly-Arg-7-starch-4-methylcoumarine; N-t-BOC-Leu-Ser-Thr-Arg 7-starch-4-methylcoumarin; N-CBZPhe-Arg 7-starch-4-methylcoumarin; Pro-Phe-Arg 7-starch-4-methylcoumarin; N-tBOC-Val-Pro-Arg 7-starch-4-methylcoumarin; and N-glutaryl-Gly-Arg 7-starch-4methylcoumarin.
[069] Suitable diacetylfluorescein derivatives include, for example, fluorescein diacetate, di- (P-D-galactopyranoside) fluorescein and fluorescein dilaurate.
[070] When the biological activity to be detected is alpha-Dglycosidase, chymotrypsin, suitable fluorogenic enzyme substrates are 4-methylumbelliferyl-alpha-D-glycoside, 7-glutarylphenylalanine-7-starch-4-methylcoumarine or 4-methylumbelliferyl heptanoate, respectively. When the biological activity to be detected is alpha-L-arabinofuranosidase, a suitable fluorogenic enzyme substrate is 4-methylumbelliferyl-alpha-L-arabinofuranoside. When the biological activity to be detected is beta-D-glycosidase, a suitable fluorogenic enzyme substrate is 4-methylumbelliferyl-beta-D-glycoside.
[071] In either embodiment of the method, the first and / or second indicator system may comprise a pH indicator dye used in conjunction with a metabolizable nutrient. The pH indicator dye can be selected according to criteria known in the art, such as pH range, compatibility with the indicator and / or target microorganisms and solubility. In some embodiments, a salt form of the pH indicator can be used, for example, to increase the solubility of the pH indicator in an aqueous mixture. Some non-limiting examples of suitable pH indicator dyes include, for example, thymol blue, tropeolin OO, methyl yellow, methyl orange, bromophenol blue, bromocresol green,
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23/60 methyl red, bromothymol blue, phenol red, neutral red, phenolphthalein, thymolphine, alizarin yellow, tropeolin O, nitramine, trinitobenzoic acid, thymol blue, bromophenol blue, tetrabromophenol blue, bromocresol green, purple bromocresol, methyl red, bromothymol blue, phenol red, Congo red and cresol red.
[072] The metabolizable nutrient can be any metabolizable nutrient known in the art to react with at least one microorganism (an indicator micro-organism and / or a target micro-organism) and to result in a change in pH (for example, a change in pH pH) in an aqueous medium that is in fluid contact with the microorganism. The nutrient can be selected from a variety of nutrient types known in the art. Non-limiting examples of nutrient types include carbohydrates (for example, sugars, polysaccharides and derivatives of these substances), fats (for example, fatty acids, fatty acid esters and derivatives of these substances), amines (for example, amino acids, peptides, oligopeptides, proteins, polyamines and derivatives of these substances), polyphosphates, purines, pyrimidines, nucleosides and nucleotides.
[073] In some embodiments, the second optional indicator system may be provided in a culture device (for example, in an agar culture medium in a petri dish; in a dehydrated culture medium in a device as a culture device) Petrifilm, for example). When supplied as a component of a hydrogel, as a component of an agar culture medium, the optional second indicator system is provided in fluid communication with the culture medium. When supplied as a dehydrated component as part of a rehydratable culture device, such as a Petrifilm plate, the optional second indicated system is placed in fluid communication with the culture medium, as indicated in step 154 of figure 1. This can be achieved , for example,
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24/60 by rehydrating the dehydrated culture medium in the device with a liquid (for example, water, a buffer, a diluent). In some embodiments, rehydratable culture media include gelling agents soluble in cold water such as agar, agarose, guar gum, xanthan gum, locust bean gum, polyvinyl alcohol and / or polyvinyl pyrrolidone, for example. Optionally, the liquid can contain a material sample, thus allowing the simultaneous performance of step 154 and the inoculation step 156 by the operator.
[074] Inoculating the culture device can be done by a variety of methods that are known in the art. Some non-limiting examples of suitable inoculation methods include pourplate techniques, surface inoculation techniques, streak-plate techniques, swab-plate techniques and surface contact techniques (for example, Rodac methods). The techniques for plating the filter membrane can be used in the present method, as long as the filter membrane does not interfere substantially in the reaction between the microorganisms and the indicator systems or interfere with the observation of the indicator systems.
[075] Methods of the present description include incubating an inoculated culture device for a period. One skilled in the relevant technique will recognize that the incubation temperature can be selected according to the microorganism to be detected. For example, if a yeast or mold is to be detected, the first incubation temperature can typically be at room temperature (about 23 ° C) at about 32 ° C. For example, if a bacterium is to be detected, the first incubation temperature can typically be room temperature at about 45 ° C.
[076] According to the present description, the incubation period can be about one hour. In some embodiments, the time for the first incubation is less than about 4 hours (for example, less than about 2 hours, less than about 3 hours, or less than about 4 hours. In
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25/60 some embodiments, the incubation period is less than about 8 hours (for example, less than about 5 hours, less than about 6 hours, less than about 7 hours or less than about 8 hours). In some embodiments, the first incubation is less than about 12 hours (for example, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In some embodiments, the incubation period is less than or equal to about 15 hours (for example, less than about 13 hours, less than about 14 hours or less than about 15 hours. In some embodiments, the incubation period is up to 48 hours (for example, less than about 24 hours, less than about 36 hours or less than about 48 hours.
[077] After the incubation period, the culture device is observed to indicate the presence of an indicator microorganism. In some embodiments, the culture device is observed visually. In some embodiments, observing the culture device may comprise using an imaging device to observe the culture device. Imaging devices for scanning and, optionally, analyzing a culture device are known in the art and include, for example, PETRIFILM (PPR) plate reader, available from 3M Company (St. Paul, MN, USA) , the PETRISCAN colony counter available from Spiral Biotech (Norwood, MA) and the PROTOCOL and ACOLYTE plate readers available from Synbiosis (Cambridge, UK).
[078] In embodiments using the optional second indicator system, an indication of the presence of an indicator microorganism can be observed by detecting a conversion of the second indicator system from a first state to a second state. For example, in some embodiments, the second indicator system may include a chromogenic reagent (for example, triphenyl tetrazolium chloride or 5-bromo-4-chloro-3-indolyl-p-Dglycopyranoside) that exists in a first colorless state until it is converted
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26/60 (for example, by indicator microorganisms) for a second colored state. In other embodiments, the second indicator system may include a fluorogenic reagent (eg, 4-methylumbelliferyl β-D-galactoside or fluorescein diacetate) that exists in a first colorless state until it is converted (for example, by indicator microorganisms) for a second fluorescent state. In other embodiments, the second indicator system may include a reagent (for example, a pH indicator) that can be converted from a first colored or fluorescent state to a second colored or fluorescent state by a product of microbial activity (for example, the fermentation of a carbohydrate for final acid products).
[079] In some embodiments, detecting a conversion of the second indicator system from a first state to a second state may comprise observing a colony of microorganisms to detect the conversion. In some embodiments, detecting a conversion of the second indicator system from a first state to a second state may comprise observing the culture medium to detect the conversion.
[080] Figure 2 illustrates various aspects of detection of an indicator microorganism according to the present description. Figure 2 shows a top view of an embodiment of an inoculated culture device that includes indicator microorganisms reacting with a second indicator system. The culture device has a culture medium 282 that includes a second indicator system that includes a fermentable carbohydrate (eg, glucose) and a reagent (eg, chlorophenol red) in a first state (the first state is illustrated as gray dark in figure 2; chlorophenol red exists in a first state of violet color in a culture medium that has a pH of about 6.8 or more). Also shown in figure 2 are colonies of indicator microorganisms 284 that have fermented glucose to become diffusible acid end products, which
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27/60 react with the reagent to convert it to a second state (the second state is illustrated as a light gray halo 286 around colonies 284 in figure 2; chlorophenol red converts to a second yellow state in a culture medium having a pH less than about 5.2). In some embodiments, colonies may be stained by the reagent and may have the same color (or similar color) as the culture medium immediately around them. In other embodiments (not shown), the second indicator system may simply comprise an enzyme substrate or redox reagent that changes from a first state to a second state and thus directly changes the color or fluorescent properties of the colonies themselves, rather than the medium. of culture around the colonies.
[081] In embodiments that do not use the optional second indicator system (for example, a culture device containing a selective agar culture medium, not shown), an indication of the presence of an indicator microorganism can be detected by observing the presence of a bacterial colony (for example, by its typical size, shape, color and / or mold, as known in the art) on or inside the culture medium.
[082] Table 1 shows several non-limiting examples of selective culture media that can be used to support the growth of Enterobacteriaceae microorganisms, an exemplifying group of indicator microorganisms that indicate the presence of target Salmonella microorganisms. Also shown in Table 1 are exemplary indicator systems (ie, second indicator systems, according to the present description) that can be used to detect Enterobacteriaceae microorganisms. The first exemplifying indicator systems that can be used to indicate the presence of Salmonella target microorganisms include enzymatic substrates of caprylic acid esterase (presented in PCT Patent Application Publication No. WO2007023185, which is hereby incorporated in full, to
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28/60 reference title); 2-deoxy-D-ribose plus neutral red (shown in US Patent No. 7,150,977; which is incorporated herein in its entirety for reference);
5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside (shown in US Patent No. 6,368,817; which is incorporated herein in its entirety for reference); propanodiol plus neutral red (presented in US patent No. 5,194,374; which is incorporated herein in full, for reference); and a combination of melibiosis, mannitol, sorbitol and neutral red (shown in US patent No. 5,786,167; which is incorporated herein in full, for reference).
Table 1
Culture medium Indicator System Red Violet Bile Agar Glucose + neutral red Enterobacteriaceae PETRIFILM Counting Plate Glucose + chlorophenol red
[083] An exemplary embodiment of a method of detecting a target Salmonella microorganism according to the present description includes using a culture device (for example, a Petri agar plate or a PETRIFILM culture device) with a culture that includes the components mentioned in Table 2. When preparing the agar culture medium, all components with the exception of sodium novobiocin and cefsulodine are mixed together in any order and thereafter boiled and cooled to form a medium basal. Sodium novobiocin and cefsulodine are added to the cooled basal medium, boiled before the plating medium is complete. In addition to the culture medium, a detection article is prepared with a first indicator system comprising 5-bromo-4-chloro-3-indolyl-α-galactopyranoside, as described in Example 1. The culture medium is inoculated with a sample and incubated over a period of time (for example, about 18 to 48 hours). After incubation, the culture device is observed to indicate the presence of at least one indicator microorganism (for example, a colony and / or generally circular red zone that indicates the fermentation of 2-deoxy-D-ribose to a
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29/60 final acid product that changes the neutral red pH indicator from colorless to red). The zone may have a visible colony inside or near the center. If an indicator microorganism is detected, the detection article is brought into contact with the culture medium and the culture medium in contact with the article can be incubated for a period of time (for example, about 1 to 5 hours ) and observed to indicate the presence of a target microorganism (that is, a colony of blue color usually in the center of a red zone that was formed from the reaction of the microorganisms with the first indicator system. Optionally, a second Additional indicator system comprising a β-galactopyranoside chromogenic substrate that produces a color other than 5-bromo-4-chloro-3-indolyl-αD-galactopyranoside) can be added to the culture medium or the detection article. The second additional indicator system can be used to further identify non-target indicator microorganisms, such as certain strains of E. coli and Citrobacter freundii. As an alternative to neutral red, other pH indicators known in the art can be used in the second indicator system to detect acid end products from 2-deoxy-D-ribose fermentation. Examples of other suitable pH indicators are presented in the present invention. In some embodiments, other second indicator systems (for example, triphenyl tetrazolium chloride) can be used in place or in addition to the 2-deoxy-Dribose / neutral red indicator system to indicate the presence of indicator microorganisms. In some embodiments, other second indicators can be incorporated into the detection article instead of the culture medium. First alternative indicator systems that could be included in the detection article may include a-galactosidase chromogenic or fluorogenic enzyme substrates, caprylate esterase chromogenic or fluorogenic enzyme substrates or indicator systems to detect phenylalanine deaminase or propanediol fermentation.
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Table 2.
Component Grams / liter Yeast extract 3.00 Proteose Peptone 10.00 Lemco Lab Powder 1.00 Sodium chloride 5.00 L-phenylalanine 3.50 Ferric Ammonium Citrate 0.50 Bile Salts # 3 0.40 Bile Salts 0.20 2-deoxy-D-ribose 12.0 Neutral Red 0.03 Agar * 15.97 Deionized water * 950 ml / liter Novobiocin sodium 0.02 Cefsulodine 0.006
[084] Some non-limiting examples of selective culture media that can be used to support the growth of Enterobacteriaceae and / or coliforms microorganisms, exemplifying groups of indicator microorganisms that indicate the presence of E. coli O157 target microorganisms: H7 include half violet bile-glucose red, half violet bile-lactose, from E. coli PETRIFILM, PETRIFILM coliform counting plates, Enterobacteriaceae PETRIFILM counting plates and PETRIFILM coliform rapid counting plates. First exemplifying indicator systems that can be used to indicate the presence of E. coli O157: H7 target microorganisms include a combination of salicin, adonitol, inositol and sorbitol with phenol red (presented in US patent No. 6,617,149; which is incorporated here in full, as a reference).
[085] Some non-limiting examples of selective culture media that can be used to support the growth of Listeria microorganisms, a group of exemplifying indicator microorganisms that indicate the presence of Listeria monocytogenes target microorganisms include modified Oxfords medium and R&F. First exemplifying indicator systems that can be used to indicate the
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31/60 presence of target microorganisms Listeria monocytogenes include phosphatidyl inositol phospholipase C enzyme substrates (shown in US Patent Application Publication No. 20070259393, which is incorporated herein in its entirety for reference) and alpha mannosidase enzyme substrates (shown US Patent No. 7,351,548; which is incorporated herein in its entirety for reference). Non-limiting examples of early indicator systems that can be used to detect Listeria monocytogenes target microorganisms include 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate, 5bromo-6-chloro-3-indoxyl myo -inositol-1-phosphate, ammonium salt, 4-methylumbelliferyl myo-inositol-1-phosphate, N-methyl-morpholine salt, 3-indoxyl-aD-mannopyranoside,
5-bromo-3-indoxyl-aD-manopyranoside, 4-chloro-3-indoxyl-aD-manopyranoside, 5-iodo-3-indoxyl-aD-manopyranoside, 5-bromo-4-chloro-3indoxyl-aD-manopyranoside, chloro-3-indoxyl-aD-manopyranoside, 5-bromo-6ch1oro-3-indoxyl-aD-manopyranoside, 6-bromo-3-indoxyl-a-Dmanopyranoside, 6-chloro-3-indoxyl-aD-manopyranoid, 6- fluoro-3-indoxyl-aD-manopyranoside, 4,6-dichloro-3-indoxyl-aD-manopyranoside, 6,7-dichloro-3indoxyl-aD-manopyranoside, 4,6,7-trichloro-3-indoxyl-aD- mannopyranoside, 5bromo-4-chloro-N-methyl-aD-mannopyranoside, 3-indoxyl-aD-mannopyranose and N-methyl-3-indoxyl-aD-mannopyranose, 6-bromo-2-naphthyl-aD-mannopyanoside, 4- methylumbeliferyl-aD-manopyranoside and 4-nitrophenyl-aD-manopyranoside.
[086] In a preferred embodiment, a thin film culture device for detecting the presence of a Listeria microorganism may comprise a dehydrated broth medium that includes proteose peptone, tryptone, casamino acids, Lab Lemco Powder / meat extract bovine, glucose, yeast extract, hydrogen dipotassium phosphate, lithium chloride, bovine serum albumin, guar gum, nalidixic acid (sodium salt), chromogenic enzyme substrates to detect enzymatic activity of glucosidase (eg, Salmon-pD-glycoside and / or Magenta-pD
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32/60 glycoside) and ceftazidime pentrahydrate. A liquid sample is used to inoculate the medium and a Listeria microorganism, if present, grows and forms colonies that are red in color due to the hydrolysis of the chromogenic enzymatic substrates disp-glycosidase. If a Listeria microorganism is detected in the culture device, the medium inoculated in the culture device can optionally be contacted with a detection article comprising a dehydrated coating that includes a chromogenic indicator to detect the enzymatic activity of phosphatidyl inositol phospholipase C (for example example, 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate), guar gum and a phosphate buffer. If one or more of the colonies includes Listeria monocytogenes microorganisms, it will hydrolyze the enzymatic substrate of phosphatidyl inositol phospholipase C, causing the colonies to turn blue. Advantageously, the detection article only needs to be used if a Listeria microorganism is first detected by observing the evidence of enzyme activity of β-glycosidase associated with a colony.
[087] Some non-limiting examples of selective culture media that can be used to support the growth of microorganisms Enterobacteriaceae, an exemplary indicator microorganism that indicates the presence of target microorganisms Cronobacter sakazaki, include violet bile glucose medium red and enterobacteriaceae PETRIFILM counting plates. First exemplary indicator systems that can be used to indicate the presence of Cronobacter sakazaki target microorganisms include chromogenic alpha glycosidase and enzymatic beta cellobiosidase substrates (shown in US patent application publication No. 2006/0257967 which is incorporated herein in full, by way of of reference).
[088] In some embodiments, a group of indicator microorganisms that is known to be found in a similar environment (eg
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33/60 (faecal material) as the target micro-organism cannot actually include the target micro-organism. An example of this condition is illustrated by the relationship of coliform indicator microorganisms and target Salmonella microorganisms. Coliform bacteria are found in fecal material and are characterized by the ability to ferment lactose to final acid products. Certain target microorganisms Salmonella (for example, Salmonella enterica Typhimurium) are found in fecal material and do not ferment lactose into final acid products. Accordingly, a method according to the present description may include providing a first indicator system (for example, 5-bromo-4-chloro-3-indolyl-aD-galactopyranoside) to detect Salmonella target microorganisms and a second indicator system (eg lactose and neutral red) to detect coliform indicator microorganisms. In this embodiment, colonies that react with the first indicator system would not react with the second indicator system.
[089] Observation of an indication of the presence of an indicator organism denotes the possible presence of a target microorganism (for example, a potential pathogen) in a sample. Thus, it is a characteristic of the method that, when an indication of the presence of indicator microorganisms is observed, a detection article can be used to confirm the presence or absence of the target microorganism in a sample. Otherwise, in some embodiments where an indication of the presence of indicator microorganisms is not observed in the culture device, the use of a detection article in the method can be avoided because the absence of an indicator microorganism in a sample denotes the absence of a micro - target organism in a sample.
[090] Methods of the present description comprise providing a detection article comprising a first indicator system. Figure 3 shows a perspective view of an article of detection
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300 in accordance with the present description. The detection article 300 comprises a solid support 310 with upper and lower main surfaces, and a coating 320 disposed thereon. The liner 320 comprises a first indicator system, as described here. The first indicator system can be selected to detect a specific target microorganism. Optionally, an adhesive layer (not shown) can be disposed on the solid support 310 between the solid support 310 and the coating 320. The optional adhesive layer must comprise an adhesive that does not substantially interfere with the reaction of the first indicator system with the target microorganism and / or interferes with the observation of the second indicator system. A non-limiting example of a suitable adhesive includes the isooctyl acrylate / acrylamide pressure sensitive adhesive (94: 6) described in US Patent No. 4,565,783; which is hereby incorporated in full, as a reference.
[091] In some embodiments, the liner 320 is substantially free of water (that is, it has no water content greater than or less than the water content of the dehydrated coating when left in equilibrium with the environment). In some embodiments, coating 320 may be applied to substrate 310 or the optional adhesive layer (not shown) in the form of a dry coating such as a powder or the coating may be applied to the substrate in the form of a liquid which is subsequently dried on the substrate , both processes as described, for example, in US Patent No. 4,565,783. In some embodiments, article 300 may be constructed, coated and dried as described for the manufacture of the composite in US Patent No. 6,022,682, which is incorporated herein in its entirety for reference.
[092] In some embodiments, coating 320 comprises a binder. There are many binders that would be suitable for use in detection article 300. Some non-limiting examples of binders
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Suitable 35/60 include agarose, guar gum, xanthan gum, locust bean gum and other natural gums. A preferred binder is guar gum.
[093] The coating 320 may also preferably include other constituents, for example, a suitable buffering agent to control the pH at a point where a reaction between the first indicator system and the target micro-organism is facilitated. The choice of specific buffering agent (for example, a phosphate buffer) and pH (for example, 7.2) may depend on the first indicator system and / or the target microorganism, as will be recognized by a person having basic knowledge of technical.
[094] The solid support 310 must be selected from materials that can be coated and does not substantially obscure the observation of the reagents or products of the first indicator system and, optionally, the second indicator system. The solid support 310 may be a polymer film, such as a polyester film. The solid support 310 can be derived from a laminar material (e.g., polymeric film, paper, nonwoven), left to cut or perforate the detection articles 300 of the desired size or shape after coating and drying. In some embodiments, the solid support 310 may be transparent or translucent, or it may become transparent or translucent when placed in contact with a hydrogel. The material used for the solid support 310 can be selected to give any degree of rigidity or flexibility to the detection article 300. In addition, the detection article 300 can be prepared in any shape (for example, circular, ovoid, square, rectangular) , etc.) or thickness, depending on what is desired for a specific application.
[095] The solid support 310 is preferably transparent or at least translucent, to allow visualization of color changes that develop when the article is placed in fluid contact with a culture device. The solid 310 support also provides article stability and protects
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36/60 against damage.
[096] The solid support 310 can be selected so that it is detachable from the coating 320, leaving the coating free for use in testing (for example, in fluid contact with a culture medium) without the solid support 310. For example, where a polyester film is used as the solid support, the solid support 310 can be peeled off the coating 320 when the coating 320 becomes hydrated after contact with the culture device.
[097] In use, the detection article is placed in fluid contact with the culture medium of the culture device. Preferably, the detection article and the culture medium will be brought into contact in the culture device (for example, by placing the detection article in contact with the culture medium in the culture device). In certain preferred embodiments, the detection article is sized so that it has substantially the same shape and surface area so that the article comes in contact with an entire surface of the culture medium in the culture device. In some embodiments, the detection article can be pre-hydrated (for example, with sterile water or buffer), although it is contemplated that the humidity in the culture medium is sufficient to hydrate the detection article, thus allowing the first indicator system to come into fluid contact with microorganisms present in the culture device.
[098] Without adhering to the theory, it is believed that fluid contact between the culture medium and the detection article allows the diffusion of the first indicator system, a metabolite produced by the microorganism and / or the microorganism (or component thereof, such as an enzyme, for example) so that a component of the first indicator system can react with the metabolite, the target microorganism or a component of the target microorganism and be converted from a first state to a second state. Thus, in some realizations, the conversion of the first
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37/60 indicator system from a first state to a second state, if present, is indicative of the presence of at least one target microorganism.
[099] In some embodiments, the conversion of the first indicator system from a first state to a second state, if absent, is indicative of the presence of at least one target microorganism. In these method embodiments, when an indicator microorganism changes the first indicator system from a first state to a second state (for example, the colony and / or the culture medium immediately around the colony becomes colored or fluorescent), then the indicator micro-organism is not the target micro-organism. On the contrary, in these achievements, if the indicator microorganism does not change the first indicator system from a first state to a second state, it is an indication that the indicator microorganism is the target microorganism. A specific non-limiting example can illustrate this point. Certain microorganisms of the Shigella genus are pathogenic to humans and thus Shigella is an example of a target microorganism. Shigella is a genus in the Enterobacteriaceae family. Since Enterobacteriaceae microorganisms (including the Shigella species) can be found in fecal material, Enterobacteriaceae microorganisms are an example of Shigella indicator microorganisms. In one embodiment of the method of the present description, a sample of material can be inoculated into the selective culture media of Enterobacteriaceae (for example, bile red violet agar or Enterobacteriaceae PETRIFILM counting plates). If indicator microorganisms are detected in the culture medium, a detection article comprising, for example, an enzymatic chromogenic substrate to detect β-glucosidase, β-fucosidase, and / or N-acetyl-βgalactosaminidase can be contacted with the medium of culture. The conversion of any of these enzyme substrates from a non-colored to a colored state is an indication that the colony is not the microorganism
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38/60 target because the Shigella target microorganisms do not comprise any of the corresponding enzyme activities of these enzyme substrates.
[0100] After placing the detection article in fluid contact with the culture medium, the culture medium in contact with the article is observed to detect a conversion of the first indicator system from a first state to a second state (for example, from a colorless state to a colored state, from a non-fluorescent state to a fluorescent state, from a first color to a second color). Such a conversion can be detected, for example, by none of the detection means discussed in the present invention to detect the conversion of the second indicator system from a first state to a second state. Optionally, in some embodiments, the culture medium in contact with the article can be incubated at a predetermined temperature for a period of time to facilitate the conversion of the first indicator system from a first state to a second state. The incubation temperature can be, for example, room temperature (about 23 ° C), up to about 28 ° C, up to about 30 ° C, up to about 35 ° C, up to about 37 ° C, up to about 42 ° C or up to about 45 ° C. In some embodiments, the culture medium in contact with the article can be incubated at the same temperature as the incubation temperature used to cultivate and detect the indicator microorganisms. In some embodiments, the culture medium in contact with the article can be incubated at a different temperature (for example, lower or higher) than the incubation temperature used to cultivate and detect the indicator microorganisms.
[0101] In some embodiments, the culture medium in contact with the article can be incubated for at least about 15 minutes. In some embodiments, the culture medium in contact with the article can be incubated for at least about 30 minutes. In some embodiments, the culture medium in contact with the article can be incubated for at least
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39/60 about 60 minutes. In some embodiments, the culture medium in contact with the article can be incubated for at least about 90 minutes. In some embodiments, the culture medium in contact with the article can be incubated for at least about 2 hours. In some embodiments, the culture medium in contact with the article can be incubated for at least about 4 hours. In some embodiments, the culture medium in contact with the article can be incubated for up to about 60 minutes. In some embodiments, the culture medium in contact with the article can be incubated for up to about 90 minutes. In some embodiments, the culture medium in contact with the article can be incubated for up to about 2 hours. In some embodiments, the culture medium in contact with the article can be incubated for up to about 3 hours. In some embodiments, the culture medium in contact with the article can be incubated for up to about 4 hours. In some embodiments, the culture medium in contact with the article can be incubated for up to 5 hours. In some embodiments, the culture medium in contact with the article can be incubated for up to 8 hours. In some embodiments, the culture medium in contact with the article can be incubated for up to 12 hours. In some embodiments, the culture medium in contact with the article can be incubated for up to 24 hours. In some embodiments, the culture medium in contact with the article can be incubated for between about 15 minutes and about 8 hours, inclusive. In some embodiments, the culture medium in contact with the article can be incubated for about 30 minutes. minutes and about 8 hours, inclusive.In some embodiments, the culture medium in contact with the article may be incubated for between about 30 minutes and about 5 hours, inclusive.In some embodiments, the culture medium in contact with the article The article can be incubated for between 1 hour and 5 hours, inclusive. In some embodiments, the culture medium in contact with the article can be incubated for between about 2 hours and about 5 hours, inclusive. In some achievements,
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40/60 the culture medium in contact with the article can be incubated for between about 2 hours and about 4 hours, inclusive.
[0102] Figure 4 shows a top view of the culture device of figure 3 after placing a detection article in contact with the culture medium (growth) in the device. The culture device has a culture medium 482 on and / or within which colonies grow. As shown in figure 2, colonies of indicator microorganisms 484 are present and changed the first indicator system from a first state (dark gray) to a second state (light gray), forming a distinct halo 486 around the colonies of micro- indicator organisms 484. Also shown in figure 4 are colonies of target microorganisms 485, which appear to be larger than colonies of indicator microorganisms 484. This may be, for example, due to the conversion of the second indicator system of a first state to a second state. In some embodiments, the second indicator system may be an enzymatic precipitable chromogenic substrate (for example, 5-bromo-4-chloro-3-indolyl-pD-glucuronide) which, when reacted with a target microorganism, can make the colony blue and make the colony appear slightly larger. Colonies of 485 target microorganisms also reacted with the first indicator system and have 486 halos around them.
[0103] Methods of the present description may optionally further comprise a step of enumerating a type of microorganism. Enumerating a type of microorganism comprises counting countless colonies (or colony-forming units) of the specific type of microorganism. The number of colony forming units listed can be used to estimate the number of microorganisms per gram (or per milliliter) in the original sample. The number of colony forming units listed can be compared with a descriptive report to determine whether the original sample is in
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41/60 compliance with a quality standard, for example. In some embodiments, all microorganisms of a specific type (for example, indicator microorganisms, target microorganisms) can be listed. In some embodiments, a portion (for example, up to a predetermined limit number) of microorganisms of a specific type can be enumerated.
[0104] In some embodiments, listing a type of microorganism comprises listing the number of indicator microorganisms. In some embodiments, enumerating a type of microorganism comprises enumerating the number of target microorganisms. In some embodiments, listing a type of microorganism comprises listing the number of indicator microorganisms and the number of target microorganisms. In some embodiments, enumerating a type of microorganism may include using an image-forming device to enumerate microorganisms.
Achievements [0105] Achievement 1 is a method of detecting the presence or absence of a target microorganism, comprising:
to provide:
a culture device including a culture medium comprising selected ingredients to facilitate the growth of a predetermined indicator microorganism;
a detection article comprising a first indicator system, wherein the first indicator system is selected to detect a target microorganism; and a sample;
inoculation of the culture device with the sample;
incubation of the inoculated culture device for time
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42/60 sufficient to allow the growth of indicator microorganisms;
observe the culture device to indicate the presence of at least one indicator microorganism;
contacting the culture medium of the culture device incubated with the detection article; and observing the culture device in contact with the article to detect a conversion of the first indicator system from a first state to a second state.
[0106] Realization 2 is a method according to realization 1 in which the conversion of the first indicator system from a first state to a second state, if present, is indicative of the presence of at least one target microorganism.
[0107] Realization 3 is the method according to realization 1, in which the conversion of the first indicator system from a first state to a second state, if absent, is indicative of the presence of at least one target microorganism.
[0108] Realization 4 is the method according to any of the previous realizations, characterized by the fact that it additionally includes providing a second indicator system and placing the second indicator system in fluid communication with the culture medium, in which to observe the culture for indicating the presence of at least one indicator microorganism comprises detecting a conversion of the second indicator system from a first state to a second state.
[0109] Embodiment 5 is the method according to any of the preceding embodiments, wherein providing the culture device further comprises providing a culture device comprising a hydrogel or a dry gelling agent soluble in ice water.
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43/60 [0110] Realization 6 is the method according to any of the previous realizations, in which contacting the culture medium with the detection article is done only when the indication of the presence of at least one micro- indicator organism is observed.
[0111] Embodiment 7 is the method according to any of the preceding embodiments, wherein providing a culture device further comprises providing a culture device that includes the second indicator system.
[0112] Realization 8 is the method of any of realizations 1 to 7, in which providing a culture medium comprises providing a selected culture medium to facilitate the growth of an Enterobacteriaceae microorganism, in which providing a detection article comprises providing a detection article to detect a microorganism of the genus Salmonella.
[0113] Embodiment 9 is the method of Embodiment 8, wherein the first indicator system comprises a reagent for detecting the enzymatic activity of α-galactosidase or caprylate esterase.
[0114] Embodiment 10 is the method of any of embodiments 1 to 7, in which providing a culture medium comprises providing a selected culture medium to facilitate the growth of an Enterobacteriaceae microorganism, in which providing a detection article comprises providing a detection article to detect a microorganism of the genus Shigella.
[0115] Embodiment 11 is the method of Embodiment 10, wherein the first indicator system comprises a reagent to detect β-glucosidase, β-fucosidase, N-acetyl-p-galactosaminidase, or a combination of any two or more of the activities previously mentioned enzyme enzymes.
[0116] Embodiment 12 is the method of any of embodiments 1 to 7, in which providing a culture medium comprises providing a culture medium selected to facilitate the growth of a microorganism
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Enterobacteriaceae, in which providing a detection article comprises providing a detection article to detect a microorganism of the genus Cronobacter.
[0117] Run 13 is the method of run 12, in which the first indicator system comprises a reagent to detect enzymatic activity of α-glycosidase and / or β-cellobiosidase.
[0118] Embodiment 14 is the method of any of embodiments 1 to 7, in which providing a culture medium comprises providing a selected culture medium to facilitate the growth of an Enterobacteriaceae microorganism, in which providing a detection article comprises providing a detection article to detect Escherichia coli.
[0119] Embodiment 15 is the method of Embodiment 14, wherein the first indicator system comprises a reagent for detecting the enzymatic activity of β-glucuronidase.
[0120] Embodiment 16 is the method of any of embodiments 1 to 7, in which providing a culture medium comprises providing a selected culture medium to facilitate the growth of a coliform microorganism, in which providing a detection article comprises providing a detection article to detect Escherichia coli.
[0121] Embodiment 17 is the method of Embodiment 16, wherein the first indicator system comprises a reagent for detecting the enzymatic activity of β-glucuronidase.
[0122] Realization 18 is the method of any of realizations 1 to 7, in which providing a culture medium comprises providing a selected culture medium to facilitate the growth of a microorganisms of the genus Listeria, in which providing a detection article comprises providing a detection article to detect Listeria monocytogenes.
[0123] Achievement 19 is the method of achievement 18, in which the
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45/60 The first indicator system comprises a reagent to detect the enzymatic activity of Da-mannopyranidasidase and / or phospholipase C specific to phosphatidyl inositol.
[0124] Embodiment 20 is the method of any of embodiments 1 to 19, in which observing the culture device or the culture device in contact with the article comprises observing the culture device visually.
[0125] Achievement 21 is the method of any of achievements 1 to 19, in which observing the culture device or the culture device in contact with the article comprises observing the culture device with the use of a formation device images.
[0126] Realization 22 is the method according to any of the previous realizations, characterized by the fact that it additionally comprises enumerating colony forming units of indicator microorganisms in the culture device.
[0127] Realization 23 is the method according to any of the previous realizations, characterized by the fact that it additionally comprises enumerating colony forming units of target microorganisms in the culture device.
[0128] Embodiment 24 is the method according to any of the previous embodiments, in which contacting the culture medium with the detection article further comprises contacting the culture medium with the detection article at a predetermined temperature .
[0129] Embodiment 25 is a detection article comprising a substrate with upper and lower main surfaces and a coating comprising a first indicator system arranged on at least one of the main surfaces, where the first indicator system is converted from a first state to a second state by activity
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46/60 enzymatic α-galactopyranoside or caprylate esterase.
[0130] Embodiment 26 is the article of Embodiment 25, in which the first indicator system includes an indicator selected from the group consisting of 5-bromo-4-chloro-3-indolyl-αD-galactopyranoside, 5bromo-6-chloro-acid 3-indolyl-caprylic, 5-bromo-4-chloro-3-indolyl-caprylic acid, and a combination of any two or more of the above indicators.
[0131] Embodiment 27 is a detection article comprising a substrate with upper and lower main surfaces and a first indicator system coated on at least one of the main surfaces, where the first indicator system is converted from a first state to a second state by the enzymatic activity of β-glucuronidase.
[0132] Realization 28 is the article of realization 27, in which the first indicator system includes an indicator selected from the group consisting of 5-bromo-4-chloro-3-indolyl-pD-glucuronic acid, p-nitrophenyl-βglycuronide, p-nitrophenyl-2,3,4-tri-O-acetyl-and-glucuronic acid methyl ester, phenolphthalein glycuronic acid, phenolphthalein mono-P-glycuronic acid, naphthyl-AS-BIβ-D-glucuronide, 4-methylumbelliferyl β- D-glucuronide, 8-hydroxy quinoline-beta-D-glucuronic acid, sodium salt, 2-naphthyl-beta-D-glucuronic acid, sodium salt, 4-nitrophenyl-beta-D-glucuronic acid, sodium salt, phenolphthaleinabeta- D-glucuronic acid, sodium salt monohydrate, 5-bromo-4-chloro-3indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt, 3-indoxyl-beta-Dglycuronic acid, cyclohexylammonium salt, 3-indoxyl- beta-D-glucuronic acid, sodium salt, 5-bromo-6-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt, 5-bromo-4-chloro-3-indoxyl-beta-D-gli acid curonic, anhydrous sodium salt and 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodium salt trihydrate and a combination of any two or more of the above indicators.
[0133] Realization 29 is a detection article comprising
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47/60 a substrate with upper and lower main surfaces and a first indicator system coated on at least one of the main surfaces, in which the first indicator system is converted from a first state to a second state by the enzymatic activity of α-mannopyranidasidase or phospholipase Phosphatidyl inositol specific C s.
[0134] Embodiment 30 is the article of Embodiment 29, in which the first indicator system includes an indicator selected from the group consisting of 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate, 5-bromine -6chloro-3-indoxyl myo-inositol-1-phosphate, ammonium salt, 4-methylumbelliferyl myoinositol-1-phosphate, N-methyl-morpholine salt, 3-indoxyl-α-mannopyanoside, 5bromo-3-indoxyl-aD -manopyranoside, 4-chloro-3-indoxyl-a-Dmanopyranoside, 5-iodo-3-indoxyl-aD-mannopyranoside, 5-bromo-4-chloro-3indoxyl-aD-mannopyanoside, chlorine-3-indoxyl-aD-mannopyanoside , 5-bromo-
6-chloro-3-indoxyl-aD-manopyranoside, 6-bromo-3-indoxyl-aD-manopyranoside, 6-chloro-3-indoxyl-aD-manopyranoside, 6-fluoro-3-indoxyl-aD-manopyranoside, 4, 6-dichloro-3-indoxyl-aD-manopyranoside, 6,7-dichloro-3indoxyl-aD-manopyranoside, 4,6,7-trichloro-3-indoxyl-aD-manopyranoside, 5bromo-4-chloro-N-methyl- -aD-mannopyranoside, 3-indoxyl-aD-mannopyranose and N-methyl-3-indoxyl-aD-mannopyranose, 6-bromo-2-naphthyl-a-Dmanopyranoside, 4-methylumbelliferyl-aD-mannopyanoside, 4-nitrophenyl-a -Dmanopyranoside and a combination of any two or more of the previously mentioned indicators.
[0135] Embodiment 31 is the article of any one of embodiments 25 to 30, characterized by the fact that it additionally comprises an adhesive layer, in which at least a portion of the first indicator system is disposed on or within the adhesive layer.
[0136] Realization 32 is the article of any of realizations 25 to 31, in which the first indicator system is covered in
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48/60 both main surfaces.
[0137] Embodiment 33 is the article of any of embodiments 25 to 32, in which the substrate is selected from the group consisting of a polymeric film, paper, a nonwoven, a filtering membrane and derivatives of any of the previously mentioned.
[0138] Embodiment 34 is the article of any one of embodiments 25 to 33, wherein the coating comprises a binder.
Examples
Materials
Monobasic Potassium Phosphate (KH2PO4) - Mallinkrodt Baker, Inc .; Phillipsburg, NJ, USA
Dibasic Potassium Phosphate (K2HPO4) - AMRESCO; Solon, OH
Guar gum - M150 guar gum MEYPROGAT, Meyhall Chemical AG
5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside - BIOSYNTH AG, Rietlistr, Switzerland
Polyester film - 0.074 mm (2.91 ml) transparent polyester film
BCIG - 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide acid Cyclohexyl ammonium salt - BIOSYNTH AG
Methyl Gluicuronide - 1-O-methyl-beta-D-glucuronic acid, Sodium Salt - BIOSYNTH AG
Example 1. Preparation of a Detection Article to detect a Salmonella target microorganism.
[0139] A coating composition was prepared by adding 13.6 g of monobasic potassium phosphate in 1000 ml of water treated with reverse osmosis in a 4 liter container and mixing with an air mixer for about 1.5 minutes followed by the addition of 3.4 g of
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49/60 dibasic potassium phosphate and mix for about 2 minutes. Then, 11 g of guar gum was slowly added and mixed for about 5 minutes. The container was covered and heated on a hot plate, under mixing, until the mixture reached 80 ° C. The container was removed from the hot plate and mixed at room temperature for about 15 minutes and then cooled until the mixture reached 40 ° C. An indicator suspension was prepared by adding 5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside to 10 ml of reverse osmosis water and vortexing the mixture to form a uniform suspension. After cooling the coating composition to about 40 ° C, the indicator suspension was mixed into the coating composition.
[0140] The mixture was then removed from the refrigerator and allowed to reach room temperature. The mixture was manually coated with a knife to a width of about 20.32 cm (8 inches) on a 25.4 cm (10 inch) by 137.2 cm (54 inch) polyester film to obtain a weight coating dryness of about 9.69 g / m ² (0.150 grams per 24 square inches, Disc 1A). The coating procedure was repeated to provide coated films having a dry coating weight of 11.95 g / m ² (0.185 g / 24 square inches, Disc 1B), 15.5 g / m ² (0.240 g / 24 square inches, Disc 1C) and 19.38 g / m ² (0.30 g / 24 square inches, Disc 1D). The coated sheets were dried in an oven set at 110 ° C (230 ° F) for 5 to 15 minutes until dry. The coated sheets were stored in plastic bags. The sheets were cut into 10.2 cm (4 inch) squares and the squares were kept in plastic bags until they were tested.
Example 2. Detect a Salmonella target microorganism with a Detection Article.
[0141] A colony of Salmonella enterica Agona ((FSD # 140)
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50/60 isolated from an agar streak plate was inoculated into 5 ml of soybean tryptcasein broth and incubated overnight at 35 ° C. The overnight culture was diluted in Butterfield's phosphate diluent to obtain a suspension that has approximately 100 colony forming units (CFU) per milliliter. Four bacterial culture devices (Enterobacteriaceae 3M ™ Petrifilm ™ counting plates; 3M Company; St. Paul, MN, USA) were each inoculated with 1 ml of the bacterial suspension according to the manufacturer's instructions and incubated at 35 ° C for 22 to 24 hours. The devices were inspected and the red colonies (approximately 1 mm in diameter) surrounded by yellow acidic zones (approximately 24 mm in diameter) The upper film of the culture plate was carefully lifted from the lower film to expose the culture medium on the plate. culture. Two square discs (Disc 1A) were inserted into the plate. The first disc was placed with its surface coated against the culture medium adhered to the lower film (that is, facing downwards). The second disc was placed on top of the first disc with the coated side of the second disc facing the opposite side of the bottom film (that is, facing upwards). The upper film was carefully lowered (using a rolling motion) to bring the culture medium adhered to the upper film in contact with the coated side of the second disc. Light finger pressure was applied to the outer surface of the closed plate's upper film to ensure contact between the surfaces of the culture device and the coated surfaces of the disks. The procedure was repeated with Discs 1B, 1C and 1D. The disk devices were incubated at 35 ° C and inspected every hour for five hours. Digital images were taken during each inspection. The images showed that, in about 3 hours, all colonies on each plate turned blue to bluish green due to the hydrolysis of 5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside by microorganisms in the colonies.
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Example 3. Preparation of a Detection Article to Detect an E. coli Target Microorganism.
[0142] A coating composition was prepared according to the procedure of Example 1. A suspension was prepared by dissolving 0.4 g of 1-O-methyl-pD-glucuronic acid (sodium salt) and 2.0 grams of 5-bromo-4-chloro-3-indoxyl-pD-glucuronic acid in 50 ml of water treated with reverse osmosis in a beaker with constant stirring at room temperature with a magnetic stir bar. The beaker containing the indicator suspension was placed in a refrigerator.
[0143] The coating composition was removed from the refrigerator when it cooled to approximately 40 ° C and was subsequently mixed with an air motor mixer to form a vortex. The indicator suspension was added to the coating composition and mixed for about 20 minutes. The resulting detection medium was then covered and cooled until use.
[0144] Discs 2A, 2B, 2C and 2D were prepared and stored according to the procedure described in Example 1 having a dry coating weight of 9.69 g / m ² (0.150 grams per 24 square inches, Disc 2A), 11.95 g / m ² (0.185 g / 24 square inches, Disc 2B), 15.5 g / m ² (0.240 g / 24 square inches, Disc 2C) and 19.38 g / m ² (0.30 g / 24 square inches, 2D Disc).
Example 4. Detect a Target E. coli Microorganism with a Detection Article.
[0145] An isolated colony of the E. coli strain (ATCC # 51813) was inoculated in 5 ml of soybean tryptcasein broth and incubated at 35 ° C for 20 hours to provide an overnight culture having a bacterial concentration of approximately 2 x 10 ⁹ cfu / ml. The culture was vortexed and 10 microliters of the culture was added to 99 ml of Butterfields phosphate buffer (Dilution 1). The suspension was shaken vigorously for about 20
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52/60 seconds and 5 microliters of that dilution was added to another 99 ml of Butterfields phosphate buffer (Dilution 2). This suspension was shaken vigorously for about 20 seconds. Sixteen bacterial culture devices (3M ™ Petrifilm ™ High Sensitivity Coliform Counting Plate; 3M Company; St. Paul, MN, USA) were each inoculated with 5 ml of Dilution 2 according to the manufacturer's instructions. Four culture devices were incubated at each of the 4 temperatures (32 ° C, 35 ° C, 37 ° C and 44.5 ° C, respectively) for 22 to 24 hours. The culture devices were removed from the incubators and a digital image of each plate was recorded with a digital camera. Each colony had a characteristic appearance (that is, a red colony center about 1 mm in diameter surrounded by a red zone about 2 to 3 mm in diameter) of a coliform colony on the High Sensitivity Coliform Count Plate .
[0146] Discs of each type (i.e. Disc 2A, Disc 2B, Disc 2C and Disc 2D) were placed as described in Example 2 in separate culture devices that were incubated at each temperature. The devices with the disks were then incubated at their respective temperatures for 5 hours. A digital image of each culture device was recorded after each hour of the 5-hour incubation period.
[0147] After the culture devices containing the disks were incubated, they were observed. Each of the red colonies on all plates turned blue after the culture device was brought into contact with the disk and incubated for 5 hours. Plates that were incubated at a higher temperature with the disks were observed to have blue colonies earlier than plates that were incubated at lower temperatures. Plates that received detection articles with higher coating weights were also observed to have blue colonies earlier than plates that received detection articles with lower coating weights.
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Example 5. Prophetic Example of the Preparation of a Culture Device for Cultivating Listeria Indicator Microorganisms.
[0148] A culture medium composition is prepared with the materials in Table 3. Protein Peptone, Tryptone, Casamino Acids and Yeast Extract can be obtained, for example, from Becton Dickinson (Sparks, MD). Lab Lemco powder / beef extract can be obtained, for example, from Oxoid (Hampshire, United Kingdom). Bovine serum albumin can be obtained, for example, from Serologicals Corporation (Norcross, GA). Beta-D-glycoside indicators can be obtained from BioSynth AG, for example. Ceftazadima can be obtained from Glaxo Wellcome, for example.
Table 3.
Component Material Grams 1 Proteose peptide 6.0 2 Triptona 19.2 3 Casamino acids 12.0 4 Lab Lemco Powder / Beef Extract 10.0 5 Glucose 5.0 6 Yeast extract 14.0 7 Potassium phosphate, dibasic 9.0 8 Lithium chloride 15.0 9 bovine serum albumin 6.0 10 Guar gum 10 11 Nalidixic acid, sodium salt 0.02 12 Salmon-beta-D glycoside 0.06 13 Magenta-beta-D glycoside 0.066 14 Ceftazidime pentahydrate 0.027
[0149] A first mixture is prepared by adding components 1 to 10 (shown in Table 3) with 1000 ml of reverse osmosis water, mix to obtain a uniform suspension, heat the mixture to 80 ° C and then cool to temperature environment. While the first mixture is cooling, a second mixture is prepared by mixing components 11 to 14 (shown in Table 1 in 10 milliliters of reverse osmosis water. The second mixture is vortexed to provide a uniform mixture, which is added to the first mixture and then mixed further to provide a uniform culture medium. The culture medium is then chilled
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54/60 for 18 to 24 h (2 to 8 ° C).
[0150] The refrigerated culture medium is left to warm naturally to room temperature and then applied the knife as a coating on a sheet of 0.074 mm (2.6 mil) thick transparent polyester film and dried at about 93 °. C (200 ° F) for 5 to 10 minutes. The knife span is adjusted to provide a dry coating weight of approximately 33.27 to 34.88 g / m ² (515 to 540 mg / 24 in. ² ). A 0.46 mm (18 mil) thick sheet of closed cell polystyrene foam is laminated to a pressure sensitive adhesive transfer tape. Circular openings of 5.1 cm (2 inches) in diameter are perforated from the foam sheet and the foam sheet is laminated to the coated side of the culture medium of the polyester film to form a structure similar to the lower portion of the culture device shown in Figure 1 of US Patent No. 4,565,783. The preparation of the culture device is completed by removing the top film from a 3M ™ Petrifilm ™ Staphylococci Express Counting Plate and affixing it to the Styrofoam sheet with double coated tape, thus producing a device that resembles the complete culture device shown in Figure 1 of US Patent No. 4,565,783.
Example 6. Prophetic Example of Preparing a Detection Article to Detect a Target Microorganism Listeria monocytogenes.
[0151] A coating composition is made by mixing guar gum, disodium phosphate and hydrogen potassium phosphate (shown in Table 4) in 1000 milliliters of reverse osmosis water to form a uniform dispersion as described in Example 1.
Table 4
Material grams 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate 0.60 Guar gum 10 Disodium phosphate, dibasic 8.0 Potassium phosphate monobasic 4.0
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55/60 [0152] The container with the coating composition is covered and the dispersion is mixed continuously while heated to a temperature of 80 ° C. It is then removed from the heat and mixed continuously at room temperature. An indicator suspension is prepared by adding 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate (which can be obtained from Biosynth AG, for example) to 10 ml of reverse osmosis water and vortexing to form a uniform suspension. After the coating composition has cooled to room temperature, the indicator suspension is mixed into the coating composition. The mixture is covered and refrigerated for 18 to 22 hours. The coating mixture is removed from the refrigerator and left to warm naturally to room temperature. A 0.074 mm (2.9 mil) polyester film is knife coated with the mixture according to the procedure described in Example 1 to obtain a film with a dry coating weight of about 5.17 to 6.14 g / m ² (80 to 95 mg / 24 in. ² ). The polyester film is rotated and knife coated on the other side, in the same way. The film, coated on both sides, is then die cut into circular discs having a diameter of about 5.1 cm (2 inches).
Example 7 - Prophetic Example of Detecting a Target Microorganism Listeria monocytogenes [0153] Enrichment cultures are prepared from food and environmental samples using standard sample collection procedures - and enrichment broth, (for example, as specified by the U.S. Department of Agriculture, US Food Safety Inspection Service or Bacteriological Analytical Manual). Eight 10-fold serial dilutions of the culture are prepared in Butterfield's diluent. One milliliter of each dilution is inoculated into individual culture devices described in Example 5. The devices are then incubated at 35 to 37 ° C for 18 to 30 hours. Devices are inspected for typical colony mold (eg, red colonies approximately 0.5 to 1.5 mm in diameter)
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56/60 indicative of Listeria (a group of indicator microorganisms that indicates the presence of Listeria monocytogenes). If typically colored colonies are seen on the culture device after incubation, the user can also insert a detection article to differentiate the colonies. The upper film of the culture device is gently pulled and a 5.1 cm (2 inch) detection article described in Example 6 is brought into contact with the culture medium. The upper film is closed again, placing it in contact with the detection article. The upper film can be pressed with light pressure of the fingers to contact the growth areas of the device with the detection article. The device is incubated for up to 5 hours at 35 to 37 ° C and inspected periodically (for example, hourly) for the presence of a blue or green-blue color in the colonies. Blue or greenish-blue colonies indicate the presence of L. monocytogenes.
Example 8 - Method of Detecting a Listeria MONOCYTOGENES Target Microorganism.
Preparation of Thin Film Culture Device - Substrate Coated with Broth:
[0154] All ingredients shown in Table 5, with the exception of guar gum, were mixed with 970 milliliters of deionized water in a stainless steel beaker. Under mixing, guar gum was added and the complete mixture was heated to 80 ° C. The heated complete mixture was stirred for about 15 minutes, covered and cooled in a refrigerator overnight. Before coating, twenty milliliters of Supplement A (bovine serum albumin, 3.2 g / 20 ml) and ten milliliters of Supplement B (ceftazidine pentahydrate, 40 mg / 10 ml) were mixed with the coating mixture. The coating mixture with supplements was applied to the knife as a coating on transparent polyester film (0.07 mm (2.91 mil) thick) using a 0.38 mm (15 mil) span. The coated polyester was dried for about 8
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57/60 minutes in an oven set at 210 ° C. The final coating weight of the coated dry mixture was 0.308 g / 154.8 cm ² (0.308 g / 24 in. ² ). Styrofoam spacers (0.51 mm (20 mil) thick), with 5.08 cm (2 inch) diameter openings, were adhered to the coated, dry film as described in Example 1 of US Patent No. 5,601. 998; which is hereby incorporated in full, as a reference. The substrate was cut into approximately 10.2 cm (4 ”) by 10.2 cm (4”) pieces, each piece having a spacer with a 5 cm opening that framed a circular area of the coated, dry broth composition.
Table 5. Composition of Coating Mix.
Component Material Grams 1 Peptone proteosis n ° 3 3.127 2 Type III casein peptide 9.6 3 Casamino acids 6.0 4 Meat peptide (pork) 5.0 5 Glucose 2.5 6 Yeast extract 7.0 7 Potassium phosphate, dibasic 4.5 8 Lithium chloride 9.0 10 Guar gum 12 11 Nalidixic acid, sodium salt 0.01
Preparation of Thin Film Culture Device - Substrate
Powder Coated:
[0155] Polyethylene coated paper (0.13 mm thick) was obtained from Schoeller Paper (Pulaski, NY, USA). 6-chloro-3-indoxyl-p-Dglycopyranoside (X-gluc, part number B5020) was obtained from Biosynth AG (Staad, Switzerland). X-gluc (139.1 mg was carefully mixed in 200 grams of adhesive (a copolymer of isooctyl acrylate and acrylamide in a 96: 4 weight ratio). The adhesive mixture was then applied as a plate coating over the polyethylene coated paper and heated in an oven at 210 ° C to obtain a dry coating weight of 0.237 g / 154.8 cm ² (0.237 g / 24 in. ² ) .The adhesive-coated film was coated with a layer of guar gum that was previously disinfected using ethylene oxide. The excess guar gum
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58/60 was stirred and the powder coated substrate was cut into pieces of approximately 10.2 cm (4 ”) by 10.2 cm (4”).
Thin Film Culture Device Set:
[0156] One piece of double tape (3M Company, St. Paul, MN, USA) was applied along one edge of the styrofoam spacer to each broth-coated substrate. A powder coated substrate was aligned (with the powder coated side facing the spacer) to overlay the broth coated substrate and the powder coated side was adhered to the double tape to form the assembled culture devices.
Preparation of Detection Items:
[0157] A coating mixture was prepared by mixing the following components in 1000 milliliters of deionized water: 13.0 grams of guar gum and a 141.3 milligram solution of 5-bromo-4-chloro-3-indoxyl- p-myoinositol-1-phosphate (X-IP, part number B7404-P00, obtained from Biosynth AG) dissolved in 2.0 ml of dimethyl sulfoxide (DMSO). The mixture was heated in a covered beaker at 80 ° C under mixing, mixed at that temperature for another 15 minutes and then cooled in a refrigerator. The mixture was warmed to room temperature before coating. The mixture was coated with a knife (span of 0.64 mm (25-mil)) in the transparent polyester film described above. The coated film was oven dried at 210 ° C to obtain a dry coating weight of 0.092 g / 154.8 cm ² (0.092 g / 24 in ² ). The coated substrate was cut into pieces of approximately 10.2 cm (4 ”) by 10.2 cm (4”).
Detection method.
[0158] A pure colony of each of the bacteria shown in Table 6 was inoculated in separate tubes of soybean tryptcasein broth containing yeast extract (TSBYE). The tubes were incubated at 35 ° C for 18 to 24 hours. Individual culture devices (prepared as described in this Example) were opened and hydrated by pipetting 1.5 ml of Butterfield buffer into
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59/60 circular area of the dehydrated broth defined by the Styrofoam spacer. The culture devices were closed, placing the powder-coated paper in contact with the buffer and spreading the buffer over the circular growth area defined by the spacer. The hydrated plates were left to rest at room temperature for 1 hour.
[0159] Ten microliters of each culture grown overnight from Listeria were inoculated by streak plate technique on individual hydrated culture devices and the inoculated culture devices were incubated at 35 ° C in a plastic bag with zipper for 24 hours. After incubation, colonies were observed and the appearance of the colony is described in Table 6.
[0160] The incubated plates were opened, causing the hydrated gel containing the bacterial colonies to remain attached to the polypropylene coated paper substrate. A detection article (described in this Example) was placed in the culture device so that the coated side of the detection article faces the hydrated gel containing the colonies. The culture device was then closed, causing the coated side of the detection article to contact the hydrated gel containing the bacterial colonies. The culture devices were then incubated at 35 ° C and observed. Observations are reported in Table 6. The results indicate that only Listeria monocytogenes microorganisms reacted (ie hydrolyzed) with the enzymatic substrate indicator (5-bromo-4-chloro-3-indoxyl-p-myo-inositol- 1-phosphate) of the detection article.
Table 6. Observations of Culture Devices Containing Listeria Microorganisms.
Microorganism Colony Appearance In Culture Device ¹ Colony Appearance after Addition to Detection Article ² Listeria monocytogenes,ATCC 19111 Reddish in color Blue in color Listeria grayi, Reddish in color Reddish in color
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60/60
Microorganism Colony Appearance In Culture Device ¹ Colony Appearance after Addition to Detection Article ² ATCC 19120 Listeria ivanovi,ATCC 19119 Reddish in color Reddish in color Listeria seeligeri,ATCC 35967 Reddish in color Reddish in color Listeria welshimeri,ATCC 35897 Reddish in color Reddish in color Listeria innocua Reddish in color Reddish in color
¹ Colony appearance - incubation after 24 hours ² - Colony appearance in 15 to 60 minutes after the detection article is brought into contact with the growth area of the culture device.
[0161] Various modifications can be made without departing from the spirit and scope of the invention. These and other achievements are in the scope of the following claims.

权利要求:
Claims (10)
[1]
Claims
1. METHOD TO DETECT THE PRESENCE OR ABSENCE OF A TARGET MICRO-ORGANISM, characterized by understanding to provide:
a detection article comprising a first indicator system, wherein the first indicator system is selected to detect a target microorganism;
a culture device including a second dehydrated indicator system in a dehydrated culture medium comprising ingredients selected to facilitate the growth of a predetermined indicator microorganism; and a sample;
placing the second indicator system in fluid communication with the culture medium;
inoculate the culture device with the sample;
incubate the inoculated culture device long enough to allow the growth of indicator microorganisms;
observe the culture device for an indication of the presence of at least one indicator microorganism;
placing the culture medium of the incubated culture device in contact with the detection article;
observing the culture device in contact with the article to detect a conversion of the first indicator system from a first state to a second state;
wherein observing the culture device for an indication of the presence of at least one indicator microorganism comprises detecting a conversion of the second indicator system from a first state to a second state.
[2]
2. METHOD, according to claim 1, characterized
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2/3 by contact of the culture medium with the detection article only when the indication of the presence of at least one of the indicator microorganisms is observed.
[3]
METHOD, according to any one of claims 1 to 2, characterized in that it provides a culture medium which comprises providing a selected culture medium to facilitate the growth of an Enterobacteriaceae microorganism, whereas providing a detection article comprises providing a detection article to detect a microorganism of the genus Salmonella.
[4]
METHOD, according to any one of claims 1 to 3, characterized in that it provides a culture medium which comprises providing a selected culture medium to facilitate the growth of an Enterobacteriaceae microorganism, whereas providing a detection article comprises providing a detection article to detect a microorganism of the genus Shigella.
[5]
5. METHOD, according to any of the claims
1 to 2, characterized by providing a culture medium that comprises providing a selected culture medium to facilitate the growth of an Enterobacteriaceae microorganism, and providing a detection article comprises providing a detection article to detect a microorganism of the genus Cronobacter .
[6]
6. METHOD, according to any of the claims
1 to 2, characterized by providing a culture medium which comprises providing a selected culture medium to facilitate the growth of an Enterobacteriaceae microorganism, and providing a detection article comprises providing a detection article to detect Escherichia coli.
[7]
METHOD, according to any one of claims 1 to 2, characterized by providing a culture medium that
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3/3 comprises providing a selected culture medium to facilitate the growth of a coliform microorganism, and providing a detection article comprises providing a detection article to detect Escherichia coli.
[8]
8. METHOD, according to any one of claims 1 to 2, characterized by providing a culture medium that comprises providing a selected culture medium to facilitate the growth of a microorganism of the Listeria genus, providing a detection article comprises providing a detection article to detect Listeria monocytogenes.
[9]
9. METHOD according to any one of claims 1 to 8, characterized in that it observes the culture device or the culture device in contact with the article comprises observing the culture device visually.
[10]
10. METHOD, according to any one of claims 1 to 9, characterized by observing the culture device or the culture device in contact with the article, comprising observing the culture device using an imaging device.

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法律状态:
2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-12-31| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-02-18| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 23/12/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201061428722P| true| 2010-12-30|2010-12-30|

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US61/428,722|2010-12-30|

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PCT/US2011/067149|WO2012092181A2|2010-12-30|2011-12-23|Articles and method for detecting a target microorganism|

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