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Extract Preparation (extract + preparation)

Distribution by Scientific Domains

Life Sciences	40%

Selected Abstracts

Detection of Viable Rotaviruses in Shellfish by means of Cell Culture and Immunofluorescence Assay

JOURNAL OF FOOD SCIENCE, Issue 5 2002
C. S. Santos
ABSTRACT: The goal of this work was to examine the use of cell culture and immunofluorescence assays to detect viable rotaviruses in artificially seeded oyster meat and to determine if the method for oyster extracts preparation affects the viability of the viruses. Oyster tissues were seeded with rotavirus SA11, followed by tissue extract preparation. For cytopathogenicity assays (CPE), non-cytotoxic dilutions of seeded oyster extracts were inoculated in MA 104 cells. For immunofluorescence assays (IFA) the monoclonal antibody Mab M60 anti-VP7 capsid glycoprotein of rotavirus was used. For CPE tests, no significant difference between the oyster extracts inoculated with rotavirus before and after extract preparation was detected. Similar results have been observed in IFA assays and the recoveries of viable rotavirus obtained were close to 100%. [source]

Interlaboratory evaluation of two Reverse-transcriptase Polymeric Chain Reaction-based methods for detection of four fruit tree viruses

ANNALS OF APPLIED BIOLOGY, Issue 1 2009
S. Massart
Abstract Recent technological development of molecular methods has led to the proliferation of new rapid PCR or reverse-transcriptase (RT)-PCR-derived diagnostic tests for plant viruses. Nevertheless, for routine use, the reliability of all these new methods is not widely established and there is still an apprehension to adopt them in official diagnostic for certification of plant material. This is partly because of the lack of confidence in the obtained results and the poor knowledge on the reproducibility and limits of the RT-PCR protocols. There is a lack of information on the adequate risk assessment in the use of this new technology. An interlaboratory evaluation of two RT-PCR duplex protocols for the detection of four different fruit tree viruses was performed to address these questions. Identical samples were sent as crude extract preparation to each of the participant laboratories. Samples were coded to ensure a double-blind test. General principles of result analysis are described, for example calculation of parameters such as specificity, sensitivity, repeatability, reproducibility, likelihood ratios and post-test probabilities. These parameters and the integration of the protocols within official certification scheme are discussed. Finally, guidelines for researchers desirous of validating their new plant virus diagnostic protocols through interlaboratory evaluation are suggested. [source]

Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free system

BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006
Marcus E. Boyer
Abstract The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37°C, Fd accumulated to >450 µg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28°C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system. © 2006 Wiley Periodicals, Inc. [source]