CDC: CDC - E. coli and Shigella: E. coli and Shigella in Isolates by Real-time Polymerase Chain Reaction
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Official Method Name
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Detection of Diarrheagenic Eshcerichia coli and Shigella Using LightCycler |
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Current Revision
| 1999 |
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Media
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WATER |
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Instrumentation
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Polymerase Chain Reaction (PCR) instrument |
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Method Subcategory
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Microbiological |
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Method Source
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Citation
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Hyytia-Trees, Eija, CDC Laboratory Assay: "Detection of Diarrheagenic Eshcerichia coli and Shigella Using LightCycler®", E.coli, Shigella, Yersinia, and Vibrio Laboratory, Foodborne and Diarreheal Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA |
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Brief Method Summary
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Procedures are described for analysis of isolates and may be adapted for assessment of solid, particulate, aerosol, liquid, and water samples. The assay uses real-time PCR for identification of Shigella. Cell lysate templates are prepared by suspending a portion of a colony in 300 µL of distilled water and boiling for 10 minutes. After centrifugation at 4,500 rpm for 2 minutes, 1 µL of the supernatant is used in the PCR reaction. Alternatively, DNA may be purified using a commercially available kit or automated DNA extraction system. PCR is performed on a LightCycler® using primers and probes designed for the ipaH-plasmid, which encodes the invasive plasmid antigen H. This gene can be found on both the chromosome and a plasmid for Shigella spp. |
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Scope and Application
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Applicable Concentration Range
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Interferences
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Quality Control Requirements
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At a minimum, the following QC checks should be performed and evaluated before using this protocol: positive control, negative control, and blank. PCR QC checks should be performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples document. |
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Sample Handling
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Maximum Holding Time
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Relative Cost
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Unknown |
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Sample Preparation Methods
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