Combination of immunomagnetic separation with flow cytometry for detection of Listeria monocytogenes

Hibi, Kyoko; Abe, Akihisa; Ohashi, Eiji; Mitsubayashi, Kohji; Ushio, Hideki; Hayashi, Tetsuhito; Ren, Hongyan; Endo, Hideki

doi:10.1016/j.aca.2006.03.022

Cited by 71 publications

(38 citation statements)

References 17 publications

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“…This enables concentration of the target cells from a mixed microflora, followed by FCM detection of the antibody-target-stain complex. Hibi et al (2006) reported the separation of viable Listeria monocytogenes from mixed cultures of pathogens using IMS followed by FCM analysis over range of 10 4 -10 8 CFU/ml. This assay was applied to analysis of cultures inoculated into seawater and involved use of a FITC-labelled primary antibody against L. monocytogenes followed by secondary antibody detection using a tetrameric antibody complex combined with magnetic beads.…”

Section: Immuno-fcm and Bacterial Enumerationmentioning

confidence: 99%

Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

Wilkinson

2018

Trends in Food Science & Technology

128

102

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Section: Immuno-fcm and Bacterial Enumerationmentioning

confidence: 99%

Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

Wilkinson

2018

Trends in Food Science & Technology

128

102

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“…The lowest initial contamination rate tested, i.e., 1 to 10 CFU per 25 g or ml, could be detected in chicken, infant formula, milk, and chocolate milk, meeting the required detection limit for Salmonella according to the methods of the USDA Microbiology Laboratory Guidebook (53) and the FDA Bacteriological Analytical Manual (54) and significantly reducing detection times, from 72 h to 24 h. HRP-conjugated LTF offers a rapid enzyme-linked sandwich detection assay. Colorimetric or fluorescent ELISA-based assays have been successfully combined with IMS for rapid identification and quantification of bacterial contaminations (12,14,15). We therefore assessed the functionality of the phage S16 LTF as a secondary probe to detect bead-bound Salmonella (Fig.…”

mentioning

confidence: 99%

Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells

Denyes

Dunne

Steiner

et al. 2017

Appl Environ Microbiol

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Bacteriophage-based assays and biosensors rival traditional antibodybased immunoassays for detection of low-level Salmonella contaminations. In this study, we harnessed the binding specificity of the long tail fiber (LTF) from bacteriophage S16 as an affinity molecule for the immobilization, enrichment, and detection of Salmonella. We demonstrate that paramagnetic beads (MBs) coated with recombinant gp37-gp38 LTF complexes (LTF-MBs) are highly effective tools for rapid affinity magnetic separation and enrichment of Salmonella. Within 45 min, the LTF-MBs consistently captured over 95% of Salmonella enterica serovar Typhimurium cells from suspensions containing from 10 to 10 5 CFU · ml Ϫ1 , and they yielded equivalent recovery rates (93% Ϯ 5%, n ϭ 10) for other Salmonella strains tested. LTF-MBs also captured Salmonella cells from various food sample preenrichments, allowing the detection of initial contaminations of 1 to 10 CFU per 25 g or ml. While plating of bead-captured cells allowed ultrasensitive but time-consuming detection, the integration of LTF-based enrichment into a sandwich assay with horseradish peroxidaseconjugated LTF (HRP-LTF) as a detection probe produced a rapid and easy-to-use Salmonella detection assay. The novel enzyme-linked LTF assay (ELLTA) uses HRP-LTF to label bead-captured Salmonella cells for subsequent identification by HRP-catalyzed conversion of chromogenic 3,3=,5,5=-tetramethylbenzidine substrate. The color development was proportional for Salmonella concentrations between 10 2 and 10 7 CFU · ml Ϫ1 as determined by spectrophotometric quantification. The ELLTA assay took 2 h to complete and detected as few as 10 2 CFU · ml Ϫ1 S. Typhimurium cells. It positively identified 21 different Salmonella strains, with no cross-reactivity for other bacteria. In conclusion, the phage-based ELLTA represents a rapid, sensitive, and specific diagnostic assay that appears to be superior to other currently available tests. IMPORTANCEThe incidence of foodborne diseases has increased over the years, resulting in major global public health issues. Conventional methods for pathogen detection can be laborious and expensive, and they require lengthy preenrichment steps. Rapid enrichment-based diagnostic assays, such as immunomagnetic separation, can reduce detection times while also remaining sensitive and specific. A critical component in these tests is implementing affinity molecules that retain the ability to specifically capture target pathogens over a wide range of in situ applications. The protein complex that forms the distal tip of the bacteriophage S16 long tail fiber is shown here to represent a highly sensitive affinity molecule for the specific enrichment and detection of Salmonella. Phage-encoded long tail fibers have huge potential for development as novel affinity molecules for robust and specific diagnostics of a vast spectrum of bacteria.

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“…Many studies have shown IMS to be a useful concentration and isolation method for bacteria (Favrin et al, 2003;Hibi et al, 2006;Power et al, 2003). Hibi et al, (2006) …”

Section: Sample Preparationmentioning

confidence: 99%

“…Concentration may involve immunomagnetic separation (IMS) which is used as a pretreatment and/ or pre-concentration step to capture and extract the target pathogen from a bacterial suspension using antibody-coated magnetic beads (Lazcka et al, 2007). Many studies have shown IMS to be a useful concentration and isolation method for bacteria (Favrin et al, 2003;Hibi et al, 2006;Power et al, 2003). Hibi et al, (2006) …”

Section: Sample Preparationmentioning

confidence: 99%

Application of Flow Cytometry to the Detection of Pathogenic Bacteria

Kennedy¹,

Wilkinson²

2017

Current Issues in Molecular Biology

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Outbreaks of infections have emphasized the necessity for rapid and economic detection methods for pathogens in samples ranging from those of clinical origin to food products during production and retail storage, and increasingly, in environmental samples. Flow cytometry (FCM) allows the rapid acquisition of multi-parametric data regarding cell populations within fluidised samples. However, the application of FCM to pathogen detection depends on the availability of specific fluorescent probes such as antibodies and RNA probes capable of detecting and isolating pathogens from these diverse samples. A particular issue for FCM methodology is the ability to recover and discriminate bacteria from the sample matrix which may pose a major technical hurdle towards accurate and sensitive analysis. This review article focuses on detection of pathogens using FCM in samples originating from food, water, environmental and clinical sources and outlines the current state of the art and potential future applications.

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Combination of immunomagnetic separation with flow cytometry for detection of Listeria monocytogenes

Cited by 71 publications

References 17 publications

Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells

Application of Flow Cytometry to the Detection of Pathogenic Bacteria

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