CDC: CDC - V. cholerae: V. cholerae in Isolates by Real-time Polymerase Chain Reaction
|
Official Method Name
|
Detection of V. cholerae ctxA, 01 rfb, and 0139 rfb in Isolates by Real-time Polymerase Chain Reaction |
|---|---|
|
Current Revision
| 1999 |
|
Media
|
VARIOUS |
|
Instrumentation
|
Polymerase Chain Reaction (PCR) instrument |
|
Method Subcategory
|
Microbiological |
|
Method Source
|
|
|
Citation
|
Hyytia-Trees, Eija, CDC Laboratory Assay: "Triplex PCR for detection of toxigenic V. Cholerae O1 and O139 using SmartCycler®", Foodborne and Diarreheal Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA |
|
Brief Method Summary
|
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 V. cholerae. Cell lysate templates are prepared by suspending a portion of a colony in 300 µL of 0.01M Tris-EDTA (TE) buffer 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® as single target assays or on a SmartCycler® as multiplex PCR using primers and probes designed for the cholera toxin ctx gene, the O1 antigen O1rfb gene, and the O139 antigen O139 rfb gene. |
|
Scope and Application
|
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. |
|
Applicable Concentration Range
|
|
|
Interferences
|
|
|
Quality Control Requirements
|
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. |
|
Sample Handling
|
|
|
Maximum Holding Time
|
|
|
Relative Cost
|
Unknown |
|
Sample Preparation Methods
|
