The FDA authorizes the use of the Ebola virus (Zaire) Real Time RT-PCR (TaqMan®) assay or the EZ1 assay if infection with Ebola Zaire virus (detected in the West Africa outbreak in 2014) is suspected based on clinical and epidemiological screening criteria. The EZ1 assay allows the in vitro qualitative detection of viral RNA isolated from human and animal samples such as whole blood, serum, or plasma. It is ideal for use on specimens from days 3 to 10 after the onset of symptoms.
In a document entitled ‘Ebola Information Packet for International Laboratories‘, the CDC provides guidance on
- Specimen receiving, accession, and storage
- Sample inactivation and nucleic acid extraction
- Master mix preparation
- Reaction mixture and sample addition
- Amplification and detection
Among other things, the document includes detailed instructions on how the assay should be performed. Briefly, the main steps involved are:
- Inactivation of the virus (cell lysis)
- Placing of sample in a designated workplace
- Freezing of sample before assay set up
- Thawing of frozen sample
- Addition of reagent (chloroform)
- Mixing in a vortexer
- Incubating and control of specimen at room temperature
- Vortexing, centrifugation and further processing to make the sample ready for PCR testing
Proper vortexing is important to the successful performance of the assay, that is, to produce a uniform suspension. Also, care should be taken to avoid over-vortexing which can cause bubbles to form that disrupt fluorescence detection, and can reduce enzyme activity which could decrease amplification efficiency.
There are various models of mixers/rotators/shakers integrated with advanced technology available today. For instance, the BenchMixer Vortexer from Benchmark Scientific features Q-drive™ technology that ensures instant vortexing of even the largest sample sizes, including full 50ml tubes. Quick results are important for early diagnosis and care. The device is designed for quieter, more efficient and longer lasting operation. It also comes with cold room/incubator compatibility and spill-proof electronic controls.