WO2014140381A1 - Procédés d'immunocapture et de concentration de bactéries dans un échantillon - Google Patents

Procédés d'immunocapture et de concentration de bactéries dans un échantillon Download PDF

Info

Publication number
WO2014140381A1
WO2014140381A1 PCT/EP2014/055348 EP2014055348W WO2014140381A1 WO 2014140381 A1 WO2014140381 A1 WO 2014140381A1 EP 2014055348 W EP2014055348 W EP 2014055348W WO 2014140381 A1 WO2014140381 A1 WO 2014140381A1
Authority
WO
WIPO (PCT)
Prior art keywords
bacteria
specific
target
coli
immunocapture
Prior art date
Application number
PCT/EP2014/055348
Other languages
English (en)
Inventor
Patrice Arbault
Nicolas Desroche
Delphine LAROSE
Original Assignee
Nexidia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nexidia filed Critical Nexidia
Publication of WO2014140381A1 publication Critical patent/WO2014140381A1/fr

Links

Classifications

    • 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
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins

Definitions

  • the present invention relates to methods for immunocapture and concentration of target bacteria in a sample, such as bodily fluids, foods or environmental fluids.
  • a sample such as bodily fluids, foods or environmental fluids.
  • the present invention relates to a method for immunocapture and concentration of pathogenic bacteria in food samples, such as Shiga Toxin-producing Escherichia coli (STEC), also known as verotoxin-producing Escherichia coli (VTEC), Listeria monocytogenes, Cronobacter sakazakii and Salmonella spp.
  • STOC Shiga Toxin-producing Escherichia coli
  • VTEC verotoxin-producing Escherichia coli
  • Listeria monocytogenes such as Listeria monocytogenes, Cronobacter sakazakii and Salmonella spp.
  • EHEC Enterohaemorrhagic E. coli
  • the pathogen transmission originates mainly from foods of animal origin, for instance from beef. Healthy cattle and other ruminants appear to be the main reservoir of Shiga Toxin-producing Escherichia coli. Nevertheless, other food matrices such as leafy greens, beansprouts and dairy products have also been involved in human infections.
  • the process of detection requires a step of enrichment of the sample in different broth medium like buffered peptone water (BPW) for Salmonella spp. , modified Tryptic soy broth (mTSB) supplemented with novobiocine for E. coli 0157: 1-17 and modified lauryl sulfate tryptose broth (mLST) supplemented with vancomycin for C. sakazakii.
  • BBP buffered peptone water
  • mTSB modified Tryptic soy broth
  • mLST modified lauryl sulfate tryptose broth
  • STEC STEC identified as pathogenic for humans are from the following mains serogroups: 0157: 1-17, 026, 045, 0103 , 01 1 1 , 0121 and 0145.
  • Others serogroups are emerging in Europe or in the US like 0104, 091 and 01 13.
  • Shiga toxin stxl and /or stx2 genes
  • an intimin eae gene
  • Shiga toxins are a family of related toxins with two major groups, Stx1 and Stx2. Shiga toxins act to inhibit within target cells. After entering a cell, the protein acts as an N-: it cleaves a specific adenine from the 28S of the 60S subunit of the ribosome, thereby halting protein synthesis.
  • Intimin is a () of pathogenic strains. It is an attaching and effacing protein which with other virulence factors is responsible for and. Intimin is expressed on the bacterial cell surface.
  • E. coli 01 57: 1-17 Several standard methods are available for the detection of E. coli 01 57: 1-17 such as ISO SO 16654:2001 ("Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Escherichia coli 01 57") and USDA FSIS MLG5B.02. These standard methods require a first step of enrichment, a step of immunomagnetic separation (IMS), a streaking step on specific agar plates and confirmation of tests for typical colonies.
  • IMS immunomagnetic separation
  • Methods of detection and identification of pathogenic STEC are usually based on molecular methods, such as polymerase chain reaction (PCR) or molecular isothermal amplification technique, allowing:
  • the key step in said methods is the selection of the relevant bacterial population in the food sample, in order to perform the molecular detection step, such as the PCR step, on these target bacteria.
  • the molecular detection step is performed after an enrichment step.
  • an immunomagnetic separation (IMS) step can be performed for confirming the presence of pathogenic bacteria.
  • the molecular detection method such as the PCR step, can be performed after isolation of the pathogenic bacteria by plating on agar-based media.
  • ImmunoMagnetic separation is a technique using magnetic beads coated with specific antibodies as support to separate cells. This technique involves the specific binding of an antibody to its antigen (here, an antigen expressed by bacteria such as the O-antigen), where the magnetic beads are conjugated to a specific antibody.
  • the magnetic beads allow the selective separation of bacteria expressing specific antigens, by applying a magnetic field.
  • kits of ImmunoMagnetic Separation are commercially available, such as the Dynabeads® from Life TechnologiesTM, CaptivateTM beads commercialized by Lab M, f. co/j-IMS "SEIKEN” commercialized by Denka Seiken, and RapidChek® CONFIRMTM beads commercialized by SDIX.
  • the IMS kits use specific monoclonal or polyclonal antibodies able to bind specifically the pathogenic bacteria.
  • several antibodies have been developed for the immunocapture of STEC (Dynabeads®, CaptivateTM, RapidChek® CONFIRMTM non-0157 STEC).
  • the present invention relates to a method for capturing and concentrating target bacteria in a sample which comprises the following steps: Immunocapturing the target bacteria with at least one specific antibody of said target bacteria, said specific antibody being fixed on a solid plastic support ;and Culturing the target captured bacteria on the solid plastic support to specifically increase the target bacterium population.
  • the present invention further relates to a kit for detection of target bacteria which comprises a solid plastic support coated with antibodies specific of the target bacteria and reagents for culturing the bacteria.
  • Pathogenic bacteria are bacteria that cause bacterial infection, in particular in humans. Although the vast majority of bacteria are harmless or beneficial, quite a few bacteria are pathogenic. In the context of the invention, pathogenic bacteria may be those responsible for foodborne illnesses, which can be caused by bacteria such as Shigella spp. , Campylobacter spp., Listeria monocytogenes, Escherichia coli, Yersinia spp. , Staphylococcus spp. , Bacillus cereus, Cronobacter sakazakii and Salmonella spp..
  • Immunocapture is the action of selectively binding a target microorganism from a medium comprising several microorganisms, by the action of specific antibodies directed to a membrane-expressed protein of said target microorganism.
  • Immunocapture is generally done on beads of agarose, or magnetic beads, or on a sepharose-column, as support of the antibodies.
  • immunocapture is done on a solid plastic support as detailed herein below.
  • plastic solid support designates any support made of plastic used in laboratories of research and biological analysis.
  • plastic solid support include but are not limited to microplates (also known as microtiter plate or microwell plate), tubes and microtubes.
  • the solid support can be suitably made of polystyrene, polypropylene or polycarbonate.
  • microplate' designates a, typically made of polystyrene, well known by the man skilled in the art.
  • the microplate may be as used in the Enzyme- linked immunosorbent assay (ELISA), a test using antibodies and color change to identify the presence of a cell specifically linked to said antibodies.
  • ELISAs combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme.
  • ELISA is a popular format of analytic that uses a solid-phase enzyme (EIA) to detect the presence of a substance (antigen or toxin for example) or a cell (bacteria, virus) in a liquid sample.
  • EIA solid-phase enzyme
  • 'immunocapture microplate' designates a plastic microplate coated with specific antibodies for the capture of specific bacteria by antigen-antibody complex formation or a microplate coated with an 'universal coating' such as protein A, which is able to fix any type of antibodies.
  • target bacteria refer to a bacterium species or a bacterium serogroup that one seeks to specifically identify in a sample, i.e. a target bacterium species or a target bacterium serogroup.
  • the target bacterium may be harmless bacterium or pathogenic bacterium.
  • concentrating as used herein means increasing the concentration of target bacteria, i.e. increasing the target bacterium population.
  • the present invention relates to a method for capturing and concentrating target bacteria in a sample.
  • the method comprises the following steps:
  • the target bacteria may be harmless bacteria or pathogenic bacteria.
  • the present invention is related to a method for capturing and concentrating pathogenic target bacteria in a sample, comprising the following steps:
  • the methods of the present invention are particularly suitable for capturing and concentrating target bacteria, such as pathogenic bacteria, in a sample comprising a low amount of said bacteria, such as a concentration inferior to 10 2 or 10 1 CFU (colony- forming unit) per milliliter or gram of sample.
  • target bacteria such as pathogenic bacteria
  • the methods of the present invention allow easier and quicker manipulation vs. currently used methods for immunocapture of bacteria.
  • the methods do not require the use of specific equipment, such as magnetic separator racks.
  • the yields of captured STEC bacteria are higher than those obtained with IMS beads commercially available.
  • This method can be used for the detection and the isolation of STEC in food samples and can be used in replacement of the currently used ImmunoMagnetic Separation methods.
  • immunocapturing target bacteria or “immunocapturing pathogenic target bacteria” it is meant capturing at least one bacterium species or at least one bacterium serogroup, which may be pathogenic or not, in a sample.
  • the immunocapture is achieved thanks to antibodies specific to the bacterium species or to the bacterium serogroup which are fixed on the solid plastic support.
  • the specific antibody(ies) may be fixed on the solid plastic support by any means well known by the skilled person.
  • the solid plastic support can be coated with antibodies or with an 'universal coating' such as protein A or a complex avidin/streptavidin wherein antibodies can be fixed later.
  • the sample to be tested is either put in contact with the solid plastic support onto which the specific antibodies are fixed or the sample to be tested is first contacted with specific antibodies and then put in contact with the solid plastic support onto which a "universal coating” is applied.
  • the step of culturing the captured target bacteria is intended to increase specifically the population of said bacteria. Said step is particularly useful for increasing the population of pathogenic bacteria which are generally present in very low amount in food samples. Furthermore, when the presence of target bacteria is to be confirmed by molecular biology methods, such as PCR, it is important to have sufficient amount of bacteria before the step of DNA extraction, in order to obtain a sufficient amount of DNA.
  • the step of culturing is also intended to increase the recovery levels of bacteria before a step of isolation which can be performed by streaking of bacteria on specific agar plate.
  • the step of culturing the captured bacteria is performed on the solid plastic support, i.e. the culture is performed directly on the solid plastic support, for instance directly in the wells of the solid plastic support when said support is a microwell plate, or in the tube or microtube when said support is a tube or microtube.
  • the method according to the invention comprises an in situ step of culture of the target bacteria on the solid plastic support after immunocapture.
  • said step of culture may be performed in an appropriate liquid medium.
  • the liquid medium may comprise antibiotics or others selective agents.
  • the man skilled in the art knows the appropriate culture media, adapted for each STEC serogroups or types of microorganisms.
  • the man skilled in the art also knows how to choose antibiotics harmless against the bacteria that have been immunocaptured on the support.
  • This step allows the inhibition of the growth of non-specifically immunocaptured microorganisms. The specificity of the method is therefore increased.
  • the novobiocine can be used as antibiotic, when E. coli 0157: 1-17 are detected with the method.
  • the step of culturing the captured bacteria is herein referred to as an "enrichment step".
  • the enrichment step is presently performed after immunocapture.
  • the method of the present invention may comprise a washing step performed before the culturing step: the solid plastic support is washed before culturing the captured target bacteria.
  • the washing step allows removing the nonspecific bacteria that may have adhered to the solid plastic support, i.e. removing the bacteria which are not specific to the antibodies fixed on the solid plastic support.
  • the washing step may be performed with a neutral dilution buffer solution, such as phosphate buffer solution (also named PBS), or with others buffer solutions containing dispersing agents such as Tween®.
  • the solid plastic support is a plate, a microplate, a tube or a microtube.
  • the solid support is coated with at least one specific antibody.
  • it may also be coated with two, three, four, or even more specific antibodies.
  • a coating with more than one specific antibody may allow immunocapture of more than one bacterium species or serogroup, such as two, three, four, or even more different bacterium species or serogroups.
  • the method of the invention can be used for determining the presence of target bacteria, such as pathogenic target bacteria, in a sample of water, or a sample of biological material such as feces.
  • the sample is a food sample, in particular a meat sample, in particular a beef sample.
  • the method may further comprise the step of isolating the immuno-captured bacteria on specific agar plates. This step allows confirming the presence and /or identification of the immuno-captured bacteria.
  • Different specific media are known by the man skilled in the art, to identify specific bacteria, such as pathogenic bacteria.
  • the specific agar plates can be CHROMagarTM STEC or Rainbow® Agar or CT-SMAC (for E. coli 01 57: H7) or CT-RMAC (for E. coli 026).
  • the method may further comprise the step of extracting DNA from the immuno-captured bacteria, and detecting specific (pathogenic) genes by polymerase chain reaction or molecular isothermal amplification techniques. After enrichment of the pathogenic bacteria population (culturing step), sufficient amount of DNA can be extracted to identify the specific genes. For instance, sufficient amount of DNA can be extracted to identify the specific genes of intimin and/ or stx1/stx2, and specific genes of serogroups.
  • a step of nonselective or even selective enrichment of the bacteria population in the sample may be performed by culture in an appropriate culture medium.
  • This step allows an enrichment of the whole bacterial population, and therefore optimizes the odds to selectively immuno-capture target bacteria, such as pathogenic target bacteria that may be present in low amount in the sample, such as food samples.
  • This step is referred herein as a pre-enrichment step.
  • the pathogenic bacteria are from the species Shiga- Toxins producing Escherichia coli, Salmonella spp., Cronobacter sakazakii, or Listeria monocytogenes.
  • the microbiological detection of all these bacteria may require a specific or a non-specific pre-enrichment step.
  • the invention is also related to immunocapture of STEC of the serogroups 0157, 026, 045, 091 , 0103, 0104, 01 1 1 , 01 13, 0121 and 0145.
  • the specific antibody is chosen from the group consisting in: an antibody anti E. coli 0157, an antibody anti E.
  • an antibody anti E. coli 0111 an antibody anti E. coli 045, an antibody anti E. coli 091 , an antibody anti E. coli 0103, an antibody anti E. coli 0104, an antibody anti E. coli 01 13, an antibody anti E. coli 0121 and an antibody anti E. coli 0145.
  • the solid plastic support may be coated with at least one specific antibody (monoclonal or polyclonal) for immunocapture of the target bacteria, such as the pathogenic target bacteria.
  • the solid plastic support may be coated with at least two specific antibodies.
  • said support can be coated with two, three, four or more different antibodies, each antibody being specific of specific target bacteria, i.e. one pathogenic bacterium species or one pathogenic bacterium serogroup.
  • the invention is also related to a kit for detection of target bacteria, such as pathogenic target bacteria, comprising a solid plastic support coated with at least one specific antibody and reagents for culturing said bacteria.
  • the kit may further comprise reagents useful for the washing step.
  • Said kit is designated in particular for immuno-capture of pathogenic bacteria from the species Shiga-Toxins producing Escherichia coli, Salmonella spp. , Cronobacter sakazakii, or Listeria monocytogenes.
  • a classical protocol of immunocapture according to the invention can be summarized as shown in figure 14. As shown in figure 13, this new method of detection and amplification of pathogenic bacteria allows an advantageous decrease of the experimental time necessary for testing several food samples.
  • Figure 1 Variation of the coating concentration of anti E. coli 0157 IgG (2.5 or 10.0 ⁇ IgG/mL). Control is a non-coated microplate with IgG.
  • Figure 3 Variation of the incubation time of specific enrichment step in mTSB+Novobiocine (3 or 5 hours), with a coating concentration of 2.5 or 10.0 ⁇ g of IgG per mL . Control is a well without IgG.
  • Figure 4 Comparison of IMS Beads protocol and immunocapture-microplate method.
  • Figure 5. Comparison of the efficiency of commercial Captivate 0157 beads; Nexidia beads and Nexidia microplate for the capture of E. coli 0157: 1-17 in an enrichment broth contaminated at 10 5 CFU per mL.
  • Figure 7 Comparison of the quantity of E. coli 0157: 1-17 captured by Nexidia beads or Nexidia microplates when beads are incubated 5 hours in mTSB+N after the classic protocol of IMS.
  • Figure 8 Approach used for the validation of Nexidia immunocapture-microplate method on ground beef initially contaminated.
  • Figure 9 Comparison of the quantity of E. coli 0157 captured by Nexidia beads, Captivate 0157 or Nexidia microplates when a sample is contaminated with about 10 cells of E. coli 0157 before the enrichment step.
  • Figure 10. Detection of E. coli 026 with microplate - Control is a well without IgG.
  • Figure 1 1 Multiplex detection of two E. coli 0157 and 026 serogroups using the IMC method
  • Figure 1 Comparison of the experimental time necessary for testing 16 or 40 samples with magnetic beads or microplates.
  • E. coli 01 57 strain 10 CFU per mL with an E. coli 01 57 strain are performed in this enriched broth after the enrichment step. This broth is used to evaluate the performances of the method.
  • specific enumerations of E. coli 01 57/H7 or other serogroups were performed by plating IMS beads suspensions or immunocapture- microplate cultures (diluted or not in phosphate buffer saline) on specific agar media plate (Sorbitol MacConkey Agar media supplemented with CT-supplement or CHROMagarTM STEC) as shown in figure 1 5.
  • Results are represented in figures 1 and 2.
  • the coating of the wells with 2.5 ⁇ g/mL or 10.0 ⁇ g/mL allowed similar yield of E. coli 01 57 captured at the three levels of artificial contamination.
  • the contact time has an impact on capture efficiencies.
  • the contact time lasting one hour allows the higher yield of immunocapture of E. coli 01 57 and therefore seems to be optimal.
  • the other times tested allow acceptable levels of E. coli 01 57: 1-17 captured.
  • Figures 4 and 8 present the protocols. Two types of artificial contamination with E. coli 01 57: 1-17 were performed: (i) initial contamination of 25 g of ground beef with E. coli 01 57: 1-17 between 1 to 10 cells and (ii) contamination of the ground beef enrichment
  • Figures 5 and 9 present the comparative results obtained with (1 ) commercialized magnetic beads CaptivateTM 01 57 (LabM), (2) immunomagnetic beads coated with the same antibodies used for the microplate coating and (3) the immunocapture-microplate method.
  • concentrations of E. coli captured with the immunocapture-microplate method were higher than the concentration of E. coli 01 57: 1-17 obtained with the standard IMS method.
  • the differences of concentration vary between 1 log to 2.5 log according to the levels of E. coli 01 57: 1-17 contamination levels.
  • Figure 6 presents the protocol used to evaluate the performances of the 2 potential protocols using the immunocapture-microplate method.
  • the protocol used is the same than the one with the microplate 01 57 (contact between IgG and enrichment broth: 1 hour; incubation in mTSB+N: 5 hours). 2.5 and 10 ⁇ g/mL are concentrations of IgG used for the coating.
  • Results are presented in figure 10. These results confirm the potential of the immunocapture-microplate method for detection and isolation of STEC on food samples.
  • a microplate was coated with two antibodies (anti-01 57 and anti-026) at the same concentration.
  • a non specific enrichment step was performed from 25 g of ground beef during 18 hours at 37° C to simulate the development of the interfering flora.
  • Artificial contaminations with a E. coli 01 57: H7 strain and a E. coli 026 strain are performed in this enriched broth after the enrichment step at three ratio (10 3 026/10 5 01 57, 10 5 026/10 3 01 57 and 10 5 026/10 5 01 57 CFU per ml_). These three artificial contaminated broths are used to evaluate the performances of the method.
  • specific enumerations of E. coli 01 57/H7 and E. coli 026 were performed by plating immunocapture-microplate cultures on specific agar media plate (Sorbitol MacConkey Agar media supplemented with CT-supplement or CHROMagarTM STEC).
  • microplates were coated with one (anti- 01 57), two (anti-01 57 and anti-026) or three different antibodies (anti-01 57, anti-026 and anti-01 1 1 ) at the same concentration. Then, a non specific enrichment step was performed from 25 g of ground beef during 18 hours at 37° C to simulate the development of the interfering flora. Artificial contaminations at 10 3 , 10 5 or 10 7 CFU per mL with one E. coli 01 57: H7 strain were performed in this enriched broth after the enrichment step. This broth was used to evaluate the performances of the multiplex microplate.
  • a microplate was coated with protein-A at two concentrations (0.1 ug/ ⁇ . and 1 .0 ⁇ g/ ⁇ L).
  • a non specific enrichment step was performed from 25 g of ground beef during 18 hours at 37° C to simulate the development of the interfering flora.
  • Artificial contaminations with one E. coli 01 57: 1-17 strain are performed in this enriched broth after the enrichment step at two levels (10 3 and 10 5 CFU per ml_). These contaminated broths are used to evaluate the performances of the method.
  • a volume of 1 mL of the contaminated broth were inoculated with 2.5 ⁇ g of IgG anti- 01 57 and incubated at room temperature for 1 hour. After the antigen/antibody reaction, an aliquot (100 ⁇ _) of this mixture capture was introduced in well-coated protein A and incubated an additional 1 hour. After washing and in s/tu-culture, specific enumerations of E. coli 01 57/H7 are performed by plating immunocapture-microplate cultures on specific agar media plate (Sorbitol MacConkey Agar media supplemented with CT-supplement). In parallel, similar assays are performed using the standard microplate protocol (microplated coated with IgG anti-01 57).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé destiné à capturer et à concentrer des bactéries visées dans un échantillon. Le procédé comporte une immunocapture des bactéries visées à l'aide d'au moins un anticorps spécifique auxdites bactéries visées, l'anticorps étant fixé sur un support en plastique solide. Les bactéries visées capturées sont ensuite cultivées sur le support en plastique solide.
PCT/EP2014/055348 2013-03-15 2014-03-17 Procédés d'immunocapture et de concentration de bactéries dans un échantillon WO2014140381A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201361786733P 2013-03-15 2013-03-15
EP13305301 2013-03-15
US61/786,733 2013-03-15
EP13305301.7 2013-03-15

Publications (1)

Publication Number Publication Date
WO2014140381A1 true WO2014140381A1 (fr) 2014-09-18

Family

ID=48698973

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2014/055348 WO2014140381A1 (fr) 2013-03-15 2014-03-17 Procédés d'immunocapture et de concentration de bactéries dans un échantillon

Country Status (1)

Country Link
WO (1) WO2014140381A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107167598A (zh) * 2017-04-28 2017-09-15 东北农业大学 一种快速检测阪崎克罗诺杆菌的试剂盒及其应用
CN110632302A (zh) * 2019-10-30 2019-12-31 中国农业科学院农产品加工研究所 一种同时检测待测样品中大肠杆菌和沙门氏菌的含量的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1253203A1 (fr) * 2001-04-25 2002-10-30 Becton Dickinson and Company Germination, développement, immobilisation et détéction rapide des microorganismes
CN101133164A (zh) * 2004-07-02 2008-02-27 海军秘书处代表的美国政府 再测序病原菌微阵列

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1253203A1 (fr) * 2001-04-25 2002-10-30 Becton Dickinson and Company Germination, développement, immobilisation et détéction rapide des microorganismes
CN101133164A (zh) * 2004-07-02 2008-02-27 海军秘书处代表的美国政府 再测序病原菌微阵列

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BYRNE BARRY ET AL: "Antibody-based sensors: principles, problems and potential for detection of pathogens and associated toxins.", SENSORS (BASEL, SWITZERLAND) 2009, vol. 9, no. 6, 2009, pages 4407 - 4445, XP002714546, ISSN: 1424-8220 *
HUEBNER I ET AL: "RAPID DETERMINATION OF MEMBERS OF THE FAMILY ENTEROBACTERIACEAE IN DRINKING WATER BY AN IMMUNOLOGICAL ASSAY USING A MONOCLONAL ANTIBODY AGAINST ENTEROBACTERIAL COMMON ANTIGEN", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 58, no. 9, 1 September 1992 (1992-09-01), pages 3187 - 3191, XP008049650, ISSN: 0099-2240 *
PINA M. FRATAMICO ET AL: "Detection of Shiga toxin-producing Escherichia coli in ground beef using the GeneDisc real-time PCR system", FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY, vol. 2, 1 January 2012 (2012-01-01), XP055082962, DOI: 10.3389/fcimb.2012.00152 *
SANDSTROM G E ET AL: "Duct ELISA for detection of bacteria in fluid samples", JOURNAL OF MICROBIOLOGICAL METHODS, ELSEVIER, AMSTERDAM, NL, vol. 5, no. 1, 1 May 1986 (1986-05-01), pages 41 - 47, XP023988666, ISSN: 0167-7012, [retrieved on 19860501], DOI: 10.1016/0167-7012(86)90022-9 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107167598A (zh) * 2017-04-28 2017-09-15 东北农业大学 一种快速检测阪崎克罗诺杆菌的试剂盒及其应用
CN107167598B (zh) * 2017-04-28 2019-02-26 东北农业大学 一种快速检测阪崎克罗诺杆菌的试剂盒及其应用
CN110632302A (zh) * 2019-10-30 2019-12-31 中国农业科学院农产品加工研究所 一种同时检测待测样品中大肠杆菌和沙门氏菌的含量的方法

Similar Documents

Publication Publication Date Title
De Boer et al. Methods for the detection and isolation of Shiga toxin‐producing Escherichia coli
Gupta et al. Detection of Yersinia enterocolitica in food: an overview
Weagant et al. An improved rapid technique for isolation of Escherichia coli O157: H7 from foods
Deisingh et al. Detection of infectious and toxigenic bacteria
Feng Commercial assay systems for detecting foodborne Salmonella: a review
RU2265061C2 (ru) Способ выявления бактерий и набор для его осуществления
Chapman et al. Use of commercial enzyme immunoassays and immunomagnetic separation systems for detecting Escherichia coli O157 in bovine fecal samples
JP2779509B2 (ja) 検出方法
CN101971032B (zh) 在液体培养基中通过凝集实时检测微生物的方法
Vermunt et al. Isolation of salmonellas by immunomagnetic separation
Almeida et al. Detection of Escherichia coli O157 by peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) and comparison to a standard culture method
US5415997A (en) Method for detecting low levels of microorganisms
Clotilde et al. Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods
Paoli et al. Single-chain Fv antibody with specificity for Listeria monocytogenes
JPH1128099A (ja) 黄色ブドウ球菌の検出法
WO2005001475A3 (fr) Appareil et procede de detection d'organismes vivants microscopiques au moyen de bacteriophage
Morlay et al. Label-free immuno-sensors for the fast detection of Listeria in food
Pongsunk et al. Rapid identification of Burkholderia pseudomallei in blood cultures by a monoclonal antibody assay
Terao et al. Potential rapid and simple lateral flow assay for Escherichia coli O111
US6004766A (en) Method for detecting low levels of microorganisms
WO2014140381A1 (fr) Procédés d'immunocapture et de concentration de bactéries dans un échantillon
Rigby Enzyme-linked immunosorbent assay for detection of Salmonella lipopolysaccharide in poultry specimens
Chon et al. Advanced methods for isolating from and confirming Campylobacter spp. in milk and dairy products
Chapman et al. Evaluation of a commercial enzyme immunoassay (EHEC-Tek) for detectingEscherichia coliO157 in beef and beef products
US20150079597A1 (en) Method for capturing and concentrating a microorganism in a biological sample

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14710330

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14710330

Country of ref document: EP

Kind code of ref document: A1