CDC: CDC - S. Typhi: Salmonella Typhi in Isolates by Real-time Polymerase Chain Reaction.
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Official Method Name
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Triplex PCR for Detection of Salmonella Typhi Using SmartCycler |
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Current Revision
| 1999 |
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Media
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VARIOUS |
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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: "Triplex PCR for detection of S. Typhi using SmartCycler®", 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 S. Typhi. 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, deoxyribonucleic acid (DNA) may be purified using a commercially available kit or automated DNA extraction system. PCR is performed on a SmartCycler® using primers and probes designed for the Vi capsular gene (viaB), the H antigen gene (fliC-d), and the tyvelose epimerase gene (tyv). This assay is also available for the LightCycler® platform as three single target PCRs. |
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Scope and Application
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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. |
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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 www.epa.gov/sam/pdfs/EPA-QA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. |
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Sample Handling
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Maximum Holding Time
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Relative Cost
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Less than $50 |
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Sample Preparation Methods
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