Indicators for Waterborne Pathogens (2004) / Chapter Skim
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Appendix C: Detection Technologies
Appendix C: Detection Technologies
Pages 287-307
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From page 287... ...
, DNA Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling (Baldi and Hatfield, 2002) , and a review article, "Rapid Methods and Automation in Microbiology" in the inaugural issue of Comprehensive Reviews in Food Science and Food Safety published by Institute of Food Technologists (Fung, 2002)
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From page 288... ...
Membrane filtration of turbid surface water can be problematic and a labor-intensive Multiple Tube Fermentation method is often used. Detection of enteric protozoa and viruses requires analysis of much larger volumes of water than the 100 mL typically used for bacterial analysis because of their lower expected concentrations.
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From page 289... ...
Viruses in this fluid may be assayed directly, but they are usually further concentrated by acid precipitation or by polyethylene glycol precipitation. Two classes of adsorbent filters have been used to concentrate enteric viruses from freshwater and sewage effluents: negatively charged and positively charged filters.
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From page 290... ...
. CONVENTIONAL AGAR PLATE METHOD, MPN METHODS, AND IMPROVED METHODS FOR MONITORING MICROORGANISMS IN WATER Viable Cell Count Methods and Related Technologies Viable Cell Count Use of selective and nonselective agars for growing live bacteria, yeasts, and molds requires water sampling, sample dilution, application of samples into petri
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From page 291... ...
. "Real-Time" Instruments for Viable Cell Count Viable cell counts can be made with the following real-time instruments: · direct epifluorescent filter technique involves vital dyes to stain live bacteria for obtaining viable cell counts in about one hour using fluorescent microscopy;
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Miniaturized Viable Cell Count Methods Using Microtiter Plates and Spot Plating Miniaturized methods have been developed to reduce the volume of reagents and media used and include the following: · loop dilution and spot plating of liquid sample on agar surfaces; and · automated pipette for dilution and spot plating liquid sample on agar surfaces. Most Probable Number Method The MPN method is a reliable but laborious method that has been used internationally for about 100 years for coliform enumeration in water.
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In general, the larger the number of viable cells in the water sample, the shorter is the detection time of these systems. A scattergram of cells versus detection time is generated that has an inverse slope.
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From page 294... ...
, which measures the color change of an agar plug at the bottom of a chamber containing nutrient, dye, and the water sample; the uniqueness of the system is that the color compounds developed during microbial growth are diffused into the agar plug and the automatic measurement of color change by the instrument is done without the interference of particles in the sample. Measure of Gases Instruments that measure specific gas development include the following: · Bactec (Johnston Laboratories, Inc., Cockeysville, Md.)
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APPENDIX C 295 Immunochemical Techniques A powerful scheme for detecting specific antigens, including both soluble proteins and whole microorganisms, is immunoassays. In this approach, antibodies (either polyclonal or monoclonal)
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Nucleic Acid Detection Genetic methods, based on DNA or RNA analysis, have been rapidly developing in all areas of applied microbiology which include food, clinical, industrial, environmental, and water in the past 20 years. DNA Sequences A variety of strategies can be employed for DNA sequence analysis.
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If the target sequence of interest is present in the sample, it hybridizes via Watson-Crick nucleotide base pairing (i.e., to the complementary probe DNA sequence attached to the surface)
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used the time consuming and laborious electrophoresis to detect PCR products. Recently, the new BAX system combines DNA amplification and automated homogeneous detection to determine the presence or absence of specific targets like Salmonella.
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During the PCR amplification, the light emission from the reporter increases exponentially if the target sequence is present -- the final level being measured by spectrophotometry after termination of the PCR. Because amplification occurs only in the presence of a particular target sequence, TaqMan assays are of particular value for the detection of specific sequences and are useful for waterborne pathogen detection.
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From page 300... ...
Nucleic acid technologies are becoming increasingly sophisticated and powerful tools for applied microbiology and the detection of waterborne pathogens and indicator organisms. Microarray Technology Recently, much attention has been directed to the field of "gene chips" and "microchips," in which microarrays of sensors or detection elements are used to detect a great variety of molecules including waterborne and foodborne pathogens and indicator organisms (Ramsay, 1998)
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From page 301... ...
In addition, they are amenable to implementation in "lab-on-a-chip" devices. Mass Spectrometry An important new area for pathogen detection is mass spectrometry.
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An example of an alarm sensor would be a trigger sensor employed for detection of various biological agents. Triggers used for bio-detection typically comprise an ultraviolet light source used to excite an air or water sample.
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Biosensors employ a biological recognition element such as an enzyme, DNA sequence, receptor, or antibody to perform the recognition and are powerful tools in applied microbiology. Ivnitski et al.
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MPN procedures can be performed in aliquots of liquid media (non-selective and selective liquid media using the conventional five-tube or three-tube MPN methods and miniaturized MPN procedures using 96-well microtiter plates and computer interphase)
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Target microbes and molecules can be separated and detected by microfluidic, proteomic, genomic, microarray, and microchip technologies. Biosensors and mass spectrometry technologies can also be used to detect many target whole cells and cell components.
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1980. Poliovirus concentration from tap water with electropositive adsorbent filters.
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2002a. Design and evaluation of oligo nucleotide-microarray method for the detection of human intestinal bacteria in fecal samples.
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