Researchers sought to develop a fast, reliable, and inexpensive procedure for the extraction, amplification, and detection of rabies virus RNA with the aim to develop faster tests for a rabies diagnosis.
Though simple and inexpensive tests for the rabies virus are available, they take a long time to develop the results and they are very dependent on high-quality samples that are sometimes not available. They are not ideal for diagnostic purposes, especially in areas where rabies is prevalent and resources are limited. As such, reliable methods of quickly detecting the virus in animals and in humans may ease the burden of rabies through increased rabies diagnosis, leading to timely and appropriate intervention. RT-qPCR and RT-RPA, two quick, relatively simple and inexpensive techniques, have shown to be effective for the detection of a number of viruses. Their applicability to rabies screening is, however unknown.
In a recent study published in the Virology Journal, researchers sought to design and assess a procedure for the detection of the rabies virus. Brain samples from 55 animals confirmed to have rabies or a related virus and eight from animals confirmed to be virus-free were used for experiments. Various methods of RNA extraction, RNA amplification and detection, and their combinations were compared in the development of the procedure.
Manual and automated RNA extraction methods, which separate certain virus-derived components (vRNA) from the rest of a sample, were assessed based on their efficiency and their ability to consistently yield detectible virus-derived components. RNA amplification and detection methods, which multiply vRNA to amounts that can be worked with and subsequently identified, were assessed based on their ability to accurately confirm the presence or absence of the rabies virus. When analyzed individually, each RNA extraction method was measured against a reference extraction method and followed up by a manual reference RNA amplification and detection method. Conversely, when analyzed individually, each RNA amplification and detection method was complemented by a manual reference method. RNA extraction and RNA amplification and detection methods were also later paired and tested.
RNA Extraction Methods
- Most consistent
- 13% more effective than the reference method
- Fastest: required only 20 minutes for extraction compared to 45 for reference
- 18% more efficient than the reference method
RNA Amplification and Detection:
- Required the least amount of time to yield results (15 minutes)
- Greatest ability to identify vRNA
- Can recognize and copy vRNA at concentrations of one vRNA strand per one μL reaction
- No false positives or false negative identifications
- Can recognize and copy vRNA at concentrations of 1000 vRNA strand per one μL reaction
The accuracy of an RNA amplification and detection method was found to be unaffected by the extraction method with which it was paired. The combination of SXT and RT-RPA was identified as the quickest, most reliable, and most cost-effective pairing.
It should be noted that all three methods of RNA amplification and detection were highly efficient and accurate, with the lowest efficiency held by EZI at 97% and the highest number of false-negatives held by RT-RPA at 3%.
The study findings suggest a combination of SXT RNA extraction and RT-RPA RNA amplification and detection may be effective as a method for rabies diagnosis. Notably, the samples in this study were from the brains of deceased animals, which have higher concentrations of rabies vRNA than body regions more relevant to diagnosis in living patients, such as circulating blood. Normal and HighSpeed RT-qPCR each possess a far greater ability to amplify and detect vRNA than RT-RPA, and are able to detect concentrations closer to those found in blood. This may render these techniques as the most suitable for detection of rabies in humans. Further research will be required to determine the appropriateness of the evaluated techniques as diagnostic tools.
Written by Raishard Haynes, MBS
Reference: Schlottau, K. et al. (2017). Development of molecular confirmation tools for swift and easy rabies diagnostics. Virol. J. DOI 10.1186/s12985-017-0853-y