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Dwivedi H.P.,North Carolina State University | Dwivedi H.P.,Biomerieux Inc. | Smiley R.D.,North Carolina State University | Smiley R.D.,U.S. Food and Drug Administration | Jaykus L.-A.,North Carolina State University
Applied Microbiology and Biotechnology | Year: 2010

The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K d value) of 292.8∈±∈53.1 nM with 47.27∈±∈5.58% cells fluorescent (bound) in a 1.48-μM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25-36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1-5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets. © 2010 Springer-Verlag.

The present invention is directed to a method for separating, characterizing and/or identifying microorganisms in a test sample. The method of the invention comprises an optional lysis step for lysing non-microorganism cells that may be present in a test sample, followed by a subsequent separation step. The method may be useful for the separation, characterization and/or identification of microorganisms from complex samples such as blood-containing culture media. The method may also be useful for the physical separation and/or enrichment of two or more different or individual microorganism species contained in a mixed test sample. The invention further provides for spectroscopic interrogation of the separated microorganism sample(s) to produce measurements of the microorganism and characterizing and/or identifying the microorganism(s) in the sample using said spectroscopic measurements.

The present invention is directed to a method for inactivation and/or extraction of fungus samples (e.g., mold or yeast samples), the method comprising the following sequential steps: (a) acquiring a test sample known to contain or that may contain fungus and suspending the test sample in a container containing water and/or suspension medium; (b) adding ethanol to the suspension; (c) centrifuging the container to pellet the fungus and removing and discarding the supernatant; (d) resuspending the fungus in formic acid; (e) adding acetonitrile to the sample; (f) centrifuging the sample; and (g) recovering the supernatant. In accordance with the present invention, the recovered supernatant can be subjected to mass spectrometry analysis for characterization and/or identification of the unknown fungus.

Pfaller M.A.,University of Iowa | Andes D.,University of Wisconsin - Madison | Arendrup M.C.,Statens Serum Institute | Diekema D.J.,University of Iowa | And 11 more authors.
Diagnostic Microbiology and Infectious Disease | Year: 2011

We reassessed the Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints (CBPs) for voriconazole. We examined i) the essential (EA: ±2 dilutions) and categorical agreement between 24-h CLSI and EUCAST methods for voriconazole testing of Candida, ii) wild-type (WT) MICs and epidemiologic cutoff values (ECVs) for voriconazole by both CLSI and EUCAST methods, and iii) correlation of MICs with outcomes from previously published data using CLSI methods. We applied these findings to propose new 24-h species-specific CLSI CBPs. Adjusted 24-h CBPs for voriconazole and C. albicans, C. tropicalis, and C. parapsilosis (susceptible, ≤0.125 μg/mL; intermediate, 0.25-0.5 μg/mL; resistant, ≥1 μg/mL) should be more sensitive for detecting emerging resistance among common Candida species and provide consistency with EUCAST CBPs. In the absence of CBPs for voriconazole and C. glabrata (and less common species), we recommend that their respective ECVs be used to detect the emergence of non-WT strains. © 2011 Elsevier Inc.

Spinali S.,bioMerieux | Van Belkum A.,bioMerieux | Goering R.V.,Creighton University | Girard V.,bioMerieux | And 5 more authors.
Journal of Clinical Microbiology | Year: 2015

The integration of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology has revolutionized species identification of bacteria, yeasts, and molds. However, beyond straightforward identification, the method has also been suggested to have the potential for subspecies-level or even type-level epidemiological analyses. This minireview explores MALDI-TOF MS-based typing, which has already been performed on many clinically relevant species. We discuss the limits of the method's resolution and we suggest interpretative criteria allowing valid comparison of strain-specific data. We conclude that guidelines for MALDI-TOF MS-based typing can be developed along the same lines as those used for the interpretation of data from pulsed-field gel electrophoresis (PFGE). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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