WO2012158041A1 - Procédés et kits diagnostiques pour déterminer la présence d'un microorganisme dans un échantillon - Google Patents

Procédés et kits diagnostiques pour déterminer la présence d'un microorganisme dans un échantillon Download PDF

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Publication number
WO2012158041A1
WO2012158041A1 PCT/NL2012/050352 NL2012050352W WO2012158041A1 WO 2012158041 A1 WO2012158041 A1 WO 2012158041A1 NL 2012050352 W NL2012050352 W NL 2012050352W WO 2012158041 A1 WO2012158041 A1 WO 2012158041A1
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Prior art keywords
microorganism
sample
phage
salmonella
lysed cells
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PCT/NL2012/050352
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English (en)
Inventor
Aldert Anthonie Bergwerff
Guiseppe CACCIATORE
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Rna Holding B.V.
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Publication of WO2012158041A1 publication Critical patent/WO2012158041A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)

Definitions

  • the invention relates to methods and kits for detecting a microorganism in a sample.
  • Agents such as phage, capable of lysing the microorganism are employed.
  • Contamination by microorganisms is a major cause of food and water-borne infections globally causing gastroenteritis, diarrhea, cramps, vomiting and often fever, such as that referred to as "food poisoning" to "life -threatening disease” .
  • Illness in humans often results from the eating of undercooked meats, milk or eggs or from cross -contamination of other foods which are eaten without cooking.
  • the most commonly recognized food borne infections are those caused by bacteria such as, Salmonella, Listeria, Campylobacter, Staphylococcus aureus and E. coli 0157:H7.
  • Salmonella is found throughout the environment, particularly in the intestinal tracts of birds, reptiles, and farm animals.
  • the principle bacterial pathogens that have been shown to cause human intestinal disease associated with food poisoning include Bacillus cereus, Salmonella enterica spp, Listeria monocytogenes, Vibrio paraheamolyticus, enterotoxigenic £ . coli, Campylobacter spp., Staphylococcus aureus, Yersinia enterocolitica, and Clostridium perfringens.
  • Conventional methods for the detection of contamination employ non-selective or selective culturing, or enrichment, followed by plating the cultures on selective media for verification of suspect colonies. This approach is time consuming and can take several days before results are obtained. Improved diagnostic methods and kits are therefore needed for the detection of microorganism in a sample, such as a bacterial contaminant in food, water, environmental, medical, agricultural, veterinary, pharmaceutical or industrial fermentation preparations.
  • the present disclosure provides rapid and highly sensitive methods and kits for the detection of microorganisms in a sample.
  • the disclosure provides methods for determining the presence or absence of a target microorganism in a sample comprising: dividing the sample into at least a first part and a second part; exposing the first part of the sample to an agent capable of lysing said target microorganism; providing conditions that permit the agent to lyse the microorganism, if present; and detecting the presence of non-lysed cells in the first and second parts.
  • said agent is a phage and the method comprises the steps of exposing the first part of the sample to a phage capable of infecting said target microorganism and providing conditions that permit the phage to infect and lyse the microorganism.
  • the disclosure provides methods for determining the presence or absence of a target microorganism in a sample comprising: dividing the sample into at least a first part and a second part; exposing the first part of the sample to a phage capable of infecting said target microorganism; providing conditions that permit the phage to infect and lyse the microorganism, if present;
  • the microorganism is a bacterium and the phage is a
  • a decrease in cells in the first part in comparison to the second part indicates the presence of the microorganism in the sample.
  • the methods further comprise exposing the sample to a binding agent immobilized on a solid substrate, the binding agent being capable of binding the target microorganism such that the microorganism becomes associated with the solid substrate.
  • the solid support is
  • the binding agent is an antibody.
  • the methods further comprise one or more growth steps.
  • one or more of the growths steps is a selective growth step.
  • the methods further comprise at least one pre-enrichment growth step and/or at least one selective growth step, preferably the selective growth step follows the pre-enrichment step.
  • the methods further comprise exposing both first and second parts to a reagent that labels non-lysed cells and detecting said reagent to determine the presence of non-lysed cells in the first and second parts.
  • the reagent that labels non-lysed cells is selected from SYBR Green, oxazole yellow, thiazole orange, and PicoGreen, ethidium bromide, fluorescein diacetate, or a labelled binding agent, such as an antibody, capable of binding the microorganism.
  • the reagent that labels non-lysed cells is detected using flow cytometry.
  • the target microorganism is Salmonella and the phage is selected from bacteriophage P27-like, P2-like, lambdoid, P22- like, T7-like, epsilonl5, KS7, Felix 01 bacteriophage, or a combination thereof.
  • the phage is Felix 01.
  • the disclosure provides diagnostic kits for the detection of a target microorganism in a sample comprising an agent that specifically lyses a target microorganism, preferably a phage, and a reagent that labels non-lysed cells.
  • the kit further comprises a binding agent immobilized on a solid substrate, the binding agent being capable of binding the target microorganism if present in the sample such that the microorganism becomes associated with the solid substrate.
  • the solid support is immunomagnetic beads.
  • the binding agent is an antibody.
  • the reagent that labels non-lysed cells is selected from SYBR Green, oxazole yellow, thiazole orange, and PicoGreen, ethidium bromide, fluorescein diacetate, or a labeled binding agent, such as an antibody, capable of binding the microorganism.
  • the kit further comprises a microorganism susceptible to lysis by the lysing agent, e.g. a microorganism susceptible to infection by said phage.
  • the kit further comprises further a microorganism that is not susceptible to infection by said phage.
  • the microorganism is a bacterium and the phage is a bacteriophage.
  • the target microorganism detected by the kit is
  • Salmonella and the phage is selected from bacteriophage P27-like, P2-like, lambdoid, P22-like, T7-like, epsilonl5, KS7, Felix 01 virus, or a combination thereof.
  • the microorganism is a bacterium and the phage is a
  • Phages are viruses that have evolved in nature to use microorganism as a means of replicating themselves.
  • a bacteriophage for example, does this by attaching itself to a bacterium and injecting its genetic material into that bacterium, inducing it to replicate the phage from tens to thousands of times.
  • Some bacteriophage, called lytic bacteriophage rupture the host bacteria releasing the progeny phage into the environment to seek out other bacteria. The total incubation time for phage infection of bacteria, phage multiplication
  • U. S. Patent 5,888, 725 describes a method utilizing unmodified, highly specific lytic phages to infect target bacteria in a sample. Phage-induced lysis releases certain nucleotides from the bacterial cell such as ATP that can be detected using known techniques.
  • U.S. Patent 6,436,661 describes a method whereby a phage is used to infect and lyse a target bacterium in a sample releasing intracellular enzymes, which react in turn with an immobilized enzyme substrate, thereby producing a detectable signal.
  • Alternative methods detect the bacteriophage amplified in a target microorganism.
  • WO 2006/083292 describes the detection of bacteriophage protein or nucleic acid. While these methods have the advantage of using unmodified phage, the sensitivity of the assays is limited as the concentration of detected markers (nucleotides or enzymes) is directly proportional to the concentration of target bacteria in the sample. These methods also often have a relatively high background level due to the presence of the detected components in the sample. For example, meat also contains enzymes and nucleotides.
  • the present disclosure provides methods and kits for detecting
  • the disclosure provides methods for detecting the presence or absence of a microorganism in a sample comprising dividing the sample into at least a first part and a second part; exposing the first part of the sample to a phage capable of infecting said target microorganism; providing conditions that permit the phage to infect and lyse the microorganism if present; and detecting the presence of non-lysed cells in the first and second parts.
  • any agent capable of specifically lysing a target microorganism may be used in the methods and kits disclosed herein.
  • Preferred agents are the phages disclosed herein.
  • microorganism include, e.g., antibiotics and bactericides.
  • said agents may be coupled to, e.g., antibodies that specifically recognize the target microorganism.
  • the methods described herein comprise additional steps that may increase the sensitivity of the assay.
  • methods for determining the presence or absence of a target microorganism in a sample comprising: dividing the sample into at least a first part and a second part; exposing the first part of the sample to a phage capable of infecting said target microorganism; providing conditions that permit the phage to infect and lyse the microorganism if present;
  • the agent preferably a phage will lyse the microorganism if present, thereby decreasing the number of intact cells in the first part of the sample in comparison to the second part of the sample.
  • the presence of a phage will lyse the microorganism if present, thereby decreasing the number of intact cells in the first part of the sample in comparison to the second part of the sample.
  • microorganism in a sample is indicated by a greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, preferably greater than 85%, or greater than 90% reduction in cell number in the first part of the sample, i.e., agent treated, as compared to the untreated part of the sample.
  • microorganisms refer to bacteria, mycoplasmas, and other microscopic living organisms.
  • the microorganisms are bacteria.
  • Target bacteria contemplated by the present methods and kits include, but are not limited to, bacterial cells that are food, water, or environmental
  • detection of bacteria relates to a single species, isolate, serovar or strain as well as groups or families of organisms.
  • Bacteria include, but are not limited to, Acinetobacter spp., Actinomyces spp., Bacillus spp. (e.g., Bacillus anthracis, Bacillus subtilis, Bacillus cereus), Bordetella pertussis, Borrelia burgdorferi, Borrelia recurrentis, Brucellae (e.g., Brucella melitensis, Brucella abortus, Brucella suis, Brucella canis),
  • Acinetobacter spp. e.g., Actinomyces spp.
  • Bacillus spp. e.g., Bacillus anthracis, Bacillus subtilis, Bacillus cereus
  • Bordetella pertussis e.g., Brucella melitensis, Brucella abortus, Brucella suis, Brucella canis
  • Brucellae e.g., Brucella melitensis, Brucella ab
  • Costridium spp. e.g., Clostridium difficile, Clostridium perfringens
  • Staphylococcus aureus Staphylococcus epidermidis
  • Shigella spp. e.g., Shigella dysenteriae
  • Spirillum minus Streptococci
  • Treponema pallidum Ureaplasma urealyticum
  • Vibrio spp. e.g., Vibrio cholerae, Vibrio vulnificus
  • Xanthomonas maltophilia e.g., Yersinia pestis.
  • bacteria are selected from Salmonella; E. coli, preferably E. coli 0157:H7; Listeria, preferably L. monocytogenes; Legionella and Campylobacter.
  • the genus Salmonella comprises two species, Salmonella bongori and
  • Salmonella enterica Salmonella enterica is divided into serovars or serotypes, based on somatic O-antigens, flagellar (H) antigens, and surface antigens. There are at least 1500 food-borne Salmonella serovars identified so far.
  • Salmonella serotypes detectable using the methods and kits described herein include, for example, Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Java, Salmonella enterica serovar Infantis, Salmonella enterica serovar Virchow, Salmonella enterica serovar Mbandaka. Salmonella enterica serovar Livingstone,
  • Salmonella enterica serovar Senftenberg Salmonella enterica serovar Hadar, Salmonella enterica serovar London, Salmonella enterica serovar Rissen, Salmonella enterica serovar Anatum, Salmonella enterica serovar Bredeney, and Salmonella enterica serovar Derby. While all Salmonella enterica serotypes are considered potentially pathogenic to humans, only a few are pathogenic to animals. They are often the inhabitants of healthy poultry and pigs, which serve as reservoirs for human transmission. Some serotypes do demonstrate host specificity. The identification of a particular subspecies or serotype can therefore provide information regarding, for example, the contamination route.
  • the disclosure provides methods as described herein for detecting the presence or absence of one or more bacterial serotypes in a sample by using one or more bacteriophages that are capable of distinguishing between one or more serotypes.
  • serotype and serovar refers to bacteria within the same species that can be distinguished on the basis of their surface antigenic properties.
  • Samples include, e.g., various food products, water, industrial or agricultural products, biological samples such as urine, feces, sputum, blood or tissue samples, and may be solid or liquid samples.
  • Phages preferably lytic phages, are employed in the methods and kits described herein due to their high degree of specificity when infecting a microorganism.
  • Lytic phages also includes phages which are normally non- lytic but have been modified to induce lysis in the host microorganism. A method for the modification of such phages is described in U.S. Patent
  • a phage capable of infecting said target microorganism refers to the ability of the phage to infect and replicate within the host and ultimately destroy the cell while releasing numerous phage progeny into the medium.
  • the term phage includes, for example, bacteriophages,
  • mycobacteriophage and mycoplasma phage.
  • the phage is a bacteriophage.
  • the specificity of a phage for its host is determined at two levels.
  • the first level of control involves the interaction of phage components with
  • a phage is specific for a given host when it is capable of infecting the given host and does not infect cells of another species or strain.
  • microorganism refers to conditions associated with, for example, time, temperature, and suitable buffering conditions that are well-known to one skilled in the art. (See for example Sands JA, et al., 1974 Biochimica et
  • Example 1 of the present disclosure The methods and kits provided herein can also be used to determine the presence or absence of more than one microorganism or more than one bacterial serotype in a single assay by using one or more phages.
  • a single phage can recognize, for example, more than one bacterial serotype.
  • a combination of phages is used that recognize different host species, different host strains, or preferably different host serotypes.
  • Phage useful in the methods and kits described herein also may include genetically modified or recombinant phage that have been altered to increase binding affinity, infectivity, burst size, multiplicity of infection or lytic ability for the microorganism to be detected.
  • the phages useful in the methods and kits described herein have not been genetically modified to express a report molecule.
  • the phage is not genetically modified.
  • a wide variety of bacteriophages are available for any given bacterial cell, for example, from the American Type Culture Collection (ATCC, P.O. Box 1549 Manassas, Va., USA) or by isolation from natural sources that harbor the host cells. A list of phage types is published as the Catalogue of Bacteria &
  • Bacteriophages (American Type Culture Collection, Rockville, Md. 1989). Over 20,000 strains of bacteria (with 7,000 being Salmonella) have been evaluated for phage identification (He and Pan, 1992, J Clin Microbiol 30(3): 590-4).
  • the phage is Felix 01.
  • Additional Salmonella bacteriophages and method for isolating phages are described in PCT Publication WO2005/024005 and Capparelli R et al., J Infect Dis. 2010 Jan 1;201(1):52-61 A skilled person is aware of additional Salmonella bacteriophages and method for isolating phages.
  • Salmonella phages which are described, e.g., on the world wide web at thebacteriophages.org/frames_names.htm.
  • Additional bacteriophages include, but are not limited to
  • Actinoplanes/Micromonospora phages (Ap3, Ap4, Mml, Mm3, Mm4, Mm5, phiUW 51); Amycolatopsis phages (W2, W4, W7, Wll); Bacillus phages (GA-1, Phi 29, SP.beta.); Campylobacter phages (e.g., NTCC 12669 , NTCC 12670 , NTCC12671 , NTCC12672 , NTCC12673 , NTCC12674 , NTCC12675 ,
  • NTCC12676 NTCC12677 , NTCC12678 , NTCC12679 , NTCC12680 ,
  • Saccharothrix phage (Wl); Sporichthya phage (Spl); Streptomyces phages (P23, P26, SI, S2, S3, S4, S6, S7, SH10, phi A. streptomycini III, phi8238, phiC31); Terrabacter phages (Tbl, Tb2); Tsukamurella phage (Tsl).
  • Other suitable phages are known to one of skill in the art and may be found, e.g., on the world wide web at thebacteriophages.org/frames_names.htm.
  • Phages specific for particular bacteria can also be selected using routine techniques in the laboratory due to the ability of the phage to rapidly mutate, thereby producing host range mutants.
  • lysed cell and “non-lysed cell” are well-understood to one of skill in the art.
  • a non-lysed cell has a cell wall significantly intact to allow its detection by various assays known to one of skill in the art.
  • these devices rely on the use of a single sensor (e.g., pH or carbon dioxide indicator) in a layer adjacent to a layer of growth medium for detecting the presence of a bacterium.
  • a single sensor e.g., pH or carbon dioxide indicator
  • the presence of non-lysed cells is detected using a reagent that labels non-lysed cells.
  • Suitable reagents include reagents which are permeable to cells, e.g., SYBR Green, oxazole yellow, thiazole orange, ethidium bromide, fluorescein diacetate,and PicoGreen.
  • reagents for labelling non-lysed cells include labelled-binding agents capable of binding the microorganism.
  • the binding agent may preferentially bind the target microorganism over non- target microorganisms or may bind microorganisms indiscriminately.
  • Binding agents include antibodies as well as non-immunoglobulin binding agents, such as aptamers, phage display-derived peptide binders, or scaffold-based binding proteins (e.g., Nanobody, Evibody, Ankyrin repeat protein, Transbody,
  • antibodies include, e.g., monoclonal antibodies; polyclonal antibodies; antigen-binding fragments including, but not limited to, Fab, F(ab'), F(ab')2, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, diabodies, triabodies, tetrabodies, artificial antibodies, and phage display-derived antibodies.
  • binding agents against various microorganisms, as well as methods of generating binding agents are well known in the art. The binding agents may recognize, for example,
  • Suitable binding agents include, e.g., anti-lipid A antibodies (AbCam # ab20001). In some embodiments, the binding agents recognize intracellular components and are thus preferably permeable to cells. Labels suitable for binding agents include fluorescent molecules.
  • the reagents that label non-lysed cells can be detected using a variety of different techniques including, e.g., flow cytometry, fluorescence microplate readers, and fluorescence microscopy. Preferably, the presence of non-lysed cells is determined using a reporter that can be detected using flow cytometery (including, e.g., fluorescent agents such as SYBR green as well as binding molecules labelled with such fluorescent agents).
  • the detection of the presence or absence of a target microorganism is determined by comparing the number of non-lysed cells between the first and second parts.
  • the presence of non-lysed cells is understood to include not only the determination of whether non-lysed cells are present or absent, but also the determination of the concentration of non-lysed cells in the first and second parts and/or the determination of the relative concentration of non- lysed cells in the first part as compared to the second part.
  • a reduction in the number of non-lysed cells in the sample part treated with the lysing agent indicates the presence of the target microorganism. It is within the purview of one skilled in the art to establish what constitutes a significant reduction of cells.
  • the reduction of cells in the first part as compared to the second part is greater than 40, 50, 60, 70, or preferably 80%, indicating the presence of a target microorganism in a sample.
  • the methods described herein comprise one or more growth steps. These steps are useful, e.g., if a greater sensitivity is needed.
  • a non-selective growth step i.e., "pre-enrichment”
  • pre-enrichment increases the number of microorganisms in a sample
  • a selective growth step preferentially increases the number of target microorganisms in the sample. It is not uncommon for a microorganism in a sample to suffer from sublethal damage, e.g., from thermal processing of food, freezing, thawing, osmotic shock, or prolonged.
  • a pre-enrichment step may be useful to restore the injured cells.
  • Suitable growth media is known to one skilled in the art and includes, for example, buffered peptone water, lactose broth, tryptone soya, brilliant green, etc.
  • pre-enrichment growth media is non-selective growth media.
  • Samples can be inoculated on agar or liquid growth media.
  • the sample is cultured for 2-24 hours, preferably for 4-8 hours, in pre-enrichment growth media.
  • a selective growth step allows the growth of the targeted microorganism, while growth of competing organisms is inhibited. Examples of selection agents include antibiotics, dyes, bile salts, detergents, and other substances known to those skilled in the art.
  • Selective growth for Salmonella can be carried out in, e.g., selenite cystine broth, tetrathionate broth, or Rappaport Vassiliadis soya peptone broth. It is within the purview of one skilled in the art to select suitable selective media depending on the target microorganism.
  • a selective growth step may also comprise varying the growth conditions, for example by altering the temperature or CO2 levels during culture.
  • the sample is cultured for 2-24 hours, preferably for 8-18 hours, in selective growth media.
  • methods are provided comprising a non-selective growth step followed by a selective growth step.
  • the growth steps can precede or follow steps dividing the sample, exposing the sample to a lysing agent, and exposing the sample to a binding agent.
  • the order of the steps depends on the sample being tested, the microorganism being tested for, as well as the non-target microorganism in the sample. It is within the purview of one skilled in the art to determine the order of the steps in the method.
  • the methods for determining the presence or absence of a target microorganism in a sample comprise culturing said sample in pre-enrichment growth media, exposing said sample to a binding agent immobilized on a solid substrate; dividing the sample into at least a first part and a second part; exposing the first part of the sample to a lytic agent, preferably a phage capable of infecting said target microorganism; providing conditions that permit the agent to lyse the microorganism, preferably conditions that permit the phage to infect and lyse the microorganism if present and culturing said samples in selective growth media; and detecting the presence of non-lysed cells in the first and second parts.
  • a lytic agent preferably a phage capable of infecting said target microorganism
  • the dividing of the sample into a first part and a second part may be performed before, during or after the culturing in pre-enrichment growth media and exposure to a binding agent steps.
  • Example 2 describes such a preferred method.
  • An additional step that may be used alone or in conjunction with additional growth steps to increase sensitivity is a step which selects for the target microorganism.
  • the methods described herein further comprise a step comprising exposing the sample to a binding agent immobilized on a solid substrate.
  • the binding agent preferentially binds the target microorganism over unrelated and/or related microorganisms. Unbound sample may then be removed by washing the solid support with an appropriate buffer.
  • Suitable binding agents include antibodies as well as non-immunoglobulin binding agents, such as aptamers, phage display-derived peptide binders, or scaffold-based binding proteins (e.g., Nanobody, Evibody, Ankyrin repeat protein, Transbody, Anticalin, Microbody fibronectin-based scaffolds).
  • antibodies include, e.g., monoclonal antibodies; polyclonal antibodies; antigen-binding fragments including, but not limited to, Fab, F(ab'), F(ab')2, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, diabodies, triabodies, tetrabodies; artificial antibodies; and phage display-derived antibodies.
  • binding agents against various microorganisms, as well as methods of generating specific binding agents are well known in the art.
  • the specific binding agent recognizes Salmonella.
  • Salmonella binding agents include RDI-TRK3S022 (Research Diagnostics) and ab31555 (AbCam).
  • Binding agents may be immobilised on a solid support using conventional methods.
  • Suitable solid supports include, e.g., fibers, fibrous filters, membrane filters, magnetic beads, non-magnetic beads, columns, and matrices.
  • the solid support is immunomagnetic beads and an
  • an immuno-separation step uses an antibody conjugated to magnetic beads.
  • a selective or pre-enrichment growth step is used after the separation step.
  • a selective or pre-enrichment growth step is before or after the separation step.
  • the methods comprise a non-selective "pre-enrichment" growth step; exposing the sample to a binding agent immobilized on a solid substrate as described herein; dividing the sample into at least a first part and a second part either before or after the sample is exposed to a binding agent; a selective enrichment growth step;
  • kits for the detection of a target microorganism in a sample comprise one or more lysing agents, preferably phages, as described herein and a reagent that labels non- lysed cells as described herein.
  • Phage compositions can take the form of relatively crude lysates of bacterial cultures or highly purified virus
  • Phage can be formulated in various ways for storage and shipping. Phage may be lyophilized into a dry powder and added to the growth media as a supplement during reconstitution of a powdered media or added later during the growth process if desired. Phage is also available in a liquid form or suspension. The phage may also be provided as a set of serial dilutions. Preferably, the kits further comprise a binding agent immobilized on a solid substrate as described herein.
  • kits further comprise one or more of the following: filter microplates, phage diluents, buffer that provides conditions to permit the phage to infect and lyse the microorganism if present in the sample.
  • the kits further comprise a microorganism susceptible to infection and lysis by the phage provided in the kit.
  • the microorganism provided has a low pathogenic potential or has been modified to reduce its pathogenicity.
  • a kit is provided comprising one or more bacteriophage capable of infecting one or more Salmonella serotype and a reagent that labels non-lysed cells, such as SYBR Green or a fluorescently labeled antibody specific for Salmonella.
  • the kit further comprises a binding agent immobilized on a solid substrate, such as anti- Salmonella antibody conjugated immunobeads, and/or bacteria capable of being infected and lysed by the one or more bacteriophage, such as Salmonella.
  • a binding agent immobilized on a solid substrate such as anti- Salmonella antibody conjugated immunobeads, and/or bacteria capable of being infected and lysed by the one or more bacteriophage, such as Salmonella.
  • the methods and kits provided herein for detection of a target provide high detection sensitivity in a short amount of time without the need for lengthy cultural growth.
  • the present methods and kits can provide for the detection of less than about 100, less than about 50, or 10 or less cells in a sample.
  • the present methods can provide for the detection of less than about five, less than about four, less than about three, or less than about two cells in a sample.
  • the methods can provide for the detection of a single cell in a sample.
  • 1 CFU is detectable in 25 grams of sample.
  • An exemplary embodiment for detecting a microorganism, such as Salmonella, in a sample, such as food, may include the following steps:
  • an element means one element or more than one element.
  • Figure 1A and IB Flow cytometric analysis of a minced meat spiked with 10 CFU/25 g of Salmonella serovar Enteriditis.
  • Figure 1A shows the analysis of the aliquot which was incubated with buffer not containing phage, (negative control).
  • Figure IB shows the analysis of the aliquot which was incubated with the phage mixture.
  • Figure 2A and 2B Flow cytometric analysis of a minced meat spiked with 10 CFU/25 g of Salmonella serovar Typhimurium.
  • Figure 2A shows the analysis of the aliquot which was incubated with buffer not containing phage, (negative control).
  • Figure 2B shows the analysis of the aliquot which was incubated with the phage mixture.
  • Figure 3 Results of a flow cytometric analysis of spiked minced meat samples.
  • Counter refers to the bacteria count from the part of a sample in which bacteriophage has been added (+) or the part of a sample in which
  • Wash medium A 0.2 ⁇ filtered PBS containing 0.05 % Tween 80 and 1% Alaska wash medium (AWM) additive
  • Wash medium B 0.2 ⁇ filtered PBS containing 1 % AWM additive Peptone saline (PFZ) solution, Biokar diagnostics (prod no. BK014HA)
  • Buffered peptone water (BPWT) with 0.05 % Tween 80, Biokar diagnostics (prod no. BK018HA)
  • TLB Tryptone soy broth
  • Oxoid Prod no. CM0129
  • Salmonella selective broth Selenite brilliant green (SBG) broth, BD
  • IMS Immunomagnetic separation
  • Assay buffer PBS 1% (v/v) horse serum containing 0.005% (m/v) Tween 20 and 5 mM EDTA
  • Salmonella enterica cultures Salmonella serovar Anatum, Salmonella serovar Bredeney, Salmonella serovar Derby, Salmonella serovar Enteriditis,
  • Salmonella serovar Hadar Salmonella serovar Infantis, Salmonella serovar Anatum, Salmonella serovar Java, Salmonella serovar London, Salmonella serovar Mbandaka, Salmonella serovar Rissen, Salmonella serovar
  • Salmonella phage mixture bacteriophage Felix 01 (obtainable by the Felix d'Herelle Reference Center, Universite Laval, Quebec, Canada); bacteriophage Ent and bacteriophage MS24. Spiking and preparation of food samples
  • the selectively grown samples were diluted 1000-fold in PFZ. 10 ml of the diluted sample and 20 ⁇ of anti-Salmonella IMS beads were pipetted in a sterile 15 ml polypropylene tube. The tube was placed in a pre-heated (37°C) magnetic separator rotator (MSR) and rotated at 5 rpm for 20 min at 37°C.
  • MSR magnetic separator rotator
  • the liquid was removed using the vacuum manifold and the wells were washed with 200 ⁇ assay buffer.
  • the IMS beads and bacteria were re-suspended in 150 ⁇ assay buffer and stained with 1.5 ⁇ SybrGreen I (diluted 1620x in assay buffer) for 15 min at room temperature.
  • the liquid was removed using the vacuum manifold and both the stained bacteria and IMS beads were re-suspended in 130 ⁇ assay buffer and measured on the Beckman Coulter Cell lab Quanta SC MPL. A quantity of 2,000 IMS beads are measured as an internal reference to compare the results of different food samples.
  • Minced meat samples were spiked with 13 different Salmonella spp. at approximately 10 CFU/25 g and all Salmonella spp. could be detected as exemplified for Salmonella Enteriditis and Salmonella Typhimurium in Figure 1 and Figure 2, respectively, and in Table 1.
  • the darker population of dots corresponding to the IMS beads used as an internal standard is encircled in Figures 1 and 2.
  • a triangle indicates the bacterial population detected in the samples.
  • Table 1 demonstrates that the addition of salmonella specific bacteriophages reduces the number of bacteria by more than 85%.
  • Wash medium A 0.2 ⁇ filtered PBS containing 0.05 % Tween 20 (PBST)
  • Assay buffer PBS containing 1% horse serum, 5 mM EDTA and 0.005 % Tween 20
  • Salmonella selective both: Selenite brilliant green (SBG) broth, BD Diagnostics ( prod no. 271510)
  • IMS Immunomagnetic seperation
  • Salmonella enterica cultures Salmonella serovar Anatum, Salmonella serovar Bredeney, Salmonella serovar Derby, Salmonella serovar Enteriditis,
  • Salmonella phage mixture bacteriophage Felix 01 (obtainable by the Felix d'Herelle Reference Center, Universite Laval, Quebec, Canada); bacteriophage Ent and bacteriophage MS24.
  • reaction tubes were placed in an IMS rack.
  • the magnets were inserted into the rack and the reaction tubes were shaken over head by hand for 3 minutes to capture IMS beads.
  • liquid inside the reaction tubes was poured out and the beads were washed with 1 ml PBST.
  • the washing step was repeated twice.
  • the magnet was removed from the IMS rack and IMS beads were suspended thoroughly in 100 microliter PBST.
  • the re-suspended sample was split into two aliquots of 50 microliter and the aliquots were transferred to two different wells of a pre- wetted filter plate. The liquid inside the well was removed using the vacuum manifold and each well was filled with 200 microliter SBG.

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Abstract

L'invention concerne des procédés et des kits de détection d'un microorganisme dans un échantillon. Des agents capables de lyser le microorganisme sont employés. Les agents de lyse préférés sont des phages. Dans des modes de réalisation préférés, un échantillon, par exemple un échantillon d'aliments, est divisé en deux parties. Un agent capable de lyser un microorganisme est ajouté à une première partie de l'échantillon dans des conditions permettant de lyser le microorganisme, si celui-ci est présent. La présence du microorganisme est détectée en déterminant les cellules non lysées dans chacune des deux parties. Une réduction des cellules non lysées dans l'échantillon traité avec un agent de lyse particulier est indicative de la présence du microorganisme dans l'échantillon.
PCT/NL2012/050352 2011-05-18 2012-05-18 Procédés et kits diagnostiques pour déterminer la présence d'un microorganisme dans un échantillon WO2012158041A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113151515A (zh) * 2021-03-31 2021-07-23 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） 一种食源性致病菌的检测试剂盒及检测方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113151515A (zh) * 2021-03-31 2021-07-23 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） 一种食源性致病菌的检测试剂盒及检测方法

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