The Experts below are selected from a list of 3303 Experts worldwide ranked by ideXlab platform
Kent J Voorhees - One of the best experts on this subject based on the ideXlab platform.
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detection of escherichia coli using Immunomagnetic Separation and bacteriophage amplification coupled with matrix assisted laser desorption ionization time of flight mass spectrometry
Rapid Communications in Mass Spectrometry, 2003Co-Authors: Angelo J Madonna, Sheila Van Cuyk, Kent J VoorheesAbstract:The application of whole cell analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has emerged as a valuable tool for rapidly identifying/detecting bacteria. This technique requires minimal sample preparation and is simple to perform, but is generally limited to purified samples of bacteria at concentrations greater than 1.0 × 106 cells/mL. In this paper, we describe a bacterial detection method that integrates Immunomagnetic Separation with bacteriophage amplification prior to MALDI-MS analysis. The developed method consists of three main stages: (1) isolation of a target bacterium by Immunomagnetic Separation; (2) infection of the immuno-captured bacterium with a lytic bacteriophage; and (3) assay of infected medium for bacteriophage progeny using MALDI-MS to produce a molecular weight signal for the virus capsid protein. With this technique, the presence of Escherichia coli in broth was determined in less then 2 h total analysis time at a concentration of ∼5.0 × 104 cells/mL. Copyright © 2002 John Wiley & Sons, Ltd.
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detection of bacteria from biological mixtures using Immunomagnetic Separation combined with matrix assisted laser desorption ionization time of flight mass spectrometry
Rapid Communications in Mass Spectrometry, 2001Co-Authors: Angelo J Madonna, Franco Basile, Edward T Furlong, Kent J VoorheesAbstract:A rapid method for identifying specific bacteria from complex biological mixtures using Immunomagnetic Separation coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been developed. The technique employs commercially available magnetic beads coated with polycolonal antibodies raised against specific bacteria and whole cell analysis by MALDI-MS. A suspension of a bacterial mixture is mixed with the Immunomagnetic beads specific for the target microorganism. After a short incubation period (20 mins) the bacteria captured by the beads are washed, resuspended in deionized H2O and directly applied onto a MALDI probe. Liquid suspensions containing bacterial mixtures can be screened within 1 h total analysis time. Positive tests result in the production of a fingerprint mass spectrum primarily consisting of protein biomarkers characteristic of the targeted microorganism. Using this procedure, Salmonella choleraesuis was isolated and detected from standard bacterial mixtures and spiked samples of river water, human urine, and chicken blood. Copyright © 2001 John Wiley & Sons, Ltd.
Angelo J Madonna - One of the best experts on this subject based on the ideXlab platform.
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detection of escherichia coli using Immunomagnetic Separation and bacteriophage amplification coupled with matrix assisted laser desorption ionization time of flight mass spectrometry
Rapid Communications in Mass Spectrometry, 2003Co-Authors: Angelo J Madonna, Sheila Van Cuyk, Kent J VoorheesAbstract:The application of whole cell analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has emerged as a valuable tool for rapidly identifying/detecting bacteria. This technique requires minimal sample preparation and is simple to perform, but is generally limited to purified samples of bacteria at concentrations greater than 1.0 × 106 cells/mL. In this paper, we describe a bacterial detection method that integrates Immunomagnetic Separation with bacteriophage amplification prior to MALDI-MS analysis. The developed method consists of three main stages: (1) isolation of a target bacterium by Immunomagnetic Separation; (2) infection of the immuno-captured bacterium with a lytic bacteriophage; and (3) assay of infected medium for bacteriophage progeny using MALDI-MS to produce a molecular weight signal for the virus capsid protein. With this technique, the presence of Escherichia coli in broth was determined in less then 2 h total analysis time at a concentration of ∼5.0 × 104 cells/mL. Copyright © 2002 John Wiley & Sons, Ltd.
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detection of bacteria from biological mixtures using Immunomagnetic Separation combined with matrix assisted laser desorption ionization time of flight mass spectrometry
Rapid Communications in Mass Spectrometry, 2001Co-Authors: Angelo J Madonna, Franco Basile, Edward T Furlong, Kent J VoorheesAbstract:A rapid method for identifying specific bacteria from complex biological mixtures using Immunomagnetic Separation coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been developed. The technique employs commercially available magnetic beads coated with polycolonal antibodies raised against specific bacteria and whole cell analysis by MALDI-MS. A suspension of a bacterial mixture is mixed with the Immunomagnetic beads specific for the target microorganism. After a short incubation period (20 mins) the bacteria captured by the beads are washed, resuspended in deionized H2O and directly applied onto a MALDI probe. Liquid suspensions containing bacterial mixtures can be screened within 1 h total analysis time. Positive tests result in the production of a fingerprint mass spectrum primarily consisting of protein biomarkers characteristic of the targeted microorganism. Using this procedure, Salmonella choleraesuis was isolated and detected from standard bacterial mixtures and spiked samples of river water, human urine, and chicken blood. Copyright © 2001 John Wiley & Sons, Ltd.
A Rodriguez - One of the best experts on this subject based on the ideXlab platform.
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evaluation of the efficacy of Immunomagnetic Separation for the detection of clavibacter michiganensis subsp michiganensis in tomato seeds
Journal of Applied Microbiology, 2008Co-Authors: L De Leon, A Rodriguez, M M Lopez, Felipe SiverioAbstract:Aims: To evaluate the effectiveness of the optimized Immunomagnetic Separation (IMS)-plating protocol in relation to other culture, serological and molecular techniques currently used for Clavibacter michiganensis subsp. michiganensis in seed-testing laboratories. Methods and results: Bacterial suspensions, tomato seed extracts spiked with the pathogen and naturally infected seeds were IMS-plated for the detection of C. m. subsp. michiganensis. These results were compared with plating on general (YPGA) and semiselective (mSCM) media, double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunofluorescent assay (IF) or polymerase chain reaction (PCR). Different seed lots and pathogen strains were also tested. IMS-plating allowed the detection of less than 10 CFU ml−1 of pathogen in all assayed samples. The mSCM medium provided positive results for 10 CFU ml−1 in naturally infected seeds, but up to 14 days was necessary for the typical colonies of the target to be come visible. By serological techniques, 103 and up to 104 CFU ml−1 were detected by IF and ELISA, respectively. DNA extraction was required to obtain positive results by PCR in seed extracts containing 103 CFU ml−1 or more. Conclusions: Among the evaluated methods, IMS-plating provided the best results regarding sensitivity and specificity for C. m. subsp. michiganensis detection, allowing the recovery of viable bacteria from seed extracts. Significance and impact of the study: IMS-plating increases isolation rates of C. m. subsp. michiganensis and could improve standard protocols currently used for routine analysis.
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detection of clavibacter michiganensis subsp michiganensis in tomato seeds using Immunomagnetic Separation
Journal of Microbiological Methods, 2006Co-Authors: L De Leon, Felipe Siverio, A RodriguezAbstract:Abstract The use of pathogen-free plant material is the main strategy for controlling bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis . However, detection and isolation of this pathogen from seeds before field or greenhouse cultivation is difficult when the bacterium is at low concentration and associated microbiota are present. Immunomagnetic Separation (IMS), based on the use of Immunomagnetic beads (IMBs) coated with specific antibodies, was used to capture C. michiganensis subsp. michiganensis cells, allowing removal of non-target bacteria from samples before plating on non-selective medium. Different concentrations of IMBs and of two antisera were tested, showing that IMS with 10 6 IMBs/ml coated with a polyclonal antiserum at 1/3200 dilution recovered more than 50% of target cells from initial inocula of 10 3 to 10 0 CFU/ml. Threshold detection was lower than 10 CFU/ml even in seed extracts containing seed debris and high populations of non-target bacteria. The IMS permitted C. michiganensis subsp. michiganensis isolation from naturally infected seeds with higher sensitivity and faster than direct isolation on the semiselective medium currently used and could become a simple viable system for routinely testing tomato seed lots in phytosanitary diagnostic laboratories.
Felipe Siverio - One of the best experts on this subject based on the ideXlab platform.
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evaluation of the efficacy of Immunomagnetic Separation for the detection of clavibacter michiganensis subsp michiganensis in tomato seeds
Journal of Applied Microbiology, 2008Co-Authors: L De Leon, A Rodriguez, M M Lopez, Felipe SiverioAbstract:Aims: To evaluate the effectiveness of the optimized Immunomagnetic Separation (IMS)-plating protocol in relation to other culture, serological and molecular techniques currently used for Clavibacter michiganensis subsp. michiganensis in seed-testing laboratories. Methods and results: Bacterial suspensions, tomato seed extracts spiked with the pathogen and naturally infected seeds were IMS-plated for the detection of C. m. subsp. michiganensis. These results were compared with plating on general (YPGA) and semiselective (mSCM) media, double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunofluorescent assay (IF) or polymerase chain reaction (PCR). Different seed lots and pathogen strains were also tested. IMS-plating allowed the detection of less than 10 CFU ml−1 of pathogen in all assayed samples. The mSCM medium provided positive results for 10 CFU ml−1 in naturally infected seeds, but up to 14 days was necessary for the typical colonies of the target to be come visible. By serological techniques, 103 and up to 104 CFU ml−1 were detected by IF and ELISA, respectively. DNA extraction was required to obtain positive results by PCR in seed extracts containing 103 CFU ml−1 or more. Conclusions: Among the evaluated methods, IMS-plating provided the best results regarding sensitivity and specificity for C. m. subsp. michiganensis detection, allowing the recovery of viable bacteria from seed extracts. Significance and impact of the study: IMS-plating increases isolation rates of C. m. subsp. michiganensis and could improve standard protocols currently used for routine analysis.
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detection of clavibacter michiganensis subsp michiganensis in tomato seeds using Immunomagnetic Separation
Journal of Microbiological Methods, 2006Co-Authors: L De Leon, Felipe Siverio, A RodriguezAbstract:Abstract The use of pathogen-free plant material is the main strategy for controlling bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis . However, detection and isolation of this pathogen from seeds before field or greenhouse cultivation is difficult when the bacterium is at low concentration and associated microbiota are present. Immunomagnetic Separation (IMS), based on the use of Immunomagnetic beads (IMBs) coated with specific antibodies, was used to capture C. michiganensis subsp. michiganensis cells, allowing removal of non-target bacteria from samples before plating on non-selective medium. Different concentrations of IMBs and of two antisera were tested, showing that IMS with 10 6 IMBs/ml coated with a polyclonal antiserum at 1/3200 dilution recovered more than 50% of target cells from initial inocula of 10 3 to 10 0 CFU/ml. Threshold detection was lower than 10 CFU/ml even in seed extracts containing seed debris and high populations of non-target bacteria. The IMS permitted C. michiganensis subsp. michiganensis isolation from naturally infected seeds with higher sensitivity and faster than direct isolation on the semiselective medium currently used and could become a simple viable system for routinely testing tomato seed lots in phytosanitary diagnostic laboratories.
L De Leon - One of the best experts on this subject based on the ideXlab platform.
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evaluation of the efficacy of Immunomagnetic Separation for the detection of clavibacter michiganensis subsp michiganensis in tomato seeds
Journal of Applied Microbiology, 2008Co-Authors: L De Leon, A Rodriguez, M M Lopez, Felipe SiverioAbstract:Aims: To evaluate the effectiveness of the optimized Immunomagnetic Separation (IMS)-plating protocol in relation to other culture, serological and molecular techniques currently used for Clavibacter michiganensis subsp. michiganensis in seed-testing laboratories. Methods and results: Bacterial suspensions, tomato seed extracts spiked with the pathogen and naturally infected seeds were IMS-plated for the detection of C. m. subsp. michiganensis. These results were compared with plating on general (YPGA) and semiselective (mSCM) media, double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunofluorescent assay (IF) or polymerase chain reaction (PCR). Different seed lots and pathogen strains were also tested. IMS-plating allowed the detection of less than 10 CFU ml−1 of pathogen in all assayed samples. The mSCM medium provided positive results for 10 CFU ml−1 in naturally infected seeds, but up to 14 days was necessary for the typical colonies of the target to be come visible. By serological techniques, 103 and up to 104 CFU ml−1 were detected by IF and ELISA, respectively. DNA extraction was required to obtain positive results by PCR in seed extracts containing 103 CFU ml−1 or more. Conclusions: Among the evaluated methods, IMS-plating provided the best results regarding sensitivity and specificity for C. m. subsp. michiganensis detection, allowing the recovery of viable bacteria from seed extracts. Significance and impact of the study: IMS-plating increases isolation rates of C. m. subsp. michiganensis and could improve standard protocols currently used for routine analysis.
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detection of clavibacter michiganensis subsp michiganensis in tomato seeds using Immunomagnetic Separation
Journal of Microbiological Methods, 2006Co-Authors: L De Leon, Felipe Siverio, A RodriguezAbstract:Abstract The use of pathogen-free plant material is the main strategy for controlling bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis . However, detection and isolation of this pathogen from seeds before field or greenhouse cultivation is difficult when the bacterium is at low concentration and associated microbiota are present. Immunomagnetic Separation (IMS), based on the use of Immunomagnetic beads (IMBs) coated with specific antibodies, was used to capture C. michiganensis subsp. michiganensis cells, allowing removal of non-target bacteria from samples before plating on non-selective medium. Different concentrations of IMBs and of two antisera were tested, showing that IMS with 10 6 IMBs/ml coated with a polyclonal antiserum at 1/3200 dilution recovered more than 50% of target cells from initial inocula of 10 3 to 10 0 CFU/ml. Threshold detection was lower than 10 CFU/ml even in seed extracts containing seed debris and high populations of non-target bacteria. The IMS permitted C. michiganensis subsp. michiganensis isolation from naturally infected seeds with higher sensitivity and faster than direct isolation on the semiselective medium currently used and could become a simple viable system for routinely testing tomato seed lots in phytosanitary diagnostic laboratories.