The development of 'real-time' RT-PCR techniques allows the quantification of this RNA in 'real-time,' giving a relatively quick and reliable method for the measuring levels of viral RNA. PCR based techniques are not currently recommended by the WHO for routine post-mortem diagnosis of rabies. However, in laboratories with strict quality control procedures in place and demonstrable experience and expertise, these molecular techniques have been successfully applied for confirmatory diagnosis and epidemiological surveys. For these reasons, it is likely that international bodies will accept their use in the future for routine rabies diagnosis. Reverse transcription PCR has been reported to confirm rabies diagnosis intra-vitam in suspect human cases, when conventional diagnostic methods have failed and post-mortem material is not available (Box 1) [57] . Rabies virus RNA can be detected in a range of biological fluids and samples (e.g. saliva, CSF, tears, skin biopsy sample and urine). Owing to the intermittent shedding of virus, serial samples of fluids such as saliva and urine should be tested but negative results should not be used to exclude a diagnosis of rabies. All positive PCR results should be sequenced to confirm the origin of the virus and rule out possible contamination. In terms of the RNA concentrations in the brain, the sensitivity especially of nested or real-time PCRs may be beyond the threshold needed for routine post-mortem testing. Also, contamination of negative samples could lead to an unjustifiable administration of a high number of costly post exposure prophylaxis and would produce false data for the rabies surveillance. However, with the introduction of accreditation for laboratories, quality control measures are being implemented in a growing number of laboratories worldwide. Such quality controls for diagnostic rabies PCRs should encompass several measures, including the inclusion of appropriate positive, negative, and inhibition controls in assay runs. The consistency and the interassay reproducibility should also be ensured over time by monitoring performance. Only if laboratories meet the required standard [58] , can PCR fulfil its full potential. The use of PCR should not be restricted only as a confirmatory diagnostic test for decomposed samples but also as a powerful tool for virus typing Figure 2 . Microarray identification of rabies virus RNA prepared from a brain sample from a confirmed case of human rabies. Total RNA was extracted using TriZol (Invitrogen) and treated with DNase I prior to conversion to double stranded DNA [45] . Non-specific amplification was achieved using a DNA polymerase (Klen Taq, Sigma) and the products were labelled through binding of Alexa Fluor 555 reactive dye (Invitrogen) to amplicons. Labelled target DNA was denatured at 95uC and chilled on ice before dilution in hybridization buffer and addition to the microarray slide. Hybridization occurred at 50uC overnight. Slides were washed and the target-probe binding was captured using GenePix Pro 6.1 software (Molecular Devices). Statistical analysis of the data was conducted using DetectiV software [64] . doi:10.1371/journal.pntd.0000530.g002