EPA-OW: AstroRT-PCR: Astrovirus serotypes by real-time reverse transcription-PCR
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Official Method Name
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Development of an Astrovirus RT-PCR Detection Assay for Use with Conventional, Real-Time, and Integrated Cell Culture/RT-PCR. |
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Current Revision
| 2004 |
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Media
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WATER |
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Instrumentation
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Acoustic Velocity Meter |
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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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Canadian Journal of Microbiology. 2004. 50: 269 - 278 |
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Brief Method Summary
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Procedures are described for analysis of clinical samples and may be adapted for assessment of solid, particulate, aerosol, liquid, and water samples. The method detects eight astrovirus serotypes. The method is a real-time reverse transcription-PCR procedure optimized for use in a real-time PCR assay using an ABI Prism® 7000 and can be integrated with sample-cell culture (CaCo-2 cells) to enhance sensitivity. Water samples are collected by filtration (1MDS filter), and viruses are eluted using a beef extract solution (1.5%, pH 9.5). Viruses are concentrated using Celite® adsorption (pH 4.0), filtration, and Celite-elution with sodium phosphate (0.15 M, pH 9.0), followed by further concentration and processing to remove inhibitors (ultracentrifugation, solvent extraction, and molecular weight [MW]-exclusion filtration). Concentrated samples are analyzed directly or indirectly (following cell culture) by a two-step real-time reverse transcription-PCR (i.e., reverse transcription followed by real-time PCR) assay using astrovirus-specific primer sets. |
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Scope and Application
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This method is for the identification of astrovisurs in water and is categorized as a screening procedure because it has not been officially validated. Determination of astrovirus serotypes by real-time reverse transcription-PCR procedure optimized for use in a real-time PCR assay using an ABI Prism® 7000 and can be integrated with sample-cell culture (CaCo-2 cells) to enhance sensitivity. |
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Applicable Concentration Range
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Interferences
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May have extraction interfences by non-target compounds. |
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Quality Control Requirements
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SAM lists these procedures for detection in solid, particulate, aerosol, liquid, and water samples. Further research is needed to develop and standardize the procedures for environmental sample types. At a minimum, the following QC checks should be performed and evaluated when using this protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to ensure reliability of the analytical results should also be performed. 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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