There is no terrestrial rabies in the UK but there are related viruses in bats
Rapid antemortem diagnosis ensures appropriate patient management
There are 55,000 deaths per year worldwide. Unfortunately 50% of these are in children under 15yrs. Mainly Asia and Africa. Post exposure treatment is available.
A reverse transcription loop mediated isothermal amplification (RT-LAMP) assay was designed to detect Classical rabies virus. The assay is performed at 68oC in a single tube containing both the reverse transcriptase (Thermoscript; Invitrogen) and the polymerase.
The assay was tested using a Genie I machine with enzymology from OptiGene. Using “tagged” loop primers detection of LAMP products was performed on lateral flow devices. Samples from Ghana, Morocco, Nigeria, Kenya, Botswana, South Africa, Turkey and Pakistan were tested from various species: human, dog, bat, jackal, mongoose and goat
The RT-LAMP was extremely fast using the enzymes from OptiGene with most signals being positive in under 20 minutes. Using the enzymes from OptiGene produced a faster result than other suppliers assessed.
Temperature annealing analysis post-amplification allows reliable identification of product – confirming a positive result. Using lateral flow devices to capture tagged LAMP products it was possible to detect positive rabies in dog brain samples from Ghana.
It was possible to detect classical rabies from a wide geographical area covering the two clades of virus (cosmopolitan and Arctic-like). RT-LAMP appears to be 1log less sensitive than the RT-PCR1 currently employed for detection of rabies virus at VLA but takes a fraction of the time.
Figure 1: Fluorescence plots of positive and negative classical rabies samples
Figure 2: Detection of the LAMP products form 10 Ghanaian positive dog samples using lateral flow devices
RT-LAMP produces extremely rapid positive signals using purified rabies RNA from clinical samples and isolated virus
The use of lateral flow devices allows detection of LAMP products in an inexpensive manner
Using enzymology from OptiGene the rabies assay performs well and a positive signal is produced more rapidly than with other enzymes assessed
The OptiGene instrument (Genie I) allows for the acquisition of a very large number of data points during the run and annealing analysis is possible confirming the identity of product
1 Wakeley, P.R., et al. 2005. Development of a Real-Time, TaqMan Reverse Transcription- PCR Assay for the Detection and Differentiation of Lyssavius Genotypes 1, 5 and 6. J. Clin. Micro. 43: 2786-2792.
Acknowledgements: Professor Tony Fooks and other members of the Rabies and Wildlife Zoonosis Group; gifts of viral strains from kind collaborators from around world.